CLINICAL BACTERIOLOGY
WEEK 2 LECTURE
Introduction to Microbiology
 Study of microorganism (microscopic
organism)
 One of the microorganism is bacteria
 Magnification of microscope is only 30
to 266 times
History
Anton van Leeunwenhoek
 first observations of bacteria at the
microscopic level
 He was able to view the presence of
microorganism from rain water
 One of the first one who made
observation of bacteria at microscopic
level
 He was able to device a drawing of
bacteria way back in 1684
 He was consider as the father of
bacteriology and protozoology
 He was the first one also who
discovered and viewed the presence of
protozoan
Huygens
 free living protozoa
Robert Hooke (1667)
 filamentous microscopic fungi
 the mold form by fungi
 two forms of fungi: mold and yeast
 ang nakita ni robert hooke ay mold
Pietro Antonio Micheli
 described 900 species of molds
*during their time they believe that diseases
were cause by animals
Cicero and Fracastorius
 one of the scientist that theory is near
to the reality
 fevers might be caused by minute
animals (contagium vivum)
 contagium vivum- mga hindi nakikitang
creatures
 this theory is nearest that we know
today
 fever- is the reaction of the body, is an
inflammatory reaction of our body
towards an infection.
 Nagiging viable ang bacteria in 37
degree Celsius
Spontaneous Generation
Louis Pasteur
 when air is filtered through cotton
wool, large numbers of microorganisms
are held back.
 They believe that living things came
from non-living things
 Spontaneous generation is disapproved
by Louis Pasteur
 He conducted a study:
 Hindi mabubuhay ang microorganism
na walang papasok na oxygen base on
their theory dati. But having the swan
neck flask will prevent the entry of any
organism that will come from the
outside.
  Set up number and set up number 2
will only prove that living things do not
arise from non-living things
John Tyndall
 existence of heat-stable forms of
certain bacteria
 removal of which involved the process
of repeated heating and rest
 thyndalization- process of repeated
heating and resting
 where are some organism that can be
still viable even in extreme condition
(more than 37 degree celcius and more)
 nadiscover niya na merong bacteria na
heat stable. Kahit iboil hindi na
mamatay ang bacteria.
In history of microbiology there were
developments
To process
 Improvement in microscopes
 Development of methods for culturing
microorganisms
 Culturing- propagate the number of
bacteria in vitro using culture medium
 Culture medium- synthetic. Imitated the
nutrients need for the growth of
bacteria
 Culture medium- to identify their
features of bacteria
 Bacteria usually thriving in human body
(most of the important medically
bacteria which is normal flora or
normal microbiota), environment
(water, soil) and animals.
 Normal flora- this are bacteria that are
thriving in our body but they are not
causing a diseases unless they become
pathogens. When they are present in
our normal state hindi sila nag cacause
ng infection. Nagiging pathogens sila if:
1. If they transfer site
2. Increase number in usual site
*major nutrients that are need by bacteria
to growth:
1. Carbon
2. Nitrogen
3. atp- serves as their energy
4. 37 degree celcius
**how do we imitate them? We are using
culture medium.
Robert Hooke (end of 16thcentury)
 Microscope with 3-500x
 Recognized cellular structures
 We are able to classify their different
groups of bacteria
 Methodology of culture
Ferdinand Cohn (1849)
 staining of histological specimens
 histological specimens- tissues
 carmine and hematoxylin- vegetable
dyes
 we use vegetable dyes for as to easily
identify the presences and cellular
features of bacteria
Robert Koch (1877)
 methylene blue to stain bacteria
 We are using it up to present
Robert Koch (1882)
 staining the tubercle bacillus with
 Tubercle bacillus- mycobacterium
tuberculosis. Causing tuberculosis.
 mycobacterium tuberculosis- cell wall is
impenetrable by methylene blue
because the cell wall is made up of
waxy envelope
 Employing for the stain to penetrate the
waxy envelope (mycolic acid)
  Acid fast staining- lalagay sa flame yung  culture media- they are classified
slide na may stain at hihintayin mag according to purpose
steam  turbid- positive ang growth
 clear- negative ang growth
Hans Christian Gram (1884)
1. General purpose- basal medium.
 Gram’s stain- differential stain Majority of non-fastidious bacteria are
capable of growing
It can differentiate two groups:
2. Selective
1. Primary stain- crystal violet: can stain 3. Differential
gram positive (purple) 4. Enrichment
2. Secondary stain- safranine: can stain Solid medium
gram negative (red)
 Introduction of gelatine and then agar
Frederick Loeffler (1890)
in 1882
 emonstrate the presence of bacterial  There is pattern of growth in bacteria
flagella using gelatin or agar. It can be color,
 ang mga bacteria ay motile but not all shape, texture
 ultraviolet microscope (1919)  Colony- pattern of growth on the
 electron microscope (1934) surface of culture media
 Belgian physicist Marton- magnification Silica gel media
of 2-300,000x
 scanning electron microscope (1965)-  Chemolithotropic bacteria such as
doble nung 300,000 thiobacillus thiooxidans

Pasteur (1860) Julius Richard Petri (1887)

 Semisynthetic medium- using  petri dish

ammonium salt, yeast ash, candy sugar
 Before it is discover they are using meat
broth
 Broth- nutrients are dissolve in water
 assistant ni Pasteur
Beijerinck (1898): enrichment culture media
McIntosh and Fildes

Ferdinand Cohn (1872)  anaerobic jar- chamber with 0% oxygen

 ang mga bacteria meron din silang
 basal medium, to which various
atmospheric requirements some of the
additions could be made
requires:
 non-fastidious bacteria- they are only 1. oxygen
have standard requirement 2. carbon dioxide
 fastidious- group of microorganism 3. little amount of oxygen / reduce oxygen
which have additional special 4. increase amount of carbon dioxide
requirement 5. do not require oxygen
example: Chamberland (1884):
Neisseria spp- require iron for growt  Autoclave- sterilization equipment
Haemophilus- hemin (x factor)
Edward Jenner (1796) Classification

 Cowpox virus used to immunized a boy  Organization of microorganisms that

against smallpox
Ignaz Semmelweiss (1844)
 Prevented Childbed fever
Louis Pasteur (1865)
 disease must be airborne
Joseph Lister
 covering wounds / wound dressing with
chemicals
Metchnikoff (1882)
 cellular immunity, phagocytes
 passive immunity, active immunity
specific and non-specific cellular
immemorial
 responsible in discovery of immunity
Ehrlich (1891)
 active and passive immunity
 responsible in discovery of immunity
Charles Calmette
share similar morphology, physiology
and genetic components into specific
group/ “taxa”
 Specie - Collection of bacterial strains
that share common physiologic &
genetic features Subspecie; biotype;
serotype; genotype
 Genus- Comprises of different species
with common and sufficiently different
features
 Family- always ending in “ceae”
example: Strepotococceae
- Genera or genous
- Genous - always starting
with CAPITAL LETTERS
- Example: Streptococcus spp.
- Genera- start with small
letters. With species
- Example: streptococcus
pneumoniae
 Order
 Class
 Division
 Kingdom

 BCG vaccine- bacille calmette guerin Nomenclature

TAXONOMY  Naming of microorganisms according to

established rules & guidelines
3 disciplines  BINOMIAL: Every organism is assigned a
 Classification genus and species name in Latin or
 Nomenclature Greek derivation
 Identification - Combination of genera and
species
Importance:
Components:
 Use a common label for every
organisms  Genus: Always capitalized
 Minimizes confusions about names  specie: never capitalized
 Genus + specie
 In print: italics
 In script: underlined
  Example: Streptococcus pyogenes or Phenotypic criteria Principles
Streptococcus pyogenes Macroscopic Microbial growth
 May be abbreviated: S. pyogenes morphology pattern in culture
 Informal designations: streptococcus media
(pertain to a group) - colonial size,
texture and
Identification pigmentation
Microscopic Size, shape,
 Describe key features of microorganism morphology intracellular
Methods: inclusions or
appendages,
 Genotypic characterization arrangement of
- Genetic makeup bacterial cells
- Genes and nucleic acids Staining Ability of the
 Phenotypic characterization characteristic organism to be
stained with a
 Readily observable
particular stain
characteristics

Workflow: either gram positive

or gram negative,
1. Specimen collection acid fast or non-acid
2. Direct microscopic examination fast
3. Culture Environmental Temperatures,
4. Bacterial identification requirements presence or absence
a. Macroscopic examination of oxygen
 Colony (growth pattern) (aerotolerance), pH
morphology levels presence of
ions and salts
 Growth in culture media
b. Microscopic examination either 20 degree
 Characteristic of bacteria under celcius or 30 or 60
the microscope *morphology shape ng bacteria:
5. Biochemical exam / test
 Coccus / cocci- spirical shape
Genotypic criteria Principles  Bacillus / bacilli- rod shape
DNA base G, C, A, T  Spirochets- foiled (zigzag)
composition ratio (nitrogenous bases)
- Used as indicator of Gram positive
relatedness
Nucleic acid base Order of bases along  Cocci
sequence analysis RNA or DNA  Bacilli
strands  Spirochets
- Measure of degree
of relatedness Gram negative

 Cocci
 Bacilli
Host-Microorganism Interaction Reservoir- origin. Cam be human, animal,
environment (water or soil)
Stages:
Host- human

Indirect- to reservoir to intervening agent to the

host

Exposure is dependent on human activities

 Reservoir
 place of origin of infecting agent

RESERVOIR Examples Examples

Human Direct Indirect
• Conatal • Nosocomial
Started in exposure- incubation period • Blood infections
transfusion (hospital
Pathogenesis • Sexual acquired)
Exposure transmission • Ingestion of
contaminated
 Dito palang magkakaroon ng mode of food or water
transmission Animal Direct Indirect
• bite • Bite of
Incubation period insect
(arthropod)
 dito nangyayare ang growth and
vector
multiplication of bacteria
• Water food
Clinical manifestation supply
• Animal for
 the patient will experience the sign and human food
symptoms of the disease Environment Inhalation of
soil and dust
Outcome particles or
inoculation
 the patient will recover or will develop
Modes of transmission
chronic infection or disability or worst it
will resolve to death  Direct
Physical encounter between host and  Indirect
microorganism - Vectors
- Vehicle

*Insects

 vector rather than reservoir

 arthropods tick, fleas, mosquito

*** useful in determining ideal specimens for

isolation & precautionary measures
Microorganism colonization of host surfaces B. Mucus membrane

HOST Structure Protective activity

Mucosal cell • Capability to slough off
 Microbial colonization • Tight intercellular junction
 Persistent survival of microorganisms in Goblet cell • Mucus production:
surface of human body • Protective lubrication
 Dependent in human defenses that • Bacterial trapping
protects internal tissues and organs • Contains Ab
a. Skin • Provides antibacterial
- Physical and chemical barrier substances:
b. Mucous membranes • Lysozyme (degradative
A. Skin enzyme), lactoferrin (compete
with bacteria for iron),
*yung skin natin na intact is inferactable to lactoperoxidase (enzyme
bacteria against bacteria)
MALT • Specific and nonspecific
Structure Protective activity immunity
Dermal layer • Physical barrier *iron- important for blood production
• Capability to slough
off MICROORGANISM
• Provide dry, acidic
and cool conditions  Colonizers/ Normal flora
Hair follicles, • Produce acids, - Microorganisms that inhabits
glands alcohols and toxic human body
lipids  Resident
Conjunctival • Tears (flushing - Survive, thrive and multiply
epithelium (eyes) action) - Presence is more permanent
SALT (skin associated • Specific and  Transient
lymphoid tissues) nonspecific immunity - Survive but not multiply on the
• immunity- our surface
system is program to  Vary with anatomic location
destroyed any
foreign material that * useful in determining clinical significance of
is detected in our microorganism isolated from patient specimens
system.
• specific- they have  Microbial colonization
specific foreign - Last step in establishment of
material that they long-lasting, commensal
target relationship between colonizer
• non-specific- as and human host
long as its foreign it - First step in developing
will be destroyed, it infection and disease
will be kill.
 Virulence factor- are characteristic of
bacteria which is use to colonized or
invade host. They are causing infection
or using for survival.
 Factors contributing to  Responses to microbial invasion
successful colonization  Nonspecific response
Survival against • Localization - Biochemical or cellular
environmental conditions in moist area - Phagocytes
• Protection - Inflammation
within
 Responses to microbial invasion
ingested
 Nonspecific response
debris
• Expression - Phagocytes
of specific - Cells that ingest and destroy
metabolic foreign particle
characteristics
PMN (polymorco Macrophage
Attachment and adherence • Pili
nuclear cells) ex. Ex. Monocytes
to host cell surface • Adherence
Neutrophil
proteins
First cell on the scene --
motility, production of Bone marrow- Bone marrow-
subs. that may circulation circulation- tissues
compete with host for Days or less in Several days to
acquisition of nutrients, survival weeks
ability to coexist w/ -- Mediates immune
other system defenses
microorganism
*ang unang cell sa site of infection is pmn and
Microorganism entry, invasion and susundan sila ng macrophages
dissemination
** effect of pmn only days or less than on the
HOST site of infection
Factors contributing
to disruption of
physical barrier
Trauma • Wounds
• Abrasions
• Burns
Inhalation • Smoking
• Toxic gases
Implantation of
medical devices
Other diseases • DM, alcoholism
Childbirth and
overuse of antibiotics
*lysosome- degradative component of
cytoplasm or cellular organelle
 Cellular B cells T cells NK cells
Residence Lymphoid Circulation
tissues &
Lymphoid
tissues
Function Produce Similar to
Ab cytotoxic but
Responses to microbial invasion not require
presence of Ag
 Nonspecific response to function
- Inflammation Subtypes • B cells • Helper T
- Swelling • Plasma cells
- Redness cells • Cytotoxic
- Heat • B- T cells
- Pain memory •
cells Suppressor
Responses to microbial invasion T cells

 Nonspecific response
- Inflammation MICROORGANISM

Infection

 Growth and multiplication of

microorganisms that result in damage to
host

Disease
Responses to microbial invasion
 Infection produce notable changes in
 Specific response (immune system) human physiology
- Antibody-mediated immunity Pathogens
- Cell-mediated immunity
 Microorganisms causing
infections/diseases

Virulence factors

 Characteristics that enable them to

cause disease

Pathogenesis

 First step in infection and disease

development
Microbial virulence factor (characteristic of
bacteria to cause infection)

 Attachment
- Microbial attachment to surface
through different MOT
- pathogens vs colonizers
 Invasion
- Traumatic factors
- Direct actions of virulence
factors
 Example:
- capsule: Klebsiella pneumoniae
- enzymes: Staphylococcus
aureus
 Surviving inflammation
- Phagocytes
- Production of capsule and
toxins
- Inhibit fusion of phagosome-
lysosome
- Resistance to lysosome
- Active and rapid replication
 Complement system
- Capsule to hide surface
molecules
- Produce substances that:
- inhibit complement activation
- Destroy specific complement
proteins
 Microbial toxins
- Biochemically active substances
released by microorganisms
that have a particular effect on
host cells
- Can cause disease in the
absence of pathogens
- Intoxication
- Ingestion of preformed
bacterial toxin
 Endotoxin
- Gram (-) bacteria
 Exotoxin
- Gram (+) bacteria
- Specific and more limited
effects than endotoxins
Microbial toxins

OUTCOME

Pathogenesis
Exotoxins- outside

Endotoxins- inside
Definition of Terms Disinfection

Acute phase  removal of microbes that may cause

disease from an environment
 Early stage of a disease preceding the
adaptive phase of the immune response Disinfectant

Anaerobe  Substance designed to be used on

inanimate objects to kill or destroy
 Organism that does not require oxygen
disease-producing microorganisms
for life and reproduction.
Etiologic agent
Antibody
 Microorganism causing a disease.
 Protein or immunoglobulin molecule
characterized by specific amino acid Fastidious
sequence produced by the host as a
 Hard to grow; requires additional
result of a specific antigenic stimulation.
growth factors.
Antigen
Genotype
 Substance that produces sensitivity and
 Genetic makeup of an organism.
initiates an immune response
Gram-positive bacteria
Antisepsis
 Bacteria that retain the crystal violet–
 Destruction of microorganisms to
iodine complex and appear blue-black
prevent infection
on Gram-stained smears.
Bacteremia
Gram-negative bacteria
 Presence of viable bacteria in the blood,
 Bacteria that do not retain the crystal
as evidenced by their recovery in blood
violet complex; stained red by the
cultures
safranin counterstain.
Bactericidal
Halophilic
 Antimicrobial that kills a microorganism
 “Salt-loving”; an organism that grows
Bacteriocin best in media with an increased
concentration of NaCl.
 Proteins produced by some bacteria that
inhibit the growth of other strains of the Immunocompetent
same organism or related specie
 ability of an immune system to mobilize
Capnophile and deploy its antibodies and other
responses to stimulation by an antigen.
 Microorganism that grows best in the
presence of carbon dioxide Immunocompromised

Capnophilic  describe an individual with deficient

function of the
immune system
 Immunosuppression describe the state
of an immune system that is suppressed
Latent phase
 permits the infection to evolve without
any obvious external symptoms.
Mesophile
 Organism that grows best in moderate
temperature, neither hot nor cold
Microaerophile
 microorganism that grows in conditions
of reduced oxygen and increased carbon
dioxide.
Microaerophilic
 Microorganisms that require
environments containing concentrations
of oxygen lower than that present in the
atmosphere
Microbial load
 Total number of organisms present
Obligate aerobe
 Microorganism that requires oxygen for
growth.
Obligate anaerobe
 Microorganism that can live and
reproduce only in a strict anaerobic
environment (0% oxygen)
Nosocomial infection
 Infection acquired within 72 hours of a
stay in a health care facility.
Opportunistic infection
 Disease caused by a microorganism with
low virulence that becomes pathogenic
in a host with low immunologic
resistance.
Pathogenicity
 Ability of a microorganism to cause
disease.
Phenotype
 Observable or measurable
characteristics of an organism.
Sepsis
 Systemic response to bacterial infection
Resistant strain
 not inhibited by the usual systemic
concentrations of the antimicrobial
agent with normal dosage schedules
Susceptible
 implies that an infection caused by the
bacterial strain tested may be
appropriately treated with the dosage of
antimicrobial agent recommended for
that type of infection and infecting
species.
Intermediate
 implying that the agent might be
effective for infections located at body
sites where the drugs are physiologically
concentrated, or when a high dosage of
drug can be used.
Zoonosis
 Disease that humans acquire from
exposure to infected animals or
products made from infected animals.
Zoonotic
 Pertains to diseases that can be
transmitted from animals to humans.
BACTERIOLOGY LECTURE
WEEK 3
Bacterial Cytology and Physiology
Classification based on cellular type:
Microbiology
 study of microorganisms
 large and diverse group of microscopic
organisms that exist as
 single cells or cell clusters
 bacteria, parasites, fungi, viruses etc
Bacteria
 unicellular organisms (One nucleus)
 lack a nuclear membrane and true
nucleus because it does have a nuclear
membrane but the component of the
nucleus that or the component of the
true nucleus is the same in what is
present in the prokaryote
 ang pinagkaiba lang ng prokaryotic and
eukaryote in terms of true nucleus is
that nuclear membrane but the
component is the same. It is cannot
seen under the microscope. For us to
determine its presence it is usually
attached in the mesosome of the
cellular membrane
 prokaryotes
 Prokaryotic cells does not have nucleus
it is having true nucleus. Why is it
called true? Because it is have a
demarcation line or membrane which
are the nuclear membrane.
 That is why when you draw cell which is
eukaryotic you will be drawing one
circle and you will drawing inner circle
inside. That means meron siyang
nuclear membrane.
 When you look at it under the
microscope you will be able to clearly
see the presences of the nucleus dahil
may membrane na naka enclose dito.
 Inside the nucleus- contains the
genome of the organism
 Genome- DNA or RNA
1. Prokaryotes
 Archaea (Archaeobacteria)
- Extremophiles- they are capable of
growing under the extreme
environmental condition. It means
when you exhaust them into
extreme conditions they can still
survive and that is their usual
habitat
- Halophiles- these are organism that
can tolerate increase salt
concentration.
- Thermophiles- the one that can
tolerate increase temperature.
- anaerobic methanogens- these are
the one that is usually in habiting in
the intestinal tract of animals.
Meaning masyadon mababa ang ph
of those habitats therefore kahit
nag anon they can still survive. They
can also found in dark level of sea
- cell walls lack peptidoglycan (this is
the major difference of archaea
that is what separate from the
eubacteria)
 Bacteria (Eubacteria)
- Has a cell wall peptidoglycan
- Peptidoglycan- urine layer
2. Eukaryotes
 Fungi, algae, protozoa, animals, and
plants
Cytology Cytoplasmic
structures
Characteristics Prokaryote Eukaryote (cont.)
Size 0.4-2 μm 10-100 μm Spores Present
(mas Malaki Endospores
dahil meron Bacillus &
silang Clostridium
cellular (use of
organelles) organism as
mitochondri also as
a, virulence
endoplasmic factor)
reticulum, Outer
golgi membrane/ Cell
apparatus Envelope
Cytoplasmic Nucleoid membrane- Structures
structures region of the bound Sterols Absent Present
cytosol nucleus (true except (example:
nucleus) Mycoplasma fungi but in
Attached to spp. (are bacteria
mesosome bacteria but walang
(sack like in they possess sterols)
structure in sterols. Most
the cell of the bacteria
membrane) walang sterol
Nucleus Present Present pero merong
Ribosomes RNA and Present exception)
protein (free (endoplasmic
in the reticulum) Plasma
cytoplasm. membrane- it
Some are provides
bound but this osmotic
are found in barriers,
the location for
cytoplasmic electron
membrane) transport
Granules Present (because hindi
Use for nakakapag
storage produce ng
deposits sariling energy
(virulence ang mga
factors and bacteria)
nutrients) Plasma No Carbohydrat
membrane carbohydrates es
Polysaccharide phospholipid glycolipids
s (glycogen, bilayer & and
lipids, protein) protein glycoprotein
s
 Outer
membrane/ Cell
Envelope
Structures
Cell wall Present Present
*Virulence factor- the characteristic of bacteria
that enable them to cause infection.
- Example: staphylococcus aureus
**when bacillus and clostridium are exposed to
extreme condition this bacterial cells will
produce or enclosed themselves in a spore.
These are they response to environmental
condition.
***may mataas na carbohydrates ay ang fungi.
Specifically tytin
Cell Wall
 Maintains shape of cell
 classify bacteria to its Gram stain or acid
fast reaction
 exception for Mycoplasma spp.
 Prevents bursting of the cell from the
high osmotic pressure
Cytology
Cell wall (maintain the shape of the cell)
 Gram positive
 thicker peptidoglycan than negative
(murein layer)
 when you apply crystal violet it will take
up crystal violet. When you apply grams
iodine it will be absorb it will remain
bare. And then decolorization. It is
resistant. It won’t decolorize. It will
remain purple.
 consists of glycan chains of alternating
N-acetyl-d-glucosamine (NAG) and N-
acetyl-d-muramic acid (NAM)
 teichoic acid (Peptidoglycan)
 lipoteichoic acid
 crystal violet- it makes the cell wall
purple
 grams iodine is a more than. Iniincrease
niya ang affinity ng stain with the cell
wall of bacteria
 acetone alcohol- decolorizer.
Tatanggalin niya dapat yung primary
stain na nilagay mo
 safranin- secondary stain also known as
the counter stain
Cell wall
 Gram negative
 thinner peptidoglycan
 thinner than gram (+)
 outer peptidoglycan (periplasmic space)
 additional & unique to gram (-)
 proteins, phospholipids, and
lipopolysaccharide
 end color is red
 mabilis ang onset manifestation
 unang magkakaroon ng clinical
manifestation.
 Mas capable mag cause ng infection.
They have more virulence factor than
gram positive
Difference:
 Urine layer- thicker in positive thinner
in negative
 The periplasmic space- only found in
gram negative. It contains degradative
enzymes (it serves as they virulence
factor) detoxifying enzyme. That is the
reason why gram negative organism
 most likely causing severe 2 functions of pili and fimbriae
manifestation.
1. to mediate DNA exchange
- mabilis ang onset manifestation
2. it attaches the bacteria to the
- unang magkakaroon ng clinical
surface(hindi siya ginagamit for
manifestation.
locomotion unflagellar)
 Presence of lipopolysaccharides- are
 nonmotile, long, hollow protein tubes
also virulence factor of gram negative.
that connect two bacterial cells
Responsible in producing fever, shock
 nonflagellar, sticky, protein, hairlike
conditions.
appendages
Cell wall  for attachment
 Acid fast Flagella
 Tubercle bacilli (discovered by Robert
 eto na yung ginagamit ng mga organism
fox hindi mapenetrate ng methylene
for locomotion. Hindi lahat ng bacteria
blue)
may flagella
 Mycobacterium & Nocardia (Gram (+))
 some of them are motile- merong
 waxy layer of glycolipids and fatty acids
flagella
(mycolic acid)
 non motile- walang flagella
 majority ng cell wall almost 60% ay
lipids. Characteristics Prokaryote Eukaryote
 Major lipid component is known as Flagella Simple Complex
mycolic acid (strongly hydrophobic component: component:
molecule) polymers of MTs and
flagellin polymers of
Characteristics Prokaryote Eukaryote tubulin with
Surfaces by rotary dynein
polymers action at
Glycocalyx As capsule Present the base coordinated
sliding
used as microtubules
Virulence
factor (this
won’t be
detected by
phagocytes)

clear area
(“halo”-like) Lophotrichous- multiple flagella arising from
Cilia Absent Present one hole or on end of bacteria
Pili and Present Absent
fimbriae Polar- from one hole or end of bacteria

Peritrichous- multiple or there are all sides of

bacteria are surrounded by the flagella.
 Characteristi Prokaryote Eukaryote Golgi body
cs
Genome 70S (50S and 80S (60S  process substances for transport out of
size 30S) and 40S) cell

Mitochondria
S- Svedberg
unit(sedimentati  generate energy
on rate.) by
theodor Lysosomes
svedverg
 provide environment for controlled
Sedimentation enzymatic degradation of intracellular
rate- unit of substances
time during high Nucleus
speed
centrifugation  provide membrane enclosure for
location nucleoid nucleus chromosomes
Electron cell membrane mitochond
transport (if present) ria and Morphology
for energy chloroplast
(inner
membrane
)
Reproductio Asexual through Sexual and
n binary fission asexual

Example:
fungi
Membrane- Absent Present
bound
organelles COCCI
Chloroplasts Absent Present
Diplococci- elongated yung spherical cell
(algae &
plants) - have gram negative (Neisseria) and
pero hindi gram positive (streptococcus
sa fungi pneumonia)

In cluster- staphylococci
Cytology
In chain- streptococci
Cellular organelles (eukaryotic cells)
BACILLI
Endoplasmic reticulum
Coccobacili- individual, spherical pero
 process and transport proteins elongated.

Pleomorphs or pleomorphic organism- usually

available and presenting themselves in various
sizes, in various forms.
*mag kakaiba ang shape magkakaiba ang sizes- Component
triomorphic organism acetone:
Absolute
**scenario- pleomorphism acetone plus
Fusiform- stapered at the end and its pointed 95% of
ethanol
Palisading- arrange in parallel to one another. Safranin Secondary 30 seconds
Aligned side by side stain
 Washing in between steps
Microscopic Examination
Gram stain
Differential dye- it can differentiate 2 groups
Components Gram Gram
 gram positive positive negative
 gram negative Crystal Violet Purple Purple
Gram’s Purple Purple
Steps:
Iodine
1. meron ka munang slide na merong Acetone Purple Colorless
specimen gagawa ka ng bacterial smear. alcohol
2. You’re going to dry, air dry. After Safranin Purple Pink
airdrying you need to fix it.
3. The fixation occurs when you pass it Acid fast stain
through flame this is a physical fixation
or alcohol. But in bacte we use heat.  Acid fast bacilli mga matataas ang
Pass it 3 times mycolic acid pwede gamitin- Ziehl-
Neelsen method or Kinyoun method
Gram Stain
 Heat or methyl alcohol
 Heat or methyl alcohol  Ziehl-Neelsen- hot method we use heat
 Kinyoun- cold method we use tergitol
Components Function Duration
Crystal Violet primary stain 1 minute Components Function Duration
Gram’s mordant or 1 minute Carbol primary stain 10 minutes
Iodine fixative Fuchsin (red)
Hintayin mag
fixative- it’s produced ng
the same in steam
increasing Heat 1 minute
the affinity. Acid alcohol Decolorizer -
Pinapakapit
yung stain sa Component:
bacterial cell hydrochloric
wall acid 3% and
Acetone Decolorizer - 95% ethanol
alcohol Methylene Secondary 1 minute
Critical step blue/alachite stain
green
 acid fast- Classification based on how bacteria meet
yung mga nutritional needs:
bacilli kulay
pula yung 2 classification
background
 Autotroph- carbon is usually from the
niya yung
carbon dioxide
counter stain
mo blue or  Heterotroph- this are organism which
green usually get their carbon from
photosynthesis
Non-acid  Organic compound- mga carbohydrates
fast- color of and proteins
bacteria is
blue or green Autotrophs Heterotrophs
 Washing in between steps carbon CO2 • photosynthesis
(“phototrophs”)
Acid fast stain • Oxidation of
organic compound
Components Acid fast Non-acid (“chemolithotrophs”)
fast energy Organic • Oxidation of
Carbol Fuchsin Red Red Compounds organic
Heat Red Red “hemolithotrophs” Compounds-
Acid alcohol Red colorless kaliangan ng oxygen
Methylene Red Blue or • Fermentation of
blue/malachite green organic
green Compounds- they
doesn’t need oxygen
Other Also known as • all bacteria that
Nutritional Requirement
characteristics “lithotrophs” inhabit
Source of: the human body
• normal flora
 Carbon- for making cellular constituents
50%
 Nitrogen- for making proteins Bacterial Metabolism
14% Fermentation (anaerobic process)
 Energy- for performing cellular
functions  They will be obtaining their energy
ATP either there energy or carbon via
fermentation process
Phosphate: nucleic acid  Glycolysis (EMP)- ang substrate ay
Phospholipids: cell membrane 4% glucose
 Common product- acid
Sulfur: protein synthesis  We use ph indicator
Mineral ions  Fermenter- mga organism na kayang
mag produce ng acid by using organic
compound as substrate para makapag
produce ng acid
  organic compound (ex. glucose)
Acid/Alcohol
Oxidation (aerobic process)
 organic compound (ex. glucose)
Acid/Alcohol
 Nitrogen Acid/Alcohol
 Oxidizer- kaya nila mag utilize ng
organic compound makakapag produce
sila ng acid pero dapat merong oxygen
 Nitrogen- inorganic source
Respiration (aerobic process)
 Kreb’s cycle
 Electron transport chain: aerobic
process
 organic compound (ex. glucose)
CO2 + H2O
*some organism are non-utilizers of
carbohydrate. Some bacteria are not capable of
utilizing carbohydrates ng gagaling yon sa ibang
sources.
Environmental Factors Influencing Growth
Environment
 influences the growth rate of bacteria
 considered when bacteria are
cultured
Factors:
 pH- common physiologic pH ng mga
bacteria for growth neutral is pH(7.0
and 7.5)
 Temperature
- cold temperatures (10° to 20° C)
(psychrophiles)
- moderate temperatures (20° to 40°
C) (mesophiles)
- high temperatures (50° to 60° C)
(thermophiles)
 Gaseous composition of the
atmosphere
- Normal atmospheric composition:
21% O2 and 1% CO2
- require 21% oxygen (obligate
aerobes)
- require oxygen but do not use it for
metabolism (aerotolerant
anaerobes)
- require small amount of oxygen (5-
6%) (microaerophiles)
- cannot grow in the presence of
oxygen (obligate anaerobes)
- grow either with or without oxygen
(facultative anaerobes)
- require enriched 5-10% CO2
(capnophiles)
Bacterial Growth
Generation Time “doubling time”
 time required for one cell to divide into
two cells
 binary fission (asexual reproduction)
 takes minutes or hours
 The duration in between this called the
doubling time. Minutes or hours for it
to complete.
 Hindi ibig sabihin multiplication
Growth Curve
 growth of a bacterial culture when
plotted during balanced growth state
 the number of bacteria is proportional
to the number of bacterial properties
 Bacterial properties- indicator of
bacterial growth. Mass ng bacteria,
yung protein contain niya, yung nucleic
acid contain niya etc.
 Para magkaroon ng balance dapat kung
gaano karami yung bacterial properties
niyo dapat equivalent lang yan sa
number ng na poproduce niyo.
  balanced growth state =
𝑏𝑎𝑐𝑡𝑒𝑟𝑖𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟
𝑏𝑎𝑐𝑡𝑒𝑟𝑖𝑎𝑙 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠
indicator of
bacterial growth
Achieved the bacterial growth
1. Its equal or proportional to bacterial
properties
2. When there’s no toxin produced
3. With enough nutrients for growth
4 Phases of Growth:
Lag phase
 bacteria are preparing for reproduction
 wala pang nangyayareng growth
 growth rate: 0
Log phase
 bacterial numbers increase
logarithmically
 growth rate: positive
Stationary/ Plateau phase
 nutrients are becoming limited (ubos
na, nagamit na lahat ng nutrients sa
culture media)
 numbers of bacteria remain constant
with decreased viability (mas marami
parin ang buhay kesa sa patay)
 growth rate: 0
Death phase
 number of nonviable bacterial cells
exceeds the number of viable cells
Bacterial Counting
Direct counting under the microscope
 estimate the number of bacteria
present in a specimen
 direct microscopic examination or
microscopic examination (bacterial
identification)
 disadvantage: cannot distinguish
between live and dead cells
Direct plate count
 by increasing dilutions of broth cultures
on agar plates (CFU/mL)
 provides a count of viable cells only
 example: bacterial cell count in urine
cultures
Density measurement
 density (cloudiness or turbidity) of a
bacterial broth culture in log phase can
be correlated to CFU/mL of the culture
 used to prepare a standard inoculum
for AST
2 types of bacterial counting
1. semi quantitative
- estimate
- usually a reporting is 1+, 2+, 3+, 4+
- specimen, gagawa ng bacterial
smear, stain, look at the microscope
(direct microscopic examination)
- can determine gram positive and
negative
- kapag 0 ang nakuha need pa rin
iculture.
2. quantitative
2 ways of quantitative
 count it using liquid or broth medium-
incubate in 37 degree celcius for 18-24
hours.
- Pag positive nag growth (turbid)
 - Pag negative ang growth (it’s clear)
2 ways to count it:
1. liquid ang ginamit- using the 0.5
mcfarland (pang compare ng
turbidity with your sample)
 Ang equivalent niya ay 1.5 x 108 colony
unit per mL
 cfu- colony forming unit
 Densitometer- determined the density
and the density will be converted to cfu
per mL.
2. Solid yung medium na ginamit.
 Specimen, usually its platted, upon
culturing you will use calibrated loop,
incubate in 37 degree celcius 18 to 24
hours.
 Sa lines of strict may mga tutubong
colony bibilangin yon
 Number ng colony x unit ng calibrated
loop = total cfu per unit

Workflow:

1. Specimen collection
2. Direct microscopic examination
3. Culture- nag iinocculate and incubate
4. Bacterial identification- perform
microscopic (characteristic ng bacteria)/
macroscopic exam (colonial
morphology)
5. Biochemical exam / test

*5-7 days pinaka maikli 3 days

BACTERIOLOGY LECTURE  pag mataas ang carbohydrate and lipid
content mas mahirap patayin
WEEK 4
 rich in dipicolinic acid and calcium
Microbial Control and Biosafety
Cell walls of mycobacteria
Before and after your work you need to
 rich in lipids
decontaminate your area. Kailangan
natin linisin ang area natin before and
after to avoid contamination

Sterilization vs Disinfection (killing of

microorganism)

Sterilization

 destruction of all forms of life including

bacterial spores (spores uses of
virulence factor and a survival factor)
 chemical or physical methods
Nonlipid viruses- naked or non-envelope. Hindi
Disinfection sila naka enclose sa envelope.

 destruction of some forms of life except
bacterial spores (lahat except spores)
 chemical or physical methods
- Disinfectant: any agent applied to
inanimate objects
- Antiseptic: applied to skin, cannot
be used as disinfectants
*envelope ginagamit na term para sa mga
viruses.
**covid-19 is envelope therefore kaya sinabi
nila pwedeng gumamit ng alcohol dahil kaya na
ng alcohol yorn.
Microbial load

*alcohol is only agent of disinfection. When total number of organisms present

alcohol is use in table tops in surfaces its cold
disinfectant but when you use in the skin before
venipuncture its called antiseptic
Factors Affecting Degree of Microbial Killing
Type of Organisms
 most likely composed of organisms with
varying degrees of susceptibility to
killing agents
 not all the organisms die at the same
time

 presents variability to withstand

chemical and physical treatment
 it depends on biochemical composition
and mechanisms to protect themselves.

