CLINICAL BACTERIOLOGY (LEC)
Introduction to bacteriology
TABLE OF CONTENTS
Bacteriology, History
Microbial Taxonomy, Nomenclature, Cell Structures
for Prokaryotic and Eukaryotic Cells
Bacterial Morphology, Stages of Growth, Metabolism,
Nutritional Requirements
Replication, Gene Transfer, Biochemistry &
Metabolism,
Microbiological Culture
Objectives:
I. Discuss the brief history of
bacteriology
II. Discuss the taxonomy and
nomenclature of bacteria
III. Discuss the cellular structure of a
bacteria
IV. Discuss about bacterial morphology
V. Discuss the phases of bacterial
growth
VI. Discus about microbiological culture
BACTERIOLOGY
 The study of single-celled microorganisms that
lack a true nucleus
HISTORY
 Hippocrates (460 – 370 BC)
o Known as the Father of Medicine
o Founded ethics in the field of medicine
 Aristotle (384 – 322)
o Proposed the Spontaneous
Generation Theory which stated the
living organisms could develop from
non-living materials
 Hans and Zacharias Janssen (1590)
o Developed the first compound
microscope, a vital instrument in the
laboratory
 Robert Hooke (1660)
o Published Micrographia which contains
drawings and detailed observations of
biological materials seen in the
microscope
 Anton Van Leeuwenhoek (1676)
o Known as the Father of Microbiology
o The first person to observe
microorganisms under the microscope
 Francesco Redi (1688)
o Refuted the idea of spontaneous
generation by showing that a rotting
meat kept from flies did not develop any
maggots
 Agostino Bassi de Lodi (1835)
o Found that the disease affecting silk
worms were caused by a fungus
1 o Fungus were the first microorganism to
be recognize as a contagious agent of
2
animal disease
4  Theodor Schwann (1836)
o Developed the Cell Theory, which states
5
6 that the cell is the basic functional unit of
all living organisms
 Louis Pasteur (1861)
o Developed the Germ Theory of Disease,
which states that a germ causes
infectious diseases
o He also refuted the Spontaneous
Generation Theory
 Robert Koch (1876)
o Named as the Father of Bacteriologic
Technique
o Developed the Koch postulates in
1884 which states:
 The agent must be present in
every case of the disease
 The agent must be isolated and
cultured in-vitro
 The disease must be reproduced
when a pure culture of the agent
is inoculated into a susceptible
 The agent must be recoverable
from the experimentally-infected
host
o The Koch postulates are the basis of the
culture methods done in the laboratory.
 Von Behring and Kitasato (1890)
o The development of vaccines
 Edward Jenner
o Known as the Father of Immunology
o First to institute vaccination against
infectious diseases
 Joseph Lister
o Developed aseptic surgery (sterile)
 Hans Christian Gram
o Employed Gram’s staining for
microscopic bacterial cell differentiation
 Jules Bordet
o Discovered Bordetella pertussis as the
causative agent for whooping cough
 Friedrich Loeffler
o Cultivated Kleb’s Loeffler’s bacilli
 Alexander Flemming
o Discovered penicillin from the mold
Penicillum nolatum
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  First syllable of first 2 letter are used when 2 or
more genera names begin with the same first
letter.
MICROBIAL TAXONOMY o ex. Ent.coli or Esch.coli
 Taxonomy is the orderly classification and
CLASSIFICATION OF BACTERIA
grouping of organisms into taxa or categories
 Phenotypic characteristics – readily observable
 Comes from the Greek word taxon meaning
 Genotypic characteristics – genetic make-up of
arrangement and nomos meaning law.
an organism based on their DNA and RNA
 3 CATEGORIES OF TAXONOMY:
structure and homology
o Classification
 Cellular type
 Categorization of organisms into
o Prokaryotes
taxonomic groups
 cell lacks a true nucleus and no
o Nomenclature
membrane-bound organelles
 Naming of an organism by
o Eukaryotes
international rules according to
 have a true nucleus and
its characteristics
organelles
o Identification
o Archaeobacteria
 Refers to the practical use of a
 Undergo extreme environmental
classification scheme
conditions and are not
LINNAEAN TAXONOMY encountered in clinical
Formal Rank Example microbiology
Domain Bacteria
Kingdom Prokaryotae PROKARYOTIC/BACTERIAL CELL
STRUCTURE
Division or Phylum Gracillicutes
Class Scotobacteria  Cytoplasmic structures
Order Eubacteriales o Nucleus – no membrane bound nucleus
Family Enterobacteriaceae o Mesosome – point of attachment of
Genus Eschecrichia chromosomes
Species or Epithet Coli o Ribosomes – consists of RNA and
Subtype Escherichia coli O157:H7 protein and is the site of protein
 Species is defined as the most basic taxonomic synthesis
group. o Cytoplasmic Granules/Inclusion Bodies-
o Further division of species: storage deposits of polysaccharides
 Subspecies (subsp) are based  Babes-Ernst Bodies/Volutin
on phenotypic differences and Granules/Metachromatic
are readily observable Granules and is found in
 Serovarieties (serovar) are Corynebacterium diptheriae
based on serologic differences  Endospores – found in Bacillus
that would involve immunology and Clostridium
 Biovarities (biovar) are based on  Cell Envelope Structures
biochemical test result o Plasma Membrane (Cell Membrane)
differences  composed of a phospholipid
bilayer
NOMENCLATURE
o Cell Wall (Murein layer) – maintains
 It is a binomial system which means that the
nomenclature of the bacteria will contain its shape of cell and has high affinity to
genus and species. dyes.
 Composed of N-
 1st letter of family name is capitalized and has
acetylglucosamine and
an –aceae ending.
Nacetylmuramic acid
o Ex. Enterobacteriaceae 1st letter of
o Gram positive – thicker cell wall and
genus name is capitalized
contains teichoic acid
 1st letter of genus name is capitalized
 Characteristics:
 Species name begins with lower case letter
 Permeability – osmotic pressure
 Both the genus and the species should be is maintained
italicized in print or underlined when written in  Plasmolysis – a cell in a saline
script solution shrinks because water
 Abbreviation – 1st letter of genus capitalized passes out
followed by a period and the species name  Plasmoptysis – cell will burst in
follows distilled water
o Ex. E.coli

