[TRANS] TOPIC 5: CONTROLLING MICROBIAL GROWTH IN VITRO

E. Psychotrophs – are psychrophiles with optimum

growth temperature of 4°C (refrigerator temperature)
OUTLINE Example: bread molds
I Factors that affect Microbial Growth F. Psychroduric organisms – they prefer warm
II Encouraging the Growth of Microbes In Vitro temperatures but can tolerate cold temperatures; can
A Types of Culture Media be preserved in a frozen state
III Inhibiting the Growth of Microbes in Vitro
IV Factors that affect the Activity of Disinfectants
V Physical Methods of Microbial Control
A Heat Table 1. List of bacterial groups according to their temperature
B Pasteurization requirements
C Filtration CATEGORIES OF BACTERIA BASED ON THEIR TEMPERATURE
D Cold REQUIREMENTS
E Desiccation Category Minimum Optimum Maximum
F Radiation
VI Chemical Methods of Microbial Control
Growth Growth Growth
A Chemical Methods of Sterilization Temperature Temperature Temperature
B Chemical Methods of Disinfection (°C) (°C) (°C)
Thermophiles 25 50-60 113
Mesophiles 10 20-40 45
FACTORS THAT AFFECT MICROBIAL GROWTH Psychrophiles -5 10-20 30
1. Availability of Nutrients
Nutrients are the various chemical compounds ➢ Clinical Laboratory incubates their bacterial culture at 35°C.
needed to sustain the life of the organisms. These serve as ➢ Pseudomonas and Campylobacter can grow both at 35°C
source of carbon, oxygen, hydrogen, nitrogen, phosphorus, and 42°C.
and sulfur as well as other elements (e.g., sodium;
potassium, chlorine; magnesium; calcium; and trace 4. pH
elements such as iron, iodine, and zinc) that are usually ➢ Most microorganisms prefer a neutral to slightly
required in lesser amounts. alkaline growth medium (pH 7.0 – 7.4)
2. Moisture A. Acidophiles – prefer a pH of 2-5
All living organisms require water to carry out their Example: Lactobacillus acidophilus
normal metabolic processes, and most will die in B. Alkaliphiles – prefer pH >8.5
environments containing to little moisture. Microbes with Example: Vibrio cholerae is the only human pathogen
spores may become dormant for years at a dry that grows above pH 8
environment (desiccation). But when they are placed in a 5. Osmotic Pressure
moisture- and nutrient-rich ➢ Osmotic pressure is the pressure that is exerted on a
3. Temperature cell membrane by solutions both inside and outside the
➢ Optimum temperature, is the temperature cell.
required by organisms to grow best. ➢ Osmosis is the movement of solvent through a
➢ When organisms are placed below their permeable membrane from a solution having a lower
minimum required temperature, they will not concentration to a higher concentration
grow. ➢ Cells survive in an isotonic solution, that means the
➢ When they are placed at a temperature above its concentration of the fluid where the cells are
maximum requirement, they will die. suspended is equal to the concentration of the fluids
On the basis of temperature requirements, organisms may be inside the cells.
grouped into the following: ➢ Hypertonic solution – a solution with increased
A. Thermophiles – organisms that grow at high concentration of solutes
temperature (they love heat). They are found in hot ➢ Plasmolysis – happens when the bacterial cell
springs, compost pits, and hydrothermal vents. membrane and cytoplasm shrink away from the
Example: Cyanobacteria bacterial cell wall as the fluid inside the cell moves out
B. Extreme Thermophiles or Hyperthermophiles – in an attempt to equalize the concentration. This
organisms that favor growth at temperatures above happens when the bacterial cells are placed in a
100°C hypertonic solution. (In humans, RNCs for example
Example: Pyrolobus fumarii (an archaeon; undergoes crenation, or shrinkage).
grows at 113°C) ➢ Hypotonic solution – a solution with decreased
C. Mesophiles – organisms that grow at moderate concentration of solutes.
temperature; grows best at 37°C; most pathogenic ➢ Plasmoptysis – happens when the bacterial cell
organisms are mesophilic organisms ruptures and the cytosol escapes. This happens when
D. Psychrophiles – are cold-loving organisms; grows the bacterial cells are placed in a hypotonic solution.
best at -13°C, has 20% salinity, and contains high The water in the solution gets inside the bacterial cells
concentrations of ammonia and sulfur causing increased pressure of fluid inside the cells.
Example: Yersinia monocytogenes, Unlike human cells, bacteria have rigid cell walls. Thus,
Yersinia enterocolitica lysis or rupture does not happen immediately.

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 TRANS: CONTROLLING MICROBIAL GROWTH IN VITRO

However, continued suspension of bacterial cells in the

hypotonic solution leads to a pressure so great that the
cells rupture.

