Clinical Bacteriology BACT211

BACTERIAL CELL COMPONENTS

2ND YEAR | SECOND SEMESTER | PRELIM DATE: FEBRUARY 17, 2024
Prof. Ma. Christy V. Gonzales mvgonzales@fatima.edu.ph
Prof. Pamela Sengson-Sevilla pssengson@fatima.edu.ph
382 235 2533 Online Lecture Discussion

CELL WALL GRAM POSITIVE VS GRAM NEGATIVE BACTERIA

Gram Positive Cell Wall

− also referred to as the peptidoglycan layer or murein
layer − has a very thick protective peptidoglycan (murein)
− peptidoglycan layer
o main component of the cell wall − purple in color
o target of the artificial staining (morphology, size) − has inner membrane
− no outer membrane
− when doing the staining method, it is the target of − teichoic acid
the artificial stain, particularly the gram stain o attracts magnesium and calcium
− other components of the cell wall o provides rigidity
o phospholipid bilayer
− lipoteichoic acid
o some proteins
o regulate autolytic enzymes
− this structure gives the bacterial cell shape and o capable of lysing its own cell
strength to withstand changes in environmental o has antigenic property – stimulate or initiate
osmotic pressures that would otherwise result in cell immune response
lysis o like muramidase
− protects against mechanical disruption of the cell o Streptococcus pneumoniae
o if the cell wall will not protect the cell, the cell  has autolytic enzymes
will be lysed  can lyse their own cell
o through fixation  need to be cultured everyday
− offers some barrier to the passage of larger  does not last long
substances  within 24 hours – subculture and propagate
− a barrier separating the external and internal  presence of autolytic ability
environments that acts like a filter o Streptococcus pyogenes – antistreptolysin O
− bacteria cannot synthesize their own food; instead,
these nutrients always come from the environment
− has external and internal environments
− protect the cell from the osmotic pressure coming
from the environment
 Gram Negative Cell Wall o determine if it is gram-positive and gram-
− thin peptidoglycan layer negative
− red in color o a differential staining technique – utilizes two
− periplasmic space stains
o can only be found in gram-negative bacteria − fixation
o gel-like substances o a bacterial smear step
o assists in the capture of nutrients o affix bacterial cell on the surface of the slide
o contains degradative enzymes o after doing so, it is now ready for staining
 bacteria can secrete degradative enzymes o physical fixation
for the process of entry or invasion  heat
 if there’s a presence of these, the nucleus  pass through flame
will be degraded; thus, leading to the  most common because of alcohol lamp that
disappearance of lining protection, enabling is readily available
the easier attachment of the bacteria  most destructive
o captures nutrient from the environment  routinely done in the laboratory

− outer membrane o chemical fixation

o proteins (porins)  methanol
o phospholipids  there’s a need for the slide to be submerged
o lipopolysaccharide (LPS) to it
o has porin-channel – door, gate − [VIAS] staining process – washing in between
o functions o [1 minute] primary – crystal violet
 acts as a barrier to hydrophobic compounds  initial stain
and harmful substances
o [1 minute] mordant – Gram’s iodine
 acts as a sieve, allowing water-soluble
 strengthen the affinity or attachment of the
molecules to enter through protein-lined
stain to the cell wall
channels called porins
 provides attachment sites that enhance o decolorizer – acetone in 95% ethanol
attachment to host cells  remove excess stain of crystal violet
 most critical and crucial step
− inner membrane
 for differentiation
o peptidoglycan
o periplasmic space o [30 seconds - 1 minute] secondary – safranin
o proteins  counterstain
 red
Gram Staining
Problems arising in Gram Staining
− vigorous rinsing of the smear after staining with
crystal violet
false -
o effect on gram-positive: false +
o effect on gram-negative: no effect

− prolonged decolorization
o effect on gram-positive: take up the safranin
only false -
o effect on gram-negative: no effect
− gram stain
o important step in the workflow of bacterial − insufficient decolorization
identification o effect on gram-positive: no effect
o effect on gram-negative: false +, not take up the
safranin
− insufficient counterstain o Mycobacterium
o effect on gram-positive: no effect o Bacillus
o effect on gram-negative: false + o Erysipelothrix
o Lactobacillus
− dead or degenerating or antibiotic treated bacteria o Listeria
o age of culture (incubation period) – 18 - 24 hours o Clostridium
o beyond that period, it is already considered dead o Rothia
o Aerobic Actinomyces
Gram Variability
o Corynebacterium
− a characteristic exhibited by gram positive bacteria o Kurthia
− in one visual field, it shows purple and red-colored
bacteria in one organism − have a gram-negative reaction not because it has a
− does not take up homogenous stain – others take up gram-negative cell wall but because they do not have
safranin, others take up the crystal violet a cell wall MUA
− does not result into a standard color expected for o Mycoplasma spp.
gram staining for the reason that it is o Ureaplasma spp.
o naturally gram variable o Acholeplasma spp.
o acquired − yeast – gram-positive
− gram variability – red and purple − spirals are very difficult to stain using gram staining,
− gram ghost however stainable spirals are usually gram negative
o colorless or faint blue – already dead, − Mycobacterium and Nocardia spp.
degenerated, treated with antibiotic, or o have a gram-positive cell wall structure, however
nonexistent cell wall because 60% of the cell wall is made of
hydrophobic lipids mainly mycolic acid, it affects
− natural gram variability its permeability this makes it difficult to gram
o Mobiluncus spp. stain
o Gardnerella vaginalis o acid-fast bacteria
− acquired gram variability (for gram positive bacteria) o lipid content – 60%
− contributing factors PUBS o fatty acid – mycolic acid
o use of old culture o waxy
o pH of staining reagents
 acidic – for basic cell components
ACID-FAST CELL WALL
 basic – for acidic cell components
− contain a waxy layer of glycolipids and fatty acids
o bacterial autolysis (mycolic acid)
 no more space for the stain to penetrate
ACID- FAST STAINING
o staining reaction time
− specifically designed for a subset of bacteria whose
 vary depending on the manufacturer’s
cell walls contain long-chain fatty (mycolic) acids
instruction
− mycolic acids render the cells resistant to
Guiding Rules in the Gram Stain Reaction of Medically decolorization, even with acid alcohol decolorizers
Important Bacteria − Mycobacteria
o most commonly encountered acid-fast bacteria
− [NVM] all cocci are gram positive except
o typified by Mycobacterium tuberculosis –
o Neisseria
etiologic agent of tuberculosis
o Veillonella
o Branhamella or Moraxella − partially acid-fast organism LRN
 cocci are round or spherical in shape o Nocardia
 purple under the microscope o Rhodococcus
o Legionella micdadei
− [MyBELLCRACK] all bacilli are gram negative except
− distinctly acid fast Summary of Acid-Fast Staining Technique
o Cryptosporidium
o Isospora
− three methods
o Ziehl-Neelsen Method
o Kinyoun Method
o Fluorochrome or Fluorescent Method

Acid Fast (Ziehl-Neelsen or Hot Method)

Reporting
Gram Stain Reporting
− [1] gram stain reaction
− [2] morphology
o cocci, bacilli, diplococci, diplobacilli, or spiral
o if it’s diplococci, diplobacilli – no need to state
the arrangement
− [3] arrangement
o singly, pair, tetrad, chain, or cluster
− heat has two purposes – it penetrates, mordant
− counterstaining: malachite green or methylene blue − example
o gram positive cocci in cluster – Staphylococcus
Kinyoun Acid-Fast Method aureus
− does not require the use of heat or boiling water, o gram positive diplococci – Streptococcus
minimizing safety concerns during the procedure pneumoniae
− because of a higher concentration of phenol in the o gram negative diplococci – Neisseria
primary stain solution, heat is not required for the meningitidis
intracellular penetration of carbol fuchsin o gram positive cocci in chain – Streptococcus
− referred to as the “cold” method pyogenes
− phenol penetrates the waxy layer o gram positive bacilli in chain – Corynebacterium
− mordant is needed, so Tergitol is added diphtheriae
− counterstaining: malachite green or methylene blue Acid-Fast Smear Reporting
− mordanting: chemical used is Tergitol
− decolorization: acid alcohol
o 3% sulfuric acid in 95% ethanol
o 0.5% hydrochloric in 95% ethanol – in other
references