Instances:

Spores higher numbers of organisms require

longer exposure times
 rich in proteins, lipids, and
carbohydrates
 exposure time- that is the time when
the object to be disinfected and the
agent are in contact with one another.
Concentration of Disinfecting Agent
 Mas mataas ang concentration mas
effective
 amount of disinfectant needed to
destroy microorganisms varies with the
different agents
Presence of Organic Material (serum, blood)
 example: blood, serum, mucus, pus
 pwede niyang ma inactivate yung agent
mo
 Organic material affects killing activity
by inactivating the disinfecting agent
 prevents full contact between object
and agent
 Bleach: easily inactivated by organic
material
Nature of Surface to Be Disinfected
 Kung iyan ay smooth surface madali
lang yan pero pag rough surface
merong mga edges or mga pores na di
pwedeng ma disinfect ng maayos.
Contact/ Exposure Time
 amount of time a disinfectant or
sterilant is in contact with the object
Temperature
 dapat nasa optimum temperature
palagi
pH
 ph is always neutral
Methods of Disinfection and Sterilization
Physical Method
A. Heat
Advantages:
 reliable effects, ease of use, shorter
time and cost effective in terms of
disinfection and sterilization
 most common method
1. Moist heat (sterilization)
 Heat under steam pressure-
autoclaves(equipmet)
 Aoutoclave ang ginagamit natin para sa
moist heat or steam under pressure
 What is the principle of auto clave?
Heat under stream pressure
 steam under 1 atm of pressure, or 15
psi (pound per square inch), 121°C for
15 minutes
 all microorganisms can be killed even
bacterial spores (except for prions)
 sterilization method of choice for heat
stable object
2. Dry heat (sterilization)
 Oven
 longer exposure times & higher
temperature (umaabot ng 180 degree
Celsius)
 sterilization method for heat stable
substances that are not penetrated by
moist heat
 mas mataas ang temperature mas
mataas ang contact time
 sterilization method for glass wares
*we used this method for heat stable
substances that cannot be penetrated by moist
heat example: oil,
3. Boiling & pasteurization (disinfection)
 disinfection but not sterilization (kasi
mas mababa ang temperature niya)
  Boiling- kills most microorganisms in  used in laboratory hoods and in rooms
approximately 10 minutes (100°C) of ICP
 Pasteurization- eliminates food-borne C. Radiation
pathogens & organisms responsible for
Ionizing (sterilization)
food spoilage (63°C or 72°C)
- reduces spoilage of food without  Gamma rays or electron beam
affecting its taste  short wavelength and high energy
 Sterilization method for disposable
Method Temperatu Duratio Applicatio
re n ns supplies (consumable)
Autoclave 121°C 15mins Sterilizes Non-ionizing (disinfection)
(moist heat) at 15
psi  UV rays
Oven (dry 160-180°C 1.5- Sterilizes  long wavelength and low energy
heat) 3hrs  disinfection method for surface
Boiling 100°C 15 mins disinfects
(steam) Chemical Method
Pasteurizati
 for sterilization
on
Flash 72°C 15 sec disinfects  used mainly as disinfectants
method  Chemosterilizers- chemical agents may
Batch 63°C 30 mins disinfects be used to sterilize
method
principles:

 Reaction with components of the

B. Filtration
cytoplasmic membrane
Liquid (sterilization)  Denaturation of cellular proteins
 Reaction with the thiol (–SH) groups of
 thin membrane filters composed of
enzymes
plastic polymers or cellulose esters
 Damage of RNA and DNA
containing pores
 pore sizes of 0.45 and 0.80 μm
(bacteria, yeast, molds)
 0.22-μm (for critical sterilizing)
 0.01 μm (small viruses)
 sterilization method for heat- sensitive
solutions

Air (sterilization)

 use of high-efficiency particulate air

(HEPA) filters
 HEPA- High Efficiency Particulate Air
(bio safety cabinet level lahat)
 remove microorganisms larger than
0.3 μm
Chemical Method  principle: degrades microbial cell walls
and cytoplasm,
A. Alcohol
 denatures enzymes, and coagulates
 60-90% ethyl or isopropyl alcohol
chromosomal material
 Disinfectant lang hindi sterilant. Di niya
 antiseptic lang
kayang pataying yung mga naked na
bacteria and mga spores *Iodine can be used in two forms as antiseptics:
 antiseptics and disinfectants
 tincture: alcohol + iodine
 principle: inactivate microorganisms by
 iodophor: iodine (5-10%) + neutral
denaturing protein
polymer carrier
 disadvantage: inactivated by organic
 advantage: less irritating, nonstaining,
materials
and more stable
B. Aldehydes
1. Formaldehyde  contact time: more than 30 seconds
D. Chlorine and Chlorine Compounds
 Formalin
Hypochlorite
 37% aqueous solution or formaldehyde
 forms:
gas
- liquid sodium hypochlorite-
 disadvantage: carcinogenic agent
household bleach
 not recommended (in any form) be
- solid calcium hypochlorite- powder
used as a disinfectant or
na chlorine
 sterilant on a routine basis
 principle: oxidative effects of
 disinfection method for biosafety
hypochlorous acid
cabinet
 disadvantages: long exposure time
 hindi ginagamit routine basis
required for sporicidal action
2. Glutaraldehyde
- inactivated by organic materials
 principle: inactivation of DNA and RNA
 0.5% to 1% sodium hypochlorite:
through alkylation of sulfhydryl and
disinfectant
amino groups
 Contact time: at least 3 mins
 Advantage: rapid killing action
 1 : 10 dilution of a 5.25% concentration
- remains active in the presence of
of sodium hypochlorite
organic materials
 recommendation of CDC for blood spills
 Disadvantage: extremely susceptible to
 disinfection method for water
pH changes activate at alkaline
 adenovirus- causing sore eyes
 2% solution for 10 mins: disinfectant
nakukuha sa pool na hindi na disinfect
 2% solution for 3-10 hrs: sterilant
ng maayos
 sterilization method for:
E. Detergents: Quaternary Ammonium
- heat labile medical equipment
Compound
- materials that cannot be sterilized
 principles: reducing the surface tension
with gas
of molecules in a liquid
C. Halogens
- disruption of the cellular
Iodophors membrane, resulting in leakage of
cell contents
 iodine (5-10%) + neutral polymer carrier
 povidone + iodine = iodophor
  disadvantages: effectiveness is reduced
by hard water & soap
- inactivated by organic materials
 disinfection method for noncritical
surface (benchtops and floors)
F. Phenolics
 principle: disruption of cell walls
leading to precipitation of proteins
** lower concentration of phenolics: disruption
of enzyme system
 disinfection method for:
- surface in hospitals
- household environments
 commonly found as ingredients of
germicidal soaps
1. Chlorhexidine Gluconate (0.5% to 4%)
 topical antiseptic
 principle: disrupts the microbial cell
membrane and precipitates cellular
contents
 advantages: strong affinity to the skin
and mucous membranes
- not significantly affected by organic
materials
 disadvantage: affected by pH
 disinfection method for
- hands of surgical personnel
- body of patients undergoing
surgery
2. Hexachlorophene
 effective against gram-positive bacteria
(3% for 15-30 seconds)
 principle: interrupts bacterial electron
transport
** at low conc: inhibits membrane-bound
enzymes
** at high conc: ruptures bacterial membranes
 disadvantage: with severe toxic effects
3. Chloroxylenol (0.5% to 4%)
 Parachlorometaxylenol [PCMX])
 principle: microbial cell wall disruption
and enzyme inactivation
 advantage: unaffected by organic
materials
 disadvantage: neutralized by nonionic
surfactants and
 polyethylene glycol
** low antimicrobial efficacy compared with
iodines, iodophors, and CHG in reducing skin
flora
 disinfection methods for
- healthcare personnel handwash
- surgical hand scrub
4. Triclosan
 Consumer and professional healthcare
prodducts
 principle: disrupts the cell wall
** good activity: gram (+) & (-) bacteria &
viruses
** fair activity: M. tuberculosis
** poor activity: fungi
 advantage: not significantly affected by
organic materials
 disadvantage: affected by pH,
surfactants and emollients
- formulation significantly affects
activity
- absorbed through intact skin
G. Heavy metals
 rarely used in clinical application
 slowly bactericidal- irreversible billing
ang effect
 (bacteriostatic) – reversible
 disadvantage: has toxic effects
1. Mercuric chloride: used as preservatives
for paint
2. 2. Silver nitrate (1% eye drop solution):
prophylactic treatment to prevent
 gonococcal (Neisseria gonorrhoeae)
conjunctivitis in newborns
H. Gases
1. Ethylene Oxide
 450-700mg of ethylene oxide per liter
of chamber space at 55-60°C for 2
hours
 Sterilization
 relative humidity of 30%: sporicidal
 principle: alkylation of nucleic acids in
spore and vegetative cell
 mixed with nitrogen or carbon dioxide Biosafety level 2
before use
 most common type of BSC used in a
 sterilization method for
microbiology laboratory
- materials that cannot withstand
 agents that pose a moderate potential
- steam sterilization
hazard for the employees and
2. Hydrogen peroxide
environment
 sterilant in the pharmaceutical and
medical device manufacturing
industries
3. Periacetic acid (gaseous form)
 sterilant in the pharmaceutical and
medical device manufacturing
industries
 combination: shortens contact time

Biological Safety Cabinets

 engineering control Biosafety level 3

 type of containment barrier that
 for infectious agents that are either
protects the worker from the
indigenous/ exotic or highly infectious
aerosolized transmission of organisms
 potential for aerosol transmission
 any procedure that has the ability to
 diseases with these agents may have
create aerosols should be performed in
a BSC serious lethal

Types:

Biosafety level 1

 agents that are well classified and are

not known to cause disease consistently
in healthy adults
 minimal threat to laboratory personnel
and environment
Biosafety level 4

 for agents that are dangerous and

exotic
 high risk of causing life-threatening
infections, can be transmitted by
aerosols, or have an unknown risk
of transmission
 located in a separate building or is
in an isolated zone within a building
 Types: cabinet and suit
BACTERIOLOGY LECTURE

WEEK 5

Workflow in Bacteriology Lecture Guide

Specimen Collection (critical step)

 critical considerations
- time of the collection- you always have to make sure that lagi nating maaabutan na nasa
early phase pa ng infection
- hindi pa nag bibigay ng antibiotic
- you have to maintain the viability of the organism with minimal contamination
 maintain the viability of these organisms with minimal contamination
 during the acute phase of an illness and/or before antibiotics are administered

WORKFLOW

 specimen collection (pre analytical)

 direct microscopic exam (analytical)
 culture (analytical)
 bacterial id (analytical)
 biochemical exam (analytical)

Patient information nakalagay sa label ng culture bottle

 name of the patient

 birthday
 age
 identification sa hospital
 site of collection
 time and date of collection

Specimen Container Patient Special Instruction Transportation Storage before

preparation to laboratory processing
Aqueous/vitreous Sterile, screw Immediately / Set up
fluid cap tube RT immediately
on receipt
Abscess (also
lesion, wound,
pustule, ulcer)

Pwede mag
perform ng direct
microscopic exam

Isang swab sa
direct microscopic
isang sa culture
Superficial Aerobic swab Wipe area with Swab along leading Within 24 hours/ RT
moistened with sterile saline or edge of wound 24hrs/RT
stuart’s or 70% alcohol If naka stuart
amie’s medium and amie
(enrichment iincubate
medium. This muna ng 18-
two will make 24hours bago
sure that your ilagay sa
organisms will culture
still be viable medium
upon
transportation
to the
laboratory,
room
temperature till
24 hours)
(kailangan may
transport
medium palagi
dahil ang swab
mabilis mag dry
out)
Deep Anaerobic Wipe area with Aspirate material Within 24 hours/ RT
transporter sterile saline or from wall or excise 24hrs/RT
(malalalim na 70% alcohol tissue
area kadalasang
di naabot ng
oxygen)

Kailangan pa rin
gummamit ng
anaerobic
transporter
Blood or bone Blood culture Disinfect Draw blood at time Within 2hrs/RT Must be
marrow media set Venipuncture of febrile eoisode; incubated at
(aerobic and site with 70% draw 2 sets from Kailangang mag 37°C on
Walang direct anaerobic alcohol and right and left arms; positive siya receipt
microscopic exam bottle) or disinfectant do not draw more within 5 days. laboratory
vacutainer tube such as than 3 sets in a 24-hr And within that
Whenever there’s with SPS betadine period; draw ≥20 5 days Specialize
presences of (anticoagulant ml/set (adult) or 1- kailangan machine na
blood even small SPS 0.025% SPS) 20 ml/set matransfer sa may
amount of blood (SODIUM (pediatric)depending culture media incubator- it
in your body or POLYANETHOLE on patients weight enhances the
inyour peripheral SULFONATE) growth of
circulation mag When you will be pathogen in
mamanifest ka ng getting blood for the blood. It
sign and Laging dalawa blood culture it takes few days
symptoms kasi or tatlo na should be extracted bago
wala naman consure bottle between intervals. makalagay sa
talaga sila dapat ang kukunin per Mag iinterval ka culture media
doon. patient muna ng 1hr bago
kumuha ulit. Primary
culture media-
Very important: naka selective
label sa blood differential
culture bottle kung culture media
saan kinuha
Gano katagal
Febrile stage, ang hihintayin
kailangan may lagnat bago ideclare
ang patient kasi na negative 5-
gusto mo na yung 7days
organism active yung depends of
mga bacteria. the laboratory
protcol
Blood fluids
Amniotic, Sterile, screw Disinfect skin Needle aspiration Immediate/RT Plate as soon
abdominal, cap tube or before as received
ascites anaerobic aspirating
(peritoneal), bile, transporter specimen
joints (synovial),
pericardial,
pleural

Bone
Sterile, screw Disinfect skin Consider rapid Immediate/RT 6hrs/RT
cap tube before the testing (e.g. gram
procedure stain, cryptococcal
antigen)
Cerebrospinal
fluid
Sterile specimen Sterile, screw Disinfect skin Consider rapid Immediate/RT 6hrs/ 37°C
Level 1 priority cap tube before testing (e.g. gram except for
aspirating stain, cryptococcal viruses which
specimen antigen) can be held at
4°C for up to
3 days

Ear
Inner Sterile, screw Clean ear Aspirate material Immediate/RT 6hrs/RT
cap tube or canal with behind drum with
anaerobic mild soap syringe if ear drum
transporter solution intact, use swab to
 before collect material
myringotomy from ruptured ear
(puncture of drum
the ear
drum)
Outer Aerobic swab Wipe away Firmly rotate swab Within 24hrs/RT
moistened crust with in outer canal 24hr/RT
with stuart’s sterile saline
or amie’s
medium

Eye
Conjunctiva Aerobic swab Sample both eyes, Immediate/RT Plate as soon as
moistened use swab pre received
with stuart’s moistened with
or amie’s sterile saline
medium
Corneal Bedside Clinician Immediate/RT Must be incubate at
scrapings incubation of should instill 28°C (Sab) or 37°C
BA, CA, Sab, local (everything else) on
7H10, thio anesthetic receipt in laboratory
before
collection

Foreign bodies
IUD Sterile, screw Disinfect skin Immediate/RT Plate as soon as
cap tube before received
removal

IV catheters, Sterile, screw Disinfect skin Do not culture foley Immediate/RT Plate as soon as
pins, cap tube before catheters, IV received
prosthetic removal catheters are
valves cultured
quantitatively by
rolling the segment
back and forth
across agar with
sterile forceps 4
times, ≥15 colonies
are associated with
clinical significance
GI tract
Gastric Sterile, screw Collect in Most gastric Immediate/RT Must be neutralized
aspirate cap tube early AM aspirates are on with sodium
before infants or for AFB bicarbonate within 1hr
patient eat of collection
 or gets out
bed 25:05
Gastric biopsy Sterile, screw Rapid urease test or Immediately / Must be set up
cap tube culture for 4°C immediately
Helicobacter pylori
Rectal swab Swab placed Alkaline Insert swab 2.5cm Within 24hrs/ 72hrs / 4°C
Gagamit parin in enteric Peptone past anal sphincter, 4°C
ng transport transportation Water feces should be
medium kasi it medium (APW)- if the visible on swab
is a lot of stool and if
normal flora the diagnosis
both rectal
and stool

culture is for
vibrio you
have use
APW

APW:
Alkaline pH
NaCl

Kapag
watery ang
stool APW

Gusto nating
imaintain
lang yung
gusto nating
pathogen
Stool culture Clean, leak- Selenite F- is Routine culture Within 24hrs/ 72hrs / 4°C
proof for other should include 4°C
Gagamit parin container, enteric Salmonella,
ng transport transfer feces organism Shigella, and 18-24hr at
medium kasi it to enteric (example Campylobacter, 37°C saka
is a lot of transport Salmonella, specially Vibrio, palang
normal flora medium if Shingella) Aeromonas, icuculture sa
transport will Plesiomonas, ibang culture
exceed 1hr Kapag semi Yersinia, Escherichia media
formed or coli 0157:H7, if
Yung other needed Suspected
enrichment characteristic pathogen na
medium selenite F lang ang
nagagamitin ang matitira
dito or gagamitin
transport
 medium ang
purpose
naman neto is
maintain the
viability of the
pathogen.

Enrichment
medium or
transport
medium hindi
na
popropagate
yung dami
nila na
mamaintain
lang kung
gano sila
karami

Genital tract
FEMALE
Bartholin cyst Anaerobic Disinfect skin Aspirate fluid, Within 24hr/RT
transporter before consider chlamydia 24hr/RT
collection and G C culture
Cervix Swab Remove Do not use Within 24hr/RT
moistened mucus lubricant on 24hr/RT
with stuart’s before speculum, use viral
or amie’s collection of chlamydia transport
medium specimen medium, if
necessary swab
deeply into
endocervical canal
Cul-de-sac Anaerobic Submit aspirate Within 24hr/RT
transporter 24hr/RT
Endometrium Anaerobic Surgical biopsy or Within 24hr/RT
transporter transcervical 24hr/RT
aspirate via shethed
catheter
Urethra Swab Remove Collect discharge by Within 24hr/RT
moistened exudate massaging urethra 24hr/RT
with stuart’s from against public
or amie’s urethral symphysis or insert
medium opening flexible swab 2-4cm
into urethra and
rotate swab for 2
seconds. Collect at
 least 1hr after
patient has
urinated
Vagina Swab Remove Swab secretions Within 24hr/RT
moistened exudate and mucus 24hr/RT
with stuart’s membrane of
or amie’s vagina
medium or
JEMBEC
transport
system
MALE
Prostate Swab Clean glans Collection on Within Swab: 24hrs/RT
moistened with soap secretions swab or 24hr/RT for
with stuart’s and water in tube swab, Tube: plate secretions
or amie’s immediately immediately
medium or if in tube/ RT
sterile screw-
cap tube
Urethra Swab Insert flexible swab Within 24hrs /RT for swab
moistened 2-4cm into urethra 24hr/RT for
with stuart’s and rotate swab for swab, Put JEMBEC at 37°C
or amie’s 2 seconds or collect immediately on receipt
medium or discharge on Within 2hrs on laboratory
JEMBEC JEMBEC transport for JEMBEC
transport system system
system

Respiration
tract
LOWER
BAL, BB, BW Sterile, screw Anaerobic culture Within 24hrs/ 4°C
top tube appropriate only if 24hr/RT
sheathed
(protected)
catheter used
Sputum, Sterile, screw Sputum: have Within 24hrs/ 4°C
tracheal top container patient collect from 24hr/RT
aspirate deep cough,
(suction) specimen should be
examined for
Microbacterial- suitability for
tatlong sputum culture by gram
sample stain; induced sputa
on pediatric or
bacterial uncooperative
culture- 1 patients may be
sputum watery because of
sample (e.g saline nebulization
pneumonia,
bronchitis) Dapat makakita ka
ng more than 25
direct Polymorphonuclear
microscopic cell per HPO sa
exam- purpose microscopic
is for bartlett’s examination
classification
Less than 10 na
bartlett’s epithelial cells per
classification- LPO (sputum to pag
kaya ka mag nakita mo tong
peperform ng dalawanng to)
dme kasi gusto
mo malaman Pero pag opposite
kung talagang ang nakita mo
sputum yan. saliva yon and
To screen out I should not be
its saliva or if cultured.
it’s a sputum.
Bacterial
smear + gram
stain

direct culture
UPPER
Nasopharynx Swab Insert flexible swab Within 24hr/RT
moistened through nose into 24hr/RT
with stuart’s posterior
or amie’s nasopharynx and
medium rotate for
5seconds; specimen
of choice for
Bordetella pertussis
Pharynx Swab Swab posterior Within 24hr/RT
moistened pharynx and tonsils; 24hr/RT
with stuart’s routine culture for
or amie’s group A
medium Steptococcus (S.
pygenes) only
Tissue
Anaerobic Disinfect skin Do not allow Within 24hr/RT
transporter or specimen to dry 24hr/RT
sterile, screw- out; moistened
cap tube with sterile,
 distilled water if not
bloody
Urine
Clean-voided Sterile, screw Female: Within 24hrs/ 4°C
midstream cap tube clean area 24hr/RT
(CVS) with soap
and water
Direct then rinse
microscopic with water;
exam hold labia a
part and
Pwedeng begin voiding
deretsyo na sa in commode;
culture after several
medium mL have
passed,
Yung unang collect
portion should midstream
be discarded
then the Males: clean
middle portion glans with
that must be soap and
collected and water, then
the latter rinse with
portion should water;
be discarded retract
foreskin;
Unang portion after several
ang pinaka mL have
contaminated passed,
collect
Basta non- midstream
sterile pwede
paring
imaintained or
itransport
within 24hrs at
4°C
Straight Sterile, screw Clean Insert catheter into Within 24hrs/ 4°C
catheter (in container urethral area bladder; allow first 2hr/4°C
and out) (soap and 15mLto pass; then
water and collect remainder
Sa mismong rinse with
catheter bag water)
mag aaspirate
ng urine
 Indwelling Sterile, screw Disinfect Aspirate 5-10mL of Within 24hrs/ 4°C
catheter container catheter urine with needle 2hr/4°C
(foley) collection and syringe
port
Material na
ginamit na
ininsert dun sa
bladder
Suprapubic Sterile, screw Disinfect skin Needle aspiration
aspirate container above the
symphysis pubis
Sterile urine through the
because you abdominal wall into
need to the full bladder
aspirate urine
direct to the
urinary
bladder

TYPES OF SPECIMEN

1. Sterile

2. Nonsterile: use selective medium

3. Aerobic (24 hours)

4. Anaerobic (48 hours)

TRANSPORT MEDIUM

1. Cary Blair: stool specimen

2. Stuart’s

- swabs from skin lesion/wound

- also as viral transport medium

3. Amies: respiratory specimens & swabs from skin lesion/wound

4. Transgrow: Neisseria spp.

5. JEMBEC: Neisseria spp.

6. Todd Hewitt: vaginal and rectal swab (S. agalactiae)

Specimen Processing

Level 1

 critical because they represent a potentially life threatening illness

  from an invasive source
 unang priority na iprocess
 9 TAT (turnaround time)- from the time of the collection until mag release ng result

Level 2

 unprotected and may quickly degrade or have overgrowth of contaminating flora

 Pwedeng idelay, pwede munang ilagay sa specified storage as long as may preservative yan,
yung unpreserved lalagyan ng preservatives lalagyan ng transport medium, ilalagay sa transport
medium, ilalagay sa refrigerator pwede pang iprocess sa susunod na araw.
 Unpreserved specimens

Level 3

 require quantitation
 Pwedeng idelay, pwede munang ilagay sa specified storage as long as may preservative yan,
yung unpreserved lalagyan ng preservatives lalagyan ng transport medium, ilalagay sa transport
medium, ilalagay sa refrigerator pwede pang iprocess sa susunod na araw.
 Specimens na kailangan nating iquantify yung colony and bacterial cell

Level 4

 in holding or transport media

 may be delayed to process more critical specimens first

SPECIMEN STORAGE

1. refrigerator temperature [4°C]

 urine, stool, viral specimens, sputa, swabs (not for anaerobes) and foreign devices
 mga non-sterile specimen
2. Ambient/room temperature [22°C]
 sterile body fluids, genital specimens, and ear and eye swabs (anaerobic bacteria)
 mga sterile specimen except csf
3. Body temperature [37°C]
 CSF
4. Freezer temperature [either or –70°C]
 –20°C: serum for serologic studies (1 week)
 –70°C: tissues or specimens for long-term storage

Quality assurance

- It will make sure that your result is RELIABLE, ACCURATE, PRECISE ETC
1. Pre-analytical- preparations palang. Patient identification, patient preparation, specimen
collection
2. Analytical- nag peperform na ng assay or procedure. Dme hanggang biochem examination
3. Post analytical- nag release na ng result
Direct Microscopic Examination

Purpose:

 determine the quality of the specimen

example: Sputum vs saliva: quantitation of WBCs or epithelial cells
 give indication of the infectious process involved
example: gram stain of a sputum:
WBCs and gram (+) diplococci is indicative of Streptococcus pneumonia (NORMAL FLORA)
- ang mga diplococci though they are respiratory tract normal flora anong indication na
naging pathogen na siya YUNG PRESENCE NG WBC
 routine culture workup can be guided by the results of the smear
- correlate the bacterial isolates with the types detected in the smear
 dictate the need for non-routine or additional testing
example: fungal elements in a specimen for bacterial culture
 Not useful for:
- Throat and nasopharyngeal specimens
- Specimens from vagina, cervix, and anal crypts
- Stool

Infectious process- malalaman na agad natin by looking at a direct microscopic exam kung anong
klaseng ng organism ang dinedeal natin1

*hindi lahat ng specimen nag uundergo ng dme

**upper respiratory exam (urt) specimen- hindi sinasama sad me kasi maraming normal flora

STAINS

1. Simple stain
 Carbol fuchsin, methylene blue, gentian violet
2. Differential stain
a. Gram stain
 For characterization of specimen
a. Bartlett’s classification: sputum sample
- >10 epithelial cells/LPO and <25 PMN/HPO: saliva
- <10 epithelial cells/LPO >25 PMN/HPO: sputum

Precautions
Crystal violet rinsed too vigorously before it is
complexed with iodine
Prolonged decolorization
Insufficient decolorization
Prolonged application of safranin
Insufficient application of safranin
Antibiotic-treated and dead or degenerating
organisms
Effects
Washed away the crystal violet and results to
falsely gram (-)
Results to falsely gram (-)
Results to falsely gram (+)
Prolonged application of safranin
Results to falsely gram (+)
Gram variable/ atypical
Gram variable/ atypical

1. All cocci are gram (+) EXCEPT: Nesseria spp., Veillonella spp., Moraxella spp.
2. All bacilli are gram (-) EXCEPT: Mycobacteria, Corynebacteria, Clostridia, Bacillus, Lactobacillus,
Listeria, Erysepilothrix, Nocadia, Actinomyces
3. All spiral organisms are reported as gram (-).
4. Yeast are gram (+).
b. Acid-fast stain
 detection of mycobacteria in clinical specimen

Method Components Duration Test result Test result

Ziehlneelsen’s Carbol fuchsin 5 mins 30 secs Red bacilli against Acid fast bacilli
method (with heat) blue background
- hot method Acidalcohol (3%
HCl in 95% Blue bacilli Non-acid fast
ethanol) 30 secs-1 min against blue
Methylen e blue/ background
Malachite green
Kinyoun’s Carbol fuchsin (no 3 mins Red bacilli against Acid fast bacilli
method - cold heat) Acidalcohol blue background
method (0.5% HCl in 95%
ethanol) 2 mins 30 secs Blue bacilli Non-acid fast
Methylen e blue/ against blue
Malachite green background
Pappenheim’s Carbol fuchsin 2 mins Red/ pink bacilli M. tubercul osis
method (with heat)
- differentiate M. Pappenhei m’s 4-5 mins Colorles s bacilli M. lacticola
tuberculosis from differentia ting
M. lacticola stain

Rosalic acid &

Methylen e blue
in glycerin and
absolute alcohol
Baumgarten’s Alcoholic carbol 5 mins
method fuchsin 1% nitric --
acid in 95%
alcohol
Gabbet’s method Alcoholic carbol 5 mins
fuchsin --
Acidalcohol (3% 20-30 secs
HCl in 95%
ethanol) Gabbet’s
methylene blue
solution
Special stain

Method Components Test result Structure seen

A. CAPSULAR
STAINING
(the stain is directed or
specific to the capsule
of the bacteria)
Hiss staining method Crystal violet/ Gentian Clear/ halo around the Capsule
violet bacterial cell

contrast stain 20% Violet (same as Bacteria

copper sulfate solution background)
Anthony’s method 1% copper sulfate Unstained Capsule
solution 1% Alcian blue
in 95% ethanol 1:1000 Deep purple within the Bacteria
diluted carbol fuchsin colorless halo. The
background is colored
purple.
Novelli’s method Alcian blue 1:1000 Blue Capsule
diluted carbol fuchsin
Pink Bacteria
METACHROMATIC
GRANULES STAINING
(Corynebacterium
diphtheriae)
Loeffler’s alkaline LAMB Dark blue Polar ends of the
methylene blue bacteria
(L.A.M.B.) method
Blue Bacteria
Albert method Albert’s stain Gram’s Blue to black Granules
iodine
Blue to blue green Bands

Green Cytoplasm
SPORE STAINING
(bacillus and
clostridium)
Acetic acid method 5% acetic acid Carbol Red/ Pink Spore
fuchsin LAMB
Blue Bacteria
Wirtz-Conklin method 5% Malachite green Green spherules Spore
0.5% Safranin
Red Bacteria
Dorner method Carbol fuchsin 10% Red Spore
Nigrosin
Colorless against a Bacterial cell
dark gray background
 CELL WALL STAINING
Dyar method Cetyl pyridium chloride Red Cell wall
Saturated solution of
Congo red Methylene Colorless Bacteria
blue
E. FLAGELLAR
STAINING
Leifson’s method Formalin Leifson’s Red/ Purple Flagella
flagellar stain
Colorless Bacteria
Fisher and Conn’s Carbol fuchsin Red Flagella
method
Colorless Bacteria
F. LIPID GRANULES Sudan Black B stain Blue black/ Dark bluish Fat globules
STAINING Xylol 0.5% safranin gray/ Black
Cells
Red
G. NUCLEAR BODY 1% mercuric chloride
STAINING 1% crystal violet (pH 6-
8) 2-5% Nigrosin (pH 3-
4)
H. POLAR STAIN
Wayson’s stain Solution A Purple Polar bodies
Basic fuchsin0.20 g
For Yersinia spp. (90% dye) Methylene
blue- 0.75 g (90% dye)
Ethyl alcohol20 mL
(95%)

Solution B
5% Phenol200 mL

Culture

MACROSCOPIC OBSERVATION

 gross appearance of the specimen

 allows the processor to determine the adequacy of the specimen and the need for special
processing
- Swab or aspirate
- Stool consistency (formed or liquid)
- Blood or mucus present
- Volume of specimen
- Fluid—clear or cloudy
INSTRUMENTS

1. Incubator
 Set at 35-37 °C
- 18-24 hrs (aerobic culture) routine
- 24-48 hrs (anaerobic culture) routine
2. Inoculating needles
 Nichrome wire, platinum
 Should not be longer >5cm
3. Pasteur pipette
 Transfer liquid

CULTIVATION

 process of growing microorganisms in culture by taking bacteria from the infection site (in vivo
sa loob ng katawan) and growing them in the artificial environment of the laboratory (in vitro
outside)
 requires that the nutritional and environmental growth requirements
- nutritional- carbon, nitrogen, energy
- environmental- pH, atmospheric requirement

Culture media

 on or in which bacteria are grown

 Bacterial cell multiply to sufficient numbers to allow visualization
- Fastidious- With complex requirements:
- Nonfastidious- Basic requirements

CULTURE MEDIA

I. Phases
a. Liquid/ Broth
- + growth- turbidity, - growth clear
- nutrients are dissolved in water
- bacterial growth (106 or 1M bacteria cells per mL) is indicated by change in broth’s appearance
from clear to turbid

Vibrio- kaya niyang mabuhay sa mataas na salt concentration and also kaya niyan mabuhay kahit
mataas yung ph. heliophobic organism to.

b. Semi-solid
- - 0.5-1% agar
- **agarose: most common solidifying agent
: melts at ≥95°C
: re-solidify at < 50°C
- used to observe motility
- one of the example is sulfide indole motility (SIM)
- butt lang
 - motility- kapag ang result is naging + hazy turbid
c. Solid/agar
- 2-3% agar
- Butt, slant, butt slant sa biochemical exam ginagamit
- May plated din hehi at tubes
d. Biphasic medium
- both liquid and solid phase
- Castañeda medium for Brucella spp
- Bottle container na merong slant na solid agar at merong broth

**colony: bacterial population derived from a single bacterial cell

Pure or mixed

Pure culture- magkakapareho ang colonial morphology isang oraganism lang ang present

Mixed culture- iba iba ang structure ng colony merong multiple organism na present

Streak/culture/ inoculate- specimen to your culture media

Subculture- culture media to another culture media. Pag tatransfer ng organism

II. Function
a. Supportive/ General purpose medium
- contain nutrients that support growth of most nonfastidious organisms
- without giving any particular organism a growth advantage
b. Selective medium
- contain one or more agents that are inhibitory to all organisms except those being sought
- select for the growth of certain bacteria to the disadvantage of others

example inhibitors:

**antibiotics: either gram (+) or (-)

**bile salts/ dyes: inhibit gram (+) bacteria

**alcohol: inhibit gram (-) bacteria

macConkey- selective because it has bile salt and crystal violet

c. Differential medium
- employ some factor that allows colonies of one bacterial species to exhibit certain culture
characteristics that can be used to distinguish them from other bacteria growing on the same
agar plate
 - Factor na kaya mag distinguish ng dalawang group in one agar plate
d. Enriched medium
- enhance growth of fastidious bacteria
- general purpose + additional special requirement to enchane the groth of pathogens
e. Enrichment medium
- contain specific nutrients required for the growth of particular bacterial pathogens
- enhance the growth of a particular bacterial pathogen from a mixture of organisms by using
nutrient specificity
- general purpose + additional special requirement to maintain the growth of pathogen

CULTURE MEDIUM PREPARATION

Broth Medium Solid Medium

Weigh Weigh
Dissolve Dissolve
Dispense Sterilize
Sterilize Dispense

METHOD OF INCUBATION

1. Incubator
 35° and 37°C and humidified atmospheres that contain 3% to 5% CO2
 Pag may candle jar pwedeng tumaas ng 5-10% CO2
 Pag tumataas ang carbon dioxide bumababa ang oxygen level
2. Candle jar
 Generate 3% CO2 concentration
 candle: uses up just enough oxygen before it goes out (from lack of oxygen)
to lower O2 tension
to produce CO2 and H2O by combustion
3. Anaerobic jars
 GasPak jar
 use an envelope gas generator & oxidation
 reduction indicator (methylene blue)
 kapag nag turn ng white ang methylene blue wala ng oxygen
 CO2 & H2O: generated when water is added
4. Anaerobic chambers
 0% oxygen
 contain:
 catalyst: palladium-coated alumina pellets
removes residual O2
inactivated by H2O and gaseous metabolic end products produced by the anaerobes
(H2S)
 desiccant: Silica gel
absorb H2O formed when H combines with free O2
  anaerobic gas:
5% H, 5% to 10% CO2 & 85% to 90% N
 oxidation-reduction indicator:
Methylene blue
- white (absence of O2)
- blue (presence of O2)
Resazurin

- Colorless (absence of O2)

- pink (presence of O2)

Culture Medium Use Principle & Results

Components
Acetate agar differential medium: Acetate: carbon source (+) growth: Escherichia spp. &
E.coli from Shigella spp Enterobacteriacea
Amm. salt: nitrogen
source (-) growth: Shigella, Proteus, and
Providencia spp.
Bromthymol blue:
green to blue
Alkaline peptone enrichment medium alkaline pH:
water for Vibrio and uninhibited replication
Aeromonas spp. from of organisms while
stool specimens temporarily
suppressing the
replication of other
organism
American Trudeau egg-based medium eggs: provide fatty
Society Medium used for the isolation acids
of M. tuberculosis
potatoes: provide a
carbon source.

Malachite green:
inhibitory for normal
bacterial flora
Bacteroides Bile selective differential Oxgall: separates bile- (+): dark brown or black colonies
Esculin Agar for isolation and resistant species
identification of (growth) from bile-
members of the sensitive ones (no
Bacteroides fragilis growth) 1%
group esculin: ability (+) or
inability (-) to
hydrolyze esculin
Bile Esculin Agar selective differential Oxgall: inhibits the (+): darkening of the medium
agar to isolate and growth of most gram-
identify group D positive organisms
 streptococci and
enterococci Esculin: ability (+) or
inability (-) to
hydrolyze esculin
Vancomycin: detect
vancomycin-resistant
streptococci &
enterococci

Azide: inhibit gram (-)

organisms
Bismuth Sulfite Agar selective medium for bismuth sulfite & S. typhi :
the isolation of brilliant green: inhibits
Salmonella spp gram (+), lactose- S.gallinarum,choleraesuis &
fermenting intestinal Paratyphi: light green
normal microbiota &
Shigella
Blood Agar, enriched medium tryptic soy agar base
Anaerobic, CDC useful for the isolation yeast extract, L-
of fastidious cysteine, hemin, sheep
anaerobes blood and vitamin K1
Blood Agar, useful enrichment Sheep blood:
Anaerobic, Brucella medium for the enrichment
Base, Wadsworth isolation of
moderately fastidious, Vitamin K1 and hemin
obligate anaerobes
Blood Agar, Blood Agar, Anaerobic,
Anaerobic, with with Kanamycin and
Kanamycin and Vancomycin (KV)
Vancomycin (KV)
Blood Agar, primary isolation of Laked erythrocytes
Anaerobic, Laked, obligate gram-negative (lysed by freezing) &
with Kanamycin, anaerobes, particularly vitamin K: enrichment
Vancomycin, and Bacteroides spp. Kanamycin &
Vitamin K (KVKL)
Vancomycin: inhibit all
cocci & facultative
gram (-) bacilli, except
the pseudomonads
Blood Agar, Rabbit enrichment medium in
recovery and the
demonstration of β-
hemolysis by
Haemophilus spp. and
Gardnerella vaginalis
Blood Agar, Sheep routine medium to infusion agar or tryptic
cultivate moderately soy agar base + 5% to
fastidious organisms 10% defibrinated
sheep, rabbit, or
human blood
Blood Phenylethyl selective enrichment yeast extract, hemin,
Alcohol Agar, medium for isolation vitamin K, and
Anaerobic, CDC (PEA) of Bacteroides, defibrinated sheep
Prevotella, and other blood: enrichment
obligate anaerobes
from specimens phenylethyl alcohol:
containing a mixture inhibits facultative
of obligate and gram (-) anaerobes by
facultative anaerobe suppressing DNA
synthesis and cell
division
Bordet-Gengou Blood selective enrichment Peptone: base medium
Agar (B-G) medium Glycerol, potato
for the isolation of B. infusion, & sterile, 15%
pertussis and 30% defibrinated
and B. parapertussis
sheep blood:
enrichment Penicillin,
methicillin, or

cephalexin: inhibitors
Brain-Heart Infusion enriched medium for Brains and beef heart:
Broth (BHI) the cultivation of non- provide nutrients
fastidious and
moderately fastidious Peptones, glucose,
microorganisms sodium chloride, and
buffers
rec. for the cultivation
of pneumococci for 6.5% NaCl: diff. salt-
the bile solubility test tolerant enterococci
from streptococci
Buffered Charcoal enrichment medium Ferric pyrophosphate:
Yeast Extract Agar useful in the isolation provide iron
(BCYE) of Legionella spp.,
Francisella and Yeast extract, α-
Nocardia spp. ketoglutarate, and L-
cysteine

Activated charcoal:
absorb toxic
compounds
Burkholderia cepacia isolate B. cepacia Crystal violet, bile (+): dull yellow to hot pink
Agar selectively from RT salts, polymyxin B & medium
specimens from ticarcillin: inhibit most
patients with cystic gram (+) and gram (-)
fibrosis organisms

Inorganic salts,
peptones, pyruvate,
and phenol red
Campylobacter Blood selective enrichment Brucella agar: base Campylobacter sp.
Agar (Campy-BA) medium for the medium
isolation and - flat, gray, nonhemolytic, raised
cultivation of sodium bisulfite: and mucoid colonies
Campylobacter spp. lowers redox
from stool specimens potential; enhancing - tan or slightly pink colonies
recovery of
microaerophilic swarming or spreading across the
organisms surface of the plate

10% sheep’s blood:

enrichment

Vancomycin: inhibit
gram (+) cocci

Trimethoprim: inhibit
swarming strains of
Proteus sp

Polymyxin B: inhibit
gram (-) bacilli

Amphotericin B: inhibit
filamentous fungi and
yeasts

Cefoperazone:
antipseudomonal
activity and inhibit
Enterobacteriaceae
Campylo-bacter less inhibitory for Preston agar with beef
Charcoal Differen-tial Campylobacter spp. extract and peptones:
Agar but more inhibitory for base medium
organisms found as Cefoperazone:
normal fecal selection agent instead
microbiota of cephazolin
Campylo-bacter selective liquid Thioglycolate broth
Thiogly-colate Broth medium used to with 0.16% agar: base
(Campy-Thio) enhance the isolation medium
of Campylobacter spp.
Vancomycin: inhibit
holding or transport gram (+) cocci
medium
Trimethoprim: inhibit
swarming strains of
Proteus sp

Polymyxin B: inhibit
gram (-) bacilli
Amphotericin B: inhibit
filamentous fungi and
yeasts

Cefoperazone:
antipseudomo
Cefsulodin-Irgasan- select for the isolation Peptones, beef, and (+): Yersinia spp.
Novobio-cin (CIN) of Yersinia yeast extracts:
enterocolitica in stool nutrients Sodium
samples desoxycholate,
cefsulodin, novobiocin,
Irgasan, and crystal
violet: inhibitor