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  Capsule – made up of
polysaccharides; act as virulence
factors; appear as clear halo
o Slime layer – similar to capsules but are
more diffused layers surrounding the
cell; also made up of polysaccharides
PROKARYOTIC/BACTERIAL CELL
STRUCTURE
 Gram negative organisms have an outer
membrane that serve as a primary permeability
barrier to hydrophilic and hydrophobic
compounds.
o The membrane is a bilayered
lipopolysaccharide
 Cell Appendages
o Flagella – are exterior protein filaments
that rotate and cause bacteria to be
motile. This is referred to as “true”
motility. It is made up of a protein called
flagellin.
 Classification according to number and
arrangement of the flagella on bacterial cell:
o Atrichous – no flagellum
o Monotrichous – single flagellum at one
pole
o Amphitrichous – flagellum on both poles
of the cell
o Lophotrichous – bundle of flagella in one
or both poles of the cell
o Peritrichous – flagella surrounds the
entire organism
 Pili (singular pilus) –are non-motile, long, hollow
protein tubes made up of pilin that connects two
bacterial cells
o 2 types of Pili:
 Sex/fertility pili – for sexual
conjugation by transferring DNA
from 1 cell to another
 Somatic/common pili – for
adhesion of bacteria to host cell
 Fimbriae (singular fimbria) – are non-flagellar,
sticky, proteinaceous, hair-like appendages that
aids in adhesion to tissues and to surfaces.
o It contributes to the virulence of the
bacteria.
EUKARYOTIC CELL STRUCTURE
 Nucleus
o has chromosomes which contains DNA.
They are covered with basic proteins
called histones. It is bounded by a
bilayered lipoprotein membrane known
as the nuclear membrane.
 Nucleolus
o round, refractile body which is the site of
ribosomal RNA synthesis. It is located
within the nucleus
 Endoplasmic Reticulum
o is a system of membranes that occur
throughout the cytoplasm
 Rough Endoplasmic Reticulum
o covered with ribosomes which gives it a
“rough” appearance. It is the site of
protein synthesis
 Smooth Endoplasmic Reticulum
o no ribosomes. It doesn’t synthesize
proteins but it synthesizes phospholipids
 Golgi Apparatus
o modify and package proteins sent by the
rough endoplasmic reticulum
 Ribosomes
o where protein synthesis occurs.
 Mitochondria
o main site of energy production
 Lysosomes
o contains hydrolytic enzymes for
degradation of macromolecules and
microorganisms within the cells
 Peroxisomes
o break down hydrogen peroxide
 Plasma Membrane
o regulates transport of macromolecules
into and out of the cell. Also contains
cholesterol which keeps the membrane
fluid. The polar heads of phospholipids
are hydrophilic and the nonpolar tails
are hydrophobic which lie in the center
of the plasma membrane
 Cell Wall
o provides rigidity and strength to the
exterior of the cell. Most eukaryotic cells
don’t have cell walls except for fungi.
 Cilia
o short projections that extend from the
cell surface and used for locomotion
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BACTERIAL MORPHOLOGY
 The morphology of a bacteria is classified
according to its trait.
COCCI
 Bacteria spherical in shape
 Singular: Coccus
o Diplococci – Neisseria gonorrheae
 cocci in pairs
 Categorized into two:
intracellular and extracellular
 Intracellular: bacteria found
within a cell, especially the WBC
Neutrophil also called the
polymorphonuclear neutrophil
o Streptococci – Streptococcus
pyogenes
 cocci in chains, causative agent
of strep throat
o Staphylococi – Staphylococus aureus
 cocci in clusters/grape-like
clusters, causative agent of food
poisoning
o Tetrads – Gafyka tetragena
 cocci in groups of four
o Sarcina – Sarcina lutea
 cocci in groups of eight
BACILLI
 Rod shaped
 Singular: Bacillus
o Diplobacilli – Mycobacterium
tuberculosis
 Bacilli in pairs
 ALWAYS answer that it is a non-
sporing diplobacilli
 Can be snapping or slipping
 Non-sporing Slipping bacilli = if
they are parallel to each other
 Non-sporing Snapping bacilli =
appears in V-shape
 Gram Positive
o Streptobacilli – Bacillus subtilis
 Bacilli in long chains
 Sporing streptobacilli have an
endospore
 ALWAYS answer that it is a
Sporing streptobacilli
o Coccobacilli – Escherichia coli
 Short, rod-shaped bacilli
 ALWAYS answer that it is a non-
sporing coccobacilli
o Fusiform bacilli – Fusobacterium
fusiforme
o Palisading bacilli
SPIROCHETES
 Spiral/helical shape
 Long axis bends when in motion
o Treponema pallidum
o Spirillum
 Long axis remain rigid when in
motion
o Vibrio
 Curve rod-shaped, comma
shaped ex. Vibrio cholera
GENERAL RULE:
 All cocci are gram positive except Neisseria,
Veillonella, and Moraxella group.
 All bacilli are gram negative except Bacillus,
Clostridium, Mycobacterium,
Cornyebacterium, Listeria, Nocardia,
Erysipelothrix, Lactobacillus, Kurthia,
Rothia.
 All cocci are non-motile and non-spore-
formers.
 All encapsulate organisms are non-motile.
 Bacillus and Clostridium are spore-forming
bacteria.
 Spiral organisms are very hard to stain but
they are gram negative.
STAGES OF BACTERIAL GROWTH
 Lag Phase/Physiologic Youth Phase – little or
no growth
 Log/Exponential Phase – maximum rate of
bacterial multiplication. During this phase, the
bacteria is most susceptible to antimicrobials
due to high metabolic activity.
 Plateau/Stationary Phase: # of bacteria alive
is equal to the # of dead bacteria
 Decline Phase/Degradation Phase/Death
Phase – increase in number of dead bacteria
NUTRITIONAL REQUIREMENTS
 Carbon – for synthesis of cellular component
o Litotroph/Autotroph
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  Carbon source is from Carbon
dioxide
o Organotroph/Heterotroph
 Carbon source is from organic
material i.e glucose, lactose
 Nitrogen
o For protein synthesis 3.
 ATP
o For energy
 Water
o Bacteria is 70% water
 Salt
o Halophilic organisms require salt for
growth
 Minerals
o Requires magnesium, sodium,