Hot Method and Cold Method

− acid fast: red
− non-acid fast: blue or green

Fluorochrome or Fluorescent Method

− Truant’s
− acid fast: bright yellow-orange bacilli against a dark
background
according to Centers for Disease Control and Prevention
− non-acid fast: no fluorescence
− there are a lot of reporting schemes to be considered
− in acid-fast, bacteria will be counted
− AFB – acid fast bacilli
− National Standard Reporting Scale (NSRS) selective permeability of the membrane to
o under OIO macromolecules
o adapted reporting scheme in the Philippines o site of electron chain transport necessary for
o has additional requirement energy production, hence maintaining the
viability of the bacterial cell
− National Tuberculosis Association o houses enzymes involved in outer membrane
o also under OIO and cell wall synthesis, and the assembly and
secretion of extracytoplasmic and extracellular
substances
− present in both gram-positive and gram-negative
bacteria and is the deepest layer of the cell envelope
− serves as an additional osmotic barrier and is
functionally similar to the membranes of several of
eukaryotic cellular organelles

MESOSOMES
− folds or invagination along the length of the
cytoplasmic/plasma membrane which serves as a
point of attachment for chromosomes
− where nucleoid region is attached to
− located beside the plasma membrane
− can be easily seen

FREE RIBOSOMES
− present in the cytoplasm of the bacterial cells
− sites of protein synthesis in bacterial cells
− has a size of 70S comprised of two subunits being 50S
and 30S
− S – Svedberg unit; centrifugation unit

INCLUSION BODIES
− serves as depot or storage deposits under certain
circumstances such as limited or excess of a
particular nutrient
− these may accumulate, precipitate out, and form an
inclusion body which is not bounded by a membrane
freely floating in the cytoplasm of the bacterial cell
− may be in the form of
PARTS INTERNAL TO THE CELL WALL o glycogen (carbohydrate reserves
CYTOPLASMIC/PLASMA MEMBRANE o polyphosphates (ATP reserves)
o poly-β-hydroxybutyric acid (lipid reserves)
− a phospholipid bilayer embedded with proteins that
envelops the cytoplasm but does not contain sterols − much granules
(except Mycoplasma/Ureaplasma) o contains lipids
− functions o Mycobacterium tuberculosis
o separates the intracellular components of the − Volutin/ Babes-Ernst Bodies/ Metachromatic
bacterial cell from the extracellular environment Granules
o acts as an osmotic barrier between the inside o contains polyphosphates or inorganic
and outside of the bacterial cell by allowing phosphates
o Corynebacterium diphtheriae
− Bipolar Bodies FLAGELLA
o prominent staining of each end of the bacilli
Yersinia pestis using methylene blue or WAYSON
stain giving it a “safety pin appearance”

BACTERIAL SPORES or ENDOSPORES

− complex multilayered highly refractile structure that
can be found within the cytoplasm of the vegetative
cell of the bacteria or in the environment when the
bacterial cell has been disintegrated
− serves as a resting or hibernating stage for bacteria
when they are exposed to unfavorable conditions
− highly resistant to desiccation, heat, physical
disruption, and chemical agents – that’s why it’s a
virulence factor
− spore forming bacteria
o Bacillus spp.
o Clostridium spp.
− main composition – calcium dipicolinate or calcium-
dipicolinic acid complex
PARTS EXTERNAL TO THE CELL WALL
PILI
− pili (plural) or pilus (singular)
− protein projections that are thinner and shorter than
flagella and are most usually found in gram negative
bacteria
− terms fimbriae (Latin, fringe) and pili (Latin, hairs)
are commonly used synonymously (Brinton, 1965;
Duguid & Anderson, 1967)
− composition: made up of protein material, pilin
− common or somatic or ordinary pili
o usually shorter, numerous, sticky hair-like
appendages
o primarily used for adherence or attachment to
one another, host cells, and environment
surfaces
o structurally speaking, it is one of the attachment
methods they use
− sex or fertility or F plus
o usually longer and singular, long and hollow
protein tubes
o used during mating
o primarily used for bacterial conjugation
 transfer or exchange of genetic materials
between bacterial cell using the fertility pilus
 new strain will be formed because there’s a
new genetic sequence
− flagella (plural) or flagellum (singular)
− exterior protein filaments or whip-like projections
which is embedded in the cell envelope with a motor
attached in a basal body responsible for its propeller-
like rotation of the flagella which makes bacteria
move
− flagellated bacteria are said to be moving of motile
− longer than pili
− for locomotion
− flagella of bacteria are different from eukaryotes
o prokaryotes – simple
o eukaryotes – complex
− propelling motions
o prokaryotes – rotary action
o eukaryotes – sliding coordinated motion
− bacteria are moving towards food; they always have
a target – either to the food or another bacterium for
fertility
− has antigenic property
− composition: made up of protein material, flagellin
− associated with H Antigen (Hauch Antigen)
o used to identify species
o very useful is serologically typing and identifying
species of Salmonella
o used in serology
− Peritrichous – multiple flagella
− Amphitrichous – two flagella at both ends
− Lophotrichous – several flagella at one end
− Monotrichous or Polar – one flagellum at one end
− bacteria have different flagella, if they are motile
− Vibrio and Campylobacter – peritrichous
GLYCOCALYX
− exterior high molecular weight appendage or
structure usually made up of polysaccharide
polymers or sometime polypeptides which are
produced be certain bacteria depending on
 environmental and growth conditions surrounding
the bacterial cell
− occurs outside the bacteria
− like a covering of the bacteria
− two forms
o capsule
 uniform and condensed organized material
 firmly attached to the cell wall of the
bacteria
 associated with K Antigen (Kapsule Antigen)
and a slight change in the capsular
 organized, and condensed layer
 anti-phagocytic
o slime layer
 loose, diffused
 not firmly attached to the cell wall

− both capsule and flagella acts for phagocytosis and

attachment
− some of the medically important capsulated bacteria NHSKB
o Neisseria meningitidis
o Haemophilus influenzae serotype b
o Streptococcus pneumoniae
o Klebsiella pneumoniae
o Bacillus anthracis