Mannitol:
differentiating agent
(+): Aeromo-nas spp.
Neutral red: pH
indicator
Cetrimide Agar select for P. Cetrimide: inhibitory (+): P. aeruginosa Under UVL:
aeruginosa in yellow-green fluor.
specimens with mixed Magnesium chloride &
microbiota potassium sulfate: because of pyoverdin prod.
differentiation of non– stimulate the stimulated by the low iron
glucose-fermenting, production of
gram - rods pyocyanin
Chocolate Agar enrichment agar useful sheep blood : hgb, (+) growth: N. gonorrhoeae &
in promoting the hemin and NAD Haemophilus spp.
growth of Haemophi-
lus and other Iso-Vitalex: chemical
fastidious bacterial supplement solution
species

5% horse blood
(hemin)
 Isovitalex (source ng
NAD)
For haemophilus spp.
CHROM-agar selective, differential, Peptone and glucose :
chromogenic for basal nutrient agar
isolation &
identification of yeast,
S. aureus, E. coli
O157:H7
Citrate Agar, Simmons differentiating gram (-) citrate: carbon source (+): growth (blue color in the slant)
enteric bacilli
ammonium salt: (-): no growth
nitrogen source

bromthymol blue: pH
indicator; green to
blue
Columbia Agar with aerobic and anaerobic Columbia agar: basal
and without 5% Sheep bacterial organisms nutrient agar
Blood
peptones: from casein,
meat, beef and yeast
extracts

5% defibrinated sheep
blood: support the
growth of more
fastidious organisms;
detection of hemolytic
reactions; provide X
factor

NADase present in the

blood will destroy the
V factor

CNA
Eosin– Methylene selective differential Eosin Y and methylene (+): fermenters colonies E. coli:
Blue Agar (EMB) medium for the blue dyes: inhibit the blue-black colonies with a metallic
isolation and ID of growth of gram (+) greenish sheen Enterobacter sp.:
gram (-) enteric bacteria form pink colonies
bacteria
lactose & sucrose: (-): non-fermenter colonies:
allow differentiation translu-cent and colorless or light
based on fermentation purple

Fermentation:
detected by color
 changes &
precipitation of dyes
as pH drops
Gelatin Medium differential medium (+): forms a gel (gelatinase-
used to determine positive)
bacterium’s ability to
produce gelatinase (-): remain liquid (gelatinase-
and thereby hydrolyze negative)
gelatin
Gram-Negative Broth selective enrichment desoxycholate and
medium used to citrate salts: inhibit the
enhance the chance of growth of gram-(+)
recovering enteric bacteria
pathogens,
(Salmonella and mannitol: enrichment
Shigella) from fecal
specimens
Hektoen Enteric Agar selective differential bile salts & dyes: (+): Fermenters one or both CHO
(HEA) medium for direct inhibit gram (+) and E. coli (bright orange to salmon
isolation of enteric gram (-) normal flora pink)
pathogens from feces
and for indirect lactose, salicin, and (-): Non-fermenters Salmonella &
isolation from sucrose: allow Shigella spp. (green to blue-green
selective enrichment differentiation based colonies)
broth on fermentation

bromthymol blue: pH
indicator

ferric salts
Lim Broth (modified isolation of peptones, yeast
Todd-Hewitt broth) S.agalactiae, usually extract, and dextrose:
from vaginal or rectal support the growth of
swabs obtained from the streptococci
pregnant women
Colistin and nalidixic
acid: inhibit the
growth of gram-
negative
Loeffler Coagulated recovery and High serum content & (+): colonies surround-ded by
Serum Slant identification of egg: detection of small holes containing liquefied
Clostridium proteolytic activity medium
diphtheriae

promotes the
development of
characteristic
 metachromatic
granules
Löwenstein-Jensen cultivate Potato flour, egg, and
Medium (LJ) Mycobacterium spp glycerol: detoxify and
supply nutrients
required for growth

Asparagine: maximum
production of niacin

Malachite green:
inhibits the growth of
other bacteria
Lysine Iron Agar measures three Lysine: amino acid
parameters useful in
identifying species of Glucose: carbohydrate
Enterobacteriaceae— source small amount
(1) lysine of protein
decarboxylation, (2)
lysine deamination, Bromcresol purple: pH
and (3) H2S production indicator

Sodium thiosulfate:
sulfur source Ferric

ammonium citrate:
H2S indicator
MacCon-key agar selective, differential, bile salts & crystal (+): fermenters (pink or red
(MAC) primary plating violet: inhibit gram (+) colonies)
medium selects for
Enterobacteriaceae lactose: sole (-): non-fermenters (colorless or
and other gram (-) carbohydrate source transparent colonies)
rods in the presence of
mixed microbiota neutral red: pH
indicator
pH indicator- indicator,
detecting the presence ferric salts
of acid
pink- kapag ang
neutral red nag
produce ng acid

colorless- neutral/
alkaline
Macconkey Sorbitol isolate E. coli O157:H7 same components as (+): pink to red colonies
Agar (doesn’t ferment MAC except the D-
sorbitol rapidly) sorbitol is substituted (-): colorless colonies
for lactose
Malonate Broth ID of species of sodium malonate: (+): Prussian blue
Enterobac-teriaceae, primary carbon source
particularly Salmonella (-): green
small quantities of
glucose and yeast
extract: nutrients

bromthymol blue: pH
indicator
Mannitol Salt Agar selective and 7.5% NaCl: inhibits (+): yellow (Staphylococcus)
differential primary most gram (-) and
culture medium useful gram (+) bacteria
in the recovery and ID except Staphylococcus (-): red
of staphylococci from spp.
specimens with mixed
microbiota Mannitol: sole
carbohydrate

phenol red: pH
indicator

acid- yellow
neutral/ alkaline- will
return to red
Methyl Red Voges- Differentiating among
Proskauer Medium Enterobacteriaceae
Middlebrook 7H10 cultivate 7H11: casein
and 7H11 Agars Mycobacterium spp hydrolysate
(stimulates growth of
Isoniazid-resistant drug-resistant
strains grow better on Mycobacterium
these media, tuberculosis)
especially
Middlebrook 7H11 7H10 & 7H11: amino
acids, glycerol, and
inorganic salts (growth
factors)

OADC (oleic acid–

dextrose-catalase):
simulates egg
components

Albumin: inhibit toxic

agents
 Malachite green:
inhibitor
Mitchison 7H11 more selective for Amphotericin B,
Selective Agar mycobacteria because carbenicillin,
of added antimicrobial polymyxin B, and
agents trimethoprim:
inhibitory to gram (-)
rods in and yeast
Modified Thayer- selective enrichment hemoglobin, vitamins,
Martin Agar medium used for diphosphopyridine
recovering Neisseria nucleotide, L-cysteine,
gonorrhoeae and NAD, and glutamine:
Neisseria meningitidis enrichment
from specimens that
have mixed microbiota Cornstarch: absorb
inhibitory substances
that might be present

Vancomycin: inhibit
the growth of gram-(+)

Colistin: inhibit gram (-

) rods

Nystatin: inhibit fungi

Trimethoprim:
prevents Proteus spp.
from swarming
Mueller-Hinton Agar testing the animal infusion, casein
susceptibility of extract, and starch:
organisms to support growth of mist
antimicrobial agents organism

testing starch Starch: a. protects the

hydrolysis organisms against
toxic substances
b. serve as an energy
source for some
bacteria
Mueller-Hinton Agar detect methicillin-
with 2% NaCl resistant
Staphylococcus aureus
(MRSA)
Mueller-Hinton Agar screen S. aureus
with 4% NaCl and 6 µg isolates selectively for
Oxacillin
 resistance to oxacillin
or nafcillin
Xylose-lysine- selective, differential, Sodium desoxycholate: (+) Yellow colonies: E. coli
desoxycholate (XLD) primary plating inhibits gram (+) cocci
agar medium used to and some gram (-) Yellow colonies with black centers:
isolate Salmonella and rods found in stool as Citrobacter and some Proteus spp.
Shigella spp. from normal biota
stool and other Colorless or red colonies: Shigella
specimens containing Sucrose and lactose and Providencia spp.
mixed biota (high conc.) Xylose
(low conc.) Red colonies with black centers:
Salmonella and Edwardsiella spp.
Phenol red: pH
indicator

Lysine: detect lysine

decarboxylation

Sodium thiosulfate:
sulfur source

Ferric ammonium
citrate:H2S indicator

Macroscopic Examination (describing colonial morphology)

I. Hemolytic pattern
 on BAP (blood agar plate)
 hemolysis: hemo (RBC); lysis (break apart)
 observed in the surrounding or underneath the colony
 caused by enzymatic or toxin activity of bacteria
 titgnan natin kung yung organism is nag rerelease ng toxin kasi nilylysis niya yung rbc. Pag
wala siyang capability maag release ng toxin walang lysis ng rbc walang hemolytic pattern
 requires transillumination: passing of bright light through the bottom of the plate
 types: α-Hemolysis, β-Hemolysis, γ-Hemolysis

β-Hemolysis (toxin)

 complete clearing of erythrocytes in BAP (appearance)

 complete lysis of RBCs
 examples: Streptococcus pyogenes, Streptococcus agalactiae & Listeria monocytogenes
 Trans illumination- makikita sa likod ng plate against the light. When you let the light pass
through in culture media
α-Hemolysis (toxin)

 green discoloration of the medium (appearance)

 partial lang na poproduce niya
 partial lysis of RBCs
 examples: Streptococcus pneumoniae and certain viridans streptococci
 kaya may greenish kaksi yung rbc may hemoglobin yung hemoglobin kapag hindi siya completely
nasisira meron siyang naiiwan na substance which is bilirubin siya yung color green

PAG WALANG TOXIN GANTO ITSURA NON HEMOLYSIS:

ALPHA PRIME

 the colony is surrounded by alpha and its further surrounded by beta

II. Size
 generally visual comparison between genera or species
 estimate as large, medium, small, or pinpoint
 Small is pinpoint, medium is pinhead

SMALL- Streptococcus spp.

MEDIUM – Staphylococcus spp.

LARGE- Haemophilus spp.

III. Margin
 edge of the colonies
 filamentous, rough or rhizoid, or irregular
  Filamentous: Bacillus anthracis
 Swarming (hazy blanket of growth on the surface that extends well beyond the streak lines):
Proteus mirabilis
 Rough: Diptheroids

Swarming- unique characteristic of prosteus spp.

IV. Elevation
 determined by tilting the culture plate and looking at the side of the colony
 raised, convex, flat, umbilicate (depressed center, concave—an “innie”), or umbonate (raised or
bulging center, convex—an “outie”)
 Umbilicate: S. pneumonia
 Convex: S. aureus
 Flat: beta-hemolytic streptococci

V. Density
 transparent, translucent, or opaque
 requires transillumination
 translucent: β-Hemolytic streptococci except group B (S. agalactiae; bull’s eye colony)
 opaque: staphylococci and other gram-positive bacteria
 shiny, similar to a half-pearl: Bordetella pertussis
VI. color
 white, gray, yellow, or buff
 white: CoNS
 gray: Enterococcus spp.
 yellow or off-white: Micrococcus spp. and Neisseria
  buff: Diptheroids
VII. consistency
 determined by touching the colony with a sterile loop
 brittle (splinters), creamy (butyrous), dry or waxy
 creamy: S. aureus
 sticky: Neisseria spp.
 brittle: Nocardia spp.
 dry or waxy: diphtheroid
 dry: β-hemolytic streptococci
VIII. pigment
 P. aeruginosa: green, sometimes a metallic sheen
 Serratia marcescens: brick-red
 Kluyvera spp.: blue
 Chromobacterium violaceum: purple
 Prevotella melaninogenica: brown-black

P. aeruginosa- pyocyanin- blue green

pyorubin- red, pyomelanin- brown, pyverbin- green

Serratia marcescens: prodigiossin pigment

IX. Odor
 determined when the lid of the culture plate is removed and the odor dissipates into the
environment
 S. aureus: old sock (stocking that has been worn continuously for a few days without washing);
this odor is evident when growing on MSA
 P. aeruginosa: fruity or grapelike
 P. mirabilis: putrid
 Haemophilus spp.: musty basement,
 “mousy” or “mouse nest” smell
 Nocardia spp.: freshly plowed field

Growth in Liquid medium

 Turbidity: refers to cloudiness of the medium resulting from growth

Thioglycollate broth

STREMAER VINES- Streotococci PUFFED BALL- Pseudomonas aeruginosa

SCUM- Pseudomonas aeruginosa BUBBLES- gas produced by enteric bacilli

Enterobacteria ceae- Escherichia coli

Microscopic Examination

Microscopic Description Organism

Gram (+) cocci
in pairs Staphylococcus, Streptococcus, Enterococcus spp.
in terads Micrococcus, Staphylococcus, Peptostreptococcus
spp.
in chain Streptococcus, Peptostreptococcus spp.
in groups Staphylococcus, Peptostreptococcus,
Stomatococcus spp.
in clusters Microaerophilic Streptococcus spp., viridans
streptococci, Staphylococcus spp.
encapsulated Streptococcus pneumoniae, Streptococcus
pyogenes (rarely), Stomatococcus mucilaginosus
Gram (+) diplococci Streptococcus pneumoniae
Gram (-) diplococci Neisseria spp., Moraxella catarrhalis
Gram (+) bacill
small Listeria monocytogenes, Corynebacterium spp.
medium Lactobacillus, anaerobic bacilli
large Clostridium, Bacillus spp.
diptheroid Corynebacterium, Propionibacterium, Rothia spp.
pleomorphic Gardnerella vaginalis
 beaded Mycobacteria, antibiotic-affected lactobacilli, and
corynebacteri
filamentous Anaerobic morphotypes, antibiotic-affected cells
filamentous, beaded branched Actinomycetes, Nocardia, Nocardiopsis,
Streptomyces, Rothia spp.
bifid or v form Bifidobacterium spp., brevibacteria
Gram (-) coccobacilli Bordetella, Haemophilus spp. (pleomorphic)
masses Veillonella spp.
Chains Prevotella, Veillonella spp.
Gram (-) bacilli
small Haemophilus, Legionella (thin with filaments),
Actinobacillus, Bordetella, Brucella, Francisella,
Pasteurella, Capnocytophaga, Prevotella,
Eikenella spp.
bipolar Klebsiella pneumoniae, Pasteurella spp.,
Bacteroides spp.
medium Enterics, pseudomonads
Large Devitalized clostridia or bacilli
Curved Vibrio, Campylobacter spp.
Spiral Campylobacter, Helicobacter, Gastrobacillum,
Borrelia, Leptospira, Treponema spp.
fusiform Fusobacterium nucleatum
filaments Fusobacterium necrophorum (pleomorphic)

Specimen collection- very important in bacteriology

Urether and reproductive organ- non sterile kasi may mga normal flora na tayo here

Urinary bladder- sterile organ, storage of urine

Acid- utilize carbohydrate

BACTERIOLOGY LECTURE Other organism that is gram positive but
catalase negative
WEEK 8
 Rothia mucilaginosa, Aerococcus,
Staphylococci
Alloiococcus otits
Characteristics
Test Staphylococci Micrococci
 Family: Staphylococcaceae Modified
 Genus: staphylococcus oxidase - +
 Part of normal flora specially in skin (Microdase)
Brand name
and mucus membrane
Anaerobic


Catalase (+)
Coagulase (+) & some are Coagulase (-)
acid
production
+ -
CoNS Fermenter Oxidizer
from glucose Obligate
 Gram (+) cocci in singly (mag Aero
(fermentation) aerob
kakahiwalay), in pairs and in clusters tolerance:
 Colonial morphology: creamy (pag bago facultative
(except: M.
pa) using blood agar plate anaerob
kristinae
: yellow (buttery looking when the age and M.
of the culture is already more than 24 varians)
hours) using bolood agar plate Anaerobic
Resembles the family: Micrococcaceace
acid
production
+ -
 Genus: micrococcus from glycerol
in
 Catalase (+)
the presence
 Coagulase (-) of
 Gram (+) cocci in pairs, tetrads, and, erythromycin
ultimately, irregular clusters Growth on
 Colonial morphology: yellow using
blood agar plate
Furoxone-
Tween 80–oil
- +
susceptible resistant
 Found in environment and human skin red O Agar
 Colonies: yellow pigment
 Staphylococcaceae and Furoxone- anti
microbial
Micrococcaceace discuss the same time
agent
because they both have same
characteristic
Resistance to R* (-) S (+)
 Microscopic: Both of them are grama bacitracin
positive cocci (0.04 U)
 Biochemical exam: they are both Resistance R (-) S (+)
catalase positive organism lysosome (50-
mg disk)
Resistance to S* (+) R (-)
lysostaphin
(200 ug/mL)
Anti- Susceptibility testing (resistance test)- Staphylococci
determine the effect of antibiotic or anti-
 Staphle meaning “bunches of grapes”
microbial agent against the organism
 They don’t have flagella
Effects: perform during the biochemical exam  nonmotile, non–spore-forming, and
aerobic or facultatively anaerobic
1. Susceptible (sensitive)
EXCEPT S. saccharolyticus (obligate
 Inhibited by the antibiotic
anaerobe)
 Growth: negative
 Normal flora of skin and mucous
 Result: Disk diffusion- meron kang
membranes of human & animals
culture medium, mag lalagay ka ng
 Colonies: medium sized (pinhead) (4 to
organism, then mag lalagay ng anti
8 mm) and appear cream-colored,
biotic disk, nest is incubate at 37°C for
white or rarely light gold, and “buttery-
18-24 hours (LOOKING AT THE
looking”
PRESENCE OF INHIBITION)
 Non fastidious- they can grow with the
2. Resistant
presence of only basic nutrient (CNE)
 Not inhibited
(18-24 HOURS)
 Growth: positive
 Fastidious strains requirements: CO2,
 Result: Walang makikitang zone of
hemin, or menadione with at least 48
inhibition
hrs of incubation

Organisms Infections
S. aureus • cutaneous
infections (folliculitis,
furuncles, carbuncles
& bullous
impetigo)
• food poisoning
• Scalded skin
syndrome (SSS)
• Toxic shock
Staphylococci exemption that is catalase syndrome (TSS)
negative: • Toxic epidermal
necrolysis (TEN)
 S. sciuri
S. epidermidis • nosocomial
 Macrococcus caseolyticus
infections
 S. lentus S. saprophyticus • UTI (in adolescent
 S. vitulus girls & young
women)
Aerotolerance micrococci exemption because
S. haemolyticus • wound
this is fermenters / facultative anaerobe:
• septicemia
 M. kristinae • UTIs
 M. varians • native valve
infections
 S. lugdunensis • catheter-related  α-hemolysin: lyse RBC, damage
bacteremia and platelets and macrophages and causes
endocarditis severe tissue damage incomplete lysin
 β-hemolysin: also known as
Staphylococcus aureus Spingomyelinase C or hot-cold lysin
 complete lysin: acts on sphingomyelin
 Commonly isolated in the plasma membrane of
erythrocytes
Virulence factors:
 : act in CAMP test
1. Enterotoxins  δ-hemolysin: less toxic than α-
 Groups A-E & G-J hemolysin or β-hemolysin
 Staphylococcal food poisoning  γ-hemolysin: (PVL) Panton-Valentine
- Cause by (BAD) leukocidin
 Toxic shock syndrome (TSS)  Staphylococcal leucocidin
- Cause by (BCGI) - (PVL) Panton-Valentine leucocidin
 Staphylococcal pseudomembranous - exotoxin lethal to PMN
enterocolitis (polymorphonuclear cells)
- Cause by (B) - suppresses phagocytosis
 Heat stable exotoxins (100° C for 30 - associated with severe cutaneous
mins) produce outside the cell wall infections and necrotizing
 Interacting with TSST-1: interact with T pneumonia
cells - often associated with community-
2. Toxic Shock Syndrome Toxin-1 acquired staphylococcal infections
 TSS 5. Enzymes
 previously known as enterotoxin F  Coagulase, protease, hyaluronidase &
 superantigen stimulating T-cell lipase
proliferation  Staphylocoagulase- use as aan
 production of a large amount of identification tool
cytokines for the symptoms - S. aureus
 low concentrations: causes leakage by  Hyaluronidase- spreading factor
endothelial cells - S. aureus
 higher concentrations: cytotoxic - hydrolyzes hyaluronic acid present
3. Exfoliative Toxin in the intracellular ground
 Epidermolytic toxin substance that makes up
 Staphylococcal SSS (scalded Skin connective tissues, permitting the
Syndrome) capable of causing ritter spread of bacteria during infection
disease  Coagulase, protease, hyaluronidase &
 Bullous impetigo- cutaneous lesion lipase
4. Cytolytic Toxin  Lipase (normal flora ng skin)
 extracellular proteins that affect red - by both coagulase (+) and CoNS
blood cells and leukocytes - act on lipids present on the surface
 Lysins & leukocidins of the skin
 S. aureus: α, β, γ, δ hemolysins
 6. Protein A
 Cellular components in cell wall of S.
aureus
 bind the Fc portion of immunoglobulin
G (IgG)- antibody
- block phagocytosis and inhibit
action of IgG
Workflow
Specimen collection (the site of infection is the
site of specimen collection)
 From the site of infection with aseptic
technique
 No special procedures
 Aspirates: ideal sample
 Swabs: less satisfactory for both culture
and smear results
 Cutaneous lesion- aspirate pag wala
swab
Direct Microscopic Examination
 Gram (+) cocci with PMN cells
Culture
 SBA (sheep blood agar), MSA (mannitol
salt agar selective 7.5 NaCl, halophilic),
CAN, PEA, CHROMagar Staph aureus
 18 to 24 hours of incubation at 35° C to
37° C
Macroscopic examination
 Colonies: hemolytic pattern either beta
or none
 S. aureus: round, smooth, white,
creamy colonies on SBA
 S. epidermidis: small to medium sized,
nonhemolytic, gray to white colonies
Biochemical Tests
Catalase (pag gram positive cocci)
 Principle: catalase mediates the
breakdown of hydrogen peroxide (30%
H2O2) into oxygen and water
Result:
 (+) bubble formation effervescence- an
escape of gas from an aqueous solution
- Staphylococci and micrococci
 (-) no or few bubble formation
Biochemical Tests
Microdase test
 Principle: oxidase enzyme reacts with
the oxidase reagent and cytochrome C
to form the colored compound,
indophenol after 2 mins
- Micrococcus can form idophenol
Result:
 (+) development of blue to purple-blue
color micrococci
 (-) no color change staphylococci
Biochemical Tests
Bacitracin test
 Principle: determine the effect of a
small amount of bacitracin (0.04U) on
an organism
Result:
 S: (+) presence of zone of inhibition
around the disk
 R: (-) no zone of inhibition

Microscopic examination

 Gram (+) cocci in singly, in pairs and in

clusters
 Majority is in cluster
  inorganic molecule (anaerobic
respiration
 may ph indicator

oxidation fermentation basal medium- merong

carbohydrats + ph indicators (bromthymol blue)

Biochemical Tests
Oxidation-Fermentation (O/F) reactions
 Principle: determine whether an
organism uses carbohydrate substrates
to produce acid by products using OF
glucose medium
Result: glucose, xylose, mannitol, lactose,
sucrose and maltose
- acid: yellow
- no acid: green
- alkaline: blue
pag tapos mag culture pwede na mag transfer
sa ofbm tube yung isa lalagyan ng sterile
mineral oil dahil yun ang mag poprovide ng
anaerobic environment. Yung isa as is then
incubate 18-24hr at 37°c.
B- aerobic condition walang mineral oil
A- anaerobic merong mineral oil

 (+) yellow medium; acid production

staphylococci
 (-) no change in color; no acid
production micocci
- S. saprophyticus
- S. auricularis
- S. hominis
- S. xylosus
- S. cohnii

*except M. kristane and M. varians

Oxidation-Fermentation (O/F) reactions

 Fermentation
 GLUCOSE glycolysis PYRUVIC ACID;
end product: acid or acid with gas
 single acid (homolactic acid fermenters)
 mixed acids (lactic acid, propionic acid
& succinic acid) Biochemical Tests
Oxidation Coagulase Test
 GLUCOSE glycolysis PYRUVIC ACID  Principle: used to differentiate
CO2 Staphylococcus aureus from CoNS by
 requires oxygen (aerobic respiration) detecting enzyme coagulase
 formation of fibrin
Result:
- (+) fibrin clot S. aureus
- (-) no fibrin clot CoNS
Coagulase Test
2 forms of coagulase enzyme:
Bound coagulase/ clumping factor
 bound to the bacterial cell wall and
reacts directly with fibrinogen in plasma
 alteration of fibrinogen precipitates
on the staphylococcal cell
 Use for screening
 Fibrin clot (clumping of cells) (+)
S.aureus, S. lugdunensis and S. schleiferi
 Slide coagulate test because you’re
doing this in the slide
 Plasma could be in human, rabbit or pig
plasma in EDTA ideal is rabbit
 Fibrinogen- magkakaroon ng
interaction with the bound coagulates
magkakaroon ng clumping
 2-3 minutes
Free coagulase/ Staphylocoagulase
 extracellular protein enzyme (free
coagulase) + CRF (coagulase-reacting
factor)
 coagulase-CRF complex + fibrinogen
 fibrin clot possibility of autolysis due to
fibrinolysin
 confirmatory
 tube coagulate test
 0.5mL of plasma plus colony from
culture
 Extend 4hr in 21-25°C
 Need to check every 30 minutes interval
because maari mag produce si staph ng
fibrinolysin
 False negative result
 Extracellular protein enzyme na mag
rereact dun sa reacting factor na meron
din sa plasma
 Nag popositive lang ditto si S. aureus
Pyrrolidonyl Arylamidase (PYR) Test
 Principle: differentiate S. aureus from
other CoNS by the presence of the
enzyme L-
pyrroglutamylaminopeptidase
 L-pyrroglutamylaminopeptidase
hydrolyze L-pyrrolidonyl-β-
naphthylamide/PYR (substrate) = β-
naphthylamine
 β-naphthylamine + N, N-
methylaminocinnamaldehyde/p-
dimethylaminocinnamaldehyde
(reagent)
Result:
 (+) Bright red color (Coagulate
negative staph CoNS) S.
lugdunensis, S. intermedius, S.
schleiferi
 (-) No color change or an orange
color S. aureus
Vogue-Proskauer (VP) test MRSA (methicillin-resistant Staphylococcus
aureus)
 Principle: determine the ability of
some organisms to produce neutral  Penicillin-resistant (β-lactam) strains of
end products from glucose S. aureus
fermentation  Requires penicillinaseresistant
 Acetly-methylcarbinol or acetoin penicillins, such as nafcillin, oxacillin, or
cefoxitin

types:

- CA-MRSA, HA-MRSA. HACO-MRSA

MRSE

TOC:

VISA (vancomycin-intermediate
Staphylococcus aureus)
Result:
VRSA (vancomycin-resistant Staphylococcus
 (+) Red color S. aureus
aureus)
 (-) Yellow color S. lugdunensis, S.
haemolyticus, S. schleiferi Macrolide Resistance

- Resistance to clindamycin
Novobiocin Susceptibility Test - modified double disk diffusion test
(D-zone test)
 Principle: determine the effect of a 5-
μg novobiocin disk on an organism

Result:

 (+) presence of zone of inhibition

around the disk other CoNS
 (-) no zone of inhibition S. saprophyticus

Diagram
BACTERIOLOGY LECTURE
WEEK 9
Streptococcus and Enterococcus
 Gram + cocci but catalase -
Characteristics
 Family : Streptococcaceae
 Catalase (-)
 Gram (+) cocci in pairs and in chains
- appear more elongated
- In chains when growth in broth
cultures
 facultative anaerobes, aerotolerant
anaerobes, capnophilic
 some of them are also consider as
fastidious because some of them are
requiring carbon dioxide or increase of
carbon dioxide
 for us to culture them ilalagay natin
sila sa candle jar
 candle jar- tumataas ang carbon
dioxide bumababa ang oxygen
 can ferment glucose (lactic acid); no gas
 Colonies: small and transparent /
pinpoint
 Weak false (+) catalase in media with
blood (peroxidase activity of
hemoglobin)
Lancefield Classification: Rebecca Lancefield
(1930)
 Antigens- C carbohydrate
(polysaccharide) in the cell wall
S. agalactiae- acid stable because it is a normal
flora of vaginal canal. Sa area nay an mababa
ang ph.
Enterococcus- previous member of
streptococcus. they still share similar
characteristic from streptococcus. kaya niya
mag survive sa stream condition
S. pneumoniae- walang lancefield group
antigen ang meron siya ay C SUBSTANCE
Viridans streptococcus
 Not a proper name
 Referring to a group of organisms which
are sharing the same characteristics
 They are all show α- hemolytic pattern
Brown smith (hemolytic pattern)
1. β (beta) - demonstrated as a,b,c
2. α (alpha) – demonstrated by
streptococcus pneumoniae and
viridans streptococcus
3. non hemolytic- group D streptococcus
(bovis group) and enterococcus
Sub group
1. S. bovis group
2. S. mitis group
3. S. mutans group
4. S. salivarius group
5. S. anginosus group
Characteristics
 Hemolytic pattern Classification:
- exotoxins that damage intact RBC

Hemolytic Description Appearance

pattern
β-hemolytic Complete Clear area
pattern lysis of RBCs around
around colony
colony

yung
exotoxins
 nung  resist phagocytosis & adherence to
organism na mucosal cells. Blocking phagocytosis
yon is  80 serotypes (M1, M2......) M1 to M80
capable of  M1 is the most common.
damaging
 Capable of attaching streptococci to
the RBC
mucosal cells to respiratory tract. Yung
completely
infection is localized infection restricted
α-hemolytic Partial lysis Greenish
pattern of RBCs discoloration lang in one area. Counter part of
around of localized is generalized kumakalat na sa
Colony area around ibang organs
colony  Counter part in staphylococcus is
Incomplete PROTEIN A
lysis. Hindi 2. Protein F (fibronectin-binding protein)
lahat ng RBC & Lipoteichoicacid
nasira  adherence to epithelial cells
around the (attachment)
colony
3. Hyaluronic acid capsule
γ-hemolytic No lysis of No change in
 prevents opsonized phagocytosis by
pattern/ RBCs around agar
nonhemolytic colony neutrophils or macrophages
Merong  component of the capsule and the
growth ng capsule similar to M protein it resist
colony sa phagocytosis by neutrophils or
agar macrophages
αʹ hemolytic Small area of Small area of  mask its antigens and remain
pattern intact RBCs intact RBCs unrecognized by its host
around around 4. StreptolysinO (SLO) and StreptolysinS
has small colony colony  Cytolysin which is toxin
intact area surrounded surrounded
surrounded by by Characteristics StreptolysinO StreptolysinS
by non a wider zone a wider zone (SLO)
hemolytic of complete of complete Hemolysis on Anaerobically Aerobically
and the hemolysis hemolysis SBA Without With oxygen
bigger is beta plates oxygen
hemolytic hemolysin O hemolysin: S hemolysin:
oxygen labile oxygen stable
Cells lysed leukocytes, Leukocytes
Group A streptococcus (GAS)
platelets, and
Streptococcus pyogenes other Occurring sa
cells as well surface ng
Virulence factor: component that enable to as RBCs agar plate
cause them disease
sa ilalim ng
1. M protein agar plate
 encoded by the genes emm Immunogenicity highly Non-
immunogenic immunogenic
 kaya mag Hindi niya Streptococcus pyogenes
initiate ng kaya mag
immune initiate ng Infections:
response immune 1. Bacterial pharyngitis
(producing response
 pharyngitis and tonsillitis (upper
antibodies) walang
respiratory tract)
antibodie
antibodies production.  strep throat
specific to  most common clinical manifestations of
streptolysin GAS infection
O 2. Pyodermalinfections
 impetigo, cellulitis, erysipelas, wound
Aantibodies infection, arthritis or scarlet fever
that detected  merong mga rashes
by: anti- 3. Necrotizing Fascitis (NF)
streptolysin
 invasive infection characterized by
O (ASO) test.
rapidly progressing inflammation and
We can use it
as a test to necrosis of the skin
detect the  flesh eating bacteria
presence of  suppurative fascitis, hospital gangrene,
streptococcus and necrotizing erysipelas
pyogenes
types:
5. Deoxyribonuclease (DNase)
 A, B, C, and D 1. causing aerobic and anaerobic bacteria
 most common: Dnase B (clastridium spp.)
 immunogenic- NAG IINITIATE NG 2. causing GAS (Streptococcus pyogenes)
IMMUNE RESPONSE 3. salt water NF (vibrio spp.)
6. Streptokinase 4. Streptococcal Toxic Shock Syndrome
 lysis of fibrin clots through the action of  produces SpeA
on plasminogen plasmin Lyses  M1 and M3
clot 5. Poststreptococcalsequelae
 Immunogenic but not specific to GAS  Post streptococcal infection
meron din ang GROUP C AND G (+)  Kadalasang nag kakaroon ng infection
7. Hyaluronidase against streptococcus pyogenes eto
 Hyaluronic acid yung nadedevelop nilang complication.
 spreading factor  Rheumatic fever (RF) and acute
 enzyme that solubilizes the ground glomerulonephritis (AGN)
substance of mammalian connective  Treatment of choice (TOC): Penincilin
tissues
8. Streptococcal Pyrogenic Exotoxins
 Pyro means hot
 cause a red spreading rash, referred to
as scarlet fever
 SpeA, SpeB, SpeC, and SpeF
Group B streptococcus (GBS)
 CHARACTERISTIC OF B ANTIGEN- acid
stable
Streptococcus agalactiae
 Normal flora of vaginal canal and rectal
area
Virulence factor:
1. Capsule
 resist phagocytosis but is ineffective
after opsonization
 Component: sialic acid
 Para iblock or iresist ang phagocytosis
2. Hemolysin, CAMP factor,
neuraminidase, Dnase, hyaluronidase
& protease
 CAMP factor- for identification
 Products not involve in virulence
Streptococcus agalactiae
Infections:
Invasive disease of newborn
 Vertical transmission (mother to the
baby)
 Colonization in vagina and rectal area
Workflow
Specimen collection
S. pyogenes
 Swab: tongue should be depressed and
the swab rubbed over the posterior
pharynx and each tonsillar area
 Swab from the throat use tongue
depressor para maiwasan ang normal
floara sa oral cavity
 Exudate: touched with swab
 avoid tongue and uvula
 transport media are still needed but not
required
S. agalactiae
 swab: vaginal and rectal material with
swabs between 35 and 37 weeks of
gestation
 need to use transport medium
specifically Todd-Hewitt broth (10
μg/mL colistin) & Lim broth (15 μg/mL
nalidixic acid)
 colistin- anti fungal. Inhibitor for fungi
 nalidixic acid- inhibitor for other gram +
and gram -
Direct Microscopic Examination
 Gram (+) cocci round or oval-shaped,
occasionally forming elongated cells
Culture
 SBA, BAP, SBA containing
sulfamethoxazole (SMZ) (for bacteria)
or colistin and polymixin B (for fungi)
 18 to 24 hours of incubation at 35° C to
37° C aerobically or anaerobically
 Capnophiles ang karamihan sakanila
kaya need ipasok sa candle jar
Macroscopic examination
 Colonies:
 S. pyogenes: small, transparent, and
smooth with a well-defined area of β-
hemolysis
 S. agalactiae: grayish white mucoid
colonies surrounded by a small zone of
β-hemolysis
- StrepB Carrot Broth: orange or red
pigment after incubation for 6
hours
Microscopic examination
 Gram (+) cocci with some short chains
Biochemical Tests
Catalase
 Principle: catalase mediates the
breakdown of hydrogen peroxide (30%
H2O2) into oxygen and water
 Result:
- (+) bubble formation staphylococci
& micrococci
- (-) no or few bubble formation
streptococci & enterococci
Bacitracin test (taxo A)
 Principle: determine the effect of a
small amount of bacitracin (0.04U) on
an organism
 Result:
- Susceptible (+) presence of zone of
inhibition around the disk
(S. pyogenes)
- Resistant (-) no zone of inhibition
(other β- hemolytic group B AND C)
(S. agalactiae and S. dysgalactiae
and equine)
Pyrrolidonyl Arylamidase (PYR) Test
 Principle: differentiate S. aureus from
other CoNS by the presence of the
enzyme L-
pyrroglutamylaminopeptidase
 L-pyrroglutamylaminopeptidase
hydrolyze L-pyrrolidonyl-β-
naphthylamide/PYR (substrate) = β-
naphthylamine
 β-naphthylamine + N, N-
methylaminocinnamaldehyde/p-
dimethylaminocinnamaldehyde
(reagent)
 Result:
- (+) Bright red color (S. pyogenes)
- (-) No color change or an orange
color (other β- hemolytic group B
AND C)
HippurateHydrolysis Test
 Principle: determine the capability of
organism to hydrolyze hippurate as
substrate
ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑧𝑒
 hippuric acid ℎ𝑖𝑝𝑝𝑢𝑟𝑖𝑐𝑎𝑠𝑒 glycine &
benzoic acid (deaminated by other
reagent which is the ninhydrin)
 Hippuricase enzyme is capable of
hydrolyzing the hippurate or hippuric
acid reagent
𝑑𝑒𝑎𝑚𝑖𝑛𝑎𝑡𝑒𝑑
 Glycine purple colored
𝑛𝑖𝑛ℎ𝑦𝑑𝑟𝑖𝑛
products
 Result:
- (+) deep purple color (S. agalactiae)
- (-) colorless or slightly yellow pink
color (other β- hemolytic group A
AND C)
CAMP Test (christie, atkin, munch petersen)
 Principle: detection of diffusible
extracellular protein (CAMP factor) that
acts synergistically with the beta-lysin
(springo myelin C) of S. aureus to cause
enhanced lysis of red blood cells
 Result:
- S. agalactiae (+) Enhanced
hemolysis indicated by an
arrowhead-shaped zone of beta-
hemolysis at the juncture of the
two organisms (nag form ng arrow
head)
- (other β- hemolytic group A AND C)
(-) No enhancement of hemolysis
(hindi nag form ng arrow head)
  pneumonia, sinusitis, otitis media
(infection sa middle ear), bacteremia
(presence of bacteria in the blood) ,
and meningitis (infecting the CNS)
(pwede kuha ditto ng CSF as specimen)
 most frequently encountered in
children < 3 years old with recurrent
otitis media

Vaccines:
Streptococcus pneumonia
PCV7
 Pneumococcus
 purified polysaccharides of seven
 Normal flora ng respiratory tract
serotypes conjugated to a diphtheria
 member of the S. mitis group
protein
 member din siya ng viridans
 for use in children
streptococcus which is S. mitis pinag
 7 sero types
kaiba niya sa mitis group wala siyang
antigen PCV13
 cell wall: C substance
 Additional serotypes (1, 3, 5, 6A, 7F, and
- reacts with CRP to form precipitate
19A)
Virulence factor  <5 years old children
 13 sero types
Capsular polysaccharide
PS23
 capsule is made up of polysaccharide
 Susceptible to opsonization  23 purified capsular polysaccharides for
 Strains that lack capsule: non- adults
pathogenic  23 sero types
 With capsule: pathogenic
Workflow