potassium, iron
 7. Additional or Other Requirements
o X Factor/hemin
 Derived from hemoglobin
degradation
o V Factor/NAD/Coenzyme 1
 Produced by some bacteria
(Staphylococcus aureus) and
yeast
o Protein
 For Mycobacteria which requires
increases amount of protein
that’s why the media is egg
based
 Environmental Requirements
o Oxygen, temperature, pH
 Oxygen
o Obligate aerobe – only survive in the
presence of 15-21% O2 and 0,03% CO2
o Obligate anaerobe – lives only in the
absence of O2
 5-10% hydrogen
 5-10% CO2
 80-90% Nitrogen
 0% oxygen
o Facultative anaerobe – generally
AEROBIC but can survive in the
absence of O2
o Aerotolerant anaerobe – an ANAEROBE
that can survive even in the presence of
O2
o Microaerophile – requires only 5% of O2
o Capnophile – requires 5-10% CO2
 Temperature
o Psychrophilic/Cryophilic
 cold temp 15-20ᵒC
o Mesophilic
 30-37ᵒC; most pathogens belong
here
o Thermophilic
 warm temp. about 50-60ᵒC
o Hyperthermophilic/Extreme
Thermophilic
 80-100ᵒC
 pH
o Most bacteria require neutral or slightly
alkaline pH around 7.0-7.5
 Acid loving: Lactobacillus
acidophilus requires pH 3.0;
tomato juice agar is used
 Alkali-loving: Vibrio requires pH
8-10; use Alkaline Peptone
Water as a culture medium
BACTERIAL REPLICATION
 Bacteria multiplies through binary fission;
creating two identical daughter cells.
 4 Stages in Bacterial Replication
o Unwinding of supercoiled DNA
 Required so that the enzymes
and cofactors involved in
replication can access the DNA
molecule
o Unzipping of DNA
 Once the replication site is
exposed, unzipping of DNA
occurs
o Synthesis of new DNA strands
 Involves DNA polymerase
enzyme
o Termination of replication
 Would occur when 2 replication
ports meet
 Results to 2 complete
chromosomes
GENE TRANSFER
 3 Mechanisms of Genetic Transfer:
o Bacterial transformation
 A free or naked DNA found in the
environment is taken by the
bacterial cell
 The DNA taken would then take
one of three courses:
 Integrated into existing
bacterial genetic material
 Degraded
 May replicate in the
cytoplasm of the bacterial
cell if it is compatible with
the plasmid
o Bacterial transduction
 Where the virus injects DNA into
the bacterial cell
 Once the virus injects DNA into
the bacterial cell, it may undergo
lytic cycle or lysogenic cycle
 Lytic cycle: the replication
of the bacterial
chromosome is disrupted
 Lysogenic cycle: the
bacteriophage DNA is
incorporated into the
bacterial genetic material
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 o Bacterial conjugation
 DNA is transferred from one cell
to another
 Can be done by the sex pilli
BACTERIAL BIOCHEMISTRY AND
METABOLISM
 Bacteria use biochemical pathways to
catabolize carbohydrates (CHO) and produce
energy by:
o Fermentation
 Anaerobic utilization of pyruvic
acid
 End product of fermentation is
lactate, butyrate, ethanol, and
acetoin which accumulates in the
culture media
o Oxidation
 Aerobic utilization of pyruvate
 Most important pathway is the
TCA or The Krebs Cycle
 Results in the production of acid
and CO2
BACTERIAL METABOLIC PATHWAYS
 The starting carbohydrate for bacterial
fermentation or oxidation is glucose. Bacteria
breaks down glucose to pyruvic acid by 3
metabolic pathways:
o Embden-Meyerhof-Parnas (EMP)
Pathway
 There is a conversion of glucose
to pyruvate and generates the
majority of ATP needed by the
bacteria
 Major pathway
 Anaerobic
o Pentose Phosphate Pathway
 Alternative to EMP
 Glucose is converted into
ribulose-5-phosphate
 Energy or ATP yielded is lower
as compared to EMP
o Entner-Duodoroff Pathway
 Conerts glucose-6-phosphate
into pyruvate and glyceraldehyde
phosphate
 Aerobic Process
MICROBIOLOGICAL CULTURE
 Method of multiplying microbial organisms by
letting them reproduce in a culture medium
under controlled laboratory conditions.
 Isolation of bacteria by the medical technologist
letting them grow in-vitro.
 One of the primary diagnostic methods in the
microbiology laboratory.
 Biochemical Identification of Bacteria:
o Utilization of variety of substances as a
carbon source.
o Production of specific end products from
various substrates.
o Production of an acid or alkaline pH in
the test medium.
 Culture Types:
o Pure Culture
 Made up of organisms from one
specie
o Mixed Culture
 Made up of organisms from
different species
o Stock Culture
 A type of pure culture used a
supply for research
 Culture Mediums
o A nutrient medium used for
growing/isolating microorganisms.
 Types of Culture Medium According to
Consistency or Physical State:
o Liquid culture medium
 No agar or any solidifying agent
o Semi-solid culture medium
 0.5-1% agar
o Solid culture medium
 2-3% agar
 Types of Culture Medium According to
Manner of Dispensing:
o Plated Culture Media
 Dispensed into a petri dish
o Tube Culture Media
 Dispensed into test tubes
 Types of Culture Medium According to
Composition:
o Synthetic/Chemically-Defined Culture
Medium
 Exact amount of components are
known
o Complex Culture Medium
 Composition varies from batch to
batch
o Tissue Culture Medium
 For organisms that can’t grow on
artificial culture media
 Uses cancer tissues (HeLa cells)
 Types of Culture Medium According to
Function or Use:
o Simple or Basal Culture Media/General
Isolation Culture Media
 For the general isolation of
culture media and for non-
fastidious organisms (don’t
require additional nutrients)
o Enrichment Culture Medium
 Would enhance the growth of
certain organisms
o Enriched Culture Medium
 For growing fastidious organisms
(require additional nutrients for
growth)
o Selective Culture Media
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  To select the growth of desired o Differential Culture Medium
organisms and inhibit the growth  Differentiates glucose fermenters
of others for Enterobacteriaceae family
o Transport Medium
 Transporting samples to and fro
form the laboratory (Stewart and
Blair medium)