SLIME LAYER

− loose or diffused, thick, viscous unorganized material

that appears to be detached from the bacterial or
not firmly attached to the cell wall of the bacteria
− another form of
− functions
o primarily it serves as a form of protection from
phagocytosis, or in some instances
o it helps the bacteria to adhere to host tissues or
synthetic implants such as prosthetic heart
valves
 Clinical Bacteriology
BACTERIAL CULTIVATION
2ND YEAR | SECOND SEMESTER | PRELIM
Prof. Ma. Christy V. Gonzales
Prof. Pamela Sengson-Sevilla
382 235 2533
BACTERIAL CULTIVATION
− nutritional requirements
− environmental requirements
− bacterial growth phase
− different culture media
BACTERIAL GROWTH
− refers to the increase in the number of bacteria
rather than in size
− basically, bacteria grow in number and not in size
− when we grow bacteria, there are several nutritional
and environmental factors that they need for them
to successfully multiply and increase in number
− each bacteria have different sets of nutritional and
environmental factor requirements
ODU − this growth is affected by various factors such as
o optimum growth requirements
o dynamics of growth
o use of a medium that can be artificially prepared
in the laboratory
− bacterial growth requirements
o nutritional – carbon, nitrogen, energy [CNE]
 some, but not all bacteria are needing co-
factors such as mineral elements
o environmental
NUTRITIONAL REQUIREMENTS CNEEMSAW
Carbon
− needed for the synthesis of cellular components or
constituents
− represents almost 50% of the dry weight of a
bacterium
− most needed out of all the requirements
− one of the major requirements
− autotroph – bacteria that gets their carbon dioxide
from the air
− heterotroph – bacteria that gets their carbon dioxide
from organic compounds in the culture media
o most normal flora in the body are heterotrophs
BACT211
DATE: FEBRUARY 10, 2024
mvgonzales@fatima.edu.ph
pssengson@fatima.edu.ph
Asynchronous Discussion
o most common organic compound used in
microbiology lab
 carbohydrates – lactose, glucose, sucrose
Energy Source
− from adenosine triphosphate (ATP)
− needed to perform metabolic and cellular functions
− phototroph – bacteria that uses light as their energy
source
− chemotroph – bacteria that uses chemical energy as
their energy source
o some are getting it from organic compounds
− some bacteria require smaller amounts of molecules
o phosphate (nucleic acids)
o phospholipids (cell membrane)
o sulfur (protein synthesis)
− these add 4% of the dry weight
Electron Source
− lithotroph – from inorganic molecule (Fe2+)
− organotroph – from organic molecule
− when electrons are needed NAD and FAD are
reduced
− NADH reduced from NAD
− FADH2 reduced from FAD
Nitrogen
− needed for the synthesis of proteins and nucleic
acids
− free nitrogen from the air or atmosphere
− anaerobic bacteria must not have any contact with
the air that’s why nitrogenous compounds (e.g.,
peptone, yeast, beef extract) must be added in the
culture media
− 14% of the dry weight of bacterium
Water/Moisture/ Humidity
− bacterial cell is 70% water
o like a sealed plate of moisture
 − humidity level must be maintained at 70%
o put a container of water inside the incubator and
monitor regularly to prevent it from drying out
o must be checked daily because there’s a
possibility of having growth of molds and fungi in
the incubator, which can then be contaminants
of the cultures
o some incubators are equipped with humidity
regulator
Mineral Elements
− needed as co-factors in various metabolic process of
the bacteria
− some bacteria need co-factors
− like Ca2+, Mg2+, Fe2+, Sulfates, Phosphates
Salt
− bacteria can tolerate salt concentration below 6%
− there are certain bacteria that can survive high salt
concentration environment hence they’re called as
BEVS halophilic bacteria or halophiles (salt-loving)
− Staphylococcus spp.
o usually grown in MSA, which has 7% of sodium
chloride
− Enterococcus spp.
− Vibrio spp. except Vibrio cholerae & Vibiro mimicus
o can tolerate up to 10% of salt concentration
− Bacillus spp.
Additional/Special Growth Requirements
− there are bacteria that are very difficult to grow,
fastidious organisms, which require special or
additional requirements to grow in culture media
− Haemophilus spp.
o blood-loving organisms
o requires X and V factor
o some requires both and some only requires one
of the factors
o when culturing, add blood to the culture
medium
− X Factor
o hemin or hematin
o degradation product of hemoglobin (protein
component of RBC)
o CAP – chocolate agar plate (lysed RBC)
 has both X and V factors
 lysed RBC is obtained when physical method
is applied like heat
 heat inactivates the NADase, therefore the
NAD will not be hydrolyzed, and will lead to
the existence or retention of the V factor
o BAP – blood agar plate (intact RBC)
 only have the X factor and have no V factors
 intact RBC have the enzyme NADase
¤ hydrolyzes the NAD
¤ therefore, the V factor is degraded, and
only the X factor remains
− when CAP is unavailable and Haemophilus spp.
requires both X and V factor, we use BAP
o satellitism is used
 when culturing suspected Haemophilus spp.,
another culture of organisms, which is
usually Staphylococcus aureus, must be
surrounding them
 Staphylococcus aureus has beta-hemolytic
factor or beta-hemolysin which can be the
source and liberate the V factor
− V Factor
o Nicotinamide Adenine Dinucleotide (NAD)
o vitamins
− source of blood
o sheep’s blood is 5% defibrinated
 can also get from
¤ horse
¤ rabbit
¤ human blood and should be type O
 type O has non-specific inhibitor
ENVIRONMENTAL REQUIREMENTS
− from the atmosphere like oxygen and carbon dioxide
GASEOUS REQUIREMENT
Aerobe
− in the environment, we usually have 18% of O2
− bacteria that grow, live, and survive in the presence
of oxygen
− strict or obligate aerobe 2B FLMMNP
o absolutely requires oxygen to grow, live, and
survive
o Micrococcus spp.
o Mycobacterium spp.
o Pseudomonas spp.
o Neisseria spp.
o Brucella spp.
o Francisella spp.
 o Bordetella spp. o Haemophilus spp.
o Leptospira spp. o Aggregatibacter spp.
− facultative anaerobe ESS o Cardiobacterium spp.
o bacteria that have the ability to grow, live, & o Eikenella spp.
survive in small concentration of oxygen o Kingella spp.
environment − some Streptococcus pneumoniae
o they can grow, live, and survive with or without
TEMPERATURE REQUIREMENT
oxygen
− most pathogenic bacteria would grow at
o Staphylococcus spp.
o Streptococcus spp. temperature between 35-37C, hence incubator in
o Family Enterobacteriaceae the laboratory is usually set and maintained within
this temperature range for routine isolation of
− microaerophilic CHAS pathogens
o bacteria that prefer small concentration of − mesophilic – 20C to 40C
oxygen environment − psychrophilic or cryophilic – 0C to 20C
o approximately 2%-10% − thermophilic – 50C to 60C
o Campylobacter spp. − hyperthermophilic or extremely thermophilic –
o Helicobacter spp. 80C to 110C, commonly the spore forming bacteria
o Arcobacter spp. − eurithermophilic – grows in wide range of
o some Streptococcus spp. temperature
Anaerobe − sternothermophilic – grows in narrow range of
− bacteria that grow, live, and survive in the absence temperature
of oxygen PH REQUIREMENT
− strict or obligate anaerobe BCFPP − most pathogenic bacteria can grow in a neutral or
o absolutely do not require oxygen to grow, live, slightly alkaline environment (pH 7.0 – 7.5), hence
and survive most culture media used in routine isolation of
o 0% oxygen pathogens is adjusted to this pH range
o many Clostridum spp. such as Clostridium noyvi − acidophilic – acid loving bacteria
o most Bacteroides spp. o Lactobacillus acidophilus
o Fusobacterium spp.
− alkaliphilic – alkali loving bacteria
o Peptostreptococcus spp.
o Gardnerella vaginalis
o Porphyromonas spp.
− osmophilic – high osmotic pressure
− aerotolerant anaerobe BCLP o Archaebacteria spp.
o bacteria that do not require oxygen but may
tolerate or withstand limited exposure to oxygen
BACTERIAL GROWTH PHASE
o some Clostridium spp. such as Clostridium
− refers to the stages of bacterial growth
perfringens
− bacteria are replicating via binary fission
o Bacteroides fragilis
o asexual reproduction
o most strains of Proprionibacterium and
o one cell divides into two cells
Lactobacillus
− generation time or doubling time
Capnophilic o time required for one cell to divide into two cells
− organism/bacteria o could take up from 20 mins (Escherichia coli) up
− capnophiles to 24 hours (slow growing bacteria like
− in the environment, there is 1-3% of CO2 Mycobacterium tuberculosis) in a culture media
− bacteria that require 5%- 10% CO2 for growth
− (NHACEK Group) − growth rate – number of generations per hour
− x axis – duration or time
o Neisseria spp.
− y axis – logarithm – number of viable (alive) cells