Specimen collection
Other products:
S. pneumonia
 hemolysin, immunoglobulin A protease,
 RT specimens (sputum, CSF if
neuraminidase and hyaluronidase
meningitis)
TOC: penicilin
Direct Microscopic Examination
Streptococcus pneumonia
 Effusions: Gram (+) pneumococci with
Infection numerous WBC
 CSF: Gram (+) cocci in pairs with
 # 1 cause of pneumonia (lower tract
numerous WBC
infection) in ICP patients
 Sputum na kinukuha here
Culture
 BHI , TSB with 5% sheep RBCs or CAP
 18 to 24 hours of incubation at 35° C to
37° C with increased CO2 for growth
during primary isolation
 Kaialangan isubculture everyday
Macroscopic examination
 Colonies:
 S. pneumoniae: round, glistening, wet,
mucoid, dome shaped appearance
with large zone of α-hemolysis on SBA
surrounding the colonies.
 Nagiging dome shaped siya pag young
yung colonies after some time
bumabagsak siya nagkakaroon ng
depression sa gitna (coin with a raised
rim)
Microscopic examination (diplococci)
 Gram (+) cocci in pairs or in singly or in
short chains
- ends of the cells are slightly
pointed, giving them an oval or
lancet shape diplococcic
 gram (-) diplococcic- Neisseria
Biochemical Tests
OptochinTest (taxo P)
 ethylhydrocupreine hydrochloride
 Principle: determine the effect of
optochin on an organism
 Result:
- Susceptible (+) Zone of inhibition is
14 mm or greater in diameter, with
6-mm disk (S. pneumoniae)
- Resistance (-) No zone of inhibition.
Less than 14mm (other α-hemolytic
streptococci) (viridans
streptococcus)
Bile Solubility Test
 Principle: bile or a solution of a bile salt
rapidly lyses pneumococcal colonies
 Bile salts lowers the surface tension
between the bacterial cell membrane
and the medium
 Chemical name of bile salt: sodium
desoxycholate
 Pinag hihiwalay niya yung colony ng
mga streptococcus mula duon sa
culture media
 Two ways to use it: agar plate (drop
bile salt) or both medium (drop bile
salt)
Broth medium appearance
(+) clear (-) turbid
 accelerates the organism’s natural
autolytic process (intracellular autolytic
enzyme)
 Result:
- (+) Lysed colonies (S. pneumoniae)
- (-) Intact colonies (other α-
hemolytic streptococci) (viridans
streptococcus)
Viridans Streptococci
 normal microbiota of the upper
respiratory tract, the female genital
tract, and the gastrointestinal tract
 normal flora ng upper respiratory tract
 viridans means “green” α-hemolysis
 β-hemolytic and nonhemolytic species
 fastidious, with some strains requiring
CO2 for growth
S. mitis group (lancefield group A,C,F,G OR N
antigen)
 S. mitis
 S. pneumoniae
 S. sanguis
  S. oralis Colonies:

 small and are surrounded by a zone of

α-hemolysis
S. mutans group (lancefield group A,C,F,G OR N
 β-hemolytic or nonhemolytic
antigen)
Microscopic examination
 S. mutans
 S. sobrinus  Gram (+) cocci in pairs and in chains

S. salivarius group (lancefield group A,C,F,G OR Biochemical Tests

N antigen)
 PYR (-)
 S. salivarius  LAP (+)
 S. vestibularis
Biochemical tests
S. bovis group (Group that has D antigen)
 Group D streptococci and S. bovis group
 S. equinus - express the D antigen
 S. gallolyticus - common na pinapakitaa nilang
 S. infantarius hemolytic pattern is none
 S. alactolyticus
LAP test
S. anginosus group (lancefield group A,C,F,G
 Principle: detection of the enzyme
OR N antigen)
leucine aminopeptidase
 S. anginosus  Substrate: leucine- β-naphthylamide
 S. constellatus β-naphthylamine + cinnamaldehyde
 S. intermedius reagent
 Result:
Infection - (+) Red color (Viridans streptococci)
 most common cause of subacute - (-) No color change or an slight
bacterial endocarditis (causing yellow color (other α-hemolytic
infection to the heart) streptococcus)
 oral infections such as gingivitis and Workflow
dental caries

Virulence Factors

 S anginosus group: polysaccharide

capsule and cytolysin
- adherence and colonization in
endocarditis
 Groups C and G streptococci: M
proteins, SLO, hyaluronidase, and
DNase, streptokinase

*V- variable
Enterococcus
 previously classified as group D
streptococci
 D antigen
 normal flora of intestinal tract of
human and animals
 nonhemolytic, α-hemolytic or β-
hemolytic
 ability to grow under extreme
conditions
 6.5% NaCl, 45°C, alkaline pH
 pseudocatalase reaction
 E. faecalis & E. faecium
 E. durans, E. avium, E. casseliflavus, E.
gallinarum, and E. raffinosus
 PYR (+)
Enterococcus faecalis
Virulence factor:
1. extracellular surface adhesin proteins,
extracellular serine protease, and
gelatinase
- colonization of the species and
adherence to heart valves and renal
epithelial cells
2. Cytolysin
- similar to bacteriocins produced by
gram-positive bacteria
Infections:
 nosocomial infections (UTI &
bacteremia)
 Endocarditis in elderly patients
 intraabdominal or pelvic wound
infections
Workflow
Specimen collection
 blood, urine, or wound specimens
 capable of producing wide various
infection
Culture
 TSB or BHI with 5% sheep’s blood
 bile esculin azide (BEA hydrolyzing
esculin, final product black compound)
, colistin– nalidixic acid, phenylethyl
alcohol, chromogenic substrates, or
cephalexin-aztreonam-arabinose agar
 35° C in the presence of CO2 but do not
require a high level of CO2 for growth
Biochemical tests
 Acid production in carbohydrate broth
 Arginine hydrolysis
 0.04% tellurite tolerance
 Pyruvate utilization
 Resistance to 100-μg efrotomycin acid
 (+) motility
*some of them are motile
 Mot- Motile
 Man- Mannitol
 Sor- sorbose
 Ara- arabinose
 Raf- raffinose
 Tel- tellurite
 Arg- arginine
  Pyu- pyruvate
 Mgp- methyl-α-D- glucopyranuside
Streptococcus-like bacteria
Abiotrophia and Granulicatella spp.
 previously known as nutritionally
variant streptococci
 require sulfhydryl compounds for
growth
 oral and gastrointestinal microbiota
 bacteremia, endocarditis & otitis media
 satellitism on SBA with S.aureus
 10 mg/L pyridoxal hydrochloride
Biochemical tests
 production of:
- α-galactosidase
- β-galactosidase
- β-glucuronidase
 hippurate hydrolysis
 arginine hydrolysis
 acid production from trehalose & starch
Aerococcus
 common airborne organism
 opportunistic pathogen associated with
bacteremia, endocarditis, and UTI in
ICP
 similar to:
- streptococci in culture
- staphylococci in microscopic exam
 weak catalase or pseudocatalase
 Growth in 6.5% NaCl
 A. viridans: bile esculin & PYR (+)
 A. urinae: bile esculin & PYR (-)
Gemella
 Similar to viridans streptococci in
colonial morphology
 α-hemolysis or nonhemolytic
 gram (-) cocci in pairs, tetrads, clusters,
or short chains
 endocarditis, wounds, and abscesses
 G. haemolysans
Lactococcus
 previously classified as group N
streptococci
 gram (-) cocci in singly, in pairs or in
chain
 physiologically similar to enterococci
 α-hemolysis or are nonhemolytic
 UTI & endocarditis
 Meron siyang N antigen
Biochemical tests
 Differentiate from enterococci
Leuconostoc
 Catalase (-), gram (+) cocci with
irregular morphology
 intrinsically resistant to vancomycin
 found on plant surfaces and vegetables,
and in milk products
  meningitis, bacteremia, UTIs, and
pulmonary infections

Pediococcus

 facultatively anaerobe, gram (+) cocci in

pairs, tetrads, and clusters
 grow at 45° C
 intrinsically resistant to vancomycin
 gastrointestinal abnormalities or
undergone abdominal surgery

Tests Leuconostoc Pediococcus

Bile esculin + +
hydrolysis
LAP - +
PYR - -
6.5% NaCl + +
Gas (+) from + -
glucose
fermentation

Globicatella sangius

 α-hemolytic
 PYR (+)
 LAP (-)
 vancomycin susceptible

Helcococcus kunzii

 wound infections
 misidentified as A. viridans

Alloiococcus otitidis

 otitis media in children

 Non-hemolytic or α-hemolytic
 •YR (+)
 LAP (+)
 Grow slowly in 6.5% NaCl
BACTERIOLOGY LECTURE BOTH GONORRHOEAE AND MENINGITIDIS
ARE:
WEEK 10
 Fastidious organisms
Neisseria and Moraxella
 Requires iron for growth
Neisseria sp. - Iron transporting protein called
transferrin
 Family: Neisseriaceae - Transferrin- transport protein of
 Kingella, Eikenella, Simonsiella, Alysiella iron
 aerobic, nonmotile, non–spore-  Neisseria sp. Receptor for transferrin
forming, capnophilic (requiring
increase carbon dioxide)
 fastidious
 can grow anaerobically if nitrite is
present (in inorganic substances)
 Gram (-) diplococci
 Except gram – bacilli:
N. elongata
N. weaveri
N. bacilliformis
 cytochrome oxidase (-) and catalase (+)
*in their family Neisseria only a diplococci the
- N. elongate
rest are bacilli
- N. baciiformis
Neisseria sp.
 normal flora: mucous membranes of
Virulence factors:
the RT and UGT (upper gastro intestinal
 Receptors for human transferrin-iron
tract) (most of them but not all of them)
 Neisseria sp. Competing for iron
Primary human pathogen  Capsule (Neisseria meningitidis)
 Pili (fimbriae)- indicator whether the
Neisseria gonorrhoeae
organism is a virulent form or a non-
 gonococci virulent form
 not part of normal flora - N. gonorrhoeae
 once they are present they are always - Colony types: Type 1-5
pathogenic - Basis: presence or absence of pili
(use for initial attachment of the
Neisseria meningitidis organism to host tissues and
 meningococci Inhibit phagocytosis not for
 commensal inhabitant of URT of locomotion)
carriers - Virulent forms: type 1 & 2 (positive
 invasive pathogen (can infect CNS) for presence of pili)
 normal flora but once it became a  initial attachment of the organism to
pathogen they invasive pathogen host tissues
 Inhibit phagocytosis
Bone marrow- immature RBCs that requiring
iron to become mature
Virulence factors:
 Proteins in CM (cell membrane)
- Antigenic variation
- Protein I: major outer membrane porin
protein (Por)
- porA & porB: N. meningitidis
- porB: N. gonorrhoeae
- Serves as nutrient passage
- Porin- pores usually use as a passage
for nutrients from outside to inside also
as passage for waste product
- Being a porin protein
 protein II (Opa)
- adherence to phagocytic and epithelial
cells
- anti-phagocytosis
- inhibiting the action of phagocytosis
 Protein III (reduction modified protein
[Rmp])
- blocks host serum bactericidal
immunoglobulin G (IgG) action
 Core lipooligosaccharide (LOS) or
endotoxin
- Major in vivo virulence factor
- mediates damage to body tissues and
elicits an inflammatory response
- blebs: outer membrane fragments
released during rapid growth containing
the LOS (indicator that the body has an
increase microbial load)
 Lipid moiety
- differentiation from the
lipopolysaccharide found in most gram
(-) bacilli
 IgA protease
- cleaves IgA on mucosal surfaces
Neisseria gonorrhoeae
Characteristics:
 Only host: human
 Not a normal flora of our body. When
its present in our body it pathogenic
 Gonorrhoea- flow of seed, clap
meaning brothel
- acute pyogenic infection of nonciliated
columnar and transitional epithelium
- urethra, endocervix, anal canal,
pharynx, and conjunctiva
- STI
- primary reservoir: asymptomatic
carrier (common source of
transmission) (direct contact)
- Incubation period: 2-7 days
 Men: Urethra
- 3-5% asymptomatic: AHU strain
(arginine, hypoxanthine, uracil)
- Ahu strain- additional requirements
 Women: Endocervix
- 50% asymptomatic
- complications: sterility, ectopic
pregnancy, or perihepatitis (fritz-hugh-
curtis syndrome)
 Inhibited by SPS
- not recovered in blood (it is only
collecting in infected sites)
- to neutralize the effect: gelatin
 Newborns:
- Ophthalmia neonatorum
- gonococcal eye infection
- Connatal infection
- result in blindness
Workflow: Neisseria gonorrhoeae
Specimen Collection
 Specimen of choice:
- Men: urethra
- Women: endocervix
 Avoid use of disinfectants
 Calcium alginate and cotton swabs:
inhibitory
- Dacron or rayon swabs- type of swabs
different from cotton and calcium
alginate
- If no have discharge: insert Dacron or
rayon swab 2cm in to the anterior
urethra slowly and rotate it to collect
specimen.
- Anal area- rectal swab 4-5cm
  extremely susceptible to drying and
temperature changes
- direct plating
 Transport systems:
- James E. Martin Biological
Environmental Chamber (JEMBEC)
plates
- Gono-Pak
 Transgrow
- contain selective media and a CO2
atmosphere
- rolled in a “Z” pattern on the medium
(suspecting the presence of N.
gonorrhoeae)
 Amies medium with charcoal
- when direct plating is not possible
- plated within 6 hours
Direct Microscopic Examination
 Urogenital specimen: Gram stain
 Pharyngeal specimens: Gram stain not
recommended (commensal Neisseria
spp.)
 Gram (-) intracellular diplococci in pairs
with adjacent sides flattened (kidney
bean shaped)
 Gram stain with > 5 PMN/ field but no
bacteria: nongonococcal urethritis (C.
trachomatis or Ureaplasma
urealyticum)
Culture
 Medium of choice: chocolate agar plate
(CAP) ASAP
- no growth in BAP because this
organism will not grow in blood agar
plate
 Plated ASAP
 Susceptible to cold temperature
- Medium should be warmed at RT
before inoculation
 Acinetobacter spp., Capnocytophaga
spp., & Kingella denitrificans
- (+) growth on gonococcal media
- oxidase and catalase tests
 Inhibitors:
- Vancomycin, lincomycin: gram +
- Trimethoprim: swarming Proteus spp.
- Colistin: gram -
- Nystatin, Anisomycin, Amphotericin B:
fungal element yeast
 Thayer-Martin
- Vancomycin, colistin, nystatin
 Modified Thayer-Martin
- Vancomycin, colistin, nystatin,
trimethoprim
 Martin-Lewis
- Vancomycin, colistin, anisomycin,
trimethoprim
 New York City
- Vancomycin, colistin, amphotericin B,
trimethoprim
 GC-LECT
- Vancomycin, lincomycin, colistin,
amphotericin B, trimethoprim
 Incubation:
- 35° C in a 3% to 5% CO2 atmosphere
- CO2 incubator
- CO2 generating pouch (gaspak jar)
Macroscopic Examination
 small, gray to tan, translucent, and
raised
- T1 & T2: bright, smaller and raised
- T3-T5: larger, flatter colonies
 autolytic enzyme
- make the isolate nonviable on
prolonged incubation
Macroscopic Examination
 Gram (-) diplococci
 Acinetobacter & Kingella sp.
- 10 U Penicillin disk (they will be
converted to gram – bacilli)
Biochemical Tests
 Oxidase test
- done on all suspected isolates of N.
gonorrhoeae
- Reagent: 1% dimethyl-p-
phenylenediamine dihydrochloride or
 tetramethyl-p-phenylenediamine
dihydrochloride
- Using filter paper (purple) or directly
dropping to culture media (black)
 Result: (+): purple to black color after
10 mins (-): no change in color
Carbohydrate Utilization
 cystine trypticase agar (CTA)
- 1% of the individual carbohydrate
- phenol red
 Result: (+) yellow: acid production
(-): no change: no acid produced
 N. gonorrhoeae- glucose
 N. meningitidis- glucose & maltose
 M. catarrhalis- assaccharolytic (do not
utilize CHO)
*most of Neisseria is Dnase & Butyrate
esterase (+)
Neisseria meningitidis
Characteristics:
 Only found in human
 commensal & invasive pathogen
 Incubation period: 1 to 10 days
 adhere to the nasopharyngeal mucosa
leading to colonization
 endemic and epidemic meningitis and
meningococcemia
- Complication: waterhouseFriderichsen
syndrome
- 12-48 hrs of onset: death
- Meningococcal pneumonia: serogroup
Y
Workflow: Neisseria meningitidis
Specimen Collection:
• CSF, blood, nasopharyngeal swabs and
aspirates, joint fluids
• Inhibited by SPS (to neutralize add gelatin)
Direct Microscopic Examination
• Gram (-) intracellular and extracellular
diplococci
• CSF: centrifuged at 1000 x g for 10 mins
Culture
• BAP & CAP
• 35° C in a 3% to 5% CO2 atmosphere
Macroscopic Examination
• medium-sized, gray, and convex, and
encapsulated strains are mucoid
• Green tinge under BAP
 Capable of producing alpha hemolytic
pattern
Microscopic Examination Microscopic Examination
• Gram (-) diplococci with adjacent sides
 gram (-) diplococci
flattened
Biochemical Test Biochemical Tests
• Oxidase (+)
• Carbohydrate utilization test: glucose &  Oxidase & Catalase (+)
maltose  Carbohydrate Utilization Test:
• γ-glutamyl aminopeptidase: (+) N. asaccharolytic
meningitides ; (-) N. gonorrhoeae, N. lactamica,  Dnase & Esterase buterase (-)
& M. catarrhalis.  Tributyrin as substrate
 N. lactamica: glucose, maltose, lactose
- ONPG (+)ortho-Nitrophenyl-ß-
galactoside
Moraxella catarrhalis
Characteristics
 Family: Moraxellaceae
 Genera: Moraxella, Acinetobacter, and
Psychrobacter
 Gram – diplococcic
 Opportunistic pathogen
 M. catarrhalis
- opportunistic pathogen
- commensal of URT
- Infections: URTI Commensal Neisseria Species
- 3rd most common cause of acute otitis
(ear canal) media and sinusitis (URT) in
children
Specimen Collection
 middle ear effusion, nasopharynx, sinus
aspirates, sputum aspirates, or
bronchial aspirates
Direct Microscopic Examination
 intracellular gram (-) diplococci
Culture
 BAP & CAP Neisseria cinerea
 Grow well at 28° C  Colonial morphology similar to T3 of N.
 Inhibited by colistin gonorrhoeae in CAP
Macroscopic Examination  CAP & BAP
 smooth, opaque, gray-to-white  Flat colonies
colonies  To differentiate from N. gonorrhoeae
 hockey puck colonies - Susceptible to 10-μg colistin disk (≥10
 wagon wheel appearance mm zone of inhibition)
 To differentiate from M. catarrhalis
- Nitrite reduction (+)
 - Dnase reaction (-)
 To differentiate from N. flavescens
Neisseria subflava
- No yellow pigment production
 “less yellow”
Neisseria flavescens
 part of the upper respiratory
 yellow-pigmented microbiota
 asaccharolytic
Neisseria elongata
 To differentiate from N. cinerea
- (+) growth on BAP & Cap at 22° C  N. weaver & N. bacilliformis
- Yellow colonies - Rod shaped neisseria
 commensals in the upper respiratory
Neisseria polysaccharea
tract
 produces large amounts of extracellular  opportunistic pathogens
polysaccharide when grown in media
with 1% or 5% sucrose
 maraming saccharide
 To differentiate from N. gonorrhoreae
- (+) growth at NA at 35° C
- production of polysaccharide from 1%
or 5% sucrose

Neisseria mucosa

 Large, mucoid colonies

 Isolated in nasopharynx of children &
young adults
 Islolated in airways of dolphins
 Carbohydrate utilization
- Glucose, maltose, sucrose, fructose
- (+) reduction of nitrate & nitrite
- Airways ng dolphin

Neisseria sicca

• dry, wrinkled, adherent, and breadcrumb-like

colonies

Neisseria lactamica

 nasopharynx of infants and children

 similar to N. polysaccharea (encounter
in meningococcal carrier surveys)
 Similar colonial morphology to N.
meningitides
 Carbohydrate utilization test:
- Glucose, maltose, lactose
 ONPG (+)
BACTERIOLOGY LECTURE - differential: xylose, lactose, pH
indicator: phenol red
WEEK 11
- positive acid: yellow
Enterobacteriaceae - negative acid: colorless/red

- Enteric bacteria Ferric ammonium citrate: as H2S indicator

- Divided into two groups: lactose
Na thiosulfate- sulfur source (whenever the gas
fermenters and non-lactose fermenters
is present the color of the colony is black)
- Lactose fermenters- capability to
utilize carbohydrate such as lactose Result of H2S- blackening of medium
- Non-lactose fermenters- not capable to
Microscopic examination:
utilize lactose
 Gram (-) non-spore-forming facultative
General characteristics:
anaerobic bacilli
Macroscopic examination:  Method of utilization of carbohydrates-
fermentation
 BAP and CAP
- Klebsiella & Enterobacter: large mucoid Classification: tribes or subfamilies
colonies (encapsulated)
I. Escherichieae
- E. coli: β-hemolytic
- Escherichia
 EMB, MAC, HE, XLD (differential agent)
- Shigella
- Ferment carbohydrate
II. Edwardsielleae
 HE & XLD
- Edwardsiella
- Produce H2S (gas colorless)
III. Salmonelleae
EMB: Eosin Methylene Blue Medium - Salmonella
IV. Citrobacteriaceae
- Selective and differential
- Citrobacter
- Selective- dyes (Eosin y methylene
V. Klebsielleae
blue), gram -, inhibit gram +
- Klebsiella
- Differential- lactose, sucrose, pH
- Enterobacter
indicator: eosin methylene blue dyes
- Pantoea
- Positive acid: pink
- Cronobacter
- Negative acid: colorless
- Hafnia
Mac conkey: selective and differential - Serratia
VI. Proteeae
- Selective- crystal violet dye - Proteus
- Differential- lactose - Morganella
- pH indicator: neutral red - Providencia
- positive acid: pink VII. Yersinieae
- negative acid: colorless - Yersinia
XLD: Xylose – lysine, desoxycholate agar

- selective and differential

- selective- bile salt (Na desoxycholate)
(inhibitory for gram +)
Enterobacteriaceae Escherichia coli

Virulence factors: Characteristics:

Plasmids  Escherich in 1885

- Colon microbiota (gastro intestinal
 Antimicrobial resistance (reason why
tract)
they are developing resistance to
- primary marker of fecal contamination
antibiotics)
in water quality testing
 They produce extendedspectrum β-
lactamases (ESBLs) (enzymes) Virulence factors:
- carbapenemases, cephalosporinases, or
 adhesive fimbriae and sex pili
metallo-β-lactamases
 O, H & K antigens
- inactivate extended-spectrum
cephalosporins Medically important species:
- cefotaxime, penicillin, aztreozam
A. Uropathogenic E. coli- urinary tract
Antigens
B. Gastrointestinal E. coli- diarrhea
O antigen
C. Extraintestinal E. coli- septicemia and CNS
 Somatic antigen
Macroscopic examination:
 heat-stable antigen in cell wall
 heat labile  MAC: lactose fermenting colony with a
surrounding area of precipitated bile
H antigen
salts
 flagellar antigen
 heat-labile antigen in flagella

K antigen

 capsular antigen
 heat-labile polysaccharide in capsule  EMB: green metallic sheen
- K1 antigen: E. coli
- Vi antigen: S. enterica subsp. Enterica

Lactose-fermenters

 Escherichia- rapid (18-24 hours utilize

Microscopic examination:
lactose)
 Klebsiella- rapid (18-24 hours utilize  Gram (-) bacilli
lactose)
Fermentation of glucose, lactose, trehalose &
 Enterobacter- rapid (18-24 hours utilize
xylose
lactose)
 Serratia- late lactose fermenter (48 -
𝐴
TSI: 𝐴 kasi dalawa yung na utilize
hours) glucose & lactose
 Citrobacter- late lactose fermenter (48 𝐴 𝐾
- LIA: : 𝐴 OR : 𝐴 LDA - & LDC -
hours)
  Indole (+) from tryptophan
 MR (+) VP (-) palaging opposite ang
reaction
 Citrate (-) Principle (px): If only glucose is fermented
 H2S, Dnase, urease, PAD (-)
- If only glucose is fermented there
Biochemical test would not be enough amount of acid to
form
TSI (triple sugar iron)

 Butt slant

Purpose

 Detect CHO (carbohydrate) utilization

- CHO content: Glucose, lactose, sucrose
- Ratio: 1 : 10 : 10  The slant is red but the butt still remain
- pH indicator: phenol red yellow
 + acid: yellow  Appearance:
𝑟𝑒𝑑
report:
𝐾 𝐴𝑙𝑘𝑎𝑙𝑖𝑛𝑒
𝑦𝑒𝑙𝑙𝑜𝑤 𝐴 𝑎𝑐𝑖𝑑
 - acid: red
 All enterobacteriace are glucose
- uninoculated culture media:
fermenters (lahat ng NLF ay kadalasang
𝐾
𝐴
)
 Non lactose fermenter

Px: if glucose + lactose or glucose + sucrose or

G+L+S)
 gas production
- result: crack, spaces, bubbles in
medium

𝑦𝑒𝑙𝑙𝑜𝑤 𝐴 𝑎𝑐𝑖𝑑
 Appearance: 𝑦𝑒𝑙𝑙𝑜𝑤 report: 𝐴 𝑎𝑐𝑖𝑑
 Lactose fermenters

Px: if no CHO is fermented

 H2s production
- Ferric citrate: H2S indicator (colorless)  Asaccharolytic organism
𝑟𝑒𝑑 𝑘 𝑎𝑙𝑘𝑎𝑙𝑖𝑛𝑒
- Na thiosulfate: sulfur source  Appearance: report:
𝑟𝑒𝑑 𝑘 𝑎𝑙𝑘𝑎𝑙𝑖𝑛𝑒
- Result: blackening of medium
LIA (lysine iron agar)

 Components: lysine (ammino acid),

glucose
 pH indicator: bromcresol purple
 + acid: yellow
  - acid: purple  Gas production
- Ferric ammonium citrate: H2S indicator  H2S production
- Na thiosulfate: sulfur source

Purpose

 Lysine deamination
- Only happens in slant
- RECQUIRING ACID ENVIRONMENT
- Red/ bourdeaux red complex
𝑟𝑒𝑑 𝑅
- Appearance: report: LDA +
𝑦𝑒𝑙𝑙𝑜𝑤 𝐴

 Lysine decarboxylation  Kailangan acid ang environment bago

- Only happens in butt magkaraoon ng process
- Requiring acid environment  Ang enetreobacteriacea isa lang ang
- Cadaverine (neutralize acid) (acid to kaya nila either deamination or
alkaline) decarboxylation or neither
- From yellow naging purple
IMVIC reaction (Indole Methyl red Vogue-
Proskauer citrate)

 E. coli: + Indole + Methyl red - Vogue-

Proskauer – citrate
 Enterobacter: - - + +
 Klebsiella: - - + +
 Salmonella: + - + -

Urease
𝑝𝑢𝑟𝑝𝑙𝑒 𝐾
- Appearance: report: LDC + Escherichia coli
𝑝𝑢𝑟𝑝𝑙𝑒 𝐾

A. Uropathogenic E. coli:

 Most common cause of urinary tract

infection in human
 Pyelonephritis in ICP
 Virulence factor:
1. Pili
- Attachment/ Adherence to epithelial
cells
- WALANG NA FORM NA RED COMPLEX
𝑝𝑢𝑟𝑝𝑙𝑒 𝐾
- UPEC: P pilus/ pap pili, type 1fimbrae
- Appearance: 𝑦𝑒𝑙𝑙𝑜𝑤 report: 𝐴 LDC - & - DAEC: Afa/ Dr adhesions, also cause GI
LDA - infections or urinary tract infection
 2. Cytolisins
- kill immune effector cells and inhibit
phagocytosis and chemotaxis
3. Aerobactins
- Chelate iron or binding with iron
B. Gastrointetinal E. coli:
5 major categories: enterovirulent E. coli or
diarrheogenic E. coli
 ETEC: enterotoxigenic Escherichia coli
 EIEC: enteroinvasive Escherichia coli
 EPEC: enteropathogenic Escherichia coli
 EHEC: enterohemorrhagic Escherichia
coli
 Enteroadherent
- EAEC: enteroaggregative Escherichia
coli
- DAEC: diffusely adherent Escherichia
coli
Enterotoxigenic Escherichia coli : ETEC
 Traveler’s diarrhea
 watery
 one of the major causes of infant
bacterial diarrhea
 106 to 1010 organisms (infective dose)
 stomach acidity
- Inhibiting colonization
- Achlorhydria: > risk
- Achlorhydria: acidity in stomach
condition
Virulence factor:
 Fimbrae: attach to specific receptors on
intestinal microvilli
 LT (heat-labile toxin): A & B fragments
- A: active portion; converts ATP to
cAMP
- B: moiety or binding portion to GM1
ganglioside of intestinal mucosal
 ST (heat-stable toxin): stimulates
guanylate monophosphate
Enteropathogenic Escherichia coli : EPEC
 Infantile diarrhea
Virulence factor:
 Pathogenicity islands
Enteroinvasive Escherichia coli: EIEC
 dysentery
 Can penetrate the intestinal mucosa
 + blood (RBC), pus (WBC), mucus
 direct penetration, invasion, and
destruction of the intestinal mucosa
 Similar to shigella infection (bacillary
dysentery)
 106 organisms (infective dose)
Enterohemorrhagic Escherichia coli : EHEC
 Hemorrhagic diarrhea, colitis, & HUS
 HUS: hemolytic uremic syndrome
 watery diarrhea to bloody diarrhea (no
leukocytes unlike shigella dysentery
Virulence factor:
 O157:H7 strain Shiga toxin- producing
E. coli (STEC)
- Verotoxin I: phage-encoded cytotoxin
identical to the Shiga toxin (Stx) type I
 Shiga toxin I
 Same to Shingella dysenteriae
 Damage Vero cell (African
monkey kidney cell)
 Neutralized by Ab against Stx
 Shiga-like toxins
- Verotoxin II: biologically similar,
immunologically different from Stx and
verotoxin I
 Shiga-like toxins
 Not neutralized by Ab against
Stx
Enterohemorrhagic Escherichia coli : EHEC
 O157:H7 strain
- MAC agar containing sorbitol (SMAC):
stool culture
 (-) ferment sorbitol in 48 hrs
 Colorless colonies
- 4-methylumbelliferyl-β-D-glucuronide
(MUG) assay
 (+) produce β-glucuronidase
- ELISA & latex agglutination
 E. coli O157:NM
- (+) ferment sorbitol in 48 hrs
- non-motile
Enteroadherent Escherichia coli
 DAEC
- UTIs and pediatric diarrheal disease
 EAEC
- Watery diarrhea
- Fimbrae adhere to HEp2 cells in
“stacked-brick” pattern
C. Extraintestinal E. coli
 septicemia and neonatal meningeal
infection
Virulence factor:
- capsular antigen K1
- immunochemically identical to capsular
antigen of N. meningitidis group B
 Escherichia hermannii : yellow-
pigmented organism in CSF, wound &
blood
 Escherichia vulneris: yellow-pigmented
organism
Klebsiella
Characteristics: ENCAPSULATED
Macroscopic examination:
 MAC: lactose fermenting and mucoid
colony
 Potassium cyanide broth
Microscopic examination:
- Gram (-) bacilli
Biochemical test
 Fermentation of glucose, lactose,
𝐴 𝐾
trehalose & xylose: TIS 𝐴𝐺 LIA: 𝐴 LDA –
LDC -
 Indole (-) from tryptophan - - + +
 MR (-) VP (+)
 Citrate (+)
 Urease (+) & motility (-)
 H2S (-)
Characteristics:
 Microbiota of GIT
K. pneumoniae
Virulence factor:
 large polysaccharide capsule
- colony: mucoid
- protection against phagocytosis &
antimicrobial absorption
 lower RTI among hospitalized patients
& ICP
K. oxytoca
 antibiotic-associated hemorrhagic
colitis
 Biochemically similar with K.
pneumonia EXCEPT indole (+)
K. pneumoniae subsp. ozaenae
 atrophic rhinitis, a tissue-destructive
disease restricted to the nose
 plasmid-mediated ESBLs
K. pneumonia subsp. rhinoscleromatis
 Rhinoscleroma
Raoutella (Klebsiella) ornithinolytica
 Indole & Ornithine decarboxylase (+)
 Isolated from the urine, RT & Blood
Raoutella (Klebsiella) planticola
 Isolated from the urine, RT & Blood
Raoutella planticola: difficult to distinguish
from K. pneumoniae
Klebsiella variicola: isolated from primarily
sterile sites
Enterobacter
Characteristics:
Macroscopic examination:
 MAC: lactose fermenting and mucoid
colony
 Potassium cyanide broth
Microscopic examination:
 Gram (-) bacilli
Biochemical test
 Fermentation of glucose, lactose,
𝐴 𝐾
trehalose & xylose TIS 𝐴𝐺 LIA: 𝐾
 Indole (-) from tryptophan
 MR (-) VP (+)
 Citrate (+)
 Urease & motility (+)
 H2S (-)
 Ornithine decarboxylase & lysine
decarboxylase (+) EXCEPT E. gergoviae
or E. cloacae
Characteristics:
 isolated from wounds, urine, blood,
and CSF
 Pantoea (Enterobacter) agglomerans
- septicemia resulting from
contaminated intravenous fluid
- P. agglomerans HG XIII
 yellow pigment, is primarily a
plant pathogen
 E. gergoviae
- RT samples & blood
 Cronobacter (Enterobacter) sakazakii
- Mucoid, yellow pigment
- Neonatal meningitis & bacteremia from
powdered milk formula
E. hormaechei
 blood, wounds, and sputum
E. asburiae
 biochemically similar to E. cloacae
 blood, urine, feces, sputum, and
wounds
E. cancerogenus
 formerly E. taylorae
 osteomyelitis after traumatic wound
Enterobacter dissolvens & Enterobacter
nimipressuralis
 newly recognized species
BACTERIOLOGY LECTURE
WEEK 14
Enterobacteriaceae
Lactose Fermenters- rapid. They can utilize
lactose within 18-24 hours
- Escherichia, klebsiella, enterobacter
- TSI: A/A YELLOW OVER YELLOW ACID
OVER ACID
- GLUCOSE + LACTOSE
Late lactose fermenters- capable of utilizing
more than the usual which is 18-24 they are 36-
48 hours
- Citrobacter, hafia, serratia
- TSI: K/A ALKALINE OVER ACID
- GLUCOSE
Non lactose fermenters
- Erwinia, Yersinia, edwardsiella,
salmonella, shigella, proteus,
providencia, morganella
- TSI: K/A ALKALINE OVER ACID
- GLUCOSE
Carbohydrate that capable to utilize
enterobacteriacae organisms- glucose
Citrobacter
Characteristics:
 Gram negative bacilli
 GIT inhabitants
 UTI & sepsis
 MAC: resemble E. coli
 Resemble Salmonella sp. EXCEPT ONPG
(+) LDC (-)
 ONPG- ortho-nitrophenyl-
galactopyranoside (test were in
citrobacter among the
enterobactteriaceae is positive)
- Unique test to differentiate citrobacter
in other enterobacteriaceae
 MR (+)
 Citrate (+)
 Slow urease (+)
- CITROBACTER, KLEBSIELLA,
ENTEROBACTER, YERSINIA, SERRATIA
- Rapid urease (+): protues, providencia,
morganella
Citrobacter
Characteristics:
C. freundii
 endocarditis in intravenous drug
abusers
 aortic valve replacement when
antimicrobial therapy failed
 H2S (+)
- Hydrogen sulfide gas: colorless
- Black: if you detected in the culture
media
- Ferric ammonium citrate: H2S indicator
- Sodium thiosulfate: sulfure source
 Endocarditis- infection sa heart
 Enterobacteriaceae producing H2S+
- salmonella, proteus, S. arizonae,
citrobacter, edwardshiella
C. koseri
 nursery outbreaks of neonatal
meningitis and brain abscesses
C. amalonaticus
 found in feces, blood and wounds
Serratia
Characteristics:
 opportunistic pathogens associated
with outbreaks in health care settings
(UTI & RTI)
 bacteremic outbreaks in nurseries and
cardiac surgery and burn units
 Slow lactose fermenters EXCEPT S.
fonticola (rapid lactose fermenter)
  o-nitrophenyl-β-D-galactopyranoside  PROVIDENCIA IS THE ONLY CPOSITIVE
(ONPG) test (+) IN CITRATE
 Dnase (+)  MORGANELLA BOTH NEGATIVE IN
CITRATE AND H2S
S. marcescens, S. rubidaea & S. plymuthica
 Proteus mirabilis & Proteus vulgaris
 Produce prodigiosin: pink to red - urine, wounds, and ear and bacteremic
pigment in RT infections
- acute glomerulonephritis
S. odorifera - urease activity of P. mirabilis: struvite
 dirty, musty odor resembling that of kidney stones
rotten potatoes  Proteus mirabilis & Proteus vulgaris
- BAP: “swarming colonies”
Hafnia - Burnt chocolate odor
Characteristics:  PAD(+) deaminate the amino acid
phenylalanine
 H. alvei  Non-lactose fermeter
 Gastroenteritis  Urease (+) RAPID
 Delayed citrate (+)  H2S (+)
- H. alvei and H. alvei biotype 1  Proteus mirabilis: indole (-) & ornithine
- H. alvei biotype 1 (+); OXK
- beer wort of brewerie  Proteus vulgaris: indole (+) & ornithine
Non-lactose-fermenters (-); OX2 & OX19
- ferments sucrose (reaction in tsi: A/A)
- Proteus
- Providencia **OXK, OX2 & OX19: plasmid which is
- Morganella virulence factor. Develop their resistance to
- Salmonella antibiotics
- Shigella Providencia
- Edwardsiella
- Erwinia Characteristics:
- Yersinia
 P. alcalifaciens, P. stuartii, P. rettgeri,
Proteus P. rustigianii, and P. heimbachae. P.
rettgeri
Characteristics:  UTI & diarrheal disease among
 Proteus, Morganella, and Providencia travelers
 normal intestinal microbiota  outbreaks in health care setting
 opportunistic pathogens  P. stuartii
 motility (+), rapid urease (+), LIA: R/A - outbreaks in burn units
RED OVER YELLOW LDA (+), PAD (+) - Isolated in urines
(PHENYL ALANINE DEAMINASE)  P. stuartii and P. rettgeri
 PROTEUS IS THE ONLY ONE WHO IS - resistance to antimicrobials
POSITIVE TO H2S AMONG THE GROUP.  P. alcalifaciens
- Diarrhea in children
  P. rustigianii Salmonella

Morganella Characteristics:

Characteristics:  Gram (-) facultatively anaerobic bacilli

 MAC: clear, colorless, non–
 M. morganii
lactosefermenting colonies
 Subtypes:
 XLD & HE: colonies with black centers
- M. morganii subsp. morganii or
 Bismuth Sulfide Agar: black colonies
Morganella Morgani
with metallic sheen
- M. morganii subsp. sibonii. or
 Non-lactose fermenter
Morganella Siboni
 Indole, VP, PAD, Urease (-)
 UTI & neonatal sepsis
 H2S (+) EXCEPT S. paratyphi or
 Motility (+)
Salmonella enterica subsp. paratyphi
**PPM (proteus, provedencia, morganella)  (-) growth potassium cyanide medium

- Motility (+), MR (+), BP (-), Lysine
deaminase (+), LIA: R/A, rapid urease
producer, PAD (+)
Salmonella: motility (+) H2S (+)
Shingella: motility (-) H2S (-)
Virulence factors:

 Fimbriae/ pili/pilus
- Adherence to GIT
 Enterotoxin
- traverse intestinal mucosa

Antigen:

Edwardsiella
Characteristics:
 E. tarda, E. hoshinae & E. ictaluri
 Human pathogen: E. tarda
- Bacterimia & wound infections
 Citrate & urease (-)
 Lysine decarboxylase, H2S, indole (+)
Erwinia & Pectobacterium
Characteristics:
 plant pathogens
 Erwinia
- Poor growth at 37° C
- (-) growth in EMB & MAC
 Somatic O antigen
- Heat stable
- lipopolysaccharide in the outer
membrane of the cell wall
 H flagellar antigen
- Heat labile
- Phase I flagellar antigen: specific
phase; occur in few strains
- determine the immunologic identity of
certain serotype
- agglutinate only with homologous
antisera
- Phase II flagellar antigen: non-specific
phase; occur in may strains
- agglutinate only with heterologous
antisera
  Capsular K antigen (Vi) 4. Carrier state
- For ID of Salmonella serotype Typhi &  Site of chronic carriage: gallbladder
Salmonella serotype Choleraesuis  Organisms secreted in the feces
continuously or intermittently
Infections:
 Important source of infection
 Acute gastroenteritis or food
Treatment:
 Typhoid fever
- most severe form of enteric fever  antimicrobial therapy
(Salmonella serotype Typhi)  cholecystectomy
- enteric fevers (Salmonella Parathypi
and choleraesuis)
 Nontyphoidal bacteremia
 Carrier state following Salmonella
infection
 Some of the patients are
asymptomatic but they are capable of
carrying the infection
 MOT: ingestion of contaminated food,
water and milk
Shigella
1. Gastroenteritis
 One of the most common forms of Characteristics:
“food poisoning”
 Escherichieae with E. coli
 From poultry, milk, eggs, and egg
 Cause: bacillary dysentery
products as well as to handling pets
- presence of blood, mucus, and pus in
 Infective dose: 106 bacteria
the stool
- 8 to 36 hours after ingestion of
 Not GI microbiota
contaminated food
 Japanese microbiologist Kiyoshi Shiga
 TOC: chloramphenicol, ampicillin &
trimethoprim-sulfamethoxazole (SXT)  Motility (-)
2. Enteric fevers  (+) gas from glucose EXCEPT S. flexneri
 Salmonella Typhi: typhoid fever  Urease, H2S, LDC (-)
 Only host: human  (-) use of acetate or mucate as carbon
 Salmonella serotypes Paratyphi A, B, source
and C and Salmonella serotype  susceptible to disinfectants & high
Choleraesuis concentrations of acids and bile
 9 to 14 days after ingestion of the Characteristics:
organisms
 1st week: blood S. sonnei
 2nd week: urine  Ornithine decarboxylase (+)
 3rd week: stool  ONPG (+)
3. Bacteremia  slowly ferments lactose
 Typhimurium, Paratyphi, and
Choleraesuis
 - MAC: delayed fermentation; pink - safety-pin appearance
colonies on MAC only after 48 hours of  Preferred growth temperature: 25°C to
inc 30°C

Y. pseudotuberculosis & Y. enterocolitica

 sporadic cases of mesenteric

lymphadenitis in humans

Y. enterocolitica
Shigella
 Natural reservoir: pig
Antigens:  MOT: direct contact with household
pets
 (+) O and K antigen
- Ingestion: contaminated pork &
 (-) H antigen vacuum-packed deli meat, beef, lamb,
Infections: chicken, chocolate milk and water
 Gastroenteritis
 dysentery  Sepsis associated with the transfusion
 24 to 48 hours after ingestion of the of contaminated packed RBCs
organisms  Mimics acute appendicitis
Yersinia Y. enterocolitica
Characteristics:  Gram (-) coccobacilli with bipolar
Y. pestis staining
 Optimal growth temperature: 25°C to
 Causative agent of plague: disease of 30°C
rodents transmitted to humans by fleas  grows better with cold enrichment
 Rat flea: Xenopsylla cheopsis  Motility (+) at 25° C not at 35° C
 3 forms:  Cefsulodin-irgasan-novobiocin (CIN)
 Bubonic/ glandular form - cefsulodin, irgasan, novobiocin, bile
- from bite of an infected flea salts, and crystal violet (inhibitors)
- symptoms appear 2 to 5 days after  Bulls eye colony
infection
- high fever with painful regional lymph Y. pseudotuberculosis
nodes known as buboes
 Pathogen of guinea pigs
 Septicemic form
 Natural reservoir: birds
- bacteria spread to the bloodstream
 Causative agent of : caseous swelling
 Pneumonic form
called pseudotubercles
- occurs secondary to bubonic plague or
 typical-looking plague bacillus
septicemic form
 Motility at 18°C to 22°C
- Primary infection through inhalation
 Urease (+)
- Fatality rate: 100% if untreated
 Ferment rhamnose
 Gram (-) short plum bacillus
 Methylene blue or Wayson stain:
- bipolar staining
 C. + + - +
diversus/koseri

Hafnia

I M V C
H. - V V -
alvei
IMVC Reactions

 Indole, methyl red, vogues- proskauer,

citrate
Proteus
Escherichia
I M V C
I M V C P. - + V V
E.coli + + - - mirabilis
P. + + - V
vulgaris
Klebsiella (- - + +) P. - + - -
penneri
I M V C
K. - - + +
pneumoniae Providencia (+ + - +)
K. oxytoca + V + +
K. ozaenae - + - V I M V C
P. + + - +
rettgeri
Enterobacter (- - + +) P. + + - +
stuartii
I M V C
E. aerogenes - - + +
E. cloacae - - + + Morganella (+ + - -)
E. V V V V
agglomerans I M V C
M. + + - -
morganni
Serratia (- - + +) subsp.
morganii
I M V C
S. - V + +
Marcescens Salmonella (- + - +)
S. - + + +
liquifaciens I M V C
Most - + - +
serotypes
Citrobacter

I M V C
C. freundii V + - V
Shigella (- + - -)  1-5°C: Serratia & Yersinia
 45° C to 50° C: E.coli
I M V C
ABC V + - - Culture: LACTOSE FERMENTERS
C - + - -

Yersinia

I M V C
Y. V + - -
enterocolitica
Y. + + - V
frediriksenii Culture: LATE LACTOSE FERMENTERS
P. penneri + + - -

Edwardsiella

I M V C
E. + + - -
tarda
Culture: NON-LACTOSE FERMENTERS

Enterobacteriaceae

Specimen collection:

 No special considerations required

 transport media: Cary-Blair, Amies or
Stuart media: rectal swab
 stool
 hindi kailangan ng transport medium
 not culture in plated medium
immediately after its arrival in the lab.
Nilalagay muna siya sa enrichment
medium after 18-24 hours saka palang
siya ilalagay sa plated culture medium

Direct Microscopic Examination:

 gram (-) bacilli

 Stool sample: not helpful but may
reveal if GI infection is toxin mediated,
invasive process

Culture

 35°C to 37°C for 18-24 hours of

incubation
Biochemical test
BACTERIOLOGY LECTURE - trauma incurred during contact with
fresh, estuarine, or marine water or
WEEK 15
associated products
Vibrio, Aeromonas, Plesiomonas, and
if the stool is watery- alkaline peptone water
Campylobacter Species
(APW)
Enterobacteriaceae vs Vibrio, Aeromonas,
semi formed & mucoid- selenite f
Plesiomonas, and Campylobacter
Vibrio spp.
Similarities
Characteristic:
- Gram – bacilli
- Causing Gastrointestinal tract infection  Non-spore forming. facultatively
anaerobe, halophilic pleomorphic gram
Difference
(-) bacilli
Enterobacteriaceae  Halophilic- requiring sodium chloride
for growth specifically 1-8% NaCl
- Majority of the member are actually
 Broth medium: polar, sheathed flagella
part of the normal flora of our GIT
 Solid medium: peritrichous,
- Oxidase –
unsheathed flagella
- Motility + except: Klebsiella, Shingella,
Yersenia Biochemical test:
Vibrio, Aeromonas, Plesiomonas, and  Catalase (-)
Campylobacter  Oxidase (+)
- Usually found in the environment  Reduce nitrate to nitrite
- Oxidase + *except V. metschniknovii
- Motility +
 String test (+) 0.5% sodium
*Pleisiomonas is under the desoxycholate
enterbobacteriaceae family but it is a oxidase +  vibriostatic compound O/129 (S)
Vibrio - S for susceptible

Characteristic: Vibrio

 Family: Vibrionaceae Characteristic:

 Are not part though they are causing Group 1
gastrointestinal tract infection in
human they are not part of our normal  V. cholera & V. mimicus
flora. Group 2
 Environment: fresh water, brackish
water, marine/ salt water  V. metscchnikovii
- Indication of Vibrio spp. Infection:
Group 3
- consumption of raw seafood
- immigration or foreign travel  V. cincinnatiensis
- cholera-like or rice-water stools
Group 4 2 biogroups:

 G. hollisae Tests Classic El Tor

VP - +
Group 5 Hemolytic - +
 P. damsel & V. fluvialis pattern
50 μg R S
Group 6 Polymixin B
Agglutination - +
 V. alginolyticus, V. parahaemolyticus, V. in chicken
vulnificus & V. harveyi RBC
Vibrio cholera

Characteristic: V. cholerae non-O1

 V. cholerae, V. parahaemolyticus, V.  Resemble toxigenic V. cholerae O1, but

fluvialis & V. vulnificus
 V. cholerae subgroups: H & O antigen
- V. cholerae O1 (based on O antigen
composition)
- Ogawa (A, B)
- Inaba (A, C)
- Hikojima (A, B, C)
 V. cholerae O139
 V. cholerae non-O1
- resemble V. cholerae but fail to
agglutinate in O1 antisera
- non-O1 kasi mag kamuha lang sila ni O1
except that pag gumamit ng antisera ng
O1 hindi nag agglutinate dahil walang
antibody na naproproduce si V.
cholerae non-O1 na kamukha ng V.
cholerae O1 but other characteristics
are the same
 V. parahaemolyticus
- O and K antigens
most lack the cholera toxin gene
 milder form of gastroenteritis or
cholera-like disease
V. cholerae serogroups O75 & O141
 (+) cholera toxin gene (+) choleragen
 sporadic, cholera-like diarrhea
Vibrio parahaemolyticus
Characteristic:
 2nd most common vibrio causing
gastroenteritis “summer diarrhea”
 Pandemic strain: V. parahaaemolyticus
serotype O3:K6
Kanagawa phenomenon
 heat-stable hemolysin effective in high-
salt mannitol medium
 Wagatsuma agar

Vibrio cholerae O1: cholera, “Asiatic cholera” Vibrio vulnificus

or “epidemic cholera” Characteristic:
- Epidemic cholera: isang pagkain lang  2nd most serious cause of Vibrio-
pinanggalingan associated type infections
 Enterotoxin: Cholera toxin or - Septicemia
cholerageb - Wound infections
Vibrio alginolyticus
 least pathogenic for humans
 strict halophile: 1-10% NaCl
 eye and ear infections or wound & burn
infcetions
Vibrio
Specimen collection:
 body fluids, pus, tissues
 Transport medium: prevent desiccation
- Cary-Blair transport medium
- glycerol
- toxic for vibrios: Buffered glycerol
saline
Time of collection:
 ASAP in the course of illness
 before antimicrobial administration
Direct microscopic examination:
 pleomorphic gram (-) bacilli
Culture & Macroscopic Examination
 NA & BAP: 0.5% NaCl
 MAC: NLF except V. vulnificus
 Oxidase test to differentiate from
other LF
 TCBS: thiosulfate citrate bile salt
sucrose
- selective: H2S indicator: ferric citrate
- sodium citrate, sodium thiosulfate, and
oxgall (bile salts)
- Inhibit gram (+) cocci & other gram (-)
bacilli not normally found in stool
 High pH
 differential medium: sucrose &
bromthymol blue. Pag may acid yellow
pag walang acid green or colorless
- differentiates sucrose-fermenting
species from the nonsucrose-
fermenting species
Culture:
Sucrose fermenters:
 V. cholerae, V. alginolyticus, V. fluvialis,
V. furnissii, V. cincinnatiensis, V.
metscchnikovii, and some V. vulnificus
strains
Nonsucrose-fermenters:
 V. mimicus, V. parahaemolyticus, P.
damsela and most V. vulnificus strains
Enrichment medium:
 Alkaline Peptone Water with 1% NaCl
 pH at least 8.5%
Microscopic examination:
 Gram (-) bacilli
Biochemical tests: Presumptive tests
 150 ug vibriostatic agent O/129 (S) &
String test (+)
- Differentiate from Aeromonas
 Ferment Inositol (-)
- Differentiates from Pleisiomonas
EXCEPT V. cincinnatiensis & some V.
metscchnikovii
 Oxidase (+)
- Differentiates from
Enterobacteriaceae EXCEPT V.
metscchnikovii
 O/F reactions (fermenter)
- Differentiates from oxidative
Pseudomonas
*vibrio spp: facultative anaerobe -> fermenter
Biochemical test: Definitive Groups:

Group 1  mesophilic group (37°C) (motile by

polar flagellum)
 V. cholera & V. mimicus
- A. hydrophila complex
Group 2 - A. veronii complex
- A. caviae complex
 V. metscchnikovii  psychrophilic group (22°C) (nonmotile)
Group 3 - A. salmonicida

 V. cincinnatiensis Infection:

Group 4  Gastrointestinal Infections A. caviae

- Acute, secretory diarrhea often
 G. hollisae accompanied by vomiting
Group 5 - Acute, dysenteric form of diarrhea
(similar to shigellosis
 P. damsel & V. fluvialis - chronic diarrhea lasting more than 10
days
Group 6
- cholera-like disease, including rice
 V. alginolyticus, V. parahaemolyticus, V. water stools
vulnificus & V. harveyi - nebulous syndrome commonly referred
to as traveler’s diarrhea (similar to
*all of a group are capable of growth in NA enterotoxigenic E. coli).
without additional NaCl except GROUP 1
 septicemia, meningitis, wound
*all of them are oxidase + , catalase - and infections & keratitis associated with
nitrate to nitrite reduction + except V. contact lens wear
metscchnikovii
Specimen collection:
Aeromonas
 No special considerations
Characteristics:
Direct microscopic examination
 Family: Aeromonadaceae
 gram-negative bacilli
 oxidase (+), glucose-fermenting, gram-
negative bacilli Culture
 Environment: freshwater, estuarine,
 large, round, raised, opaque colonies
and marine env.
with an entire edge and a smooth,
 vibriostatic compound O/129 (R)
often mucoid, surface
- R for resistant
 BAP (beta hemolysis): A. hydrophila, A.
 Indole +
veronii biovar sobria, and A. jandaei
 CIN II w/ 4 μg of cefsulodin instead of
15 μg
- pink-centered colonies (mannitol
fermentation), with an uneven, clear
 apron resembling Yersinia
enterocolitica (oxidase (-))
- Oxidase (+)
Bichemical tests:
 Indole (+)
 Oxidase (+)
- Differentiates from Enterobacteriaceae
EXCEPT Plesiomonas shingelloides
 String test (-) & O/129 (R)
- Differentiates from Vibrio spp.
 Growth in NB with 0% NaCl (+) but (-) in
6% NaCl
- Differentiates Aeromonas &
Pleisiomonas spp. from Vibrio spp.
 Inositol fermentation (-) & (+) Glucose
fermentation w/ or w/out acid
- Differentiates from Pleisiomonas spp.
Plesiomonas
Characteristic:
 Family : Enterobacteriaceae
 (-) gas production from glucose
 Motile, oxidase-positive, glucose
fermenting, facultatively anaerobic,
gram-negative bacilli
 P. shigelloides
- O/129 (S)
 Inositol +
Characteristic:
 Motile: monotrichous or two to five
lophotrichous flagella
 P. shigelloides
- cross-agglutinate with Shigella sonnei,
S. dysenteriae & S. boydii
- Anti-sera -.Ab specific to shingella react
with Ag of P. shingelloids
Virulence factors:
 O & H antigen
Microscopic examination:
 Gram (-) bacilli in singly, in pairs, or in
short chains or filamentous forms
Culture & Macroscopic examination:
 shiny, opaque, nonhemolytic colonies
appear, with a slightly raised center
and a smooth and entire edge
 MAC: may be LF or LLF
- Oxidase test (+)
 Inositol Brilliant Green Bile Agar
- white to pink colonies
 (+) growth in CIN
- colonies with an opaque apron
 (-) growth in TCBS

Infection:

 Gastroenteritis
- watery or secretory diarrhea
- subacute or chronic disease that lasts
from 14 days to 2 to 3 months
- more invasive, dysenteric form that
resembles colitis
 bacteremia and meningitis
Campylobacter & Campylobacter-Like Species - Buffered glycerol saline (toxic to
campylobacters)
Vap vs Campylobacter
 H. pylori
VAP - gastric biopsy (juice)
- Transport medium: Stuart medium
- Glucose +
- Tissue samples: Cysteine-Brucella
- FA broth with 20% glycerol (frozen at −70°
Campylobacter C)

- Asaccharolytic Culture & Macroscopic examination:

- Microaerophile
 Selective media for Campylobacter spp.
- capnophile

Characteristic:

 Previously classified under vibrios

(oxidase +)
 Asaccharolytic
 Family: Campylobbacteraceae
- Camplyobacter, Arcobacter,
Sulfurospirillum
 Microaerophile
Culture & Macroscopic examination:
- 5% oxygen
 Family: Helicobactereaceae  CAP or Brucella agar with 5% horse red
- Helicobacter and Wolinella blood cells & Skirrow’sagar
- H. pylori
Characteristic:
 C. jejuni and other enteric
 From the environment campylobacters grow optimally at 42◦C.
 Campylobacter spp. - EXCEPT: C. fetus subsp. fetus (37◦C)-
- abortion in domestic animals blood
- most common cause of bacterial  Microaerophilic and capnophilic
gastroenteritis (Campylobacter jejuni) environment
- 4th most common cause of foodborne - Campylobacter spp.
gastrointestinal illness (Foodborne - 5% O2, 10% CO2, and 85% N2 EXCEPT:
Diseases Active Surveillance Network) C. rectus & C. curvus (strict anaerobes)
 Helicobacter pylori - Helicobacter spp.
- gastric, peptic, and duodenal ulcers, GI - 5% to 10% O2 and 5% to 12% CO2
carcinoma  GasPak jar
- For campylocaters
Specimen collection:
 Evacuation replacement system for
 C. fetus subsp. Fetus 72hours
- Blood culture - strict anaerobic condition
 Campylobacter spp. - anaerobic jar
- Stool samples & rectal swab - Pressure: 15 inches Hg
- Transport medium: Cary-Blair - Gas: 10% CO2, 90% N2
 - 5% CO2, 10% H2, 85% N2 - oral administration of 13C- or 14C-
- 10% CO2, 10% H2, 80% N2 labeled urea
 Candle jar - release of 13CO2 or 14CO2
- least ideal environmental condition - detected in the exhaled breath by a
scintillation counter
Microscopic examination:
Nonfermenting Gram Negative Bacilli
 Campylobacter spp. & Arcobacter spp.
- curved, non–spore-forming, gram (-) Characteristic:
bacilli
 fail to acidify
 Campylobacter jejuni & other enteric
- OF media when it is overlaid with
campylobacter
mineral oil
- long spirals or ‘S’ or seagull-wing
- TSI agar butts k/k
shapes
 Grow in aerobic environment
 Gram stain: stain poorly
 Some oxidize CHO to derive energy for
 carbolfuchsin : recommended counter
their metabolism: oxidizer
stain (safranin 2-3 mins extension)
 Some are inert or biochemically
 Hanging drop preparations or Phase
inactive: nonoxidizers or asaccharolytic
contrast microscope:
 Most are Oxidase (+): differentiate
- “darting” motility single polar
from Enterobacteriaceae
flagellum
- Pleisiomonas shingelloids: only
- Brucella tryptic soy broth not in dist.
member of Enterobacteriaceae w/cc is
water
oxidase (+)
 H. pylori
- motile Pseudomonas genus
- curved, non–spore-forming, gram (-)
Characteristic:
bacilli
- multiple flagella at one pole  Pseudomonads
- Gram (-) bacillus or coccobacillus
Biochemical tests:
(diplococcus)
 Cambylobacter spp. - Strictly aerobic metabolism
- Oxidase (+) - Motile usually with polar or polar tufts
- Microscopic examination of flagella
- Differentiates from Aeromonas & - Oxidase(+) EXCEPT: P. luteolus and P.
Pseudomonas oryzihabitans
- Oxidase (+) & (+) growth at 42° C in a - Catalase (+)
microaerophilic environment - Grows on MAC agar
 Hippurate hydrolysis test (+) - Oxidizer & asaccharolytic
- C. jejuni
 Helicobacter pylori
- identified by nonculture methods
- Gastric biopsy
- Rapid urease (+)
- incubated at 37° C for 2 hours
- urea breath test
Pseudomonas aeruginosa - water-soluble and fluoresces under short-
wavelength UVL
Infections:
Characteristic:
 leading cause of nosocomial
respiratory tract infection  P. aeruginosa
 3rd most common cause of bacteremia - Produce pyocyanin: blue & water-
(with ecthyma gangrenosum of the soluble pigment
skin)  PYOCYANIN + PYOVERDIN= green color
 nosocomial UTI characteristic of P. aeruginosa
 ventilator-associated- pulmonary  Pyorubin (red) & pyomelanin (brown)
infection (patients w/ CF)  BAP
 otitis externa (swimmers o divers) - β-hemolytic, flat spreading colonies
“swimmer’s ear” with a characteristic metallic sheen
 Jacuzzi or hot tub syndrome:  2-aminoacetophenone: fruity,
necrotizing skin rash grapelike odor or corn-taco like odor
 denitrification of nitrates and nitrites
Virulence factors :
 arginine dihydrolase (ADH) (+)
 Endotoxin  growth at 42° C (differentiate from
- LPS other pseudomonads)
 Motility, pili, capsule  Citrate(+)
 Exotoxins  Acetamide utilization (+), Cetrimide
- Exotoxin A Agar growth (+)
- Block protein synthesis
Pseudomonas fluorescens & Pseudomonas
- Similar to diphtheria toxin
putida
- proteases, hemolysins, lecithinase,
elastase & DNase Characteristic:
 Alginate
 produce pyoverdin but not pyocyanin
- Overproduction causes colonies to be
mucoid  Grows at 42° C
 Cannot reduce nitrate to nitrogen gas
Characteristic:  Produce acid from xylose: separate
them from other fluorescrnt
Fluorescent group
pseudomonads
 P. aeruginosa  Gelatin hydrolysis
 P. fluorescens - Pseudomonas fluorescens (+)
 P. putida - Pseudomonas putida (-)
 P. veronii
Pseudomonas stutzeri
 P. mosselii
 P. monteilii Characteristics:

These are all:  wrinkled, leathery, adherent colonies

producing light-yellow or brown
- produce pyoverdin: yellow-green or yellow-
pigment
brown pigment
 ADH (-)
  starch hydolysis (+) Burkholderia genus
 6.5% NaCl (+)
Burkholderia cepacia:
 Nitrate to nitrite reduction (+)
 Oxidase (weak & slow), ONPG & LDC (+)
Pseudomonas mendocina
 ODC & nitrate to nitrite reduction (-)
Characteristics:  Motile (lopothricous)
 Pink colonies in MAC: lactose oxidizers,
 nonwrinkled, flat colonies that may
Maltose & mannitol
appear with a yellowish-brown pigment
 Yellow on :
 Oxidase and ADH (+)
- OFPBL (Oxidative/Fermentative
 not produce pyoverdin. Not member of
Polymixn B Bacitracin Lactose)
fluorescent group
- PC agar ( Pseudomonas cepacia agar)
 Acetamide & starch hydrolysis (-) - BCSA (B. cepacia selective agar)
 Motile (single polar flagellum)  Nonwrinkled colonies: differentiate it
 Oxidizes glucose and xylose from P. stutzeri
Acinetobacter genus  produce a nonfluorescing yellow or
green pigment that may diffuse into
Characteristics: the media
 Cause of 1-3% nosocomial infection Burkholderia mallei:
next to P. aeruginosa
 strictly aerobic  Causes glanders (RT zoonosis; horses,
 Gram (-) coccobacilli or gram (-) cocci in mules & donkeys)
blood culture  Also causes Farcy (most severe
 Resembles Neisseria dessiminated type of glanders)
 Oxidase (-)  Only nonmotile pseudomonad
 Catalase (+)  Potential bioterrorism agent
 Nonmotile  O/F= +/- (glucose, maltose & lactose)
 Can grow in MAC  Nitrate to nitrite reduction & ADH (+)
 A. baumannii: saccharolytic & A. Burkholderia pseudomallei:
lwoffii: asaccharolytic.
 Causes
Stenotrophomonas maltophilia - meliodosis or Glanders-like
Characteristic: - Vietnamese time bomb
 Nonfermentative wrinkled colony:
 3rd most common non-fermentative ashdown mmedium
gram (-) bacillus - P. stutzeri does not use lactose
 Oxidase (-) - with colistin
 Catalase, DNase, esculin and gelatin - deep pink colonies due to absorption of
hydrolysis, and lysine decarboxylase (+) neutral red
 Nonfermentative - exhibit an earthy odor
 Bluish colonies in MAC  O/F= +/- (lactose oxidizer)
 susceptible to SXT
- Drug of choice
Shewanella putrefaciens *reside in human oral mucosa

Characteristic: *causes endocarditis

 TSI: K/K with H2S Haemophilus spp.

 Oxidase (+)
Characteristics:
Alkaligenes faecalis
 derived from the Greek word meaning
Characteristics: “blood-lover”
 Requirements:
 Oxidase & Catalase (+)
- X factor (from RBC hemin or hematin)
 O/F= -/- asaccharolytic (“X for unknown”)
 Odor: apple-like or fruity odor - V factor (nicotinamide adenine
 Motile (peritrichous) dinucleotide [NAD]) (“V for vitamin”)
 UTI, wound & diarrhea  BAP
Moraxella lacunata - V factor dependent Haemophilus spp.
growth (-)
Characteristic: - RBC are still intact and contains
NADase (hydrolyze V factor)
 Causes Blepharoconjuctivitis
 Lacunae (pitting of agar)
 Oxidase & Catalase (+)
 O/F= -/- (asaccharolytic)
 (-) growth in MAC
 Gram (-) coccobacilli
- Mistaken as Neisseria

Haemophilus and other Fastidious Gram -

Negative Bacilli  CAP
- lysing of the red blood cells by heat
Pasteurellaceae family
- releases both the X factor and the V
Characteristic: factor
- inactivates NADases
 Genera: Haemophilus, Actinobacillus,
Pasteurella, and Aggregatibacter
 gram (-), pleomorphic, coccoid to rod-
shaped cells
 nonmotile & facultatively anaerobic
 Catalase & nitrate to nitrite reduction
(+)
 Oxidase (-)
- Haemophilus
- Aggregatibacter
- Cardiobacterium
- Eikenella
- Kingella
Satellitism
 phenomenon w/c helps in the
recognition of Haemophilus spp. that
require V factor
 occurs when S. aureus, S. pneumoniae,
or Neisseria spp. produce V factor
- Organisms: source of V factor
- BAP: source of X factor
 H. ducreyi
- Only require X factor
Haemophilus influenza (Pfieffer’s bacillus)
Virulence factor:
1. Capsule
 capsular polysaccharide: plays the most
significant role
 antiphagocytic property and
anticomplementary activity
 Serologic groups: a, b, c, d, e & f
 Most invasive: serotype b (Hib)
- composed of ribose, ribitol, and
phosphate (polyribitol phosphate)
 Nontypable H. influenzae (NTHi)
- Non-encapsulated
 Blood: fibrinogen -> plasma protein
coagulation
2. Immunoglobulin A Proteases
 Only genus that produces IgA
protease: H. influenza
 cleave secretory IgA
3. Adherence Mechanisms
 NTHi strains: adherent to human
epithelial cells
- Localized infection
 Serotype b: not adherent to human
epithelial cells
- systemic infections
Infection:
Encapsulated strains Non encapsulated
strains
Septicemia Otitis media and
effusion
Septic arthritis Conjunctivitis
Meningitis Sinusitis
Osteomyelitis Bacteremia
Cellulitis Pneumonia*
Pericarditis
Pneumonia
epiglottitis
Haemophilus aegyptius
 acute conjunctivitis
 Konch-Weeks bacillus
 Commonly referred as “Pink eye”
Haemophilus influenzae Biogroup aegyptius
 conjunctivitis in pediatric patients
 Nonencapsulated
 Cause systemic disease known as
Brazilian pupuric fever (BPF)
Haemophilus ducreyi
 strict human pathogen & smallest
pathogenic bactreia
 causative agent of chancroid (highly
communicable ST genital ulcer disease
(GUD))
- commonly referred as soft chancre
- Hard chancre (syphilis)
 Not part of microbiota
 Suppurative, enlarged, draining,
inguinal lymph nodes (buboes)
Haemophilus parainfluenzae
 Endocarditis
 Mitral valve as primary site of infection
Workflow
Specimen collection:
 blood, CSF, middle ear exudate, joint
fluids, upper and lower RT specimens,
swabs from conjunctivae, vaginal
swabs, and abscess drainage
 Lower RT: bronchial washing
  Genital sites
- cleaned with sterile gauze moistened
with sterile saline
- swab pre-moistened with sterile
phosphate-buffered saline at the base
of ulcer
- aspirate from buboes
 Direct plating bedside instead of
transport medium
Culture:
H. influenzae
 CAP incubated between 33° C & 37° C
with 5% to 10% CO2 at 18-24 hrs of
incubation
 capnophiles
- With 300 ml/L bacitracin
- excellent medium for the isolation of
Haemophilus spp. For RT specimens
H. ducreyi & H. aegyptius
 CAP with 1% IsoVitaleX or Vitox
(additional sources of X & V factor)
H. ducreyi (grows best at 33° C)
 Nairobi biplate
- GC agar base with 2% bovine
hemoglobin and 5% fetal calf serum w/
vancomycin
- Mueller Hinton agar with 5%
chocolatized horse blood w/
vancomycin
Colonial morphology:
H. influenzae
 CAP: translucent, tannish, moist
colonies with a distinct “mousy” or
bleachlike odor
 Do not grow on MAC
H. Parainfluenzae & H. parainfluenzae
 CAP: tannish and drier with a medium
to large size compared with H.
influenza
BAP with horse or rabbit blood
 H. influenza: nonhemolytic
 H. parahaemolyticus: B-hemolytic
H. ducreyi
 CAP: small, flat, smooth, nonmucoid,
transparent to opaque colonies
- colonies can be pushed intact using a
loop
- difficult to pick up
- produce a “clumpy” nonhomogeneous
appearance when suspended in saline
Microscopic Examination:
 small, gram-negative coccobacilli to
long filaments
H. ducreyi
 pale staining gram-negative coccobacilli
arranged singly or in groups
 Commonly referred as “school of fish “
or “railroad tracks” or “fingerprints”
Biochemical tests:
X Factor and V Factor Requirement
 Haemophilus Quad Plate
- Media with X factor only
- Media with V factor only
- Media with X and V factors
- Media with X and V factors with horse
red blood cells
Porphyrin Test
 alternative method for differentiating
the heme-producing (x factor) species
of Haemophilus
  Principle:
- based on the ability of the organism to
convert the substrate δ-aminolevulinic
acid (ALA) into porphyrins or
porphobilinogen (intermediates in
synthesis of X factor)
 Reagent: p-
dimethylaminobenzaldehyde (Kovacs’
reagent)
 Result:
- Red color in lower aqueous phase
(presence of porphobilinogen)
- Reddish orange fluorescence in UVL
detection at 360 nm (presence of
pophyrins)
Characteristics:
 gram-negative bacilli
 common fastidious nutritional
requirements & growth with increased
CO2
 ENDOCARDITIS
 normal biota of the oral cavity
 Opportunist organisms
Aggregatibacter aphrophilus
Characteristics:
 Greek aphros and philia: foam loving or
desiring high concentration of CO2
 does not require CO2
 dental plaque and gingival scrapings
 Previously known as H. aphrophilus and
H. paraphrophilus
 Catalase (-)
 Utilize glucose, maltose, sucrose &
lactose

Aggregatibacter actinomycetemcomitans

Characteristics:

 “star shape with four to six points” in

the center of the colonies after 48 hrs
of incubation
 Catalase (-) & oxidase (v)
HACEK Group  (-) growth in MAC
Haemophilus spp.  Urease (-): differentiate from
Actinobacillus
Aggregatibacter actinomycetemcomitans  Utilize glucose
(Actinobacillus actinomycetemcomitans)
Cardiobacterium hominis
Cardiobacterium hominis
Characteristics:
Eikenella corrodens
 C. hominis and C. valvarum
Kingella spp.
 form rosettes or sticklike structures in
*capnophilic yeast extract
 (-) growth in MAC
*dysgonic  “pitting” on agar
 Catalase (-) and indole (+): differentiate
from Aggregatibacter spp.
  Oxidase (+)

Eikenella corrodens

Characteristics:

 “clenched fist wounds” or after the

skin has been broken by human teeth Pasteurella spp.
 “pit” or corrode the surface of the agar Characteristics:
 Odor: chlorine bleach-like odor
 Catalase (-)  infection: Pasteurellosis (zoonosis-
 Oxidase & ornithine (+) animal bites)
 Asaccharolytic: similar to Moraxella  Agent of Shipping fever in cattles
 normal microbiota of the oral cavity of
Kingella spp. birds and mammals
Characteristics:  P. multocida
 Capsule, non-motile, bipolar stain
 Kingella kingae, K. denitrificans, K. “safety pin”
oralis & K. potus  oxidase and catalase (+)
 coccobacillary with squared ends in  Utilize glucose
pairs or short chains  Ornithine, Indole, Urease (+)
 Catalase (-): differentiate from  (+) BAP & (-) MAC
Moraxella spp. and Neisseria spp.  P. canis (dogs)
 Oxidase (+)  P. stomatis & P. dagmatis (dogs and
 Utilize glucose, maltose cats, humans)
 (+) growth in Neisseria selective agars
Legionella spp.
Capnocytophaga spp.
Characteristics:
Characteristics:
 L. pneumophila: legionnaires’ disease
 Family: Flavobacteriaceae & Pontiac fever
 C. ochracea, C. gingivalis, C. sputigena,  L. micdadei: Pittsburrgh pneumonia
C. haemolyticus & C. granulosa  L. bozemanni: Wiga’s agent of
 normal microbiota of the oral cavity of pneumonia
humans  Sources: lakes, rivers, hot springs, mud,
 SEPTECEMIA with neutropenia hot water systems, water-cooling
(mababa ang neutrophil count) towers for air-conditioning &
 fastidious, facultatively anaerobic, evaporative condensers
gram (-) fusiform bacilli  resist water treatment (3 mg/L
 require increased CO2 for growth chlorine)
 produce gliding motility on solid agar - multiply over 20° C to 43°
 ferment sucrose, glucose, maltose, and - adhere to pipes, rubber, plastics, and
lactose sediment
 reduce nitrates and hydrolyze esculin - survive and multiply within free-living
 oxidase and catalase (-) protozoa
  MOT: aerosolized water particles
Isolation:
 Sputum: diluted 1 : 10 with 0.2 N KCl-
HCl (5 mins)
 Incubate: 37° C in air for at least 7 days
 (-) growth in BAP
 Require L-cysteine
 Best medium: BCYE agar with L-
cysteine or Feeley Gorman medium
 BYCE- Buffered Charcoal Yeast Extract
 Colonies: grayish white or blue-green,
convex, and glistening colonies.
“ground-glass” or “cut glass”
appearance
 Direct Fluorescent Antibody (DFA)
- Legionella Ag
 Stain
 Deiterle silver stain: black
Francisella tularensis
 Characteristics:
 infection: Tularemia (zoonosis-
rodents, rabbits, deerfly, water
trapper’s disease)
 MOT: ingestion, inhalation, arthropod
bite or contact with infected tissues
 Non-motile, capsule, obligate
anaerobe
 Requires cysteine, cystine, or
thiosulfate
 Catalase (+)
 Oxidase, Urease, MAC (-)
 Culture:
- Glucose Cysteine Blood Agar (GCBA)
- Peptone Cysteine Agar (PCA)
- Cysteine Heart Agar (CHA)
Bordetella spp.
Characteristics:
 Gram (-) bacilli, obligate aerobe
 Grows best at 35° C to 37° C
 Toluidine blue: Bipolar granules
Virulence factors:
 B. pertussis
- Filamentous hemagglutinin (FHA) &
pertactin: attachment
- Pertussis toxin (PT): interferes with
signal transduction
 B. parapertussis & B. bronchiseptica
- Adenylate cyclase toxin: inhibits host
epithelial and immune effector cells
- Tracheal cytotoxin: inhibiting DNA
synthesis & promoting cell death
Bordetella spp.
Characteristics:
 B. pertussis: whooping cough or
pertussis
 B. parapertussis: milder form of
whooping cough; pertussis-like
syndrome
 B. bronchiseptica: inhabits respiratory
tract of canines (kennel’s cough)
Infection:
 Pertussis
 3 stages:
1. Catarrhal phase: general flu-like symptoms
2. Paroxysmal phase: repetitive coughing
episodes followed by the characteristic
“whoop” at the end of the coughing spell
3. Convalescent phase: recovery period
 Specimen: Nasopharyngeal asiparates
or swab (calcium alginate or Dacron
polyester)
  Culture medium
- Bordet-gengou (Potato-Blood Glycerol)
( mercury drop or pearl-like colonies)
- Regan-Lowe transport medium
(charcoal agar w/ 10% horse blood &
cephalexin)
- Jones Kendrich (charcoal, yeast extract)
- Charcoal-Cephalexin Blood Agar (CCBA),
Stainer & Scholte, Casamino Broth

Brucella spp.