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CLINICAL BACTERIOLOGY (LEC)
SAFETY IN THE CLINICAL MICROBIOLOGY
LABORATORY
Most Common Practices in the Clinical
Microbiology Laboratory
I. Sterilization and Disinfection
II. Chemical and Physical Methods of Disinfection and
Sterilization
III. Common Disinfectants and Antiseptics Used in Healthcare
Settings
IV. Principles and Applications of Disinfectants and antiseptics
 Sterilization- destruction of all forms of life
Definition of Terms:
including bacterial spores (endospores)
 Autoclave- a high pressure device used to
sterilize equipment and supplies by subjecting
them to high pressure saturated steam at 121°C
for 15-20 mins. In a clinical microbiology
laboratory, that is where culture media is placed
during media preparation (some media
preparations requires autoclaving)
 Disinfectant- a disinfectant is a chemical or
physical agent that is applied to inanimate
objects to kill microbes
 Antiseptic- a chemical agent that is applied to
living tissue to kill microbes
 Not all disinfectants are antiseptics because an
antiseptic additionally must not be so harsh that
it damages living tissue
 Disinfection- reducing the number of viable
microorganisms present in a sample
 Not all disinfectants are capable of sterilizing.
 Quaternary ammonium compounds (Quats)-
a type of disinfectant that is a cationic detergent
 Bactericidal- an antimicrobial that kills a
microorganism. e.g., formalin, UV rays
 Bacteriostatic- a condition where the
multiplication of the bacteria is inhibited without
killing them. e.g., alcohol
 Chemical disinfection- use of chemicals to kill
or inhibit the growth of microorganisms
 Physical disinfection- use of methods such as
heat, UV light, and filtration to kill or inhibit
organisms
 Gas sterilization- uses ethylene oxide gas;
used to sterilize objects that can’t be autoclaved
like plastic products; relative humidity of 30%
can destroy spores
 Blood-borne pathogens- are microorganisms
in the blood or other body fluids that can cause
illness and disease in people
 Material Safety Data Sheets (MSDS)- a form
with data regarding the properties of a particular
substance. It may include instructions for the
safe use and potential hazards associated with a
particular material or product. It also includes on
the proper storage of the material or product.
 Section 1: Product Identification- It
includes all the relative information
about the product and its NFPA
hazardous rating
 Section 2: Chemical Composition- We
need to know the chemical composition
of a certain product to know their proper
storage temperature
 Section 3: Emergency and First Aid
Procedures
 Personal Protective Equipment- includes all
types of equipment used to increase individual
safety while performing potentially hazardous
tasks e.g., face masks, face shields, respirators,
hazmat suits, etc.
 Standard Precautions- treat all blood and body
fluids as potentially infectious and capable of
transmitting HIV or other blood-borne diseases.
 Occupational Safety and Health
Administration (OSHA)- a US federal agency
charged with the enforcement of safety and
health legislation in the laboratory.
 Biological Safety Cabinets (BSC)- these are
hoods that are a type of containment barrier that
protects the medical laboratory scientists from
aerosol transmissions of microorganisms;
handling of microbiological specimens should be
performed in a BSC
Factors that Affect the Degree of Killing Organism
1. Types of organisms- spore-forming bacteria are
highly resistant to disinfectants because of the thick
protein coat of spores and endospores contains calcium
dipicolinate which is responsible for its resistance to
disinfection
2. Number of organisms (microbial
load/bioburden)- higher numbers of organisms
require longer exposure to disinfectants
3. Concentration of Disinfecting Agent- the
higher the concentration the better the disinfectant. It
is effective to use pure disinfectant rather than those
that are diluted but pure disinfectants are more
dangerous than the diluted ones.
4. Presence of Organic Material- blood, mucus,
and pus inactivates disinfecting agents and it
prevents full contact between object to be killed and
disinfectant. It should be removed first before
disinfecting.
5. Nature of Surface to be Disinfected- certain
materials require certain methods of disinfection
6. Contact Time- the length of time needed for a
disinfectant to be in contact with the microorganism
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 to be destroyed. Ex. Contact time for iodine and
alcohol: 1-2 mins.
7. Temperature- directly proportional to the degree
of disinfection
8. Biofilms- a community of bacteria that forms a
protective material over them that protects them
from environmental factors. Ex. Catheters, water
pipes
9. Compatibility of Disinfectants- when more
than one disinfectant is used, their compatibility should
be considered. Ex. Bleach and quats negate each other
Physical Method of Disinfection/Steralization
1. Heat- the most reliable method of sterilization of
articles that can withstand heat. Heat acts by
oxidative effects as well as denaturation and
coagulation of proteins.
 Factors affecting heat as a physical method of
disinfection
1. The nature of heat- moist heat is more
effective than dry heat
2. Temperature and time- inversely proportional
3. Number of microorganisms present
4. Nature of microorganisms- depends on
species and strain of microorganism, those with
endospores or not
5. Type of material- contaminated materials
needs higher temperature to be disinfected
 Dry Heat
 Hot air oven: Articles to be sterilized are
exposed to high temperature in an
electrically heated oven.
 Dry heat acts by protein denaturation,
oxidative damage and toxic effects of
elevated levels of electrolytes.
 Different temperature-time relations for
holding time are:
 60 minutes at 160°C
 40 minutes at 170°C
 20 minutes at 180°C
 Moist Heat
 Moist heat acts by coagulation and
denaturation of proteins. It is superior to dry
heat
 Boiling: Boiling water (100°C) kills most
vegetative bacteria and viruses immediately.
 Certain bacterial toxins such as
Staphylococcal enterotoxins are also
heat resistant.
 Some bacterial spores are resistant to
boiling and survive; hence this is not a
substitute for sterilization.
 Steam at 100°C: Instead of keeping the articles
in boiling water, they are subjected to free steam
at 100°C for 90 mins.
 An autoclave can also serve the
same purpose.
 The temp. of the autoclave is set at
121°C with 1/15 psi for medical
apparatus and 132°C for medical
waste.
 Advantages of steam: It has more
penetrative power than dry air and it
moistens the spores (moisture is
essential for coagulation of
proteins). Very effective way of
sterilization, quicker than hot air
oven.
 Disadvantages: Drenching and
wetting of articles may occur;
trapped air may reduce the efficacy
and it takes long time to cool.
2. Radiation- “cold sterilization” because it does
not generate heat.
 Non-Ionizing Radiation are low energy rays
with poor penetrative power.
 Mode of action: inhibits DNA replication
 UV radiation (doesn’t kill spores)
 Ionizing Radiation are high-energy rays with
good penetrative power.
 Mode of action: damages the nucleic acids
of microorganisms
 Electron beams and gamma rays
(Sporicidal)
3. Filtration- does not kill microbes, it only
filters/separates them out.
 Membrane filters with pore sizes between 0.2-
0.45 μm are commonly used to remove particles
from solutions that can't be autoclaved.
 HEPA filters are at least 99.97% efficient for
removing particles >0.3 um in diameter.
4. Sonic and Ultrasonic Vibrations
 Sound waves of frequency >20,000
cycle/second kills bacteria by exposing them for
one hour.
 This method is not reliable since many viruses
are not affected by these waves.
Chemical Methods of Disinfection:
Chemical Disinfectants
1. Alcohols
 Mode of action: Alcohols dehydrate cells,
disrupt membranes, and cause coagulation of
protein.
 Examples: Ethyl alcohol, isopropyl alcohol, and
methyl alcohol
 For blood culture, cotton balls should be sterile
and alcohol should be filtered through a 0.22 μm
filter
2. Aldehydes
 Mode of action: Acts through alkylation of
amino-, carboxyl- or hydroxyl group, and
damages nucleic acids. It kills all
microorganisms, including spores.
 Examples: Formaldehyde, Glutaraldehyde
(have irritating fumes)
 2% glutaraldehyde is germicidal in 10
mins and sporicidal in 3-10 hrs.
3. Phenol
 Mode of action: Act by disruption of
membranes, precipitation of proteins, and
inactivation of enzymes.
Giangan, Joyce Ann/ Ramas, Amiel. | Page 9 of 18
  Examples: 5% phenol, 1-5% Cresol, 5% Lysol
(a saponified cresol), hexachlorophene,
chlorhexidine, chloroxylenol (Dettol)
4. Halogens
 They are commonly employed as surface
disinfectants
 Mode of action: They are oxidizing agents and
cause damage by oxidation of essential
sulfhydryl groups of enzymes.
 Examples: Chlorine compounds (chlorine,
bleach, hypochlorite) and iodine compounds
(tincture iodine, iodophors)
 For tabletops with blood spills, use 1:10
dilution of 5.25% sodium hypochlorite
5. Heavy Metals
 Mode of action: precipitation of proteins and
oxidation of sulfhydryl groups. They are
bacteriostatic.
 Examples: Mercuric chloride, silver nitrate,
copper sulfate, organic mercury salts
 Eye drop solution with 1% silver nitrate
to treat the eyes of newborns to kill N.
gonorrheae that may have been
acquired through passage of the birth
canal
6. Hydrogen Peroxide
 Mode of action: It acts on the microorganisms
through its release of oxygen. Hydrogen
peroxide produces hydroxyl-free radical that
damages proteins and DNA.
7. Ethylene Oxide (EO):
 Mode of action: It is an alkylating agent. It acts
by alkylating sulfhydryl-, amino-, carboxyl- and
hydroxyl- groups.
 A chemisterilant
8. Quaternary Ammonium Compounds- are
detergents. They reduce surface tension of
molecules in a liquid
 Pseudomonas aeruginosa is resistant to quats
 Mechanisms of action of chemical agents:
a) Protein denaturation
b) Membrane disruption
c) Nucleic acid damage
d) Inhibition of metabolism
2)
Physio-Chemical Method of Sterilization:
 Mode of action: A physio-chemical method
adopts both physical and chemical method. Use
of steam formaldehyde
General Laboratory Safety
1. Chemical Safety
 Labs should have a chemical hygiene plan that
includes guidelines on proper labeling of
chemical containers, MSDSs, and chemical
safety training.
 Use fume hoods (exhausts chemical vapors to
the outside) when working with toxic chemicals
(HCl, methanol, etc.)
2. Fire Safety- Labs should post a fire evacuation
plan and conduct fire drills
3. Electrical Safety- No extension cords should be
used in the lab, wirings should be checked regularly.
Chemical Hazard Communications Plan
 Develop written hazard communication
program.
 Maintain inventory of all chemicals with chemical
and common names, if appropriate.
 Manufacturer must assess and supply
information about chemical or physical hazards
(flammability, explosive, aerosol, flashpoint,
etc.).
 Employers must maintain Material Safety Data
Sheets (MSDS) in English.
 MSDS must list all ingredients of a substance
greater than 1%, except for known carcinogens
if greater than 0.1%.
 Employers must make MSDS available to
employees upon request.
 Employers must ensure that labels are not
defaced or removed and must post appropriate
warnings.
 Employers must provide information and training
(“right-to-know”).
 Employers must adhere to Occupational Safety
and Health Administration permissible exposure
limit, threshold limit, or other exposure limit
value.
 Designate responsible person(s) for the
program.
Spills Should Be Cleaned Up Using PPEs Like:
 Fume masks
 Gloves
 Goggles
 Impenetrable apron
 Respirators
 Face shields
Methods of Acquiring Infection in the Laboratory
1. Rubbing the eyes or nose with contaminated
hands
 Once in gloves, never touch your face or any
part of the body not protected by lab gown or
hazmat suit.
2. Inhaling aerosols.
3. Accidental ingestion of bacteria by putting pens
or fingers in the mouth.
 When using ballpens frequently used in the lab,
never use them bare handed and never let them
go near the mouth and disinfect them from time
to time
4. Needlesticks- accidental needle puncture (treat
all blood and body fluids as potentially infectious)
Giangan, Joyce Ann/ Ramas, Amiel. | Page 10 of 18