Growth Rate (Constant)
− refers to the number of generations per hour
− zero, positive, or negative
Lag Phase
− growth rate: constant or zero
− the bacteria are adjusting to the environment
− no cell division and no increase in number
− there is cellular activity – protein and DNA synthesis
which are needed for cell division
Log or Exponential or Logarithmic Growth Phase
− growth rate: positive
− has cell division and increase in number
− rapid generation and doubling time
− the bacteria are most metabolically active, so they
are susceptible to antimicrobial agents
Maximum Stationary or Plateau Phase
− growth rate: constant or zero
− the nutrients are depleted
− they produce toxins that are very harmful to those
microorganisms
− rate of cell division equates to the rate of cell death
− has cell division but they don’t increase in number
Decline or Death Phase
− growth rate: negative
− due to unfavorable environment, there’s no
bacterial growth
− there are some bacteria that still divides, but there is
a larger number of those who are dying than those
who are viable
− number of non-viable is greater than viable
CULTURE MEDIA
− an artificial preparation in the laboratory which
contains basic foundation of nutrients and a
solidifying agent (if needed) to support the growth of
microorganisms
− additional substances may be added to enrich the
media for growth of microorganisms that are very
difficult to grow, especially if it is a fastidious
organism
− artificially mimicking the environment and nutrition
that the bacteria need, that’s why different culture
media is used depending on what organism is
subjected for growth
TERMINOLOGIES
Culture
− noun: growth of microorganism
− verb: to grow/to cultivate microorganism
Inoculate or Plant or Cultivate
− introducing the microorganism to the culture media
Transplant or Subculture
− transfer of microorganisms from one culture
medium to another
− example – from nutrient agar to MacConkey agar
CLASSIFICATION OF CULTURE MEDIA
ACCORDING TO COMPOSITION
Synthetic or Chemically Defined
− composed of known and exact amounts of pure
chemical substances
− for autotrophic microorganisms
− usually used for bacteria
Non-Synthetic or Non-Chemically Defined or Complex
− composed of complex materials that are rich in
vitamins and nutrients that are not usually
represented by a chemical formula such as
 o peptones Tubed
o beef or yeast extract
o plant extracts
− combinations of both chemicals and complex
materials
− usually used for bacteria
− usually, container in glass tubes such as Wassermann
Tissue Culture Media tubes with different volume capacity (3mL, 5mL,
− live cells harvested from organs of humans and 10mL) or in a tube with a flat bottom and a screw cap
animals that supports the growth of obligate − have different orientations
intracellular organisms that cannot grow in o slant – solid
artificially prepared culture media o slant/deep of butt slant – solid
− usually used for viruses o deep or butt – solid
o broth – liquid
ACCORDING TO PHYSICAL STATE/CONSISTENCY Bottled
Liquid − culture media contained in a glass bottle that is
− a culture medium that doesn’t contain a solidifying usually used for blood culture
agent − Bacte Alert (most common) with SPS, which is an
− seen in tube medium anticoagulant for blood
− nutrients are dissolved in water with no solidifying
agent
ACCORDING TO FUNCTION/USE
− most common solidifying agent is agar
General Purpose or Primary or Basic or Basal or
Semi-Solid Supportive or General Isolation Culture Media
− commonly for motility − contains basic nutritional requirements to support
− seen in tube medium the growth of non-fastidious microorganisms
− a culture medium that contains 0.5%-1% agar − called as basal because it is a base medium in the
− liquid with 0.5%-1% agar preparation of other culture media
− agar is used as solidifying agent − example
o not easily degraded by the bacteria o Nutrient Agar (NA)
o can be melted and resolidify upon cooling at o Nutrient Broth (NB)
32C, and still be melted when boiled o Tryptic Soy Broth (TSB)
o Tryptic Soy Agar (TSA)
− Sulfide Indole Motility (SIM) Medium – used for
observation of hydrogen sulfide gas production, Enriched Culture Media
indole production, and motility (Enterobacteriaceae) − contains the basic nutritional requirements to
support the growth of non-fastidious
Solid
microorganisms with additives, enriching
− a culture medium which contains 1.5-3% (2-3%) agar
substances, or supplements to support the growth of
− seen in plated and tube medium fastidious microorganisms
− general purpose with the addition of additives –
ACCORDING TO MANNER OF special requirements of bacteria like blood
DISPENSING/FORMATION − example
Plated o Blood Agar Plate (BAP)
− usually contained in a container that can be made of o Chocolate Agar Plate (CAP)
glass (Pyrex) or disposable plastic (petri dishes)
Enrichment Broth
− 100mm in diameter – standard
− a primary media used to support or favor the
− 150 mm – used for Anti-Susceptibility Testing (AST)
selective growth of pathogens in a specimen, such as
 stool or sputum, where the number of normal floras
outnumber the pathogens
− example
o watery stool is a characteristic of Vibrio spp.
 placed on Alkaline Peptone Water (APW)
 high salt concentration, high pH level
(alkaline)
 not an inhibitor but becomes favorable of
the growth of those pathogens that you
would like to grow
 tolerated by Vibrio spp.
 not supporting other microorganisms like
Escherichia coli and Enterococcus that are
also present in stool
 if inhibitor is added, then it is already
selective
− examples of Enrichment Broth
o Alkaline Peptone Water (APW)
 used to selectively favor the growth of Vibrio
while inhibiting all other normal intestinal
flora due its high pH
 high salt concentration
 unfavorable for the normal flora of the
intestine
o Selenite F Broth
 used to selectively favor the growth of
Salmonella while inhibiting all other normal
intestinal flora
o Thioglycolate Broth (THIO)
 an enrichment broth for anaerobic bacteria
but should be used solely in the isolation of
anaerobic bacteria since it can also grow
aerobes and facultative anaerobes
 anaerobic bacteria usually grow at the
bottom of THIO
o GN Broth (Gram Negative Broth)
 used to selectively favor the growth of
Salmonella and Shigella while inhibiting all
other normal intestinal flora
o Todd-Hewitt Broth
 a liquid enrichment recommended for the
production of Streptococcal haemolysin
(virulence factor of Streptococcus) and the
cultivation of streptococci prior to
serological grouping
Transport Culture Media
− a primary isolation culture media which maintains
the viability of bacteria without allowing rapid
multiplication if there is an anticipated delay in
bringing the specimen collected bedside or remotely
to the laboratory
− used when we are anticipating some delay in
transportation or collection
− example
o Stuart’s Transport Medium (Neisseria spp.)
o Cary-Blair (Vibrio spp.)
o Amie’s (Neisseria spp.) – usually with charcoal
o brands
 Transgrow
 JEMBEC – John E. Martin Biological
Environmental Chamber
Selective and Differential Culture Media
− not all selective culture media is differential culture
media, but all differentials are selective
− selective culture media
o favors the growth of the organism of interest
using inhibitors added in the culture media
o addition of inhibitors
− differential culture media
o contains indicators which change in color as a
result of a product produced be a chemical
reaction in the components of the media such as
glucose
o it is important to remember and understand that
not all selective culture mediums are differential
but all differential culture media are selective
o contains two characteristics
o example
 there is mixed organism in a specimen
 we are looking for lactose fermenter
 we should use culture media that can
differentiate those who can ferment lactose
from those who can’t
 frequently, we are looking for the color
 in McConkey Agar
¤ lactose fermenter would appear color
pink
¤ not lactose fermenter would appear
colorless
 INHIBITORS IN CULTURE MEDIA
Inhibitors for Gram Positive Bacteria
− selective for gram negative
− dyes – crystal violet, eosin, methylene blue, brilliant
green, etc.
− chemicals – bismuth sulfite, bile salts (sodium
deoxycholate), thiosulfate, citrate, etc.
− antibiotics – Vancomycin
Inhibitors for Gram Negative Bacteria
− selective for gram positive
− dyes – basic fuchsin and thionine for Brucella abortus
− chemicals – potassium tellurite, sodium azide,
phenylethyl alcohol
− antibiotics – Colistin, Nalidixic Acid, Trimethoprim
(Proteus)
Inhibitors for Fungi
− antibiotics – Nystatin, Anisomycin, Ampothericin B.
INDICATORS IN CULTURE MEDIA
− dyes and chemical substances such as pH indicators
(acid and alkaline products)
− not an inhibitor but is an indicator for a specific
reaction
− most commonly used indicator – pH indicator
− added in any kind of culture media either selective or
differential media
− bacteria metabolize substances like CO2 from organic
materials like carbohydrates
− bacterial metabolism
o ferment – without O2
o oxidize – with O2
− organic compounds’ products are usually acids or
alcohol
− example
o we are looking for bacteria that can ferment
lactose
o we can detect those who have the ability of
lactose fermentation with detecting an acid with
the use of pH indicator
o there are microorganisms that can produce H2S
o hydrogen sulfide gas is colorless, and we can
detect the presence of its production with the
use of H2S indicator
MOST COMMONLY AND ROUTINELY USED SELECTIVE
& DIFFERENTIAL CULTURE MEDIA IN THE LABORATORY
Eosin Methylene Blue (EMB) Agar
− original color: dark violet
− selective for: gram negative enteric bacilli
o for Enterobacteriaceae family
o can be categorized
 Rapid Lactose Fermenter (RLF) – 18-24 hours
produces dark violet
 Late Lactose Fermenter (LLF) – 36-72 hours
still produces dark violet
 Non-Lactose Fermenter (NLF)
o when using organic compound, there are
bacteria that can withstand fermentation, while
others can endure oxidation
o most Enterobacteriaceae are using
fermentation, meaning even without the use of
oxidation, they can still utilize organic
compounds such as lactose
o product is acid
− inhibitors: eosin and methylene blue
o inhibits gram positive
− differential indicators: eosin & methylene blue (pH)
o for it to be differential, we need lactose to be the
differential medium or component
o lactose is also used as carbon source
Differentiation of Family Enterobacteriaceae
Based on Lactose Fermentation
− RLF (have characteristic colony
upon growing in EMB)
o Escherichia
 medium-sized dark violet
colonies with greenish
metallic sheen
o Enterobacter
 medium-sized dark violet colonies with or
without dark center
o Klebsiella
 large sized colonies with mucoid dark violet
colonies with or without dark center
− LLF
o Hafnia, Serratia, Citrobacter
o Salmonella arizonae
o Shigella sonnei
o Yersinia enterocolitica