Characteristics:

 Gram (-) bacilli, obligate aerobe

 No capsule, zoonotic bacteria
(erythritol: enchnace the growth)
 Medium: Casteneda broth, TSB,
Wilconsin medium, CAP
 H2S (+): B. abortus & B. suis
 Fuchsin and thionine dye inhibition test
 Serologic test: Rose Bengal +
Mercaptoethanol Agglutaion

*melitensis hours <4

Natural host:

B. abortus- goat/sheep

B. melitensis- cattle

B. suis- swine (pig)

B. canis- dog
BACTERIOLOGY LECTURE Laboratory diagnosis:

WEEK 16 Specimens:

Aerobic Gram Positive Bacilli  Cutaneous anthrax

- Vesicular fluids
Bacillus anthracis
- Swabs from under the edge of the crust
Characteristics: of the eschar
 GI & Respiratory anthrax
 Largest bacteria - Blood
 Known as Anthrax bacillus
 2-5um Gram (+) bacilli in chain, Laboratory diagnosis:
bamboo square end
Culture:
 Non-motile, zoonotic bacteria
 Spore-forming (central or subterminal)  nonhemolytic colonies on BAP
 Appearing as Medussa head
Virulence factors:
 Tube gelatin medium Inverted pine
 Plasmids (pXO1 and pXO2) tree
 pXO1  Stand up like an egg white when
- lethal factor (LF), edema factor (EF) and teased with a loop
protective antigen (PA)  Motility (-)
 pX02
Selective medium
- Poly-D-glutamic acid capsule
- Inhibits phagocytosis  PLET (polymyxin-lysozyme-EDTA-
thallous acetate)
Infections:
Laboratory diagnosis:
Malignant pustule
 Catalase (+): differentiates from
 vesicle ruptures, resulting in a necrotic
Clostridium spp. (anaerobic gram (+)
lesion that continues to grow into the
bacillus)
characteristic black eschar
 String of pearl test (0.05 U of PEN) on
 also known as Cutaneous anthrax
BAP (S)
Woolsorter’sdisease  Ascoli test
- Serologic precipitation test
 Respiratory infection - Diagnostic: (+) precipitin ring
 also known as Rag picker’s disease  PCR, Fluorescence Ab test, ELISA
 mediastinitis- inflammation in the  Penicillin susceptibility test
mediastinum - (S) in 10 U Penicillin
Gastroenteritis Bacillus cereus
 Bloody diarrhea Characteristic:

 Known as Fried rice bacillus

 Food poisoning
 Diarrheal
 - meat or poultry, vegetables, and pastas  QC
(8-16 hrs) - Oven
- indistinguishable from watery diarrhea - ETO gas
by Clostridium perfringens
Corynebacterium diphtheria
 Emetic
- ingestion of fried rice Characteristics:
- nausea and vomiting 1 to 5 hours after
ingestion of contaminated food  Klebs-Loeffler bacillus
 highly pleomorphic
Infective dose:  Gram (+) bacillus appearing in palisades
 105 B. cereus cells/gram of food (“V” and “L” formations)
- Club-shaped, barbed, pallisade (picket
 100,000 bacterial cells
fence), Chinese character (X, Y, V, L)
Virulence factors:  Methylene blue: beaded appearance
Babes-Ernst granules
- Exotoxin (similar to cholera)
- Metachromatic or Volutin granules
Laboratory diagnosis: - indicates the accumulation of nutrient
reserves
Culture:
 Agent of diphteria
 β-hemolytic “frosted glass-appearing
Virulence Factor:
colony”
 Diphtheria toxin
Tests B. anthracis B. cereus
- strains of C. diphtheriae infected with a
Motility - +
lysogenic β-phage, which carries the tox
Capsule + - peritrichous
gene for diphtheria toxin
Hemolysis on - +
BAP - C. ulcerans & C. pseudotuberculosis
Growth on - + Culture:
45°C
Penicillin G S R  facultative anaerobe but grows best
Lecithinase + - aerobic conditions (37°C)
Salicin - +  Loeffler serum or Pai agars: enhance
fermentation pleomorphism & metachromatic
Gelatin HOH - + granules
and growth  Cystine-tellurite blood agar (CTBA)
on PEA - Potassium tellurite: inhibits many
noncoryneform bacteria
Bacillus subtilis - black or brownish colonies: reduction
of tellurite
Characteristic: - brown halo: cystinase activity
 Gram (+) bacilli in chain - C. diphtheriae, C. ulcerans & C.
 Spore: central pseudotuberculosis
 Common laboratory contaminant  Urease test
- (+): C. ulcerans & C. pseudotuberculosis
 Opportunistic pathogen
- (-): C. diphtheriae
 Source pf antibiotics
  Ferments glucose and maltose (no gas)
& reduces nitrate to nitrite
- C. diphtheria
Laboratory diagnosis:
Colonial types:
 Gravis type
- 1-2 mm colonies on blood agar
- Largest colonial type
 Mitis type
- fried egg appearance on blood agar
(clear colonies with white centers)
- Bleachlike odor on tellurite medium
 Intermedius type
- Small colonies (0.5 mm)
- Black colonies with gray borders on
tellurite medium
Corynebacterium pseudodipthericum
 Norma flora of throat
 Known as Hoffman’s bacillus
Corynebacterium xerosis
 Conjunctiva
Corynebacterium acne
 Skin
Corynebacterium minutissimum
 Agent of erythrasma (skin infection)
 “coral red fluorescence” on Wood’s
light
- Presence of porphyrins
Corynebacterium spp.

Laboratory diagnosis: Tests C. C. C.

diphtheriae ulcerans pseudotuberculosis
Toxigenecity test: urease - + +
In vivo nitrate + - +/-
reduction
 Animal Inoculation (guinea pig) starch/ - + -
 (+): dead gelatin
 (-): alive hydrolysis
 C. ulcerans
In vitro - Mastitis in cattles
 Eleks test: (+) precipitin line Corynebacterium spp.
- Definitive test
 Schick test Tests C. xerosis C. C. jeikeium
pseudodipthericum
- susceptibility for diphtheria
Urease - + -
- Arm + diphtheria toxin = (+) redness
CHO G-M-S None G
erythema
fermentation (Glucose-
 Roemer test: Maltose-
- Animal (pig) virulence test Sucrose)
 Catalase, Dnase, Glucose & maltose nitrate + + -
fermentation (+) reduction
Corynebacterium jeikeium

 Formerly group JK
 Resistant to a number of antibiotics
 Associated with prosthetic valve
endocarditis, pneumonia & peritonitis
Listeria monocytogenes Tests L. E.
monocytogenes rhusiopathiae
Characteristic: Catalase + -
 Gram (+) rod Motile + -
 Motile at RT (25°C)
Hemolysis Beta Alpha
- Hanging drop: Tumbling motility
VP + -
- Semisolid medium (SIM, at 25°C):
H2S - +
Umbrella like or Inverted Christmas
Esculin & + -
tree-like
Hippurate
 Major of infection is contaminated Gluconate + -
food (cabbage, fruit, dairy products) Media McBride, Cold BAP
 Agent of Human listeriosis enrichment
(granulomatosis infanseptica)
- Meningitis, sepsis, food poisoning, fetal
abortion Lactobacillus acidophilus
 Culture: McBride medium & cold Characteristic:
enrichment at 4°C
 Normal flora of the mouth, GI & vaginal
 Virulence test: canal
- Ocular test of Anton / Anton test  Known as Doderlein bacillus
- Organism inoculated in conjunctiva of  Non-pathogenic and occupational
rabbit hazard for meat, poultry & fish handlers
- (+) purulent conjunctivitis Laboratory diagnosis:
Tests Listeria Corynebacteria
 Culture: Tomato Juice Agar
Motility + -
Fermentation + - Lactobacillus casei
of salicin
 Shirota strain
 YAKULT
Erysipelothrix rhusiopathiae
Gardnerella vaginalis
Characteristic:
Characteristic:
 Gram (+) rod, non-motile
 Agent of erysipeloid  short, pleomorphic gram (+) bacillus or
- Cutaneous inflammation of hands and coccobacillus
fingers (seal finger or whale finger)  stains gram-variable or gram-negative
- Common in fish vendors and butchers  Associated with Bacterial vaginosis
- Butcher’s cut (foul smelling, grayish vaginal discharge)
 Previously known as Haemophilus
Laboratory diagnosis: vaginalis & Corynebacterium vaginalis
 H2S (+) - Amsel & Nugent scoring system: used
 Catalase (-) to diagnose BV
 Gelatin stab: Test tube brush or Bottle
brush growth
Laboratory diagnosis:
 Cytology/ Pap’s: Clue cells (EC studded
by bacteria) (direct microscopy exam)
 Whiff or Sniff test: vaginal discharge +
10% KOH
- (+) fishy amine-like odor
 Culture: Human Blood tween 80 agar
- Columbia CNA
Streptobacillus monoliformis
Characteristic:
 Agent of:
 Rat bite fever
- from animal bite/ scratch
 Haverhill fever
- ingestion of contaminated milk
Nocardia
Characteristic:
 Non–Spore-Forming, Branching
Aerobic Actinomycetes
 Fungus like-bacteria
 Gram (+), aerobic bacteria
 Partially acid fast
 N. asteroides
- Most common specie (N.cyriacigeorgica
and N. farcinica)
- Primary pulmonary infection resembling
tuberculosis
- no sulfur granules (masses of
filamentous organisms bound together
by calcium phosphate)
 N. barasiliensis
- actinomycotic mycetomas
- granulomatous chronic infection
- yellow / orange sulfur granules
 N. otitidiiscaviarum
Actinomadura
Characteristic:
 Non–Spore-Forming, Branching
Aerobic Actinomycetes
 Etiologic agent of mycetoma
- Similar to nocardia
 Most common: A. madurae
 cellobiose (-) and xylose (+)
- Differentiates from Nocardia spp.
Streptomyces
Characteristic:
 Non–Spore-Forming, Branching
Aerobic Actinomycetes
 saprophytes found as soil inhabitants
and resemble other aerobic
actinomycetes
- Similar withmorphology and the
diseases
- Actimycotic mycetoma
 Most common: Streptomyces
somaliensis
Rhodococcus equi
 formerly Corynebacterium equi
- RT infections in human
 salmon-pink pigment
 Gram stain showing characteristic
diphtheroid gram (+) bacilli with traces
of branching.
Tropheryma whipplei
Characteristic:
 Non–Spore-Forming, Branching Aerobic
Actinomycetes
 Agent of Whipples disease
 facultative intracellular pathogen first
identified by PCR from a duodenal
biopsy specimen in 1991
 gram (+) actinomycete, most closely
related to the genera Rothia,
 Rhodococcus, Arthrobacter, and
Dermatophilus
 human feces, saliva, and gastric
secretion
Microscopic examination:
 Specimen: endoscopic biopsy
specimens
 (+) PAS- histologic stain
Anaerobic Gram Positive Bacilli
Clostridium spp.
Characteristic:
 gram (+) bacilli
 Spore-forming anaerobic anaerobically
(differentiate from Bacillus spp.)
 Catalase (-) (differentiate from Bacillus
spp.)
 Habitat: human & animal
 Saccharolytic EXCEPT
- C. tetani- swarming
- C. septicum- swarming
3 types:
• Neurotoxic: C. tetani & C. botulinum
• Histotoxic: C. septicum & C. perfringens
• Enteric: C. difficile
Clostridium perfringens
 C. welchii & Bacillus aerogenes
 Chopped meat: (+) growth with gas
 Agent of Myonecrosis (gas gangrene)
 Food poisoning (Pork poisoning)
 Necrotic enteritis: Pig bell (Type C)
 Encapsulated, non-motile bacillus box
car
 Chopped meat: (+) growth with gas
Laboratory diagnosis:
BAP: double hemolysis
 Inner hemolysis: complete zone
hemolysis due to teta toxin
 Outer hemolysis: incomplete zone
hemolysis due to alpha toxin &
lecithinase
Reverse CAMP
Nagler Reaction (lecithinase test)
- Due to alpha, lecithinase C,
phospholipase C
- (+) ppt/opalescence around the
colonies o the side w/out anti-toxin
- (-) NO ppt/opalescence on the side w/
anti-toxin (Mc Clung or Neomycin Egg
yolk)
Clostridium botulinum
Characteristic:
 Known as Canned good bacillus
 Agent of food and wound botulism, as
well as infant botulism
Virulence factor: “pre-formed toxin”
- toxin labile: block the release of
acetylcholine (flaccid paralysis)
- Botulinum toxin: neurotoxin; most
potent/ powerful exotoxin
 Spores: Oval and subtermiinal
Infections:
 Wound botulism: spore in wound
 Infant botulism: grow in GUT & Honey
bee (Floppy baby syndrome)
- Sudden infant death syndrome (SIDS)
Laboratory diagnosis:
 Not cultured; Cytotoxin assay (serum);
Lipase (+)
Clostridium tetani Clostridium spp.

Characteristic:
Tests C. C. C. C.
 Known as Tuck head or Drumstick / perfring botulinu tetani difficil
Lollipop bacillus ens m e
 Agent of Tetanus (“lock jaw”) Motility - + + +
 Risus sardonicus: Devil’s grin Lecithinase + - - -
 Opisthotonus (arching of the back) Lipase - + - -
- Through direct inoculation in to the Lactose + - - -
wound Glucose + + - +
- Spore: Round and terminal
- Asaccharolytic Gram (+) anaerobic bacilli
Virulence factor Actinomyces spp.
 Tetanospasmin (neurotoxin)  Nonspore-forming
- Exotoxin associated with spastic - A. bovis: lumpy jaw
contractions/ lock jaw - A. israeli: draining sinus tract with
Laboratory diagnosis: sulfur granules

Bifidobacterium dentium- periodontal molar

 Based on clinical findings
tooth colony disease
Clostridium difficile
Eubacterium lentum- periodontal molar tooth
Characteristic: colony disease

 Agent of antibiotic-associated Propionebacterium acne- skin flora

pseudomembranous colitis
Gram (-) anaerobic bacilli
 Clindamycin
 Spore: Bacteroides fragilis
- Oval and subterminal
 Needs 20% bile (black colonies)
Culture:  GIT normal flora (foul odor)
- CCFA (Cycloserine-Cefoxitin Fructose Porphyromonas asaccharolytica
Agar)
 Black pigment
Ferments:  Red fluorescence on UVL
- Fructose: yellow colonies (horse  no bile needed for growth
manure) Prevotella melaninogenica
Direct detection of toxin in stool  Black pigment
- EIA  Red fluorescence on UVL
- Cytotoxin assay  Saccharolytic
 no bile needed for growth
Fusobacterium nucleatum  “Much granules”

 Breadcrumb colonies 3 Groups:

 Fusiform bacilli
a. M. tuberculosis
Fusobacterium necrophorum b. M. bovis
c. M. africanum
 Vincent’s angina
 M. tuberculosis complex: causing Tb
Bacteroides ureolyticus  M.O.T.T. (Mycobacteria other than
tuberculosis) or NTM (non-tuberculosis
 Pitting of the agar mycobacteria)
Gram (+) anaerobic cocci  M. leprae: Hansens disease

Peptostreptococcus anaerobius Mycobacterium tuberculosis

 SPS sensitive Characteristics:

 Indole (-)  Robert Koch in 1882
 catalase (-)  Koch bacillus
Peptostreptococcus asaccharolyticus  Obligate aerobe
 Require CO2 for growth (5-10%)
 catalase (-)
Virulence factor:
Peptococcus niger
- Cord factor & sulfatides: sticky
 Staphylococcus like response
Gram (-) anaerobic cocci Laboratory Diagnosis:
Veillonella parvula  Gram stain: qualify specimen (Bartlett’s
 Red fluorescence in UVL classification)
- > 10 EC with < 25 PMN: saliva
 Nitrate (+)
- < 10 EC with > 25 PMN: sputum
Megasphera
*EC: LPO PMN: HPO
Acidaaminococcus
Specimens:
Mycobacterium
 Sputum (3 consecutive days)
Characteristic:  Secretions obtained by bronchoscopy
 Blood
 Slender, slightly curved or straight, rod-
shaped organisms  Urine
 Non-motile, strict aerobes & non-spore  CSF: Pellicle of web-like clot
formers  Pleural, pericardial, peritoneal fluid:
 Slow growers EXCEPT: M. fortuitum & increased ADA (Adenosine Deaminase)
M. chelonei Laboratory Diagnosis: Sputum: contains mucin
 Cell wall: high lipid content & mycolic & organics debris
acid
 Resist decolorization: AFB
Methods for decontamination & Digestion  Spengler’s: for color blind individuals
(MTb: black)
Purpose of decontamination- remove normal
 Pappenheim’s: MTb- red; M.
flora & other contaminating organism
smegmatis: blue
Purpose of Digestion- break disulfide bond in  Baumgarten’s: MTb- blue; M. leprae:
mucus trap MTb red

 2-4% NaOH: digestant and AFB Grading:

decontaminating agent
CDC Method of AFB Reporting
 N-Acetyl-L-cysteine (NALC): liquefying
agent # of AFB seen Report
- Combination: NALC or dithiothreitol + 0 Negative
NaOH : most common 1-2/300 fields +/-, Repeated on 2nd
 Benzalkonium Chloride (Zephiran) slide
- digestant-decontamination 1-9/100 fields 1+
- Benzalkonium chloride + trisodium 1-9/10 fields 2+
phosphate (Z-TSP) 1-9/ field 3+
- Benzalkonium chloride- shortens >9/ field 4+
exposure time
- trisodium phosphate (Z-TSP)- liquefies AFB Grading: Philippines
sputum, requires longer time of
exposure National Tuberculosis Association Method
 5-6% Oxalic acid # of organism seen Report
- decontaminate specimens 1-2 per slide Report # & request
contaminated with Pseudomonas another specimen
aeruginosa 3-9 per slide Rare (1+)
10 or more per slide Few (2+)
Laboratory diagnosis:
1 or more per OIF Numerous (3+)
 Gram stain: gram ghost or neutral
 Acid Fast stains
AFB Grading: in the Philippines
- Carbolfuchsin (1° stain), acid-alcohol
(decolorizer) & methylene blue/ National Standard Reporting Scale (RITM)
malachite green (counter stain)
# of AFB seen Report
- Ziehl-Neelsen : heat
0 No AFB seen in 300
- Kinyoun stains: tergitol
visual fields
 Fite-Faraco’s: hematoxylin instead of +n 1-9 AFB/ 100 visual
methylene blue as counterstain fields
 Auramine-rhodamine fluorochrome 1+ 10-99 Afb/ 100 visual
stain (Truant’s) fields
- AFB smear: 2cm x 3cm 2+ 1-10 AFB/ OIF in at
- more sensitive than the carbolfuchsin least 50 visual fields
stains 3+ More than 10 AFB/
- bright, yellow-orange bacilli against a OIF in at least 20
dark background visual fields

Mycobacterium tuberculosis
Culture:
 Requires increased protein
A. Agar based
 Duboi’s Oleic Acid Albumin Medium
 Mitchison’s Medium
 Middlebrook 7H10-7H11: Anti-
susceptibility test
B. Egg-based: Inhibitor: Malachite green
 Petragnani medium: used or heavily
contaminated specimen (increased
conc. of MG)
 Lowenstein-Jensen medium
 American Thoracic Society
 Dorset Egg medium
C. Liquid Media
 Bactec 12B, Septi-Check, Middlebrook
7H9
- Use in automated machines
Culture
Colonies:
 Tan to buff colonies
 Dry, rough, warty & granular
 Resembles: Cauliflower
 Culture maintained for 8 weeks or
2months
Anti-tuberculosis Agents
 Primary drugs:
- Rifampicin, Isoniazid, Pyrazinamide,
Ethambutol, Streptomycin
 Secondary drugs:
- Ethionamide, Capreomycin,
Ciprofloxacin, Ofloxacin, Kanamycin,
Cyclosrine, Rifambutin
Mycobacterium spp.
1. Niacin Test
Principle:
 Niacin + Niacin Ribonucleotide + Aniline
Dye + Cyanogen Bromide
Result:
 (+) yellow : MTb
 (-) no color change: M. bovis
2. Catalase test at 68°C (heat stable)
 Medium: Tween 80 & Reagent: 30%
H2O2
Principle:
 Tween 80 + Mycobacteria + 30% H2O2+
heat at 68°C (20 mins)
Result:
 (+) 45mm height of gas bubbles
 (+) no bubbles M. kansasii (-) MTb
3. Nitrate Reduction Test
Principle:
 HCL + Sulfanilamide + n-
napthtylethylene diamine
Result:
 (+) Pink/red color (M. kansasii, M.
szulgai, M. fortuitum & M. tuberculosis)
 (-) no color change (M. intracellulare)
4. Tween 80 HOH
 (+) red: M. kansasii
 (-) no red/ amber: M. avium
5. Tellurite reduction test
 Tellurite: black metallic tellurium
 (+) smooth fine black precipitate (M.
avium)
 (-) gray clumps (M. kansasii)
 6. Aryl sulfatase test  (-) M. gordonae
 For rapid growers 11. Sodium Chloride Tolerance

Principle: Principle:

 Tripotassium phenolphthalein  High salt concentration (5% NaCl) in

disulfide/ sulfate acted upon by egg-based media inhibits the growth of
arylsulfatase to produce free most mycobacteria
phenolphthalein

Result:
Result:
 (+) Pink/red color (M. fortuitum-
 (+) growth (M. flavescens, M. triviale,
chelonei)
and most rapidly growing
7. TCH (Thiophene-2-Carboxylic Acid
Mycobacterium spp.)
Hydrazide) Susceptibility Test
 (S) M. bovis MOTT
 (R) MTb
Characteristics:
8. Iron uptake

Principle:  Rounyon’sClassification

Group I: Photochromogens
 convert ferric ammonium citrate to an
iron oxide  Light: pigmented
Result:  Dark: non-pigmented

1. M. kansasii: Yellow bacillus (consisting of

 (+) rusty brown colonies (rapid growers)
 (-) no color formation (M. chelonae)
9. Pyrazinamidase
Principle:
 Pyrazinamidase hydrolyzes
pyrazinamide= pyrazinoic acid &
ammonia in 4 days
beta carotene); nitrate (+)
2. M. marinum: Swimming pool granuloma
3. M. simiae: niacin (-)
4. M. asiaticum: niacin (-)
Group II: Scotochromogens

Result:  Light/dark: pigmented

 (+) red pigment (MTb & M. marinum) 1. M. scrofulaceum (scrofula): cervical lymph
adenitis (neck region); niacin (+); Nitrate (-)
 (-) no color formation (M. bovis & M.
kansasii) 2. M. szulgai:
10. Urease
3. M. xenopi
Principle:
4. M. gordonae: Tap water bacillus; Tween 80
 detection of urease activity (+)
Result: 5. M. flavescens
 (+) M. scrofulaceum 6. M. thermoresistable
Group III: Nonphotochromogens Treatment: Dapsone, Sulfone

 Non-pigmented Skin test: Lepromin test

1. M. avium-intracellulare complex: AIDS  Fernandez or Early Reaction

Battery bacillus - 24-48 hrs
 Mitsuda or Late Reaction
2. M. avium: Tb in birds & chicken
- 3-4 weeks
3. M. ulcerans: Innert bacillus; Buruli ulcers in
Mantous test
skin
- Skin test for MTb
4. M. xenopi: Hot, cold water taps at 42°C
- Tuberculin skin test
5. M. triviale - Intradermal injection “wheal”
- (+) >10mm erythema after 48-72 hours
6. M. heamophilum
Spirochetes
7. M. terrae: Radish bacillus
Borrelia spp.
8. M. malmoense
 Helically coiled bacteria transmitted
9. M. gastri: J bacillus
through arthropod vectors
Group IV: Rapid Growers - Lice & ticks
 Flexible twisted organisms resembling
1. M. fortuitum- grows on MAC w/out crystal stretched spiral
violet
Borrelia recurrentis
2. M. chelonei- grows on MAC w/out crystal
violet  Agent of Louse-borne relapsing fever
 Vector: Human louse (Pediculus
3. M. phlei: provide CO2
humanus)
4. M. smegmatis: confused with MTb in urine  High fever, muscle and bone pain and
confusion
Mycobacterium leprae
Borrelia hermsii/ Borrelia parkeri
Characteristics:
 Tick-borne relapsing fever
 Agent of Hansen’s disease or leprosy
 Vector: Ornithodoras ticks
 AFB
 Described as Cigarette packet / picket- Borrelia burgdorferi
fence
 Agent of Lyme disease
 Hydrolize 3,4-hydroxyl-phenylalanine
 Vector: Ixoda ticks & Deer ticks
(DOPA)
 3 stages:
 Tropism to peripheral nerves
- I: appearance of lesion; Erythema
Laboratory Diagnosis: chronicum mograns (bull’s eye rashes)
- II: dissemination through blood;
 Specimen: Earlobe or nasal scraping bones, CNS, heart & liver
 Culture: Armadillo pads - III: neurological abnormalities, arthritis
 Stain: Fite Faraco stain & skin lesion (chronic stage)
Borrelia spp.
 Laboratory diagnosis:
 Culture:
- Kelly’s medium
- Barbour Stoenner-Kelly’s (BSK)
 • Serological tests
- ELISA- detect antibody
- Westernblot: gold standard
- DNA: WB (western blot)
- RNA: NB (northern blot)
Treponema spp.
 Tightly twisted organism resembling
cork screw
Treponema pallidum subsp. Pallidum
 Agent of Venereal syphilis
 Great pox, Evil pox, French/Italian pox,
Spanish disease
 Transmitted by sexual contact, direct
transmission & transplacental route
(vertically)
 Stages:
- Primary syphilis: hard chancre (painless
& firm)
- Secondary syphilis: condylomata lata
(wart-like lesions)
- Latent syphilis: absence of clinical
symptoms (+ serologic tests)
- Tertiary syphilis: gummas
(granulomatous lesion), neurosyphilis
Congenital syphilis
Hutchinsonian Triad
- Notched teeth
- Keratitis
- eczema
Laboratory diagnosis:
 Direct microscopic examination using
Dark field microscope
 Serological tests
Treatment:
 Heavy metals
- Ex: Arsenic, Arsphenamine, Salvarsan
 Drug of choice
- Penicillin
• Jorisch-Herxheimer reaction
 Large quantities of toxins are released
as the bacterium dies during treatment
Treponema pallidum subsp. Pertenue
 Agent of Yaws (chronic nonvenereal
disease of skin and bones)
 Transmission: direct contact of
traumatized skin with infected lesion
Treponema pallidum subsp. Endecume
 Agent of Bejel (Non-vereal syphilis &
endemic syphilis) (lesion in oral cavity,
oral mucosa, skin, bones &
nasopharynx)
 Transmission: mouth to mouth by
utensils
Treponema carateum
 Agent of Pinta (ulcerative skin disease)
 Transmission: direct contact of
traumatized skin with infected lesion
Leptospira spp.
 Tightly twisted with one or both ends
bent into a hook
Leptospira biflexa
 Non-pathogenic
 Found in water & soil
Leptospira interrogans
 Agent of leptospirosis (human and
animals)
 Zoonosis
 - Parasitic in vertebrates other than
human
- Rodents, cattle, dogs, cats, raccoons &
bats
 Shed in urine of animals
 Transmission: direct contact with urine
of animals
 Infection may involve kidney, liver &
central nervous system
 Weil’s disease: severe form of
leptospirosis
Leptospira interrogans serologic variants
Principal leptospiral diseases:
 Icterohemorrhagiae: Weil’s disease
 Canicola: Infectious jaundice
 Autumnalis: Fo’rt Bragg of Pretibial
fever
 Grippotyphosa: Marsch fever
 Hebdomadis: 7-days fever
 mitis/Pomona: Swine-herd’s disease
Culture
 Specimen: Blood (early infection);
Urine (2nd week)
 Media:
- Elllinghausen-McCullough-Johnson-
Harris
- Fletcher’s & Stuart’s (6-8 weeks)
Chlamydia, Mycoplasma & Ricketssia
Chlamydia spp.
 Formerly Bedsonia (“large virus”)
 Obligate intracellular
 Gram (-) like CW; binary fission
 Infectious particle Elementary bodies
(diagnostic feature / marker)
Chlamydia psittaci
 Agent of Psiittacosis / ornithosis
(disease of birds, parrots, parakeets &
cockatoos)
 Transmission: inhalation of
contaminated aerosols or fomites
Chlamydia pneumoniae
 Associated with mild respiratory tract
infections
 TWAR (Taiwan Acute Respiratory)
strain
Chlamydia trachomatis
Subtypes:
 A, B, Ba, C: Endemic trachoma
(multiple/ persistent infections that
leads to blindness)
- Inclusion conjunctivitis
 D-K: Urethritis, cervicitis, pelvic
inflammatory disease, epididymitis,
infant pneumonia
 L1, L2, L3: Lymphogranuloma
venereum
Laboratory diagnosis:
 Culture: McCoy’s cell
 FREI’s test: delayed hypersensitivity
skin test for LGV
Mycoplasma spp.
 Smallest free-living organisms (gram -)
 Found in plants & animals
 Formerly known as pleuropneumonia-
like organisms (PPLOs)
 Causing pleuropneumonia in cattles
Mycoplasma pneumoniae
 Eaton’s agent
 Cause of community-acquired
pneumonia & tracheobronchitis in
children and young adults
 Primary atypical pneumonia/ Walking
pneumonia
Mycoplasma hominis & Ureaplasma
urealyticum

 Genital mycoplasmas
 Colonize adults asymptomatically
 Cause of nongonococcal urethritis in
males
 M. hominis
- Agent of salpingitis (inflammation in
fallopian tubes) & postpartal fever in
females

Laboratory diagnosis:

 Culture: Shepard’s/ A7B/ Ɛ-Agar

- “fried egg” colonies
 Serologic tests
- Cold agglutinin (Anti-I)

Ricketssiae

 Genera: Rickettsia, Ehlichia, Coxiella &

Rochalimea
 Gram (-) obligate intracellular bacteria
 Vector transmitted (lice, fleas & ticks)
 Coxiella: cannot survive outside animal
host or insect vector EXCEPT C. burnetti

Afipia felis

 Associated with cat-scratch disease

(CSD)
 Despite its rare isolation, indirect
evidence suggests that the organism
may be more commonly linked to CSD
than is currently appreciated
 Due to lack of appropriate laboratory
methods for detection
Ricketssiae
GROUP Specie Transmission Infection
Spotted R. rickettsii Ticks Rocky Mountain
fever Spotted Fever
R. conorii Ticks Boutonneuse fever

Mediterranean &
Israeli Spotted

Fever

Indian tick typhus

Kenya tick typhus

Spotted R. australis Ticks Australian/
fever Queensland tick
typhus
R. akari Mites Rickettsialpox
Typhus R. prowazekii Lice Epidemic typhus
Flying
squirrels Sporadic typhus

Brill-Zinsser disease
(latent)
R. Typhi Fleas Murine typhus/
Endemic typhus
Scrub typhus R. Mites, Scrub typhus
tsutsugamushi chiggers
O.
tsutsugamushi
Q fever C. Burnetti Ticks Q fever
Ehrlichiosis E. chaffeensis Ticks Human monocyte
ehrlichiosis
E. Ticks Human granulocyte
phagocytophila ehrlichiosis
E. owingii
Neorickettsia Ticks Sennetsu fever
sennetsu
Rochalimeae R. quintana Lice Trench fever
Bartonella spp

Organism Habitat MOT Infection

B. quintana Small rodents Human Trench fever
body
Human louse
B. Human Sand flies Carrion’s
bacilliformis disease

Oroya fever
B. henselae Domesticated Bites or Cat-scratch
cats scratch disease
from
domestic Peliosis
cats hepatitis

Cat fleas
B. Domestic Bites or Bacteremia
clarridgeiae cats scratch
from Cat-scratch
domestic disease
cats
B. Rats Fleas Endocarditis
elizabethae
 CLINICAL BACTERIOLOGY area is easy to move around in to prevent
different accidents
WEEK 2: LABORATORY SAFETY
Aside from Safety hazard we also have to be
SAFETY familiarize about different Safety
Precautions
A laboratory personnel must  Do not eat,drink , and smoke inside the
learn to know: laboratory.
• what hazards exist inside the laboratory  While working in the laboratory make sure
• the basic safety precautions that you tie your hair. (especially for girls)
• how to apply the basic rules  Observe proper disposal.
of common sense
How to apply the basic rules of common
sense

 As a medical technologist/microbiologist
we should be properly trained and equip
with proper protective equipment (PPE).

CHEMICAL SAFETY

 Hazard Communication Standard

- ensure that all laboratory personnel have a
thorough working knowledge of the hazards of
the chemicals with which they work.
“ Employee right to know
- These hazard communication standard
mandate or ensures that all hazardous chemicals
Biological Hazard- Are exposure to infectious in the laboratory must have a label. We use the
agents. NFPA label or The National Fire Protection
Sharps hazard- We collect blood ang Association Label.
ginagamit sa pang collect ng blood ay
needles,syringe,lancets,and glass wares.
Chemical Hazards - Exposure to different
chemicals,preservatives, and reagents inside the
laboratory. And take note that *every
chemicals in the laboratory are different.
There are chemicals that are poisonous,
corrosive, flammable and carcinogenic.
Radioactive Hazards- Exposure to a
radioactive isotopes.
Electrical Hazards- Nowadays in the
laboratory we use high tech machines and
equipment and some of the laboratories are fully
automated.
Fire/Explosive Hazard- Open flames, organic
chemicals.We are exposed because we are using
an alcohol lamp.We use this for sterilization.
Physical Hazard- Wet floors, heavy boxes, and
patients.We make certain that the laboratory
Chemical Hygiene Plan-
includes guidelines on:
 proper labeling of chemical containers
(NFPA Label)
 manufacturers’ material safety data sheets
(MSDSs)
- Information regarding chemicals
(Hazards,proper handling and disposal)
 written chemical safety training and
retraining programs
 Work with toxic or noxious chemicals
should always be performed while wearing
nitrile
gloves, in a fume hood or while wearing a fume
mask.
 Fume hoods - provided in the laboratory to
prevent inhalation of toxic fumes.
- dito nag didispense ng mga chemicals so that
we don’t inhale the toxic fumes. They filter out
the chemical odor.
 Spills should be cleaned up using a fume
mask, gloves, impervious (impenetrable to
moisture) apron, and goggles.
FIRE SAFETY
 Every employee should know the location
of fire alarm and fire exit and they should
know how to operate fire extinguisher.
 Fire is a type of accident so we do not know
when it will happen.
When a fire is discovered, all employees are
expected to take the actions in the acronym
RACE:
 Rescue —rescue anyone in immediate
danger
 Alarm—activate the institutional fire
alarm system
 Contain—close all doors and windows to
potentially affected areas so that the fire ay
hindi mabilis na kakalat.
 Extinguish/Evacuate —attempt to
extinguish the fire, if possible or evacuate,
closing the door.
How to use a Fire Extinguisher?
 P- Pull the pin in the handle
 A - Aim the nozzle at the base of the fire
 S - Squeeze the lever slowly
 S - Sweep from side to side
ELECTRICAL SAFETY
 Electrical cords should be checked
regularly for fraying and replaced when
necessary.
 All plugs should be the three-prong,
grounded type.
 All sockets should be checked for
electrical grounding and leakage at least
annually.
 No extension cords should be used in the
laboratory.
BIOSAFETY
• Understanding how microorganisms are
transmitted (chain of infection) is essential to
preventing infection.
CHAIN OF INFECTION
 INFECTIOUS AGENTS- consist of
bacteria, fungi, parasites, and viruses
 RESERVOIR- location of potentially
harmful microorganisms, the place where
the infectious agent can live and possible
multiply.
Categorize into three:
1. Human resorvoir - Inside our body
2. Animal reservoir -
3. Environment -
 PORTAL OF EXIT- way to exit the
reservoir to continue the chain of infection.
Through the nose,mouth,mucus membrane.
 MODE OF TRANSMISSION- It
transmitted when coughing,sneezing, you
have a physical contact to the infected
person.
- Direct Contact - Have physical contact.
Person to person.Through kissing,sexual
intercourse,shaking hands
- Indirect Contact - Through Droplets,
airborne, vector borne (mosquitoes),vehicle
borne (Food or inanimate objects).
 PORTAL OF ENTRY- same as the portal
of exit, which includes the mucous
membranes of the nose, mouth, and eyes,
breaks in the skin, and open wounds.
 SUSCEPTIBLE HOST- can be another
patient during invasive procedures (because
of having weak immune system), visitors,
and healthcare personnel when exposed to
infectious specimens or needlestick injuries.
- Newborns - Their body are not fully develop.
- Immuno- compromise person
The goal of a healthcare professionals is to
break the infections. Precautions:
1. Proper Disinfection- Before or after of
working we have to disinfect the working
area. (Using bleach for disinfection but
must be diluted. 1 : 10 ratio)
- Hand Hygiene
2. Proper Disposal
3. Safety Precautions - Wearing of PPE
4. Vaccine/Immunization
5. Isolation and Quarantine - If you are
exposed, you have to be quarantined. And
if you know that you have been tested
positive, you now have to isolate yourself.
6. Following the Healthy lifestyle
 Proper hand hygiene, correct disposal of
contaminated materials, and wearing
personal protective equipment (PPE) are
of major importance in the laboratory
- Infectious waste with contact to the
infectious agent for example, cotton with
alcohol, gloves, urine container must be dispose
to infectious waste.
HANDWASHING
1. Stand in front of the sink. Do not lean on the
sink with clothes.
2. Use paper towel to cover the water control
and turn on the water.
3. Wet hands thoroughly. Allow the water to
flow from arms to fingertips.
4. Apply soap to hands.
5. Wash the palm, back, and wrist of each hand
using strong, frictional, circular
movements.
6. Interlace fingers and thumbs and move hands
back and forth for ten seconds.
7. Rub nails against the palm
8. Rinse hands thoroughly
9. Dry hands well
10. Use paper towel to turn the water off
When do we perform handwashing?
 Before and after working, contact with
patients, putting PPE, going to bathroom,
and anytime when you know that you have a
close contact to contaminated areas.
-Take note that we should remove any kind
of accessories or jewelries when performing
hand washing.
-When removing the PPE, we should first
remove the gloves because they are the most
contaminated.
- The inside of the Gloves are sterile, while
the outside are contaminated.
- When removing our lab gown, sa butones lang
dapat humawak.Then we only have to touch the
inside of the lab gown when removing it.
DISPOSAL OF HAZARDOUS WASTE
 All materials contaminated with potentially
infectious agents must be decontaminated
before disposal.
1. Biohazard containers (yellow)
2. Sharps containers (red)
3. Discard shelves, carts, bins, etc.-
contaminated culture tubes and glassware
used to store media and other glassware
 should be placed in these areas for
decontamination and washing.
4. Trash cans—any non contaminated
materials, paper, or trash should be
discarded in these containers. Under no
circumstances should laboratory waste be  When the air comes inside the
disposed of in trash cans. cabinet, it filters it so that the air
that flows inside the cabinet is
BIOLOGICAL SAFETY CABINET sterile. And the air that comes out
is sterile also.
 a device that encloses a workspace in such  Class III cabinets- Air coming
a way as to protect workers from aerosol into and going out of the cabinet is
exposure to infectious disease agents. filter sterilized, and the infectious
 Used when dealing infectious agent. material within is handled with
 Class I cabinets- allow room rubber gloves that are attached and
(unsterilized) air to pass into the sealed to the cabinet.
cabinet and around the area and
material within, sterilizing only the air
to be exhausted
 They have negative pressure and
may be ventilated to the outside or
exhausted to the work area and are
usually operated with an open
front.

- The difference between a class II

cabinet and a class III cabinet is that
the class III cabinet is closed and it has
readily available gloves.
- It usually used in infectious agent that
life threatening.

 Class II cabinets- sterilize air that flows

over the infectious material, as well as
air to be exhausted.
 The air flows in “sheets,” which
serve as barriers to particles from
outside the cabinet and direct the
flow of contaminated air into the
filters.
 called vertical laminar flow BSCs

WEEK 4: BACTERIOLOGY LAB
THE COMPOUND MICROSCOPE
 An optical instrument that is used to
observe tiny objects, often objects that
cannot be seen at all with the unaided
human eye (the “naked eye”)
 Anton van Leeunwenhoek the first
person to see live bacteria and protozoa
“animalcules”
o “Father of Microbiology”
o “Father of Bacteriology”
o “Father of Protozoology”
1. Bright field microscope - Used
to observe morphology of
microorganisms such as
bacteria, protozoa, fungi, and
algae in living (unstained) and
nonliving (stained) state.
a. Common microscope
2. Dark field Microscope
 Unstained organisms are observed
against a dark background
 Useful for examining thin
spirochetes.
 Kapag darkfield Microscope ang
nakikita sa background ay dark.But
the organism being focused is clear
and light.
 Is used for the observation of
unstained organism
3. Phase- contrast
 Microscope can be used to observe
unstained living microorganisms.
 Mas mataas ang contrast compare to
darkfield.
4. Fluorescence Microscope
 Fluorescent dye attached to organism
 Primarily an immunodiagnostic
technique (immunofluorescence)
 Used to detect microbes in cells,
tissues, and clinical specimens
 Dark Background when we attached
to an organism it fluoresce. It can
turn in any different colors depends
to what color of dye we used.
 MECHANICAL SYSTEM– Base,
Arm, Stage, Sub stage, Mechanical
Stage.
 LENS SYSTEM – Nosepiece,
Objectives, Eyepiece, Focal Length
o To magnify the focus
  DIOPTER ADJUSTMENT-
 THE REVOLVING NOSEPIECE-
Where the objective lenses attached
 OBJECTIVE LENSES- We have four
objective lenses:
o SCANNER (red)- It has 4x
magnification
o LOW POWER OBJECTIVE
(yellow)- It has 10x
magnification
o HIGH POWER
OBJECTIVE(blue)- It has 40x
magnification
o OIL IMMERSION OBJECTIVE
(white) - It has 100x
magnification.
 We use oil, we use
cedarwood oil
 TOTAL MAGNIFICATION=
(eyepiece x objective lenses)
Ex.
Scanner = (10 x 4= 40x)
LPO = (10 x 10=100x)
HPO (10 x 40= 400x)
OIO (10 x 100 = 1000x)

 STAGE- Holds the specimen in place

 OTHER parts - On/OFF switch, o In the stage there is a hole and it
Fine/course adjustment Knob, Iris is called the Aperture, it pulse the
Diaphragm, condenser, Light source light coming from illumination.