Universal/ Standard Precaution:
 W-Wash hands before and after each medical
procedure.
 W-Wear gloves whenever there is a possibility of
coming in contact with blood or other potentially
infectious materials (body fluids and tissues)
 W-Wear full-body gowns whenever there is a
possibility of blood splashing
 W-Wear face masks, face shields, and eye
protection whenever there is a possibility of
blood splashing into the face
 D- Dispose of all contaminated sharp objects in
an appropriate puncture-proof container and all
hazardous materials in an appropriate container
marked for bio-hazardous waste (trashcan with
yellow plastic bag)
 E- Environmental control should be adequate
and provide procedures for routine care and
cleaning of surfaces.
Disposable of Infection Waste:
 Infectious waste from microbiology laboratories
is autoclaved onsite or sent for incineration
before disposing.
 Infectious waste should be placed into 2 leak-
proof plastic bags for sturdiness.
 Pipettes, swabs, other glass objects should be
placed into rigid carbon containers before
disposal.
 Sharp objects, scalpels and needles are placed
in a sharps container which is autoclaved or
incinerated when full.
EPA (Environmental Protection Agency) waste reduction
through the following methods:
1. Substitute less hazardous chemicals when possible
2. Develop procedures that use less of a hazardous
chemical.
3. Recycle chemicals when possible.
4. Segregate infectious wastes from uncontaminated
trash.
5. Substitute micromethodology in antibiotic susceptibility
testing and identification of organisms to reduce
volume of chemical reagents as well as infectious
waste.
WHO Classification of Infectious Microorganisms by Risk
group:through the following methods:
 Risk Group I- no or low individual risk
 e.g., E. coli, B. subtilis
 Risk Group II- moderate individual risk, low
community risk
 Can cause mild disease, difficult to contract
by aerosol
 Treatment and preventive measures are
available
 e.g., Hepadna virus, Influenza A, Dengue
fever, HIV
 Risk Group III- high individual risk, low
community risk
 Can cause serious disease
 Can spread from one person to another
 Treatment and preventive measures are
available
 e.g., Anthrax, SARS, TB, Yellow fever
 Risk Group IV- high individual and community
risk
 Can cause serious disease
 Readily transmitted from one individual to
another
 Effective treatment and preventive
measures are not usually available
 e.g., Marburg virus, Ebola, Hanta, Lassa
Biosafety Cabinet (BSC)
 These are hoods that are a type of containment
barrier that protects the medical laboratory
scientists from aerosol transmission of
microorganisms.
 Only the hands with gloves and lab gowns would
be placed inside the BSC together with the
specimen.
 UV light- used to decontaminate the BSC after
processing the specimen inside the BSC
 Comparison of BSC Characteristics
 BSC Class I- it allows room air to pass into
the cabinet, it only sterilizes the air going
inside the cabinet
 BSC Class II (most common in microbiology
lab)- it sterilizes air that recirculates (inside)
and exhausted (outside) by the cabinet
 BSC Class III- it completely protects the
worker and is enclosed with negative
pressure.
Giangan, Joyce Ann/ Ramas, Amiel. | Page 11 of 18
 Airflow Application
BSC
Exhaust System
Class Nonvolatile toxic chemicals Volatile toxic chemicals and
Recirculated Exhausted
and radionuclides radionuclides