− NLF
Salmonella-Shigella (SSA) Agar
o all Salmonella except S. arizonae
− original color: light orange
o all Shigella except S. sonnei
− selective for: Salmonella and Shigella spp.
o all Yersinia except enterocolitica
o Salmonella spp. is H2S producers
o Proteus
o Providencia − inhibitors: brilliant green, bile salts, citrate
o Morganella o some gram positive and gram negative
o Edwardsiella − differential indicators:
Mc Conkey (Mc/Mac) Agar o lactose fermenter
o pH indicator: neutral red
− original color: light pink
o H2S Indicator: ferric ammonium citrate
− selective for: gram negative enteric bacilli
o Sulfur Source: sodium thiosulfate
o for Enterobacteriaceae family
o acid: red or dark pink
− inhibitors: crystal violet, bile salts, citrate  lactose fermenter (H2S-) like Escherichia,
− differential indicators: neutral red (pH) Klebsiella, and Enterobacter [EKE – rapid
o lactose fermenter lactose fermenter]
o acid: red or dark pink
o no acid: colorless
o no acid: colorless
 Shigella (H2S-) and Salmonella (H2S+) spp.
o alkaline: yellow
o H2S+: black because of indicator and source
− Escherichia coli and Enterobacter aerogenes are both
RLF – color pink
− Proteus vulgaris and Salmonella typhimurium are
both NLF – colorless
− Staphylococcus aureus does not ferment
carbohydrate in MAC – remain the same, no growth,
and unable to utilize carbohydrates (light pink)
− once the carbohydrate is fermented by bacteria, the
product will be acid; thus, lowering the pH

Hektoen Enteric (HEA) Agar

− original color: dark green
− selective for: gram negative enteric bacilli
− inhibitors: bile salts, citrate
− differential indicators:
o lactose fermenter
o pH indicator: bromthymol or bromothymol blue
− (BTB)
 acid: orange
 no acid: green
 alkaline: blue
o H2S Indicator: ferric ammonium citrate
 o Sulfur Source: sodium thiosulfate
o can detect H2S production
o still blackening because the components used
are the same
Bismuth Sulfite (BSA) Agar
− selective for: Salmonella spp. (Salm. typhi has
distinct appearance)
o Salm. – genera
o enterica – specie
o typhi – subspecie
o in the nomenclature, if the specie is not written,
the subspecie should not be in italicized or
underlined format
− inhibitors: bismuth sulfite
− CHO incorporated: glucose
− Salmonella typhi colonies appear as black colonies
with silver metallic sheen
Brilliant Green (BGA) Agar
− selective for: Salmonella spp. except for Salmonella
typhi
− inhibitors: brilliant green
− CHO incorporated: lactose
− Salmonella spp. colonies appear as white colonies,
resembling a snowflake surrounded by brilliant red
medium
Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar
− selective, differential agar
− original color: light green or olive green
− selective for: Vibrio spp.
− inhibitors: thiosulfate, citrate, bile salts
o for some gram positive and gram-negative
bacteria
− differential indicators: bromothymol blue (BTB)
o sucrose fermenter – yellow
o non-sucrose fermenter – blue to green
o if carbohydrate, it has a partner of pH indicator
Mannitol Salt (MSA) Agar
− original color: light or salmon pink
− selective for: Staphylococcus spp.
− inhibitors: high concentration of salts (7.5%)
o can be tolerated by Staph.
o mannitol fermenter
o most bacteria don’t like salt
− differential indicators: phenol red
o acid: yellow
o no acid: red, dark pink
Lowenstein Jensen (LJ) Medium
− original color: light green
− selective for: Mycobacterium spp.
− inhibitors: malachite green
o respiratory tract normal flora
Sputum Sample needs to undergo two processes:
− decongested/digested
o to dissolve the thick mucus/mucin that might be
trapping the bacteria in the sample
o N-acetyl-L-cysteine (NALC) is usually used
− decontaminated
o to eliminate normal flora that contaminates the
sample
o NaOH is usually used as a partner of NALC
− Mycobacterium spp. have groups
o photochromogen – have pigments when
incubated with light, slow grower
o scotochromogen – produce pigments when
incubated without light, slow grower
o non-photochromogen – does not produce
pigments with or without light, slow grower
o rapid growers
Mycobacterium spp.
− Middlebrook 7H10 or 7H11 Medium
CHARACTERISTIC/BIOCHEMICAL CULTURE MEDIA
− used in the fifth step
− determine characteristics
Sulfide Indole Motility Medium (SIM)

Selective Medium for Neisseria spp.
− Proteus swarms or having concentric rings
− Proteus contamination will overwhelm the
morphology of Neisseria
− usually composed of chocolate agar base with
antibiotics
CULTURE MEDIA FOR ANTIBIOTIC
SUSCEPTIBILITY/SENSITIVITY TESTING (AST)
− sixth step
− determine what panel of antibiotic is capable of
inhibiting bacteria
− two reactions
o resistant – not capable of inhibiting bacteria
o susceptible – capable of inhibiting bacteria
− used for observation of hydrogen sulfide gas
production, indole production, and motility
o for motility, usually in a semi-solid media in butt
orientation
o inoculate organism using the inoculating needle
up until the middle part of the tube only
o then incubate it at 37oC for 18-24 hours
o if positive, there’s a presence of turbidity or
haziness around the line of stab
 meaning, from the line of stab, the bacteria
will move away from it because of the
presence of flagella for they are motile
− pink right at the top of the medium will be visible
when there’s an addition of Erlich or Kovac’s reagent
− indole+ – pink ring upon the addition of Erlich or
Kovac’s reagent
− H2S+ – blackening
Methyl Red (MR)

Most bacteria
− Mueller Hinton Agar (MHA) and Mueller Hinton
Broth (MHB)
o plated agar – presence of colonies on the surface
− used for the detection of bacterial pathogen that
of the medium signifies growth
metabolize glucose using the mixed acid pathway
o broth – turbidity signifies growth
− +MR, +VP (left tube)
Haemophilus spp. − should be in separate medium – one for MR, one for
− Mueller Hinton with Chocolate Agar Base or VP
Haemophilus Test Medium (HTM) Agar − using MR VP broth
Voges-Proskauer (VP)
− used for the detection of bacterial pathogen that
metabolizes glucose using the butylene glycol
pathway

Simmon Citrate Agar (SCA)

Moeller’s Broth
− used to detect lysine decarboxylation, ornithine
decarboxylation, and arginine dihydrolysis