 EYE PIECE/OCULAR LENS- This is  CONDESER- It focuses the light.

where to view the organisms in focus. o In the condenser it has Iris
o Has a magnification, can magnify diaphragm, it adjust the amount
the organisms. of light that pass through
o 10x magnification microscope.
  RHEOSTATS/ Brightness adjustment-
 FINE ADJUSTMENT KNOB – Used to
sharpen the image being in focus.
 COURSE ADJUSTMENT KNOB- To
move the stage up and down.
 STAGE CONTROLS – To move the
stage side by side and Up and Down.
1.Turn on the microscope and adjust light
intensity using rheostat. If you are going to
focus on the microscope you have to focus first
the SCANNER followed by the LTO, HPO, and
then OIO.
2. Switch to 10x objective, place the specimen
on stage and focus using coarse adjustment knob
3. Adjust the condenser and iris diaphragm until
desired light focus and area is acquired
4. Switch to other objectives, make sure that the
specimen is focused before moving to another
objectives.
5. If using oil immersion objective, make sure to
apply oil needed for the said objective before
moving.
 Keep the area clean and organized,
make sure that the cord will not cause
any harm to others
 Keep lenses clean by using lens paper
after each use
 Turn off the microscope after use and
do not keep the light on all day
 Return the objective lenses on the
lowest magnification and set the stage
at its lowest part after use
 Always use the revolving nosepiece
in changing objective lenses
 Hold the microscope by using two
hands; one on the arm while other on
the base
1. Carry microscope with two hands,
supporting the base with one hand.
2. Always hold the microscope in
vertical position.
3. Only clean optical surfaces with good
quality lens tissue and commercial
lens cleaner.
4. Do not use the 10x and 40x
objectives with oil.
5. Clean the oil immersion after use
6. Always remove slides with the low-
power objective raised.
7. Store the microscope with the scanner
objective in position and the stage
centered.
 A microscope should be left in a
permanent position on a study
laboratory table in an area where it
will not get jammed.
 If the microscope must be moved, it
should be held securely with one
hand supporting the base and the
other hand holding the arm.
 The microscope should be placed
gently on tabletops, to avoid jarring

 When the microscope is not being

used, it should be left with the low
power objective in position
 The stage should be centered.
 The microscope should be stored in a
plastic dust cover.
 Use the coarse adjustment only with
the low power objective
 Use oil each time the oil immersion
lens is used
 Use oil immersion oil with the oil
immersion objective only
 Clean all oculars and objectives with
lens paper after each use
 Move or transport the microscope
with one hand under the base and the
other hand gripping the arm. Avoid
jarring or bumping the
microscope
 Store the microscope covered in a
protected area.

 Specimen collection and transportation
o Collect the specimen from the
patient
o Ang goal is to collect the
specimen, to maintain the
viability of the organisms with
minimal contamination.
o Kaylangan maingat and practice
the aseptic technique.
 Direct examination of specimen-
microscopic
o Once na collect na gagawa na ng
smear and check it under the
microscope and with that we see
what is the cause of the
infection.
 Selection and inoculation of media -
culture media
o Culture media- artificial
preparation in the laboratory
 It contains nutrients so
that the bacteria will
grow and survive.
 Evaluation of bacterial growth
o Macroscopic in which we are
going to observe the PHYSICAL
appearance of the COLONY (the
growth of microorganisms).
 Biochemical testing
 Antibiotic susceptibility testing
o We use different kinds of
antibiotic to determine if the
antibiotic is effective for the
treatment.
 Collection of specimen: during acute
phase of the infection and before
antibiotics are administered
o Acute Phase meaning during the
early stage of infection
 Selection of correct anatomic site
o Correct anatomic site is where
the site of infection
 Proper techniques and supplies (with
minimal contamination from normal
biota)
o Normal biota/normal flora
means a bacteria that are
already present in our body that
do not cause harm to their host.
It can cause harm if the number
of it is increase or decrease in
the body of the host.
 Quantity
o Ex. Collect urine at least 50 ml
 Container or transport medium
o We have to use sterile container
o Transport medium is used to
maintain the viability of the
specimen
o *Specimen must be transported
within 30 mins up to 2 hrs
within collection.
 Labeling – Write appropriate label
 Specimen transport

 Collect in sterile container to prevent
any contamination – except STOOL
o Ang kailangang I- isolate sa stool
are the one who is pathogenic.
 Swabs are not recommended –
o It doesn’t provide efficient
quantity and swab are easily
contaminated and easily dry out.
o We only recommended it when
the specimen is coming from
UPPER RESPIRATORYN TRACT,
EXTERNAL EAR, EYES and
GENITAL AREA.
o We do not use cotton tip swabs
– they have excessive fatty acid
and it should be toxic for the
bacteria.
o Dacron, Rayon, Calcium Alginate
are recommended swab.
 For lesions, wounds, abscesses - area
should be cleansed to eliminate the
number of normal flora.
Patient-Collected Specimens
 Urine
 clean-catch midstream
urine specimen
 Midstream clean catch is
we avoid to collect the
first portion of the urine,
we collect the middle
portion of it also the last
portion are not included.
 Because the first portion
of the urine has
contaminants of genital
area and epithelial cells.
 Preferred specimen: First
morning urine
 Because it provides more
concentrated sample
 SPUTUM
 First early
morning specimen is
preferred and we
collect it on a deep
cough collection.
 Why Deep Cough?
To ensure that we
collect the phlegm not
the saliva.
 For the patient who have
dentures it should be removed
first.
 Rinse mouth with water and
expectorate with the aid of a
deep cough directly into a sterile
container (expectorated
sputum).
 Remove the dentures first.
 Single specimen: for detection of
bacterial lower respiratory tract
infection.
  For fungal or mycobacterial  Urine, Sputum, etc., Should
infections: three separate early maintain in a refrigerated
morning specimens temperature = 4’ Celsius

 STOOL  Fetal specimens, CSF should be

 We used stool to detect the in room temperature =
gastrointestinal pathogens. 35’Celsius
 Rectal swab: can be submitted
 PRESERVATIVES
for bacterial culture as long as
fecal material is visible on the  BORIC ACID - to maintain
swab. accurate urine colony counts
 Bacterial infection: three
 REFRIGERATION – for stool
specimens should be collected—
samples
one per day for 3 days.
 Clostridium difficile toxin
assay
 Name  For delays longer than 2
 Identification number hours: Cary-Blair
 Room number transport medium
 Physician
 ANTICOAGULANTS
 Culture site
 Date of collection  used to prevent clotting of
 Time of collection specimens

 Sodium polyanethol sulfonate

(SPS)- most common
anticoagulant
 SPECIMEN STORAGE  Concentration: should
not exceeded 0.025%
(w/vol)

 Heparin- for viral cultures and

for Mycobacterium spp.

 Citrate and
ethylenediaminetetraacetic acid
(EDTA) should not be used
  HOLDING OR TRANSPORT MEDIA
 Maintain the
Viability of the
specimen
 Stuart or Amie
transport medium-
is commonly used.
 Charcoal is added to these media
to absorb fatty acids present in
the specimen that could kill
fastidious (fragile) organisms
such as Neisseria gonorrheae &
Bordetella pertussis
 SPECIMEN PRIORITY
Levels of Prioritization
 Level 1- Critical/Invasive
o This specimen represents life
threatening illnesses coming
from an invasive source
o Mga specimen na mahirap i-
collect.
 Level 2 – Unpreserved
o Madaling madegrade
 Level 3 – Quantitation required
o Urine colony count
 Level 4 – Preserved
o Usually hold D or transport
media
 The requisition does not match the
information on the specimen label
 No patient identification on specimen
container.
 Not in appropriate transport container
or the container is leaking.
 The quantity of the specimen is
inadequate.
 Transport time: more than 2 hours and
the specimen has not been preserved.
 Received in a fixative such as formalin
except for stool.
 The specimen is dried up
 One swab was submitted with multiple
requests for various organisms.
 Gram stain of expectorated sputum
reveals fewer than 25 white blood cells
(WBCs) and more than 10 epithelial cells
per low-power field and mixed bacterial
flora.
  We observed it if it is bacteria or
fungi? , is it cocci or bacilli?
 Purposes:
 It can be used to determine the
quality of the specimen
 It can give the microbiology
technologist and the physician
an indication of the infectious
process involved.
 The routine culture workup can
be guided by the results of the
smear.
 It can dictate the need for
nonroutine or additional testing.

Primary Inoculation

Types of Culture Media

 Nonselective media/ general culture

media
 Support the growth of most non
fastidious microbes.
 Non fastidious microbes
are bacteria that are easy
to grow.
 EX. Sheep blood Agar
 Selective media
 Gross appearance of the specimen  Support the growth of one type
 Swab or aspirate or group of microbes but not
 Stool consistency (formed or another.
liquid)  Inhibitors – it can be
 Blood or mucus present antimicrobials, dyes, alcohol
 Volume of specimen  We have a culture media that
 Fluid—clear or cloudy are selective for gram positive
meaning lahat ng maggrow doon
ay gram positive lang and also
vice versa.
  EX. MacConkey Agar- is only
selective for enteric gram
negative bacilli (possible that all
organisms grow in there are all
enteric gram negative bacilli)
 Differential media
 Contains indicator
 Allow grouping of microbes
based on different
characteristics demonstrated on
the medium.
 Ex. MacConkey agar are not only
a selective media but also
differential media because
MacConkey agar has lactose
 also it considered
differential because it can
differentiate if it has
lactose fermenter or
non-lactose fermenter
 We can differentiate
lactose fermenter if the
color is dark pink
colonies
 If not lactose fermenter
it will be colorless
 Enriched media
 Contain growth enhancers that
are added to nonselective agar
to allow fastidious organisms to
flourish.
 EX. Chocolate agar- Considered
enriched media because we add
blood on it.
 Enrichment broth
 Is a liquid medium designed to
encourage the growth of small
numbers of a particular organism
while suppressing other flora
present?
 Broth media
 Can be used as a supplement to
agar plates to detect small
numbers of most aerobes,
anaerobes, and microaerophiles.
 Routine Primary Plating Media
 Nonselective agar plate.
 Enriched medium- for fastidious
organisms for normally sterile
body fluids or a site in which
fastidious organisms are
expected.
 Selective and differential
medium- for enteric gram-
negative bacilli for most routine
bacterial cultures.
 Selective medium- for gram-
positive organisms for specimens
in which mixed gram-positive
and gram-negative bacteria are
found.
 Additional selective media or
enrichment broths- for specific
pathogens as needed.
 We used this as
confirmatory
 Broth medium- may be used as
a supplement with specimens
 from sterile body fluids, tissues,
lesions, wounds, and abscesses
 swab, tissue, or fluid
 Sterile body fluids, pus, urine,
and sputum- inoculated directly
onto selected media.
 Large volumes of sterile body
fluids (peritoneal, pleural,
continuous ambulatory
peritoneal dialysis) are
concentrated to increase the
recovery of bacteria.
 If the volume of fluid is greater
than 1 mL, the specimen can be
centrifuged for 20 minutes at
3000 × g.
 Two swabs: one is used for the
culture media, and the other is
used to make the direct smear.
 The specimen is applied by rolling the
swab or placing a drop of liquid
specimen onto a small area at the edge
of the plate.
 The inoculating loop is sterilized and
allowed to cool thoroughly before
streaking the agar.
 Growth in the first quadrant can be
graded as 1+, or light growth; growth in
the second or third quadrant can be
graded as 2+ to 3+, or moderate growth;
and growth in the third or fourth
quadrant can be graded as 4+, or heavy
growth.
 1st quadrant are organism na makapal
ang tubo
 3rd and 4th are medyo light
 Most bacteria cultures are incubated at
35° C to 37° C.
 Aerobes: grow in ambient air
o They need oxygen
 Anaerobes: cannot grow in the
presence of oxygen and require an
anaerobic atmosphere.
 Capnophiles: require an increased
concentration of CO2
 Microaerophiles: grow with reduced
oxygen and increased CO2
 Most routine bacterial cultures are held
for 48 to 72 hours.
 Anaerobes and broth cultures may be
held for 5 to 7 days
WEEK 5: BACTERIOLOGY LAB
ASEPTIC TECHNIQUE
 No contaminating organisms will be
introduce to the culture materials.
Work Area Disinfection
 The work area is
first treated with a
disinfectant to kill any
microorganisms that
may be present.
Loops and Needles
 A loop or needle
is sterilized by inserting it into a
Bunsen burner or incinerator flame
until it is red-hot.
o We can use the alcohol lamp/
candle to disinfect the
inoculating loop after the
sterilization, Icool down ang
loop before it touch the
microorganism.
 This will incinerate any
contaminating organisms that may
be present.
 Allow the loop to cool completely
before picking up inoculum. This will
 Ensure that viable cells are
transferred.
 Hold the inoculating loop at least
60° angle and use your dominant
hand.
 If the inoculating loop turns red hot
that means it is sterilized.
 Inside the alcohol lamp we use
denatured alcohol
Culture Tube Flaming and
Inoculation
 Prior to inserting a cooled loop or
needle into a culture tube, the cap is
removed and the mouth of the tube
may be flamed.
 If the tube is a broth tube, the loop is
inserted into the tube and twisted
several times to ensure that the
organisms on the loop are delivered
to the liquid.
 If the tube is an agar slant, the
surface of the slant is inoculated by
drawing the loop up the surface of
the slant from the bottom of the
slant to its top.
 For stab cultures, a needle is inserted
into the agar medium by stabbing it
into the agar.
 Sterilization of Culture tube. Upon
opening of culture tube we have to
past through flame the mouth of
the culture tube.
 How to remove the cotton plug?
Yung culture tube ang
ipanghahawak is and non-dominant
 hand and then ang ipangaalis po
natin ng cotton plug is ung Pinky
finger of the dominant hand.
 Once we open the test tube, we put
it through flames at the mouth of it.
Petri Plate Inoculations
 The plate cover is raised and held
diagonally over the plate to protect
the surface from any contamination
in the air.
 The loop containing the inoculum is
then streaked gently over the
surface of the agar.
 It is important not to gouge or
disturb the surface of the agar with
the loop.
 The cover is replaced and the loop is
flamed.
 We hold the plate using non
dominant hand and open the petri
dish using one hand
 The petri dish should be inverted
when doing the inoculation
 The petri dish should not fully open.
 We only allowed to touch the surface
of the bacteria.
Final Flaming of the Loop or Needle
 After the inoculation is complete,
the loop or needle is flamed to
destroy any organisms that remain
on these implements.
 The loop or needle is then returned
to its receptacle for storage.
 It should never be placed on the desk
surface
Final Disinfection of the Work Area
 When all work for the day is
complete, the work area is treated
with disinfectant to insure that any
organism that might have been
deposited during any of the
procedures is killed.
 Before and after the procedure we
have to sterilize all the materials and
the working area.
Transfer from Broth Culture to
another Broth
 Materials
1. broth culture of Escherichia
coli
 2. tubes of sterile nutrient broth
3. inoculating loop
4. Bunsen burner or incinerator
5. disinfectant for desktop and
paper towels
6. marking pen
 PROCEDURE:
1. Prepare your desktop by
swabbing down its surface
with a disinfectant. Use a
sponge or paper towels.
2. With a marking pen, label a
tube of sterile nutrient broth
with your initials and E. coli.
3. Sterilize your inoculating loop
by flaming it until it becomes
bright red. The entire wire
must be heated.
4. Using your free hand, gently
shake the tube to disperse the
culture
5. Grasp the tube cap with the
little finger of your hand
holding the inoculating loop
and remove it from the tube.
Flame the mouth of the tube.
Note: if an incinerator is used,
the tube is not flamed.
6. Insert the inoculating loop
into the culture
7. Remove the loop containing
the culture, flame the mouth
of the tube again, and recap
the tube. Place the culture
tube back on the test-tube
rack.
8. Grasp a tube of sterile
nutrient broth with your free
hand, carefully remove the
cap with your little finger, and
flame the mouth of this tube.
9. Without flaming the loop,
insert it into the sterile broth,
inoculating it. To disperse the
organisms into the medium,
move the loop back and forth
in the tube.
10. Remove the loop from the
tube and flame the mouth.
Replace the cap on the
 11. Sterilize the loop by flaming
it. Return the loop to its
container.
12. Incubate the culture you just
inoculated at 37C for 24–48
hours
into the tube. Pick up some of
the culture on the loop and
remove the loop from the
tube.

Transfer of Bacteria from a Slant

 Materials
1. agar slant culture of E. coli
2. sterile nutrient agar slant
3. inoculating loop
4. Bunsen burner or incinerator
5. marking pen
 PROCEDURE:
1. If you have not already done
so, prepare your desktop by
swabbing down its surface
with a disinfectant.
2. With a marking pen, label a
tube of nutrient agar slant
with your initials and E. coli.
3. Sterilize your inoculating loop
by holding it over the flame of
a Bunsen burner until it
becomes bright red. The
entire wire must be heated.
Allow the loop to cool
completely.
4. Using your free hand, pick up
the slant culture of E. coli and
remove the cap using the little
finger of the hand that is
holding the loop.
5. Flame the mouth of the tube
and insert the cooled loop
WORKING WITH AGAR PLATES o Inoculating loops or needles
must be flamed in the same
manner that you used when
working with previous tubes.
One difference when working
with plates is that plates are
never flamed!
 Plate Labeling and Incubation
o Petri plates containing
inoculated media are labeled
on the bottom of the plate.
o Inoculated plates are almost
always incubated upside
down. This prevents moisture
from condensing on the agar
surface and spreading the
inoculated organisms
 PROCEDURE
1. If you have not done so, swab
your work area with
disinfectant. Allow area to
dry.
2. Label a sterile nutrient agar
slant with your name and
organism to be transferred.
3. Flame an inoculating loop
until it is red-hot. Allow the
loop to cool.
4. Raise the lid of a petri plate
sufficiently to access a colony
with your sterile loop. Do not
from gouge the agar with your loop
a plate culture, the cover as you pick up organisms.
should be only partially Simply allow the loop to
opened. gently glide over the gelatin-
 Flaming Procedures like surface of the agar. Do
not completely remove the lid
 while inoculating or removing  The bacteria are evenly
organisms from the agar spread out on the slide in
plate. This will expose the such a concentration that
agar surface to air and they are adequately
potential contamination. separated from one another
Always close the lid once you  The bacteria are not washed
have removed organisms off the slide during staining,
from the plate.  Bacterial form is not
5. With your free hand, pick up distorted.
the sterile nutrient agar slant  Good smears are critical for
tube. Remove the cap by discerning:
grasping the cap with the  (1) the morphology of cells- cocci,
little finger of the hand that is bacilli
holding the loop  (2) The arrangement of cells- single,
6. Flame the mouth of the tube in pairs, clusters, chains, tetrads, etc.
and insert the loop into the  (3) internal structures- cell
tube to inoculate the surface inclusions, endospores
of the slant, using a
serpentine motion. Avoid
disrupting the agar surface
with the loop.
7. Remove the loop from the
tube and flame the mouth of
the tube. Replace the cap on
the tube.
8. Flame the loop and place it in
its container.
9. Incubate the nutrient agar
slant at 37C for 24– 48 hours

SMEAR PREPARATION
BACTERIAL SMEAR
 A dried preparation of bacterial cells
on a glass slide.
 Properly processed smear:
 Pwede tayong kumuha ng bacteria
coming from the tube and petri dish
 The size of the smear must be
thumb size or 1 inch x ½ inch
 In spreading the culture tube we
must spread it evenly starting from
inside to outside and wait it to dry.
 Once na magdry na we have to heat
fix it (pass through flame the slide)
 Why we perform heat fixing?
 To adhere the bacteria onto
the slides.
 And to prevent washing of
during staining.
 In order to kill the bacteria
 For petri dish to slides.Before we
inoculate in the slides we have to
add onto the slides a sterile distilled
water ( 1 drop of it) or 1 drop of
Normal saline solution (NSS)
From Broth Cultures
1. Wash a slide with soap and
hot water, removing all dirt
and grease. Handle the clean
slide by its edges.
2. Write the initials of the
organism or organisms on the
left-hand side of the slide with
a marking pen.
3. To provide a target on which
to place the organisms, make
a circle on the bottom side of
the slide, centrally located,
with a marking pen. Later on,
when you become more
skilled, you may wish to omit
the use of this “target circle.”
4. Shake the culture vigorously
and transfer two loopfuls of
organisms to the center of the
slide over the target circle. Be
sure to flame the loop after it
has touched the slide.
5. Spread the organisms over
the area of the target circle.
6. Allow the slide to dry by
normal evaporation of the
water. Don’t apply heat.
7. After the smear has become
completely air dried, place the
slide in a clothespin and pass
the slide several times
 through the Bunsen burner burner. Avoid prolonged heating of
flame. the

SIMPLE STAINING
 The use of a single stain to color a
bacterial cell
 Commonly used dyes for performing
simple staining are methylene blue,
From Plates and Slants
basic fuchsin, and crystal violet.
1. Wash a slide with soap and hot  These are referred to as basic dyes
water, removing all dirt and grease. because they have color-bearing
Handle the clean slide by its edges. ionic groups (chromophores) that
2. Write the initials of the organism or are positively charged (cationic).
organisms on the left-hand side of  To observe the morphology of the
the slide with a marking pen. microorganism
3. Mark a “target circle” on the bottom
side of the slide with a marking pen.
4. Flame an inoculating loop, let it cool,
and transfer two loopfuls of water to
the center of the target circle.
5. Flame an inoculating needle and
then let it cool. Pick up a very small
amount of the organisms, and mix it
into the water on the slide. Disperse
the mixture over the area of the
target circle. Be certain that the
organisms have been well emulsified
in the liquid. Be sure to flame the
inoculating needle before placing it
in its holder.
6. Allow the slide to dry by normal
evaporation of the water. Don’t
apply heat.
7. After the slide has become
completely dry, place it in a
clothespin and pass it several times
through the flame of a Bunsen
 When washing of the slide we have the presents of Peptidoglycan
to hold the slides ang ipapatama sa layer
tubig ay ung gilid/dulo lang ng slides
and dapat nakaslant siya. Huwag
directly sa mismong smear baka kasi
mawash off siya.

o Gram positive have thick

Peptidoglycan layer whereas
the gram negative has thin
peptidoglycan layer.
o Gram negative has outer layer
consist of lipids
(Lipopolysaccharide layer)
Several factors can affect the outcome
of the procedure:
 1. It is important to use cultures that
are 16–18 hours old
 2. It is critical to prepare thin
smears (not too thin and not too
GRAM STAINING thin)
 3. Decolorization is the most critical
step in the Gram stain procedure.
 Differential Stain
 We have to use 2 or more dye or
stain.
 Gram staining is developed by Hans
Christian Gram
 Two kinds of cells, gram positive
and gram-negative, are
differentiated based on the structure
of their cell walls
o Difference of gram positive
and gram negative: Based on
Gram Stain Procedure
 Gram staining steps and Reagents:
1. Primary Stain - Crystal violet-
Apply it for 30 secs then wash it
with water – no need to air dry
o When putting a crystal
violet in gram positive it
become purple while gram
negative become purple or
violet also
2. Application of Mordant -Gram’s
Iodine- for 1 min- wash it with
water
o Strengthen the affinity of
the Primary stain.
o The color of the bacteria
upon adding gram’s iodine
is still purple
3. Decolorization - Acetone Alcohol
– 5 to 15 seconds / quick on rinse
– wash with water
-95% alcohol, acetone
o Upon addition of the
alcohol in the gram
positive bacteria will
remain purple because of
its thick peptidoglycan
layer.
o While in the gram negative
it will appear colorless
because of its thin
peptidoglycan layer and in
its outer layer has
lipopolysaccharide that
are soluble to alcohol. And
lipid membrane will be
dissolve.
4. Secondary staining/ Counter
Staining - Safranin red – 1 min -
water
o Gram positive will remain
purple
o Gram negative will appear
color red/pink because
they will take up the color
of secondary stain
5. air dry
 smears. Note: Thick smears can take
longer for decolorization.
 5. Stop decolorization by washing
the slide with a gentle stream of
water.
 6. Cover the smear with safranin for
1 minute.
 7. Wash gently for a few seconds,
blot dry with bibulous paper, and air-
dry.
 8. Examine the slide under oil
immersion.

 1. Cover a heat-fixed smear with

crystal violet and let stand for 30
seconds
 2. Briefly wash off the stain, using a
wash bottle of distilled water. Drain
off excess water.
 3. Cover the smear with Gram’s
iodine solution and let it stand for 1
minute. (Your instructor may prefer
only 30 seconds for this step.) Wash
off the Gram's iodine.
 4. Hold the slide at a 45-degree
angle and apply the decolorizer,
allowing it to flow down the surface
of the slide. Do this until the
decolorizer is colorless as it flows
from the smear down the surface of
the slide. This should take no more
the 15 seconds for properly prepared
WEEK 7: Clinical Bacteriology
(Laboratory)

ACID-FAST STAINING
 An important diagnostic tool in the
identification of Mycobacterium
tuberculosis, the causative agent of
tuberculosis, and Mycobacterium
leprae, the bacterium that causes
leprosy in humans.
MYCOLID ACID
 A complex lipid that is composed of
fatty acids and fatty alcohols that
have hydrocarbon chains up to 80
carbons in length.
 Affects the staining properties of
bacteria and prevents them from
being stained by many of the stains
routinely used in microbiology.
ZEIHL-NEELSEN METHOD
MICROBIOLOGICAL CULTURE
MEDIA PREPARATION and
STERILIZATION
Cultivation
 Process of growing microorganisms
in culture by taking bacteria from the
____________ (in vivo) and growing
them in the ____________ of the
laboratory (in vitro)
 3 main purposes of cultivating
bacteria:
1. To grow and isolate all
bacteria present in the clinical
specimen.
2. To determine which of the
bacteria that grow is most
likely causing infection and
which are likely causing
infection and which are likely
contaminants.
3. To obtain sufficient growth
of clinically relevant bacteria
to allow identification and
characterization
NUTRITIONAL REQUIREMENTS
Water
Ions
Nitrogen
Gases
Sources of carbon
Latter requirements: ____________ &
______________
TERMS TO REMEMBER:
1. _____________ - nutrients prepared
for bacterial growth
2. _____________ - suspension of
microorganism
3. _____________ - introduction of
bacteria into culture medium
4. _____________ - bacteria growing on
culture medium
 _____________ contains only
one specie
 _____________ contains
several species
 _____________ contains
unwanted species or
organisms
5. _____________ - visible growth of
microorganism on the surface of
culture media
6. _____________ - nutritional needs
are relatively complex and
exceptional components used for the
growth
7. _____________ - nutritional needs
are relatively basic and
straightforward.
CULTURE MEDIA
 nutrient preparations that are used for
culturing microorganisms
 solid, liquid or semi-solid designed
to support the growth of
microorganisms
A.CLASSIFICATION ACCORDING TO A.CLASSIFICATION ACCORDING TO
CONSISTENCY: CONSISTENCY:
1. Solid - solidifying agent is added 2. Liquid / Broth - dissolved in water
(1.5-3% of agar)
Uses:
Agar is the most commonly used as
 can be used to propagate large
solidifying agent
numbers of microorganisms in
Melt: ≥95°C fermentation studies
 for various biochemical tests
Resolidify: <50°C
Uses:
 for the surface growth of
microorganisms in order to observe
colony appearance
 for pure culture isolations
 storage of cultures
 to observe specific biochemical
reactions. 3. Semi-solid -solidifying agent is
added (<1.5% of agar)
Solid media can be poured into either
a test tube or petri plate (dish). Uses:
 In tube:  fermentation studies
1. Agar Slant - the medium in the test  in determining bacterial motility
tube is allowed to harden in a slanted  promoting anaerobic growth
position
2. Agar deep - the tube is allowed to
harden in an upright position
In Plate:
Agar Plate - containing about 15 to 16 ml of
media

CLASSIFICATION OF CULTURE
MEDIA:

II. According to Chemical

composition:
 1. Synthetic Example: ________________
- chemically defined 4. Supportive / Nonselective
- use to culture algaes or Medium
nonfastidious - contain nutrients to
heterotrophs support growth of most
2. Non-synthetic (complex) nonfastidious organisms
- Chemical composition is without giving any
not specifically defined; particular organism a
extracts of yeasts, meat or growth advantage.
plants. (bacteria are 5. Differential Medium
usually grown in this - used to distinguish them
type of media) from other bacteria
growing on the same agar
Ex: Nutrient broth, Nutrient agar,
plate
EMB
Example: _______________
III. According to Function
1. Enrichment media
- contains specific nutrients
required for the growth of
particular bacterial
pathogens
- used to enhance the
growth of particular
pathogen from a mixture
of organism by using
nutrient specificity
Example: __________________________
that contains_____________ required for
the growth of Legionella pneumophilia CULTURE MEDIA PREPARATION
STEPS:
2. Selective Media
- Favor the growth of a TUBE (WDDS) PLATE
particular bacterium by (WDSD)
inhibiting the growth of 1. WEIGH 1. WEIGH
undesired bacteria and
2. DISSOLVE 2. DISSOLVE
allowing the growth of
desired bacteria. 3. DISPENSE 3. STERILIZE
4. STERILIZE 4. DISPENSE
Example: ________________
3. Enriched Medium
- Enriched usually by
adding blood, or eggs.
 boil in order to completely dissolve
the agar.

Sterilization
 The process of rendering a medium
or material free of all forms of life.

Three Basic Ways of

Sterilization of Media:
 Autoclaving
o exposure to steam at 121°C
and 15 lbs. of pressure for 15
minutes or longer, depending
on the nature of the item.
o microorganisms, even
endospores, will not survive
longer than about 12 to 13
minutes
BLOOD AGAR PREPARATION:
1. WEIGH
2. DISSOLVE
3. STERILIZE
4. Make sure the agar is cooled down
5. Add 5% defibrinated blood (avoid
bubbles) : Blood is agitated
continuously using glass beads
(marbles)
How to compute the 5%?
6. Dispense
 If the medium lacks agar, the powder
will usually dissolve without heating.  Ultraviolet (UV) radiation
 If it contains agar, you must heat the o Around 260 nm is quite lethal
medium until it starts to simmer or to many microorganisms but
 does not penetrate glass, dirt
films, water, and other
substances very effectively.
 Ethylene oxide
o Both microbicidal and
sporicidal and kills by
covalently attaching to cell
proteins.
o It is a particularly effective
sterilizing agent because it
rapidly penetrates packing
materials, even plastic wraps.

LIQUID MEDIA – Plain

Broth, TSB
Material: Inoculating loop
Manner of incoulation: Twirl
/Mixing
SLANTED MEDIA-
Simmon's Citrate, Urea
Material: Inoculating needle /
Wire loop
Manner of incoulation: Streak
/ Line
SEMI SOLID BUTT
MEDIA - SIM
Material: Inoculating needle /
Wire loop
Manner of incoulation: Stab
Halfway
BUTT SLANTED
MEDIA - TSI, LIA
Material: Inoculating needle /
Wire loop
Manner of incoulation: Stab
& Streak
 cotton plug then, passed it through
flame.
 After that dip the inoculating loop in 5
seconds. Avoid touching the side of the
Streak-Plate Method test tube.
 After that passed through flame the test
tube and return the cotton plug.
 Streak the plate, holding it as shows. Do
not gouge into the medium with the
loop. (Huwag idiin ang inoculating loop)
 Lastly, flame the loop before placing it
down.

 Same procedure as bacterial smear

procedure we have to disinfect first the
area.
 If turbid there a presence of bacterial
growth.
 Sterilize the inoculating loop using your
dominant hand under 360° angle and
then place it on the alcohol lamp and
wait for it until it turns red hot.
 Tinawag siyang quadrant streak because
 Then after that get your bacteria
suspension make sure that na mix na it has four quadrant.
siya. 1) Streak one loopful of organisms over
 Get the bacterial suspension using the Quadrant 1 near edge of the place.
dominant hand then open it/ remove the
 Apply the loop lightly. Don’t gouge into
the medium.
2) Flame the loop, cool 5 seconds, and
make 3 to 4 streaks from Quadrant 1
through quadrant 2. Momentarily
touching the loop to a sterile area of the
medium before streaking insures a cool
loop.
3) Flame the loop again, cool it, and make
5 to 6 streaks from Area 2 through Area
3.
4) Flame the loop again, and make as 6 to
7 streaks as possible from Area 3, and
for Area 4 make as many streaks as
possible, using up the remainder of the
plate surface.
5) Flame the loop before putting it aside.
1) Streak one loopful of organism back and
forth over Area 1 starting at point
designated by “s”. Apply loop highly.
Don’t gouge into the medium.
2) Flame the loop, cool 5 seconds, and
touch the medium in a sterile area
momentarily to insure coolness.
3) Rotate dish 90° while keeping the dish
closed. Streak Area 2 with several back
and forth strokes, hitting the original
streak in a few times.
4) Flame the loop again. Rotate the dish
and streak Area 3 several times, hitting
the last area several times.
5) Flame the loop, cool it, and rotate the
dish 90° again. Streak Area 4, contacting
Area 3 several times and drag out the
culture as illustrated.
6) Flame the loop before putting it aside.
1) Spread a loopful of organisms in a small
area near the edge of the plate in Area 1.
Apply the loop lightly. Don’t gouge into
the medium.
2) Flame the loop and allow it to cool for 5
seconds. Touching a sterile area will
insure coolness.
3) From the edge of Area 1 make 7 or 8
straight streaks to the opposite side of
the plate.
4) Flame the loop again, cool it sufficiently,
and cross streak over the last streak,
starting near Area 1.
5) Flame the loop before putting it in its
receptacle.
A. Starting at the edge of the plate (Area A)
with a loopful of organisms, spread the
organism in a single continuous
movement to the center of the plate. Use
light pressure and avoid gouging the
medium.
B. Rotate the plate 180° degrees so that the
uninoculated portion of the plate is away
from you.
C. Without flaming loop, and using the
same face of the loop. Continue
streaking the other half of the plate by
starting at Area B and working toward
the center.
D. Flame your loop before putting it aside.
1. LIQUID MEDIA- Plain Broth, TSB
Material: Inoculating loop
Manner of incoulation: Twirl
/ Mixing.
o Get a colony from bacterial
suspension, same procedure as
the streak- plate method, Then
dip it in liquid media and the
twirl or mix the inoculating loop.
2. SLANTED MEDIA- Simmon's
Citrate, Urea
Material: Inoculating needle/
Wire loop
Manner of incoulation: Streak
/ Line
o Streak inside from bottom to tip
3. SEMI SOLID
BUTT MEDIA –
SIM
Material: Inoculating
needle / Wire loop
Manner of incoulation: Stab
Halfway

4. BUTT
SLANTED MEDIA -TSI,
LIA
Material: Inoculating
needle / Wire loop
Manner of incoulation:
Stab (butt) & Streak
(slant)
 Stab halfway
WEEK 10: CLINICAL Specimen Collection and Handling
BACTERIOLOGY (LAB)
 Clinical materials collected from
infected sites should be transported to
the laboratory without delay to
Staphylococcus prevent drying, maintain the proper
environment, and minimize the
 Derived from the Greek term
growth of contaminating organisms.
“staphie”, meaning “bunches of
grapes.” Microscopic Examination
 Catalase-producing, Gram positive
(Purple color) cocci that appear in  Gram positive cocci in clusters
singly, in pairs, and in cluster  ** Gram stains should be performed
 nonmotile, non–spore-forming, and on young cultures
aerobic or facultatively anaerobic
except for S. saccharolyticus, which
is an obligate anaerobe
 Toxin induced diseases, such as food
poisoning, scalded skin syndrome
(SSS), and toxic shock syndrome
(TSS), are also associated with this
organism.
 Causes UTI, Boils or pigsa, CULTIVATION
Cutaneous Infections.
Media of Choice
Micrococcus
 Grow on 5% sheep blood and
 catalase-producing, coagulase- chocolate agars (this are examples of
negative, gram-positive cocci general culture media)
 They are often recovered with  They also grow well in broth-blood
staphylococci and can be culture systems and common nutrient
differentiated easily from coagulase- broths
negative staphylococci (CoNS)
 Selective Media:incubation of 48-72
hours

 Phenylethyl alcohol (PEA)

 Columbia colistin-nalidixic acid

(CNA) agars

 Mannitol Salt Agar

 CHROMagar- Methicilin- resistant

staphylococcus aureus
 Staphylococci produce round,
smooth, white, creamy colonies on
SBA after 18 to 24 hours of
incubation at 35°C to 37° C
 S. aureus can produce hemolytic
zones around the colonies and may
rarely exhibit pigment production
with extended incubation.
 Yung mga nakapalibot ay called as
Hemolytic Pattern
Catalase Test
 In this picture Beta Hemolytic are
present.
 Difference of Beta and Alpha – when
Alpha means incomplete hemolysis
(greenish discoloration) while Beta
means complete. When we says
Gamma Hemolysis means lack of
hemolysis
 When Gram Positive Cocci the only
suspected organism are
Staphylococcus and Streptococcus
and Enterococcus
 This test differentiates catalase-
positive micrococcal and
staphylococcal species from catalase-
negative streptococcal species
 Principle: The enzyme, catalase, is
capable of converting hydrogen
peroxide to oxygen and water. The
presence of enzyme in bacterial
isolate causes rapid elaboration of
bubbles.
 Reagent: 30% hydrogen peroxide
(Baileys); 3% hydrogen peroxide
(delost)
 Results:
Positive: Bubble formation/
effervescence (Staphylococci)
Negative: no bubble
formation (Streptococci)
 In Catalase test when negative we
expect Streptococci and when
positive we expect staphylococci
 Cause of False positive reaction:
1. Entrococci – Peroxidase
(Slowly catalyzes the
breakdown of (H202) – weak
bubble formation
2. Blood Agar
3. Platinum Wire - Nichrome
wire

COAGULASE TEST
 The test is used to differentiate
Staphylococcus aureus (positive)
from coagulase-negative
staphylococci (negative) CONS – S.
lugdunesis, S. schleiferi, S.
Intermidius, S. epidermis, S. wameri,
S. simulans, S. haemolyticus.
 Principle:
 Slide: Bound coagulase, or
“clumping factor,” is bound to the
bacterial cell wall and reacts directly
with fibrinogen.- Formed Fibrin clot
 The presence of bound coagulase
correlates with free coagulase, an
extracellular protein enzyme that
causes the formation of a clot when S.
aureus colonies are incubated with
plasma
 Reagent: rabbit’s plasma