I Hard duct 0 100 YES When exhausted outdoors 1,2

II, Exhaust to room or

70 30 YES (minute amounts) NO
A1 thimble connection

II, Exhaust to room or When exhausted outdoors

70 30 YES
A2 thimble connection (minute amounts) 1,2

II,
Hard duct 30 70 YES YES (small amounts) 1,2
B1

II,
Hard duct 0 100 YES YES
B2

III Hard duct 0 100 YES YES (small amounts) 1,2

 Selection of Safety Cabinet through Risk Assessment

Protection Provided

Biological Risk Assessed BSC Class

Personne
Product Environment
l

BSL 1-3 Yes No Yes I

BSL 1-3 Yes Yes Yes II (A1, A2, B1, B2)

BSL-4 Yes Yes Yes III, II- when used in suitroom with suit

Giangan, Joyce Ann/ Ramas, Amiel. | Page 12 of 18

 Biosafety Levels
 BSLs consist of combinations of laboratory practices and techniques, safety equipment, and laboratory
facilities for proper handling of biohazardous materials.
 Most molecular labs are categorized as BSL-3 because it involves negative airflow. Before the medical
technologist goes into the molecular lab, they wear their PPEs (in a donning area) first and once they are
done with their work in the molecular lab, they would doff their PPE (in a doffing area) before going
outside the lab.

BSL AGENTS PRACTICES SAFETY EQUIPMENTS FACILITIES

Not known to consistently

cause diseases in Standard microbiological Open bench top, sink
1 None required
immunocompetent adult practices required
humans

Primary barriers: Class I or

BSL 1 practices plus: II biosafety cabinets or
· Limited access other physical containment BSL 1 plus:
Associated with human
·Biohazard warning devices used for ·Non-fabric chairs
disease. Hazard:
signs manipulation of agents that and other furniture
2 percutaneous injury, mucous
·Sharps precautions cause splashes or aerosols of that’s easy to clean
membrane exposure,
· Biosafety manual infectious materials; ·Autoclave
ingestion
defining waste PPE: laboratory coats, ·Eyewash readily
decontamination or gloves, face protection is available
medical surveillance needed

BSL 2 plus:
·Physical separation
from access
BSL 2 practices plus: corridors
Indigenous or toxic agents
·Controlled access ·Hands-free
with potential for aerosol
·Decontamination of handwashing sink
3 transmission; disease may Same as BSL 2
all wastes ·Self-closing double
have serious or lethal
·Decontamination of door access
consequences
lab clothing before · Exhaust fan
laundry ·Negative airflow
into laboratory
·Eyewash readily
available in lab

Primary barriers: All BSL 3 plus:

BSL 3 practices plus:
Dangerous/toxic agents which procedures conducted in ·Separate building
·Clothing change
pose high risk of life- Class III biosafety cabinets or isolated zone
before entering
4 threatening disease, aerosol- or Class I or II biosafety ·Dedicated
·Shower on exit
transmitted lab infections; or cabinets in combination supply/exhaust,
·All material
related agents with unknown with full-body, air supplied vacuum and
decontaminated on
risk of transmission positive pressure suit decontamination
exit from facility
system

Standard Microbiological Practices 4. Contact lens users wear safety glasses,

goggles or face shields.
1. Lab access limited/restricted when 5. Food stored outside lab in designated
experiments or work with cabinets/refrigerators.
cultures/specimens are in progress. 6. Mechanical pipetting devices are used
2. Lab personnel should wash hands after (i.e., no mouth pipetting).
handling viable materials, removing 7. Sharps handling policies/practices in
gloves, or leaving lab. place.
3. No eating, drinking, smoking, handling 8. Procedures minimize splashes/aerosols.
contact lenses, applying cosmetics, or
storing human food in lab.
9. Work surfaces are decontaminated at
least daily and/or at completion of work.

10. Work surfaces are decontaminated after

any spill/splash of viable material.