Stuart’s Urea Broth or Christensen Urea Agar

− used for the detection of bacterial pathogen that can
utilize citrate as a sole source of carbon
− growth or the medium became color blue, it is
positive for SCA
− growth+ and green medium – positive for SCA or
utilization of citrate
− growth+ and blue medium– positive for SCA or
utilization of citrate − used to detect bacterial pathogen that hydrolyze
Triple Sugar Iron Agar (TSI) urea substrate
− most common
− does the bacteria has urease enzyme
− yellow – negative
− salmon pink – positive
− can be a broth or solid medium, but still tubed

− used for the determination of bacterial pathogen’s

ability to ferment
o glucose/dextrose (1 part)
o sucrose (10 parts)
o lactose (10 parts)
− can also detect sulfide production and gas
production

Lysine Iron Agar (LIA)

− used for the determination of bacterial pathogen’s
ability to decarboxylate or deaminate the amino
acid, lysine
− can also detect glucose fermentation, sulfide
production, and gas production
 Clinical Bacteriology BACT211
BACTERIAL MORPHOLOGY
2ND YEAR | SECOND SEMESTER | PRELIM DATE: FEBRUARY 17, 2024
Prof. Ma. Christy V. Gonzales mvgonzales@fatima.edu.ph
Prof. Pamela Sengson-Sevilla pssengson@fatima.edu.ph
382 235 2533 Online Lecture Discussion

BACTERIAL MORPHOLOGY − propagation is done in order for the characteristics

− 0.25 to 1 μm in width
− 1 to 3 μm in length (0.4-2 um –Mahon)
− staining procedure separates almost all medically
relevant bacteria into two general types
o gram positive
o gram negative
− common bacterial cellular morphologies
o cocci – round, spherical, circular
o coccobacilli – ovoid or oval
o bacillus – rod-shaped or elongated
o fusiform – pointed ends
o curved
o spiral shapes
 spirochetes
syphilis ¤ Treponema pallidum
¤ Leptospira
Bacterial Identification
Step 1: Specimen Collection
− it is our responsibility to collect majority of the
specimens like urine, stool, blood, swab
− not all specimens can be collected by medical
technologists, others are to be done by physicians
like the CSF
− general rule – the site of infection is where the
specimen will be collected
o respiratory tract – sputum, nasopharyngeal
swab, bronchial washing
o urinary tract – urine
Step 2: Direct Microscopic Examination
− bacterial smear directly from the specimen
− example – urine
o using the inoculating loop, a specimen will be
collected, then it will be evenly spread out to the
slide; air dry, perform fixation, then stain it using
gram stain
to be identified easily
− standard incubation period – 18-24 hours
− standard incubation temperature – 37°C
− must know culture media for every bacterium
because they are not similar to one another
Step 4: Bacterial Identification
− once there’s already growth, proceed to the
bacterial identification
− macroscopic examination – describe the bacterial
morphology first
− microscopic examination – bacterial smear
preparation
o gram stain reaction
o morphology
o arrangement
 pleomorphism or pleomorphic bacteria
¤ appear in various forms
¤ different arrangement, size, and form
¤ Corynebacterium diphtheriae
Step 5: Biochemical Test
− Staphylococcus aureus
o specimen collection: usually skin
o direct microscopic examination: observe gram
positive cocci in cluster
o culture: BAP, PEA, NSA, and high chromagar
o bacterial ID: colonial morphology like medium-
sized, smooth, greeny, opaque, gray to white or
golden yellow colonies and have hemolytic
pattern usually beta or non-hemolytic
o microscopic exam: gram stain reaction
o biochemical test: catalase reaction, coagulase
positive, PYR positive
Step 6: AST
− antibiotic to be used for them

Step 3: Culture
− inoculation or streaking – spreading the specimen
 MICROSCOPIC SHAPES
− Thiomargarita namibiensis
o largest bacteria found in ocean sediments
o has a diameter of 0.1 to 0.3 mm
Cocci (Coccus)
− round or spherical shaped bacteria
− resulting arrangement of cocci depends on the plane
of division
− [diplococci] two common types
o Neisseria spp.
 usually, gram-negative diplococci
 appear kidney or horseshoe
o Streptococcus pneumoniae
 usually, gram-positive diplococci
 appear lancet or flame shaped
− [chain] Streptococcus spp. – gram-positive cocci in
chain
− [tetrads] Micrococcus tetragena – packets of four
− [sarcinae] Micrococcus luteus – two tetrads
− [clusters] Staphylococcus spp. – gram-positive cocci
in clusters
Bacillus (Bacilli)
− rod shaped, cylindrical, or elongated bacteria but it’s
interesting to know that this is not always true to all
bacilli since some of them also varies in
morphologies
− fusiform
o pointed ends
o Capnocytophaga
− palisade
o in parallel with one another
o Corynebacterium diphtheriae
− Bacillus anthracis
o has a box car arrangement like an old car that are
wide and long
o spore is the colorless just like the car’s
windshield
− Mycobacterium tuberculosis
o acid-fast organisms are being counted
− fusobacteria are bacilli in chain
− in acid-fast, we are talking about bacilli per visual
field
− reporting of gram stain is easier than acid-fast
− in specimen, some epithelial cells will still be present
− in sputum, there will still be PNM
− Corynebacterium diphtheriae
o various forms like Chinese characters, parallel to
one another, or in X form
 − many variants (serovariety)
o Leptospira interrogans serovar.
icterohaemorrhagiae
o Leptospira interrogans serovar. grippotyphosa
o Leptospira interrogans serovar. mutans
− many subspecies
o Treponema pallidum subsp. pallidum
o Treponema pallidum subsp. endemicum

STAINING
− imparts an artificial coloration not only to bacteria
but for other material found on clinical specimen
smear that allows them to be visualized better using
the magnification of microscope
− four categories of staining
o Direct/ Simple Stain – one stain
o Differential Stain – two stains and have
decolorizer
o Selective/ Special Stain – have a target specific
component like capsule, spore, or flagella
o Indirect/ Negative/ Relief Stain – background
have stain and the bacteria will have no stain

Direct or Simple Stain

Spirals
− helical or twisted bacteria
− Spirillum spp. which is helical but rigid while the
Spirochetes which are helical as well but more
flexible in movement Crystal Violet is used in the image and has no intention to
differentiate it, and determine its absence or presence
− usually contains one specific active chromogen in the
stain which enhances the appreciation of bacterial
size, shape, and arrangement
− one dye or stain
− commonly used simple stains
o crystal violet, gentian violet, methylene blue,
malachite green
Differential Stain
− contains two or more chromogens which further
differentiate specific component within the bacterial
cell which aids in the differentiation or grouping of
bacteria
− two or more dyes to differentiate bacterial into
group
− also includes a decolorization step which is the most
critical step in the process
− gram stain
o differentiates gram positive bacteria which stain
purple or violet from gram negative which stains
red or pink
− acid fast stain
o differentiate acid fast organism such as
Mycobacterium tuberculosis, which stains red
from nonacid fast organisms, which stains blue
or green depending on the counterstain used in
the process using the Ziehl-Neelsen or Kinyoun
Staining Methods
o other stains for acid fast organisms
 Pappenheim Stain
¤ Mycobacterium tuberculosis
 red or pink
 resist the decolorization of rosolic
acid and absolute alcohol
¤ Mycobacterium lacticola (smegmatis)
 blue
 will be decolorized, retaining the
color of methylene blue
 rosolic acid, methylene blue,
glycerin, absolute alcohol
 Baumgarten Stain
¤ uses rosolic acid as a decolorizer
¤ Mycobacterium tuberculosis – blue
¤ Mycobacterium leprae – red
¤ have specific expected color depending
on the specie of mycobacterium
− fluorochrome stain
o uses fluorescent dyes such as auramine or
rhodamine or combination of both
o these dyes remain in the cell wall of acid-fast
organism even after decolorization
Selective or Special Stain
− stains that specifically highlight or emphasize certain
bacterial cell structures or components which aids in
the presumptive identification of the bacteria
− have specific color
− remember the colors and method
− stain for cell wall
o Victoria Blue Dye – cell wall stains blue
− stains for capsule (6)
o HISS – capsule stains pale brown
o TYLER – capsule stains light violet
o MUIR – capsule stains light blue
o GIN – capsule is unstained by the bacteria will be
stained with its margins delineated by the ink
o WADSWORTH – capsule stains pinkish and
bacteria stains blue
o WELCH – capsule stains pale violet
− stains for Metachromatic Granules or Babes Ernst
Bodies or Volutin (6)
o Loeffler’s Alkaline Methylene Blue (LAMB) –
granules stain red
o Albert – granules appear blue black
o Neisser – granules appear dark blue
o Lindegran – granules appear reddish brown
o Burke’s Technique – a modified gram’s staining
technique; granules appear dark violet
o Ljubinsky – granules stain dark violet
− stains for Bacterial Spores or Endospores (3)
o Fulton-Schaeffer – spores are green
o Dorner – spores are red
o Wirtz-Conklin – spores are green
− stains for Flagella (4) − bacteria or structure (capsule) – unstained
o contains tannic acid – important component in − background – colored or stained
flagellar stain, which coats, swells, and − India Ink or Nigrosin – background is black
precipitates the flagella, enhancing its − Congo Red – background is red
visualization − Anthony – background is purple
 Leifson
 Gray
 Silver METHODS OF STUDYING BACTERIA
 Fisher-Conn − after the standard incubation of 18-24 hours,
inoculated plates are retrieved from the incubator
− stains for Rickettsia and the colonial or cultural characteristics of the
o Castañeda – stains blue bacterial colonies that grew in each culture media for
o Machiavelo – stains red each specimen is examined, this is referred to as
o Giemsa – stains blue plate reading
− stains for Chlamydia – sexually transmitted infection COLONIAL/CULTURAL CHARACTERISTICS
o Gimenez – elementary bodies stains red Shape CCRRIFS
o Machiavelo – stains red
o Giemsa – stains purple