 We incubate 1 to 4 hours and check it

every 30 minutes.
 If there are no clot formation after 4
hours, we still incubate it for 24
 hours, if there are still no cloth it
means negative.
 We can use human plasma if there are
no available rabbit’s plasma.
Cause of False positive reaction:
1. Citrate
2. Colonies from high salt concentration
culture media
Bacitracin Susceptibility Test
 It is used to distinguish staphylococci
species (resistant) from micrococci
(susceptible).
 Principle:
 The antibiotic bacitracin inhibits the
synthesis of bacterial cell walls. A
disk (TaxoA) impregnated with a
small amount of bacitracin (0.04
units) is placed on an agar plate,
allowing the antibiotic to diffuse into
the medium and inhibit the growth of
susceptible organisms.
 Results:
 Positive: Susceptible (micrococci)
 Negative :Resistant (Staphylococcus)
Procedure:
1. Using an inoculating loop, streak two
or three suspect colonies of a pure
culture onto a blood agar plate.
2. Using heated forceps, place a
bacitracin disk in the first quadrant
(area of heaviest growth). Gently tap
the disk to ensure adequate contact
with the agar surface.
3. Incubate the plate for 18 to 24 hours
at 35°C to 37°C in ambient air for
staphylococci and in 5% to 10%
carbon dioxide (CO2) for streptococci
differentiation.
4. Look for a zone of inhibition around
the disk.
Expected Results
Positive: Any zone of inhibition greater
than 10 mm; susceptible
Negative: No zone of inhibition; resistant
NOVOBIOCIN DISK TEST
 Used to differentiate Coagulase-
Negative Staphylococci
 5ug
 Incubate for 18 to 24 hours
 (+) presence of zone of inhibition
around the disk SUSCEPTIBLE-
 Coagulate Negative Staphylococcus
(CoNS)
 (-) no zone of inhibition-
RESISTANT – S. saprophyticus
Streptococci and Enterococci
 gram-positive cocci
 facultative anaerobes and generally
considered non-motile
 occur singly or in pairs, however, they
are best known for their characteristic
formation of long chains
 Some species are capnophilic (needs
the presence of carbon dioxide)
 Pyogenic causing bacteria ( fast
producing bacteria)
 Rebecca Lancefield
HEMOLYTIC PATTERNS
 We can only use if the culture media
with blood
Beta-Hemolytic Groups
 Group A Streptococci (GAS)
– Streptococcus pyogenes
– In order to differentiate S.
pyogenes from other
streptococci and
enterococci, isolates are
tested for resistance to
bacitracin.
– If a bacterial isolate is
beta-hemolytic and
sensitive to bacitracin, it
is presumed to be S.
pyogenes.
 Group B Streptococci (GBS)
– S. agalactiae – neonatal
infection
– This pathogen may be
found in the pharynx,
skin, and rectum;
however, it is more likely
to be found in the genital
and intestinal tracts of
healthy adults and infants.
– S. agalactiae colonies are
large, with a narrow zone
of beta-hemolysis
– Preliminary identification
of this species relies
 heavily on positive
CAMP reaction.
 Group C Streptococci
– Uncommon human
pathogens but may be
involved in zoonoses.
– S. dysgalactiae produce large
colonies with a large zone of
beta-hemolysis on blood
agar.
– Presumptive differentiation
of S. dysgalactiae from other
beta-hemolytic streptococci
(S. pyogenes and S.
agalactiae) is based primarily
on resistance to bacitracin
and a negative CAMP test.
Alpha-Hemolytic Groups
 Streptococcus pneumoniae – – E. faecium: alpha- hemolytic.
bacterial pnuemonia
– A significant human
pathogen.
– Colonies appear smooth,
mucoid, and surrounded by a
zone of greenish discoloration
(alpha-hemolysis).
– In culture these cells usually
grow as diplococci, but they
can also occur singly or in
short chains.
– Presumptive identification of
S. pneumoniae can be made
with a positive optochin
susceptibility test. TaxoP
 Viridans Streptococci Group
– Blood Agar: very small, gray
to whitish gray, and opaque.
– Culture: rod-like and grow in
chains.
– The viridans group can be
differentiated from the
pneumococci and enterococci
by a negative result in the
bile esculin hydrolysis test,
the salt-tolerance test, and
the optochin susceptibility
test.
 Group D Enterococci
– Blood agar: large colonies
that can appear nonhemolytic,
alpha-hemolytic, or rarely
beta-hemolytic.
– Culture: diplococci in short
chains.
– E. faecalis appear either
nonhemolytic or beta- hemolytic,
depending on the strain and the type
of blood agar used
– Group D Nonenterococci
– S. bovis
– Large, mucoid (many strains
have a capsule), and either
nonhemolytic or alpha-
hemolytic.
– Culture: in pairs and short
chains.
– Key reactions for this group
are a positive bile esculin test
and negative salt broth test.
LABORATORY DIAGNOSIS
1. GRAM STAIN – Gram positive
cocci in pairs or in chains
2. Growth on BAP & CAP
GRP A – Grayish white
– Transparent to translucent
– Matte or glossy
– Large zone of beta hemolysis
GRP B – Larger than Grp A
– Translucent to opaque
– Flat or glossy
 – Narrow zone of beta OPTOCHIN TEST
hemolysis
GRP C – Grayish white
– Glistening
– Wide zone of hemolysis
3. CATALASE TEST - NEGATIVE

 Exclusively for S. Pneumoniae

Media: 5% sheep blood agar
ALPHA HEMOLYSIS
Antibiotic: Optochin/ TaxoP
/ethylhydrocupreine hydrochloride –
interferes the ATPase (walang
maggrow sa palibot ni optochin)
 Incubate for 18 to 24 hours
 Results:
 Positive: Zone of Inhibition,
SUSCEPTIBLE
 Negative: Other Alpha-
Hemolytic Streptococcus

– Test: Optochin test if positive

they are Streptococcus
pneumoniae, If negative
nagnegative siya doon next
test ay iyong Bile esculin test,
If nagnegative ulit this are
viridans. Pag nagpositive
naman sa bile esculin test
proceed sa 6.5% NaCl and
kapag positive they are
Enterococcus spp., if negative
it is Group D streptococci.
 Exclusively for S. Pneumoniae
Media: 5% sheep blood agar
Reagent:
Plate: 10% sodium
deoxycholate
Tube: 2% sodium
deoxyxcholate
 Incubate for 35°C to 37°C for 30
minutes
 Lysis – presence of autolytic
enzymes (Amidase)

 Enterococci- halophilic (salt

loving) (positive)

 Media: Heart infusion broth with

6.55 NacL

 Indicator for acid production:

 Pag resistant we can use BILE
ESCULIN TEST
 Enterococci
 Media: Bile Esculin Agar (slant)
 Grows on a 4% bile – they will be
able to hydralyze esculin to
esculetin
 Esculetin + Ferric = dark brown to
black precipitate
 Incubate for 48 hours look for the
presence of black agar
 Positive: Growth and Blackening
of the agar slant
 Negative: Growth and no
blackening
Glucose and bromcresol purple
METHOD
1. Inoculate one or two colonies from an
18- to 24-hour culture into 6.5% NaCl
broth.
2. Incubate the tube at 35°-37°C in
ambient air for 48 hours.
3. Examine tubes for turbidity after 24
hours and if negative again at 48 and
72 hours
Expected Results
Positive: Visible turbidity in the
broth, with or without a color change
from purple to yellow. Note:
Turbidity alone is indicative of a
positive test
Negative: No turbidity and no color The antibiotic bacitracin inhibits the
change after 72 hours of incubation. synthesis of bacterial cell walls by
interfering the peptidoglycan
synthesis of bacteria. A disk
impregnated with a small amount of
bacitracin (0.04 units) is placed on an
agar plate, allowing the antibiotic to
diffuse into the medium and inhibit
the growth of susceptible organisms.
After incubation, the inoculated
plates are examined for zones of
SCHEMATIC DIAGRAM FOR inhibition surrounding the disks.
DIFFERENTIATION Method
OF GAS FROM GBS 1. Using an inoculating loop, streak two
or three suspected colonies of a pure
culture onto a blood agar plate.
2. Using heated forceps, place a
bacitracin disk in the first quadrant
(area of heaviest growth). Gently tap
the disk to ensure adequate contact
with the agar surface.
3. Incubate the plate for 18 to 24 hours
at 35°-37°C in ambient air for
staphylococci and in 5% to 10%
carbon dioxide (CO2) for streptococci
differentiation.
4. Look for a zone of inhibition around
the disk.
Expected Results
BACITRACIN
Positive: Any zone of inhibition greater than
SUSEPTIBILITY
10 mm; susceptible.
Purpose
The test is used for presumptive Negative: No zone of inhibition; resistant
identification and differentiation of
beta-hemolytic group A streptococci  Antibiotic: Bacitracin (0.04) – TaxoA
(Streptococcus pyogenes–  Media: 5% blood agar
susceptible) from other beta-
hemolytic streptococci. It is also used
to distinguish staphylococci species
(resistant) from micrococci
(susceptible).
Principle
 CAMP Test Hippurate Hydrolysis
 S.Agalactiae
 CAMP stands for: Christie, Atkins,
Munch, Peterson
 Media: 5% sheep’s blood agar
 CAMP factor + beta – lysin S.
aureus= enhanced lysis of RBC

 Hippuricase – glycine and benzoic

acid
 Glyzine – OXIDIZE – Ninhydrin =
purple color
 Reagent: 0.2 ml n=Ninhydrin
Reagent
 S.agalactiae
Method
1. Add 0.1 mL of sterile water to a 12
×75 mm plastic test tube.
2. Make a heavy suspension of the
organism to be tested.
3. Using heated forceps, place a rapid
hippurate disk in the mixture.
4. Cap and incubate the tube for 2 hours
at 35°C; use of a water bath is
preferred.
5. Add 0.2 mL ninhydrin reagent and
reincubate for an additional 15 to 30
minutes. Observe the solution for the
development of a deep purple color
Expected Results
Positive: A positive test is indicated by the
appearance of a deep blue/violet color in 30
minutes.
Negative: Colorless or slightly yellow pink
color.
Limitations
 A false-positive result may occur if
incubation with ninhydrin exceeds 30
minutes.
WEEK 14: Biochemical
Identification of Gram Negative
Bacteria
Family Enterobacteriaceae- Enterics
 Gram negative bacilli and
cocobacilli, non-spore forming,
facultatively anaerobic
 DO NOT produce cytochrome C
oxidase negative except for
Pleseimonas (an oxidase positive)
 ALL ferment glucose
 MOTILE at body temperatures
except for Klebsiella, Shigella, and
Yersinia
Laboratory Diagnosis
Specimen Collection
 To ensure isolation of both
opportunistic and fastidious
pathogens, laboratories must provide
appropriate transport media, such as
Cary Blair, Amies, or Stuart media.
 Should be kept at 4°C
Gram Stain
 ** Direct smear examination of stool
samples is not particularly helpful in
identifying enteric pathogens but may
reveal the presence of inflammatory
cells.
Culture
 grow at an optimal temperature of
35°C to 37°C,
 some species can tolerate low
temperatures (1°C to 5°C, such as
Serratia and Yersinia)
 or tolerate high temperatures(45°C
to 50°C, such as E. coli)
 visible after 18-24 hours of
incubation
 BAP, CAP, MacConkey Agar and
other selective medium
Colonial Appearance
o All Enterobacteriaceae produce
similar growth on blood and
chocolate agars; colonies are large,
gray, and smooth.
o Klebsiella or Enterobacter may be
mucoid- because of the presence of
polysaccharide capsule
o P. mirabilis, P. penneri, and P.
vulgaris “swarm” on blood and
chocolate agars.
o Y. pestis on 5% sheep blood agar are
pinpoint at 24 hours but exhibit a
rough, cauliflower appearance at
48 hours.
o Broth cultures of Y. pestis exhibit a
characteristic “stalactite pattern”
o Y. enterocolitica produces bull’s-eye
colonies
Bull’s eye
colonies
 Morganella
Edwardsiella

MUCOID

MacConkey Agar: Escherichia coli,

Enterobacter aerogenes, Proteus vulgari,
Salmonella typhimurium, Staphylococcus
aureues

MacConkey Agar
Best used to characterize gram negative rods
Lactose fermenters can be differentiated with
non-lactose fermenters
Lactose fermenter: Dark pink colonies
Non-Lactose fermenter: Clear colonies
Rapid Lactose Fermenter: 18 to 24 hrs of
incubation
Escherichia, Enterobacter, Klesiella
Late Lactose fermenters: 48 to 72 hours of
incubation
Hafnia, Serratia, Citrobacter, Salmonella
arizonae, Shigella sonnei, Yersinia
enterocolitica
Non-lactose fermenters:
All Salmonella except S. arizonae
All Shigella except S. sonei
All Yersinia except Y. enterocolitica
Proteus
Providencia
BIOCHEMICAL TESTS sterile mineral oil to create an
anaerobic environment (closed), and
the other tube is left aerobic (open),
without mineral oil overlay.
Reactions:
Acid produced on both tubes: Oxidizer
and fermenter
Acid only in closed tube: fermenter and
possible obligate anaerobe
Acid in open tube: Oxidizer

A. CARBOHYDRATE UTILIZATION
Two enzymes are necessary for a bacterium
to take up lactose and to cleave it into
monosaccharides:
A.2
A. B -galactoside permease (lactose
permease) Transport enzyme
Triple Sugar Iron (TSI)
B. B-galactosidase- hydrolyzes lactose into
glucose and galactose
LFs - possess both β-galactoside permease
and β-galactosidase
NLFs - do not possess either enzyme
LLFs - lack β-galactoside permease but
possess β-galactosidase
A.1 Oxidation (aerobically) -Fermentation
(anaerobically) Tests
OF Basal Medium
o pH indicator: bromthymol blue
o Uninoculated Medium: green
o Acid Environment: yellow
o Alkaline Environment: blue

o When O/F tests are performed, two

tubes of Hugh-Leifson OFBM are
inoculated; one is overlaid with

Method
1. With a straight inoculation needle,
touch the top of a well-isolated
colony.
2. Inoculate TSI by first stabbing
through the center of the medium to
the bottom of the tube and then
streaking the surface of the agar
slant.
3. Leave the cap on loosely and
incubate the tube at 35°-37°C in
ambient air for 18 to 24 hours.
4. Examine the reaction of medium.
Figure 12-41: Triple sugar iron
agar. A, Acid slant/acid butt with gas, no
H2S (A/A). B, Alkaline slant/acid butt, no
gas, H2S-positive (K/A H2S+). C, Alkaline
slant/alkaline butt, no gas, no H2S
(K/K). D, Uninoculated tube.
A.2 Triple Sugar Iron (TSI)
o used to determine glucose and
lactose or sucrose utilization and
H2S production, gas production
Composition: 10 part lactose, 10 parts
sucrose, 1 part glucose and peptone
pH indicator: Phenol red – yellow (ph 6.8)-
acidic
H2S Indicator: Ferrous sulfate and sodium
thiosulfate
** Reactions should be read within an 18-to
24-hour incubation period; otherwise,
erroneous results are possible.
Aerobic-
lactose/sucrose
Anaerobic-
Glucose
Reactions on TSI agar
Galactopyranoside Test (ONPG Test)
1. A/A (Acid (yellow) slant/acid
(yellow) butt) – Lactose/sucrose,
Glucose fermenter
2. K/A (Alkaline (red) slant/acid
(yellow) butt) – glucose fermenter
3. K/K (Alkaline(red)
slant/alkaline(red) butt) – no
fermentation- not a member of
enterobacteriaceae family
4. H2S production- (K/A, H2S) or
(A/A H2S) black precipitate –
requires an ACID ENVIRONMENT
5. Gas production- formation of
bubbles or splitting of the medium
in butt
H2S producer- Salmonella, Proteus,
Arizona, Citrobacter, Edwardsiella
(SPACE)
A.3 Ortho-Nitrophenyl-β-D-
o Β-Galactosidase hydrolyzes ONPG,
a colorless compound, into galactose
and o-nitrophenol, a YELLOW
compound. LACTOSE
FERMENTER
o ONPG remains colorless if the
organism is an NLF.
o The test can be performed by making
a heavy suspension of bacteria in
sterile saline and adding
commercially prepared ONPG disks
or tablets.
o The suspension is incubated at 35°
C, and positive results can generally
be seen within 6 hours.

B. Glucose Metabolism and Its

Metabolic Products
B.1 Methyl Red Test
If glucose is metabolized by the mixed acid
fermentation pathway, stable acid end
products are produced, which results in a
low pH.
 Negative: Yellow (acidic- glucose is
fermented)

Reagent: Methyl Red

Positive Result: Red color
Negative Result: Yellow
Anaerobic
B.2 Voges-Proskauer Test
o Detects the organism’s ability to Anaerobic
convert the acid products to
acetoinand 2, 3-butanediol. Glucose- acid (yellow)
(Butylene glycol pathway)
o After incubation, a- napthol is added
first as a catalyst or color intensifier.
Next, 40% potassium hydroxide
Lysine Iron Agar
(KOH) or sodium hydroxide
(NaOH) is added METHOD:
Positive Result: Red Color 1. With a straight inoculating needle,
inoculate LIA by twice stabbing
Negative Result: Yellow
through the center of the medium to
C. Amino Acid Utilization the bottom of the tube and then
streaking the slant.
2. Cap the tube tightly and incubate ate
C.1 Lysine Iron Agar 35-37C in ambient air for 18-24 hrs.

Purpose: To differentiate gram-negative A. Alkaline slant (purple)/alkaline butt

bacilli based on decarboxylation or (purple) K/K
deamination of lysine and the formation of Lysine decarboxylation and no fermentation
hydrogen sulfide (H2S). of glucose
pH indicator: Bromcresol purple B. Alkaline slant (purple)/acid butt
Lysine Deaminase (Slant) (yellow)

Positive: Burgundy/ Red Glucose fermentation

Negative: Purple C. Red slant/acid butt (yellow) R/A

Lysine Decarboxylase (Butt) – Lysine deamination and glucose

CADAVERINE - purple fermentation

Positive: Purple (CADAVERINE) Deaminase- Proteus, Providencia,

Marganella
H2S- (SACE)
Salmonella
Arizona
Citrobacter
Edwardsiella
C.2 Phenylalanine Deaminase (PAD) test
o Determines whether an organism
possesses the enzyme that
deaminates phenylalanine to
phenylpyruvic acid.
o Contains 0.2% concentration of
phenylalanine.
o The surface of the slant is inoculated
with a bacterial colony. After
incubation, addition of a 10% ferric
chloride reagent results in a green
color if phenylpyruvic acid is present
D. Miscellaneous Tests
D.1 SULFIDE INDOLE MOTILITY
AGAR (SIM) – semi solid media
Sulfide (H2S)
o Black precipitate
Indole
o Organisms that possess the enzyme
Trytophanase are capable of
deaminating tryptophan with the
formation of the intermediate
degradation products of indole,
pyruvic acid, and ammonia.
o Reagent:0.5 ml Ehrlich’s reagent
o pink to red color
Motility
o Cloudiness spreading from the
inoculation line
 o Positive: Change in color from
Light orange (pH 6.1) to Magenta
D.2 Citrate Utilization (pink) (pH 8.1) (positive result)
Purpose: It determines whether an o Negative: No change in color
organism can use sodium citrate as a sole
carbon source.
o part of a series referred to as IMViC
(indole, methyl red, Vogues-
Proskauer, and citrate)

***Simmon's Citrate Agar

Contains ammonium salts as the sole
nitrogen source

pH indicator: Bromthymol blue

Green to blue

Positive Result: Color blue/ growth into the

medium Rapid Urease Producers
Negative result: Green
(PPM)
**use light inoculum. Proteus
Providencia
Morganella
Slow Urease Producers
(CKEYS)
Citrobacter
Klebsiella
Entrobacter except E. gergoviae
D.3
Urease Test Yersinia
o Determines whether a Serratia
microorganism can hydrolyze
D.4 DNA Hydrolysis (DNAse Test Agar)
urea.
o Urease hydrolyzes urea to form Purpose: This test is used to differentiate
ammonia, water, and CO2. organisms based on the production of
o Christensen's Urea Agar deoxyribonuclease.
 o to distinguish Serratia sp. (positive) D.6 Malonate Utilization
from Enterobacter sp.,
o pH indicator: bromthymol blue
Staphylococcus aureus (positive)
o Bacteria able to use malonate as a
from other species, and Moraxella
sole carbon source also use
catarrhalis (positive) from Neisseria
ammonium sulfate as a nitrogen
sp.
source.
POSITIVE: Medium will turn colorless
around the test organism. o A positive test results in increased
alkalinity from utilization of the
NEGATIVE: If no degradation of DNA
ammonium sulfate, changing the
occurs, the medium remains green.
indicator from green to blue

D.5 Gelatin Liquefaction

Numerous bacteria produce
Gelatinases —proteolytic enzymes that
break down gelatin into amino acids.
Gelatinase activity is detected by loss of
gelling (liquefaction) of gelatin.
D.7 Nitrate and Nitrite Reduction
Positive Result: Liquefaction
o The nitrate reduction test determines
whether an organism has the ability
to reduce nitrate to nitrite and reduce
nitrite further to nitrogen gas (N2).
o After 24 hours of incubation, N, N-
dimethyl-α-naphthylamineand
sulfanilic acid is added.
o A red color indicates the presence of
nitrite.
D.8 Oxidase Test
The oxidase test determines the presence of
the cytochrome oxidase system that
oxidizes reduced cytochrome with molecular
oxygen.
Kovac’s oxidase test uses a 0.5% or 1%
aqueous solution of tetramethyl-ρ-
phenylenediamine dihydrochloride.
Positive: development of a lavender/purple
color within 10 to 15 seconds.
 Media Selective Differential Nutritional Purpose
Blood agar Hemolysis of RBCs: Routinely used to Screening colonies
(sheep) (BA) Beta: Complete lysis cultivate for oxidase enzyme
Alpha: partial, moderately
greening fastidious
Gamma: organisms;
nonhemolytic trypticase soy agar
with 5%-10%
defibrinated blood
Bismuth sulfite Selective inhibition Production of Beef extract, Isolation of
agar of most gram- hydrogen sulfide peptones and Salmonella spp.
positive and gram- (H2S); ferrous sulfate. dextrose
negative bacteria Positive reactions
(bismuth sulfite appear as brown to
and brilliant green) black precipitate
Brilliant green Selective; brilliant Lactose and sucrose Enzymatic digests Isolation of
agar green is inhibitory fermentation; positive of animal tissue, Salmonella spp.
to most gram- (phenol red) casein, lactose and
positive and gram- fermenters appear as sucrose
negative bacteria yellow to green
colonies with bright
yellow to green halos.
Salmonella spp.
Appear as white to
pink or red colonies
surrounded by bright
halo
red Selective inhibition Fermentation of Pancreatic digests Isolation and
centerCefixime- of most non- sorbitol in the of gelatin, peptone, identification of
tellurite- vercytotoxigenic E. presence of neutral and sorbitol E.coli O157-H7
containing coli and non- red. Positive colonies
MacConkeys sorbitol fermenters appear pink and
(CT-SMAC) ( sorbitol, cefixime, nonfermenters will
and tellurite) appear colorless.
Cefsulodin- Selective inhibition Fermentation of Isolation of
irgasan- of gram- negative mannitol in the Yersinia
novobiocin agar and gram-positive presence of neutral enterocolitica
(CIN) organisms red. Macroscopic
colonial appearance:
colorless or pink
colonies with red
center.
 Citrate agar, Citrate as the sole Detect organisms
Simmons (CIT) carbon source, capable of citrate
ammonium salt as utilization
nitrate. Ammonium
salt alteration changes
pH to alkaline,
bromothymol blue
shifts from green to
blue.
Decarboxylases Incorporate amino Differentiate
(ornithine, acid as differential fermentative and
arginine, lysine) media (e.g., lysine, nonfermentative
arginine, or gram-negative
ornithine). bacteria
Decarboxylation
yields alkaline, pH-
sensitive bromocresol
purple dye. Basal
medium serves as a
control.
Incubate for up to 4
days. Fermentative
organisms turn media
yellow, using glucose.
(H+) increases,
making optimal
conditions for
decarboxylation.
Conversion of the
amino acids for free
amine groups raising
the pH, reversing the
yellow to purple.
Nonfermenters turn
the purple a deeper
color.
Eosin/methylene Eosin Y and Lactose and sucrose Identification of
blue agar (EMB) methylene blue for differentiation gram-negative
dyes inhibit the based on bacteria.
growth of gram- fermentation. Sucrose Escherichia coli:
positive bacteria. is an alternate energy lactose fermenter,
source for slow forms blue-black
lactose fermenters,
 allowing quick with a metallic
differentiation from green sheen
pathogens. Other coliform
fermenters: form
pink colonies
Nonfermenters:
Translucent, either
amber or colorless
Gram-negative Deoxychocolate Increasing Enhances the
broth (GN) and citrate salts mannitol, which recovery of enteric
inhibit gram- temporarily favors pathogens from
positive bacteria. the growth of fecal specimens
mannitol-
fermenting, gram-
negative rods (e.g.,
Salmonella and
Shigella spp.)
Hektoen enteric Bile salts inhibit Differential lactose, Detection of
agar (HE) gram-positive and salicin, and sucrose enteric pathogens
many gram- with a pH indicator from feces or from
negative normal bromothymol blue selective
intestinal flora. and ferric salts to enrichment broth
detect hydrogen
sulfaide (H2S). Most
pathogens ferment
one or both sugars
and appear bright
orange to salmon pink
because of the pH
interaction with the
dye. Nonfermenter
appear green to blue
green. H2S
production produce a
black precipitate in
the colonies.
 MacConkey Bile salts and Lactose serves as the Selection for gram-
agar (MAC) crystal violet sole carbohydrate. negative organisms
inhibit most gram- Lactose fermenters and differentiating
positive organisms produce pink or red Enterobacteriaceae
and permit growth colonies; precipitated
of gram-negative bile salts may
rods. surround colonies.
Non-lactose
fermenters appear
colorless or
transparent.
MacConkey- Same as regular Used to isolate
sorbitol (MAC- MacConkey except Escherichia coli
SOR) D-sorbitol is O157:H7
substituted for
lactose. Sorbitol-
negative organisms
are clear and may
indicate E. coli
O157:H7.
Motility test Nonmotile organisms Determine motility
medium grow clearly only on for an organism
stab line, and the Identification and
surrounding medium differentiation of
remains clear. Motile Enterobacteriaceae
organisms move out Shigella and
of the stab line and Klebsiella spp.:
make the medium nonmotile;
appear diffusely Yersinia sp.: motile
cloudy. at room
temperature
Listeria
monocytogenes
(not an
Enterobacteriaceae
): umbrella-shaped
motility
Salmonella- Bile salts, sodium Lactose is the sole Select for
Shigella agar citrate, and brilliant carbohydrate, and Salmonella spp.
(SS) green, which neutral red is the pH And some strains
inhibit gram- indicator. Fermenters of Shigella from
positive and some produce acid and stool specimens.
lactose- change the indicator
fermenting, gram- to pink-red. Sodium
negative rods thiosulfate is added as
normally found in a source of sulfur for
the stool. the production of
hydrogen sulfide.
Also includes ferric
ammonium citrate to
react with H2S and
produce a black
precipitate in the
center of the colony.
Shigella spp. Appear
colorless. Salmonella
spp. Are colorless
with black center.
Selenite broth Selective inhibition Enzymatic Selective
of gram-positive digestion of casein enrichment for the
and many gram- and animal tissue growth of
negative and lactose. Salmonella spp.
organisms.

Urea agar Urea is hydrolyzed to Identification of

form carbon dioxide, Entrobacteriaceae
water, and ammonia. species capable of
Ammonia reacts with producing urease.
components of the (Citrobacter,
medium to form Klebsiella,
ammonium carbonate, Proteus,
raising the pH, which Providencia, and
changes the pH Yersinia spp.)
indicator, phenol red,
to pink. Limited
protein in the medium
prevents protein
metabolism from
 causing a false-
positive reaction.

Xylose-lysine- Sodium Sucrose and lactose in Selective media

deoxychocolate deoxychocolate excess concentrations used to isolate
agar (XLD) inhibits gram- and xylose in lower Salomonella and
positive cocci and amounts. Phenol red Shigella spp. From
some gram- is the pH indicator. stool and other
negative rods. Lysine is included to specimens
Contains less bile detect containing mixed
salts than other decarboxylation. flora.
formulations of Sodium
enteric media (e.g., thiosulfate/ferric
SS, HE) and ammonium citrate
therefore permits allows the production
better recovery. of H2S.
The following types
of colonies may be
seen:
Yellow: Fermentation
of the excess
carbohydrates to
produce acid: because
of the carbohydrate
use, the organisms do
not decarboxylate
lysine, even though
they may have the
enzyme.
Colorless or red:
Produced by
organisms that do not
ferment any of the
sugars.
Yellow to red:
Fermentation of
xylose (yellow), but
because it is in small
amounts, it is used up
quickly, and the
organisms switch to
decarboxylation of
lysine, turning the
medium back to red.
Black Precipitate is
formed from the
production of H2S.

DEFINITIONS:
 Standards that describe these methods
 ANTIMICROBIALS are compounds
that kill or inhibit microorganisms.
 It can be a antibiotics, antiviral,
antifungal or anti parasitic
 ANTIBIOTICS are antimicrobials,
usually of low molecular weight,
produced by microorganisms that inhibit
or kill other microorganisms
 First line of defense
lowest antimicrobial concentration that
completely inhibits visible bacterial
growth
are published and frequently updated by
the Clinical and Laboratory
Standards Institute (CLSI), formerly
the National Committee for Clinical
Laboratory Standards [NCCLS]) –
Nakalagay dito ang mga procedure,
different concentrations of antibiotics
and the different microorganism to be
tested.

The standardized components of

antimicrobial susceptibility testing
include:
 Bacterial inoculum size – Disk Diffusion
(mga nasa petri dish) – 1.5x10^8
CFU/mL
 Broth microdilution - 5x10^5
CFU/ml
 Growth medium (typically a Mueller
Hinton base)
Antimicrobial Susceptibility Testing  pH – 7.2 -7.4
 Cation concentration –
 Performed on bacteria and fungi
Calcium - 25mg/L, Mg –
isolated from clinical specimens to
12.5mg/L
determine which antimicrobial agents
 Blood and serum supplements
might be effective in treating infections
 Thymidine content
caused by these organisms
 Incubation atmosphere – ambient air
 PRIMARY GOAL: to determine
 Incubation temperature - 35°C
whether the bacterial isolate is capable
 Incubation duration – 16 – 18 hours
of expressing resistance to the
antimicrobial agents selected for
 Disk Diffusion:16- 18 hours
treatment
 Broth microdiffusion: – 16-20 hours
o To determine what
antimicrobial agent is needed for
the treatment of infection.
 Often performed by a disk diffusion or
dilution (minimal inhibitory
concentration [MIC]) method. –
 Antimicrobial concentrations – Magbase
sa nasa CLSI Standard but our goal is to
get the lowest concentration of those
antibiotics
Reasons and Indications for
Performing Antimicrobial
Susceptibility Tests
 Antimicrobial susceptibility testing
should be performed on a bacterial isolate
from a clinical specimen if the isolate is
determined to be a probable cause of
the patient’s infection and the
susceptibility of the isolate to
particular antimicrobials cannot be
reliably predicted based on previous
experience with the bacteria at a
specific health care facility.
 Susceptibility testing of isolates can also
provide information on decreases in
the susceptibility of bacteria to
antimicrobials
Factors to Consider When Determining
Whether Testing Is Warranted
 Body site from which the bacterium was
isolated
 Presence of other organisms and quality
of the specimen from which the organism
was grown
o We have to use PURE CULTURE
(contains only one specie)
 Host’s status
Traditional Antimicrobial
Susceptibility Test Methods
1. DISC STORAGE
 Long term storage: -20°C or below in
a non–frost-free freezer
 A working supply of disks can be
stored in a refrigerator at 2°C to 8°C
for at least 1 week.
 Disks should always be stored in a
tightly sealed container with
desiccant.
 The container should be allowed to
warm to room temperature before it is
opened to prevent condensation
Traditional Antimicrobial
Susceptibility Test Methods
 Inoculum Preparation and Use of
McFarland Standards
 Inoculum Preparation
 One of the most critical
steps in susceptibility
testing.
  Prepared by adding cells from volumes of 1% sulfuric acid and
four to five isolated colonies of 1.175% barium chloride
similar colony morphology  McFarland 0.5 standard
growing on a non-inhibitory agar  99.5 mL of 1% sulfuric
medium to a broth medium and acid
then allowing them to grow to the  0.5 mL of 1.175% barium
log phase – Bacteria is actively chloride
multiplying
 Can also be prepared directly by
suspending colonies grown
overnight on an agar plate
directly in broth or saline.
 This direct inoculum suspension
preparation technique is preferred
for bacteria that grow
unpredictably in broth. Because it
does not rely on growth in an
inoculum broth, the use of fresh
 (16- to 24-hour) colonies is  Kapag sumobra ang turbidity we need to
imperative. do dilution ( add more broth or organism)
 Use of a standard inoculum size is to achieve the standard turbidity.
as important as culture purity and
is accomplished by comparing the Traditional Antimicrobial
turbidity of the organism Susceptibility Test Methods
suspension with a turbidity
 Inoculum Preparation and Use of
standard.
McFarland Standards
Traditional Antimicrobial  Inoculum Standardization
Susceptibility Test Methods  The inoculated broth or direct suspension
is vortexed thoroughly.
 Inoculum Preparation and Use of  Under adequate lighting, the tube is
McFarland Standards positioned side by side with the
 McFarland Turbidity McFarland 0.5 standard against a white
Standards card containing several horizontal black
 We are comparing McFarland lines
Standard to the inoculum
 False-susceptible results may METHODS OF Antimicrobial
occur if too few bacteria are Susceptibility Testing:
tested, and false-resistant results
A. DIFFUSION METHOD
may be the outcome of testing too
1. Kirby-Bauer Diffusion Method (most
many bacteria.
common)
 McFarland standards can be
2. Agar Cup Diffusion Method
prepared by adding specific
3. Agar Cylinder Diffusion Methods
 4. Epsilometer or Gradient Diffusion  Results are compared to
Method determine susceptibility or
B. DILUTION METHOD resistance
1. Macrobroth Method or Tube
STEP-BY-STEP PROCEDURE:
Dilution Method ( 1mL or
greater)  Get 4 to 5 isolated colonies from pure
2. Microtube Dilution Method ( culture then Inoculate it using broth
0.05-0.1 mL) 1. Preparation of pure inoculum, using
any of the following:
KIRBY-BAUER METHOD
a) Mueller-Hinton Broth
b) Trypticase Soy Broth
c) Sterile Distilled Water
d) Natural Saline Solution
e) Brain Heart Infusion Broth
2. Standardize pure inoculum, using 0.5
McFarland Standard ( comparing)
i. If standard more turbid than
inoculum → add colonies to
inoculum or incubate inoculum
ii. If inoculum more turbid than
1. Also known as Agar Diffusion Method standard → add distilled water to
or Disk Diffusion Method inoculum
 Used to determine the sensitivity 3. Streak the pure inoculum into the
or resistance of a bacterium to an medium (MHA)
antimicrobial. i. Use a sterile cotton swab and
1. Principle: streak with no space in between
 A standardized suspension of
organism is inoculated into MHA
(Mueller-Hinton Agar)
 If the turbidity matches
the standard turbidity of
McFarland standard the
next step is to stand the
inoculum for at least 15
mins to inoculate
 Paper disk impregnated with
specific antibiotics concentration
are placed into the agar
 After 16-20 hours incubation, the
diameters of the zone of
inhibitions are measured
 RESULT

4. Apply antibiotic discs

i. Using forceps aseptically
ii. Space – at least 15mm each
antibiotic Measuring the zone of Inhibition in
5. Incubate millimeters
i. Normally 35°C for 16-20 hours.
6. Measure the zone of inhibition
i. Instrument: ruler or microcaliper
ii. Unit: mm

7. After that we need to record the result

Test Standardization
Guidelines from CSLI and EUCAST
 Must use fresh, pure overnight
cultures
 Inoculum density ( McFarland 0.5)
 Incubation temperature, time, and
atmosphere
 Disc content
 Media Composition
o Type of agar, plate thickness,
freshness
 o Specific components (NaCl, correlates with a resistance mechanism that
divalenr cations, glucose-6 indicates questionable successful treatment.
phosphate)
 Quality Control
o Reference stains with well-
established susceptibility
profiles

Definitions of Susceptibility Testing

Interpretive Categories
Susceptible (S)
Indicates that the antimicrobial agent
in question may be an appropriate choice for
treating the infection caused by the organism.
Bacterial resistance is absent or at a clinically
insignificant level.
Intermediate (I)
Indicates a number of possibilities,
including:
 The potential utility of the antimicrobial
agent in body sites where it may be
concentrated (e.g., the urinary tract) or if
high concentrations of the drug are used
 Possible effectiveness of the
antimicrobial agent against the isolate,
but possibly less so than against a
susceptible isolate
 Use as an interpretive safety margin to
prevent relatively small changes in test
results from leading to major swings in
interpretive category (e.g., resistant to
susceptible or vice versa)
Resistant (R)
Indicates that the antimicrobial agent
in question may not be an appropriate choice
for treatment, either because the organism is
not inhibited with serum-achievable levels of
the drug or because the test result highly
Dilution Susceptibility Testing Methods
 used to determine the MIC ( lowest
concentration)
 Various concentrations of an
antimicrobial agent are added to broth or
agar media.
 organism is interpreted as nonsusceptible,
susceptible, intermediate, or resistant to
each agent
 We are going to check the lowest
concentration of the tube that has no
turbidity
Dilution Susceptibility Testing Methods
 Antimicrobial Stock Solutions
 must be prepared from reference
standard antimicrobial powders,
not from the pharmaceutical
preparations administered to
patients
 frozen in non–frost-free freezers
 Optimal temperature: below −60° C ,
−20° C storage is acceptable for some
agents
Dilution Susceptibility Testing
Methods
 Broth Macrodilution (Tube Dilution)
Tests
 a twofold serial dilution series, each
containing 1 to 2 mL of antimicrobial
agent, is prepared
 Mueller-Hinton broth
 A standardized suspension of test
bacteria is added to each dilution to
obtain a final bacterial concentration of 5
× 10^5 CFU/mL.
 A growth control tube (broth plus
inoculum) and an uninoculated control
tube (broth only) are used with each test.
 After overnight incubation at 35° C, the
MIC is determined visually as the lowest
concentration that inhibits growth, as
demonstrated by the absence of
turbidity

 Broth Microdilution Tests

 Plastic trays contain between 80
and 100 (usually 96) wells.
 Wells are filled with small
volumes (usually 0.1 mL) of
twofold dilution concentrations of
antimicrobial agent in broth.
 An intermediate dilution of this
inoculum suspension is prepared
in water or saline, and a
multipronged inoculator or other
type of inoculating device is used
 Agar Dilution Tests
to inoculate the wells to obtain a
final concentration of
 Specific volumes of antimicrobial
solutions are dispensed into
approximately 5 ×105CFU/mL (5
premeasured volumes of molten
× 104 CFU/0.1-mL well).
and cooled agar, which is
 A growth control well and
subsequently poured into standard
uninoculated control well are
Petri dishes
included on each tray.
 After overnight incubation at 35°  A series of plates containing
C, the tray is placed on a tray- various concentrations of each
reading device to antimicrobial agent and growth
 facilitate visual examination of control plates without
each well antimicrobial agent are prepared.
 Growth: turbidity, a haze, or a  The agar is allowed to solidify,
pellet in the bottom of the well. and then a standard number of test
 bacteria (104 CFU for aerobes)
are spot inoculated onto each
plate using a multipronged
replicating device
 After overnight incubation, the
MIC is read as the lowest
concentration of antimicrobial
agent that inhibits the visible
growth of the test bacterium (one
or two colonies are ignored)