Giangan, Joyce Ann/ Ramas, Amiel. | Page 13 of 18

11. Disinfectants are labeled for agents
being used.
12. Cultures/stocks/regulated wastes are
decontaminated by approved method
(e.g., autoclaving) before disposal.
13. Materials decontaminated outside of lab
are transported in durable, leak-proof,
closed containers.
14. Biohazard signage posted at lab entrance
when infectious agents are present
(signage lists agents and Person in-
charge name/phone).
15. Insect/rodent control program in effect

Giangan, Joyce Ann/ Ramas, Amiel. | Page 14 of 18

CLINICAL BACTERIOLOGY (LEC)
SPECIMEN COLLECTION, TRANSPORT, AND
PROCESSING Specimen Processing
Objectives:
Know proper specimen collection procedures 1
for different specimen types
Enumerate the criteria for specimen rejection 2
State the evidence for bacterial growth 4
Know the indications for culture of different
type of specimens
5 lab, the time and date are noted and recorded
Specimen Collection, Handling, and Transport
 For optimal detection of pathogens
responsible for a disease, specimens should
be collected in the acute phase of an illness
and before giving antibiotics.
 The acute phase of the patient is
characterized by the following:
 Immediate pain
 Tenderness
 Swelling
 Inflammation and edema
 Contour deformity
 Bleeding
 Loss of normal function of the injured
area
 The volume of specimen should be adequate
for the performance of the studies requested.
 Specimen should be obtained from site of
infection with minimal contamination from
adjacent tissues and organ secretions.
 All specimens should be collected in a sterile
container (except stool) and labeled with:
 Name
 Hospital ID #
 Source of specimen
 Time of collection • After specimen
collection, place the specimen in a
biohazard bag and transport it to the lab.
If there is delay, refrigerate it to prevent
overgrowth of normal flora.
 Appropriate swab for collection: polyester-
tipped swab with a plastic shaft.
 Cotton-tipped swabs cannot be used
because it is toxic to Neisseria
gonorrheae, and wooden-shaft swabs are
toxic to Chlamydia trachomatis.
 The use of swab is not appropriate/not
optimal for anaerobes and mycobacteria
collection.
 The tissue sample/aspirate is superior to
swab specimens for the recovery of
pathogenic organisms but the collection
requires invasive procedures unlike via
swab which is a non-invasive procedure.
 Once a specimen has been received in the
by the medical technologist.
 The medical technologist should also
determine if it is a critical specimen. Process
critical specimens first:
1. CSF
2. Tissue
3. Blood
4. Other sterile body fluids
 In the case of urine, throat, sputum,
stool, and wound drainage, they may be
processed later but do not take too much
time in not processing the specimen
because we are dealing with
microorganisms.
 In the case of QNS (quantity not
sufficient) specimens, immediately call
the clinician to prioritize the testing and
do not reject the specimen immediately.
 Gross Examination of Specimen
 All processing should begin with gross
examination of the specimen.
 Areas with blood or mucus should be
located and sampled for culture and
DME.
1. Volume
2. Color- colorless, pinkish,
greenish, etc.
3. Viscosity- slightly viscous,
viscous
4. Appearance- turbid, cloudy,
clear, bloody
 Direct Microscopic Examination (DME)
 Purpose of DME:
 To assess the quality of the specimen
 Gives the med tech an early indication of
what might be wrong with the patient
 The work up of the specimen can be
guided by comparing what grows in
culture to what is seen on the smear
 Example: In performing DME, 3
morphologic types were seen but in the
culture result, only 2 grew out because
the other organism may be an anaerobe
and the culture media was incubated in
an aerobic environment.
 DME are usually not performed on throat,
nasopharyngeal, or stool specimens.
 Generally acceptable transport time of
specimen is within 2 hours after collection
but is better if transported immediately.
Criteria for Specimen Rejection
Giangan, Joyce Ann/ Ramas, Amiel. | Page 15 of 18
  Any specimen received in formalin.
(formalin spx belongs to histopathology)
 24 hour sputum collections
 Specimens placed in containers with leaks
(dealing with sterile spx)
 Out-dated or dried out specimens
 Specimens contaminated with barium,
chemical dyes, or oily chemicals ▪
Specimens from the tip of Foley catheters
 Duplicate specimens (except blood)
received in 24 hr. period
 Info on the label doesn’t match the info on
the request slip (mislabeling)
 Quantity Not Sufficient (QNS) (ask the
physician/clinician first)
 Blood catheter tips submitted for patients
without concomitant positive blood culture
Criteria for Specimen Rejection for Anaerobic Culture:
 Gastric Washings
 Urine (except for suprapubic aspirate)
 Stool (except for the recovery of Clostridium
difficile)
 Oropharyngeal specimens (except deep tissue
samples obtained during a surgical procedure)
 Sputum
 Swabs of ileostomy or colostomy sites (a tube
inserted into the belly opening of the patient to
remove wastes when the colon/rectum are not
functioning properly)
 Superficial skin specimens (not appropriate for
anerobic culture)
Different Types of Specimens Commonly Encountered
in Microbiology
Blood
 Indications for blood culture: bacteremia, sepsis,
infections of prosthetic valves, vascular grafts, mycotic
aneurysms, suppurative thrombophlebitis (vein
inflammation).
 When collecting blood for blood culture, disinfect with
70% ethyl alcohol first followed by iodine. Dry for 1-2
mins. before initiating venipuncture.
 It is best to collect blood just before chills and the best
method for blood collection is through the closed
evacuated tube system. Collect 1-5 ml of blood. Invert
blood culture bottles. Transport to the lab ASAP and
never refrigerate blood culture bottles. Incubate the
bottles for 7 days before reporting a negative result.
 The phlebotomist can also collect via the
open system (syringe) if the phlebotomist is
having a hard time doing the venipuncture
procedure. Once blood is collected using the
syringe, it is then punctured directly to the
blood culture bottle. Remove the cap first
and upon removal of the cap, it is disinfected
with an alcohol swab.
 For the closed system, the blood is directly
collected to the blood culture bottle.
Blood Culture Bottles
The ratio of blood to broth is 1:10 to
neutralize the serum bactericidal activity
because serum has antibodies, WBCs, and
antibiotics. This may impede the recovery of
microorganisms.
 Blood culture bottles also have antibiotic
resin device (ARD) to remove the
antimicrobials from the blood, especially if
the patient has taken antibiotics prior to
blood collection. Blood culture bottles also
have Fastidious antibiotic neutralization
(FAN).
 The cap of the blood culture bottles are color
coded.
❖ Blue cap indicates a plain blood culture
bottle; Green cap- the blood culture bottle is
incorporated with ARD; Yellow cap- for
pediatric patients; Maroon cap- anaerobic
culture.
 Collect 2-3 separate blood cultures from
different sites are recommended for
bacteremia with a time interval of 30-60
mins. per collection. This is for optimal
recovery of the microorganisms.
 Anticoagulant for blood culture bottles:
0.025-0.05% of SPS (sodium
polyanetholsulfonate)
 Aside from being an anticoagulant,
SPS will also prevent phagocytosis
and inactivate aminoglycosides.
 Volume: For infants, 1-5 ml of blood
is adequate. For adults with
bacteremia and CFU/mL is low,
collect 20-30 ml of blood.
 When blood culture bottle is positive,
subculture it to the following culture media:
 Culture media for recovery of aerobic
pathogens in blood (incorporated to the
blood culture bottle): TSB (Trypticase
Soy Broth), Supplemented peptone,
BHIB (Brain-heart Infusion Broth)
 Culture media for anaerobes in blood:
THIO (thioglycollate broth), anaerobic
heart infusion broth.
Manual Blood Culture
 Biphasic System
❖ There is a broth in the bottle and blood is
mixed in this broth.
Giangan, Joyce Ann/ Ramas, Amiel. | Page 16 of 18
 ❖ When it is mixed with the blood collected, it
is then sub cultured into the agar above (where
the blood-broth mixture is sub cultured). The
bottle is inverted to sub culture it into the
culture media placed above the bottle.
 Lysis Centrifugation Blood Culture System
❖ Consists of a tube containing SPS and
provides a cushion for the microorganisms
during centrifugation.
❖ Centrifuge the tube for 30 mins at 3000 G
and discard the supernatant.
❖ Vortex the sediment to lyse the RBCs then
plate the sediment on appropriate culture
media.
 Evidence of Bacterial Growth
Turbidity
Hemolysis
Gas production
Presence of discrete colonies
Automated Blood Culture
 BacT alert system
 Touch to input necessary information of the
patient. Scan the barcode of the blood
culture bottle. Load it into the cell.
 Positive blood cultures are indicated 12-36
hrs after incubation.
 Fastidious organisms of the hacek group
(Haemophilus, actinobacillus,
cardiobacterium, eikenella, and kingella)
may not be detected after 3-5 days (longer
incubation time).
 Principle: It is a colorimetric detection of
CO2 produced during microbial growth.
 Blood culture bottle used for the BacT alert
system has a CO2 sensor on the bottom of
the bottle which is permeable to carbon
dioxide. If bacteria is present in the blood of
the patient, carbon dioxide will be released
from the broth medium through the CO2
sensor.
 It would then combine with water to form
carbonic acid (H2CO3), causing decrease in
pH.
 The decrease in pH will increase the
reflectance (sensor changes from green to
yellow; yellow indicates growth of bacteria).
Once reflectance is increased, growth curve
enters in log phase. The bottle would then
flag as positive.
 Color changes are monitored every 10 mins.
 Bactec system
 Principle: CO2 detection by fluorescence
 CO2 generates hydrogen ions causing a
subsequent decrease in pH which in turn
increases the fluorescence output of the sensor
changing the signal transmitted to the machine
 Another automated system
 A sensor detects growth of
microorganisms by measuring gas
consumption and/or gas production
 Gas measured: CO2, N2, H2