− stains for Spirochetes

o Fontana-Tribondeau – spirochetes stain dark
brown or black
o Levaditi Silver Impregnation – spirochetes stain
black
o India Ink Negative Stain – spirochetes are Size PLSM
unstained; background is black or indirect stain

− stains for Mycoplasma

o Dienes – stains blue

− stains for Bipolar Bodies (Yersinia pestis)

o Wayson (safety pin appearance) – bipolar bodies
stain red

INDIRECT/NEGATIVE/RELIEF STAIN − relative size of the bacterial colony

Margin SUFFRIL

− type of staining which actually provides coloration to

the background of the smear while rendering the
bacteria and covering structure such as capsule
unstained
− useful in the identification of medically important
capsulated bacteria as well as capsulated strains of
Cryptococcus spp. especially in cerebrospinal fluid
sample in cases of meningitis − appearance of the edge of the colony
Elevation

− height of the colony

−  hemolysis [bottom left] – partial hemolytic pattern
Texture or Consistency o Streptococcus pneumoniae – colorless
o greenish discoloration in the surface
o shiny, round structures are the colonies
o surrounding the colonies, there are brownish-
greenish discoloration
o their RBCs are incompletely lysed

−  hemolysis [right, fourth] – clear zone at hemolysis

Hemolytic Pattern
− exhibits the bacteria’s ability to lyse RBCs in the
culture media
− can only be described in blood agar plate, an
enrichment medium because there’s an addition of
blood to the basal medium in order to demonstrate
the organism’s capability to lyse the red blood cells
− there are bacteria that
o can undergo complete hemolysis
o partial hemolysis
o cannot hemolyze
− BAP is used in hemolytic pattern
o Staphylococcus aureus – translucent
o the surrounding has intact red blood cells
o bacteria were able to lyse the RBCs completely
−  hemolysis [upper right, left] – non-hemolytic pattern
o Klebsiella pneumoniae (mucoid)
o Enterococcus faecalis – translucent
o there is no discoloration or clear zone
surrounding the colony
o no hemolysis
Pigmentation

− ability of the bacteria to produce unique coloration

their colony

Odor CRAMFU
− certain bacteria produce characteristic odor in
culture media
− butter-like – Staphylococcus spp.
− apple like – fruity – Alcaligenes faecalis
− apple like grape like – Pseudomonas spp.
− burnt chocolate – Proteus spp.
− earthy – Burkholderia mallei
− each bacteria have specific color, odor (not all), and
artificial color

Density

− optical property to pass light through the bacterial

colony

ANTIGENIC DETERMINATION BY SEROLOGICAL TYPING

− O Antigen OH K VI
o associated with the cell wall
o used in Widal Test – for diagnosis of typhoid
fever
− H Antigen – associated with the flagella
− K Antigen – associated with the capsule
− Vi (Virulence) Antigen – specific capsular antigen of
Salmonella typhi
 Clinical Bacteriology BACT211
STERILIZATION AND DISINFECTION
2ND YEAR | SECOND SEMESTER | PRELIM DATE: FEBRUARY 10, 2024
Prof. Ma. Christy V. Gonzales mvgonzales@fatima.edu.ph
Prof. Pamela Sengson-Sevilla pssengson@fatima.edu.ph
382 235 2533 Asynchronous Discussion

STERILIZATION VERSUS DISINFECTION − examples

Sterilization and Disinfection o spore forming bacteria
− the term was originated more than 100 years ago  spores are coated with proteins, lipids, and
− this was when Joseph Lister introduced the concept carbohydrates as well as dipicolinic acid and
of aseptic surgery calcium
o he used carbolic acid (phenol)  able to resist heat, chemical means, or other
Sterilization methods
− refers to the destruction of all forms of life, including
o Mycobacterium spp.
bacterial spores
 cell wall is high in lipid which enables them
− all or nothing process
to become resistant to most environmental
− complete removal of microorganisms
stress such as desiccation
Disinfection
− refers to a process that eliminates a defined scope of o biofilm forming bacteria
microorganisms, including some spores  certain bacteria can aggregate into
− physical or chemical methods may be used, but most communities of bacteria which makes then
disinfectants are chemical agents applied to resistant to chemical and physical means of
inanimate objects destruction
− only reduces the number of microorganisms  microorganisms living together in
Antiseptic communities
 provide protection against physical and
− substance applied to the skin for the purpose of
chemical means of destruction
eliminating or reducing the number of bacteria
present o prions
− do not kill spores and cannot be used as disinfectants  naked pieces of protein
− agent, substances that destroys or inhibits the  similar to viruses but without nucleic acid
growth of microorganisms  most resistant to the action of heat,
radiation, and chemicals
 agents that are able to cause degenerative
FACTORS THAT AFFECT THE DEGREE OF KILLING OF
diseases of the nervous system
MICROORGANISMS
TYPES OF ORGANISMS NUMBER OF ORGANISMS
− different organisms have varying ability in − this factor basically refers to the amount of
withstanding and chemical and physical treatment organisms present in the object to be treated
due to the referred to as microbial load (bioburden)
o different biochemical composition of these − in principle, the higher the number of organisms, the
organisms longer the exposure time needed to eliminate 99.9%
o various mechanisms that they use to protect of the microorganisms
themselves
CONCENTRATION OF DISINFECTING AGENT
− amount needed to destroy microorganisms based on
the agent to be used
− it is therefore important to follow, the correct
preparation and dilution as prescribed by the
manufacturer
− simply, concentrated agents do not necessarily mean
that it would work better
o others believe that the more concentrated an
agent is, the more effective it will be, but it is not;
it will depend on the agents used
− in some instances, dilution is performed because it is
believed to be effective as per research
PRESENCE OF ORGANIC MATERIAL
− examples of organic materials that may prevent the
full contact of the agent to the organisms, hence
limiting its action
o blood
o pus
o mucus
− most common example and frequently used in the
laboratory is bleach (sodium hypochlorite)
o easily inactivated by organic material
o [1:10] 1 part of bleach: 9 parts of water –
effective in inactivating organic material
NATURE OF SURFACE TO BE DISINFECTED
− some instrument that we use in the laboratory
sometimes are made up of biomaterial which
exempts them to disinfection or sterilization due to
possible damage
− example is endoscopic instruments which can’t be
autoclaved
CONTACT TIME
− it is critical to observe proper contact time of the
agent and the object to be disinfected or sterilized
− in principle, contact time may be affected by all
previous factors already mentioned as well as
temperature
− too little contact time does not allow the agent to
work properly
− example, alcohol and betadine have to be in contact
for about 1-2 minutes to work properly
− spore forms may need more contact time than its
vegetative counterpart
TEMPERATURE
− generally, disinfectants are usually used at room
temperature (20°C to 22°C)
− however, their activity may increase at a certain
degree by a corresponding increase in temperature
or may decrease when temperature is decreased
− too high or low temperature may inactivate
disinfectants and sterilant
− example – disinfection of blood spills in a refrigerator
o longer as compared to the blood spills in room
temperature
pH
− it is also important to consider the pH of the material
to be treated and he agent itself
− manufacturers usually optimize this factor to achieve
maximum activity
− take note of the pH in which the agent will be
activated, or the material to be exposed to the agent
BIOFILMS
− certain bacteria have to ability to form communities
of layers of bacteria with protective shield which is
called as biofilm
− it is important to consider that biofilm formation
may require longer contact time or increase in the
concentration of the agent
− biofilms – community of bacteria
− can be found in inanimate or animate objects
− in hospitals, the most critical place where we can find
biofilm is in the catheter
COMPATIBILITY OF DISINFECTANTS
− some disinfectants may inactivate the action of
another; hence it is also important to consider the
compatibility of the disinfectants
− myth or wrong – two disinfectants are better than
one
− before using more than one disinfectant, make sure
to check their compatibility first
− some disinfectants may inactivate the other one
− example is bleach and quaternary ammonium
compounds which may negate each other
METHODS OF STERILIZATION
PHYSICAL METHODS
MOIST HEAT
− coagulation of bacterial proteins including bacterial
enzymes
− heat – the simplest means of sterilizing materials,
provided that the material is resistant to heat
damage
Autoclave
− operates based on the principle of steam under
pressure
− usually used in the microbiology for disinfecting glass
petri dish and culture media
− effective indication
o sterilization: 121°C for 15lbs/in2 for 15 minutes
o decontamination: 135°C for 30lbs/in2 for 30
minutes
o biological indicator: Bacillus stearothermophilus
Tyndallization
− principle: fractional discontinuous sterilization
− effective indication: 100°C for 30-60 minutes
− instrument: Arnold’s Sterilizer
Inspissation
− principle: thickening through evaporation
− effective indication: 75-80°C for 2 hours
− instrument: Inspissator
− used for heat-stable substances that are not
penetrated by heat
− usually used in order to sterilize glasswares
Direct Flame
− direct application of flame in aseptic technique
− just like when sterilizing the inoculating loop
Dry or Hot Air Oven
− used in the sterilization of heat resistant materials
− effective indication: 160-180°C for 1.5 to 2 hours
− biological indicator: Bacillus subtilis var. niger
Incineration
− burns materials into ashes; used in the disposal of
biological wastes
− effective indication: 870-980°C for 2 seconds
IONIZING RADIATION
− works by alkylation of nucleic acid of bacteria using
high energy short wavelength deep penetrating
gamma rays
− used for heat sensitive materials
− usually used in the medical field for the disinfection
of disposable supplies like syringe, catheters, and
gloves
− biological indicator: Bacillus pumilis

FILTRATION
− based on membrane gradient by differences in
particle size
− very common for liquids
− used for the sterilization of heat sensitive materials

− sterilization – autoclave, oven
− disinfection – boiling water, pasteurization
DRY HEAT
− oxidation of bacterial components
− requires longer exposure time and
temperature than the moist heat
Water/Liquid Solutions/Antibiotics/Vaccines
− membrane filters – usually made of plastic polymers
or cellulose esters, and contain spores that vary in
sizes
− usually uses a thin membrane filter of cellulose
acetate with different pore size depending on the
intended purpose:
o 0.45-0.80μm – most bacteria, yeasts, and molds
are retained but may allow passage of
Pseudomonas- like organisms
o 0.22 μm – used to filter Pseudomonas- like
organisms; used for critical sterilization of pa
renteral solutions
higher
o 0.01 μm – able to retain small viruses
Air: High Efficiency Particulate Air Filter (HEPA) Non-ionizing Radiation
− has a pore size of 0.3 μm − form of ultraviolet rays
− usually used in Biological Safety Cabinet (BSC) and − uses low energy long wavelength ultraviolet rays to
rooms of immunocompromised patients disinfect heat sensitive materials as well as large
spaces
− damage DNA by forming thymine and cytosine
CHEMICAL METHODS
dimers
− not only used for sterilization, but also for
disinfection
Peracetic Acid
− for disinfection and sterilization of surgical CHEMICAL METHODS
instruments Alcohol
− usually, in the hospital, alcohol is used
Formaldehyde Vapor/Vapor Phase H2O2 hydrogen peroxide
− most effective alcohol used – (70%) ethyl alcohol and
− for HEPA filters and large spaces isopropyl alcohol
Glutaraldehyde − has an excellent in-vitro bactericidal activity against
both gram-positive and gram-negative bacteria
− for medical instruments (e.g., bronchoscopes, etc.)
− acts as liquid desiccants
− for disinfection, sterilization, and preservation
− MOA: dehydration, lipid dissolution, and protein
Ethylene Oxide (ETO) Gas denaturation
− the recommended concentration is 450 to 700 mg of − 70% Alcohol not 90%
ethylene oxide per liter of chamber space at 55°C to − minimum contact time: 1-2 minutes or until
60°C for 2 hours completely evaporated
− this method is also used extensively by the
Halogens
manufacturing industry for the sterilization of low-
− MOA: inhibits protein function and acts as strong
cost thermoplastic products
oxidizing agents
− for sterilization and disinfection
− oldest and most commonly used disinfectants –
− biological indicator: Bacillus subtilis var. globijii
chlorine and chlorine compounds; usually in the
form of hypochlorite
METHODS OF DISINFECTION − Chloride (Cl) in NaOCl: used as disinfecting agents in
PHYSICAL METHODS many laboratory and hospitals spaces, surfaces, and
Boiling also in treating water for portability Iodine (I2) in
− most done betadine used as a household antiseptics and
− destroys vegetative cells of bacteria but not their surgical antiseptics
spores Heavy Metals
− effective indication: 100°C for 15-30 minutes
− usually, slowly bactericidal, but their action is
Pasteurization primarily bacteriostatic
− well-known method − because of their toxic effects, they are not utilized
− used for the preservation of alcoholic beverages such anymore
as beers, wines, and dairy products such as milks and − MOA: denaturation of enzymes and other essential
yogurt bacterial proteins
− types − Mercury (Hg): active ingredient or merthiolate but
o batch: 62.5°C for 30 minutes this is already banned in the market due to its known
o flash: 72°C for 15 seconds toxicity
o ultra-high temperature (UHT): 72°C – 110°C for 5 − Copper (Cu): CuSO4 crystals are used as algaecide in
seconds swimming pools and aquarium
− Silver (Ag): 1% AgNO3 (silver nitrate)
 o used as prophylactic agent in Crede’s Prophylaxis
in suspected cases of Ophthalmia neonatorum
o or the 1% eyedrop solution
o to prevent gonococcal infection
o its replacement is erythromycin drops

Quaternary Ammonium Compounds (QUATS)

− their action is through the disruption of cellular
membrane, thus leading to the leakage within the
cell contents
− resistant for gram-negative bacteria such as the
Pseudomonas aeruginosa
− MOA: enzyme inhibition, protein denaturation, and
disruption of plasma membrane
− Zephiran: Benzalkonium chloride
− Cepacol: Cetylpyridium chloride

Phenol/Phenolic Compounds/Bisphenols
− used for the disinfection of hospital and household
environment
− usually found in germicidal soaps
− MOA: plasma membrane destruction and enzyme
denaturation
PHADHAG