 Positive growth in blood cultures
 Smear the broth then do Gram stain to
check for morphology under
microscope
 Subculture to THIO, TSB, BHIB
 Hemolysis Pattern on Blood Agar Plate
 After incubation, hemolysis pattern is
then observed using the blood agar
culture media.
Cerebrospinal Fluid (CSF)
 CSF is a clear, plasma-like fluid that circulates
outside of the brain, in the cavities within the
brain (ventricles), and the space surrounding
the spinal cord.
 By obtaining CSF is to lumbar tap/lumbar
puncture in the L3-L4 region. This procedure is a
sterile technique to reduce risk of infection to
the patient. 6-15 mL of spinal fluid is collected
and less volume in babies and children.
 Upon collection, it is divided among 3 tubes, 2-4
mL each and glass tubes are not acceptable
because they would cause cell adhesion which
would result into low cell count and low
differential count.
 Tube 1 (first tube collected) for clinical
chemistry section; tube 2 for
microbiology; tube 3 for hematology;
tube 4 is indicated for other tests (e.g.,
cryptococcal antigen test, or serologic
test for syphilis or cytology)
 Indications for CSF Culture: To diagnose
meningitis and viral encephalitis
 Transport to the lab ASAP and process ASAP. Do
not refrigerate except for viral cultures because
the fastidious organisms (e.g., Haemophilus
influenzae, Neisseria meningitidis) may not
survive the cold temperature. If delay is
Giangan, Joyce Ann/ Ramas, Amiel. | Page 17 of 18
 unavoidable, keep it at room temperature for
isolation of non-fastidious organisms.
 More than or equal to 1 mL is sufficient.

 Processing CSF in Micro lab:

1) Centrifuge the CSF for 15 minutes. Decant the
supernatant into a sterile tube leaving 0.5 mL of
sediment and fluid.
2) Vortex the sediment or vigorously aspirate it up and
down using a pipette.
3) Using the sediment, make a smear and do Gram
staining.
4) Do CSF culture using appropriate culture medium.
 The supernatant is stored for rapid diagnosis
(latex agglutination test indicated for
Streptococcus agalactiae, Streptococcus
pneumoniae, Neisseria meningitidis, Escherichia
coli, and Haemophilus influenzae Type B).
 These tests are useful in the diagnosis
of partially treated meningitis and it
confirming a positive gram stain smear.
 These rapid tests are discouraged
because of low sensitivity and is quite
expensive.
 Common organisms causing meningitis:
Streptococci, Escherichia coli, Listeria
monocytogenes, Neisseria meningitidis.
Also do a CSF culture to diagnose
leptospirosis (Leptospira interrogans)
and neurosyphilis (Treponema
pallidum).
 Other Body Fluids (Peritoneal Fluid, Thoracic
Fluid, Pericardial Fluid, Synovial Fluid)
 Sample volume requirement: 1-5 mL
 Sample Processing:
1. Centrifuge the body fluid
for 20-30 mins. Decant the
supernatant.
2. Mix sediment thoroughly
and make smears for Gram
staining and culture

Giangan, Joyce Ann/ Ramas, Amiel. | Page 18 of 18