ENCOURAGING THE GROWTH OF

MICROBES IN VITRO
Culture Media – artificial or synthetic media used in the
laboratory to grown microorganisms
6. Salinity
TYPES OF CULTURE MEDIA
A. Halophilic organisms – “halo” means salt; organisms
A. According to Consistency
that grow best at a salty environment
A.1. Liquid (broth culture medium)
Example: Vibrio spp.
A.2. Semi-solid
B. Haloduric organisms – organisms that DO NOT
A.3. Solid
prefer a salty environment but CAN SURVIVE
A.4. Biphasic medium (contains both solid and liquid
Example: Staphylococcus aureus
phase)
7. Barometric Pressure
B. According to Composition
➢ Bacteria survive at normal barometric pressure, about
B.1. Complex or non-synthetic (undefined contents for
14.7 pounds per square inch (psi)
example, meat broth/extracts)
➢ Piezophiles – microorganisms that survive deep in the
B.2. Synthetic or defined (commercially prepared with
ocean or in oil wells where the atmospheric pressure is
known contents)
very high
B.3. Tissue or cell-containing medium
8. Gaseous Atmosphere
A. Obligate aerobes
C. According to How the Media is Dispensed
✓ Organisms which require Oxygen to live
C.1. Plated in Petri dish
✓ Require 21% Oxygen & 0.03% CO₂
C.2. Tube, which may have a slant part only (ex. Simmon’s
✓ Contain Superoxide dismutase, the enzyme
Citrate agar), a butt part only [ex. Sulfide-Indole-Motility
that protect them harmful O₂ (SIM) medium], or both a slant and butt parts [ex. Triple
✓ Examples: Mycobacterium & Micrococcus Sugar Iron (TSI) Agar, and Lysine Iron Agar (LIA)]
B. Obligate anaerobes
✓ Bacteria that grow on the absence of D. According to Purpose
atmospheric or free Oxygen and obtain D.1. Transport medium
Oxygen from Oxygen-containing compound ✓ Transports the microorganisms to prevent drying
instead and disintegration
✓ Unable to grow and multiply in the presence ✓ Examples: Stuart, Amie’s Cary-Blair, TransGrow
of Oxygen D.2. Simple or General Isolation Medium
✓ Require: 0% Oxygen, 5-10% CO₂, 80-90% ✓ Supports the growth of most non-fastidious
Nitrogen, and 5-10% Hydrogen microorganisms
✓ DO NOT have Superoxide dismutase, ✓ Examples: Nutrient Agar (NA), Nutrient Broth,
catalase, and Cytochrome oxidase Trypticase Soy Agar (TSA), Trypticase Soy Brith
C. Facultative aerobes (TSB)
✓ Fundamentally an ANAEROBE, but can grow D.3. Enrichment Media or Broth
in the presence of atmospheric Oxygen ✓ Enhances the growth of one organism while
D. Facultative anaerobes inhibiting the growth of another organism (normal
✓ Fundamentally an AEROBE, but can grow in flora)
the absence of atmospheric Oxygen ✓ In liquid form
E. Capnophiles ✓ Examples: Gram negative (GN) broth, Selenite F
✓ Require increased Carbon dioxide; 5-10% broth, Tetrathionate broth, Thioglycollate, Alkaline
Carbon dioxide and 15% Oxygen Peptone Water (APW)
✓ Example: Haemophilus and Neisseria D.4. Enriched Medium
F. Aerotolerant ✓ Is derived from general isolation medium but with
✓ Does not grow well, but survives in the added nutrients such as blood, serum, peptone,
presence of atmospheric (free) Oxygen and vitamins
G. Microaerophiles ✓ Examples: Blood Agar Plate, Chocolate Agar
✓ Requires reduced Oxygen; 5% O₂, 10% CO₂, D.5. Selective Medium
85% N₂ ✓ A medium is selective when substances are added
✓ Example: Campylobacter, Helicobacter to selectively grow bacteria, or added with
bacteriostatic agents that prevent the growth of
unwanted microorganisms
✓ Examples of selective agents:
o Gentian violet – inhibits Gram positive
bacteria

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o Sodium desoxycholate and other bile 8. Lyophilization

salts inhibit Gram positive bacteria ✓ Freeze-drying
o Alcohol and Chloral hydrate – prevent ✓ The process of dehydration (drying) followed by
spreading of Proteus freezing to preserve the bacteria for future use and
o Sodium azide inhibits Gram negative study
bacteria 9. Sepsis
o Malachite green (found in Petragnami ✓ Refers to the presence of pathogen in blood or
and Lowenstein media for M. tissue
tuberculosis) prevents the growth of ✓ ASEPSIS = absence of pathogens
contaminants 10. Antisepsis
✓ Most selective media are also differential media. ✓ Is the prevention of infection
Thus, they are known as Selective Differential 11. Degerming/Degermation
Media ✓ Mechanical removal of microbes rather than killing
✓ Examples of Selective Differential Media: from a limited area, for example, the skin around
MacConkey (Mac) Agar, Mannitol Salt Agar, an injection site
Thiosulfate-Citrate Bile Salts Sucrose (TCBS)
Agar FACTORS THAT AFFECT THE ACTIVITY OF
D.6. Antibiotic Susceptibility Medium DISINFECTANTS
✓ Mueller-Hinton Agar is used for Kirby-Bauer Disk 1. Type of organism present
Diffusion Antibiotic Susceptibility testing ✓ Bacillus are most resistant, followed by
D.7. Differential Medium Mycobacteria, Non-lipid viruses (ex. Poliovirus),
✓ Provides distinct colonial characteristics of the fungi, vegetative (non-sporulating bacteria), and
organism finally lipid viruses (Ex. Herpes simplex virus, most
✓ Can differentiate one group of organisms to susceptible)
another (ex. Lactose fermenters from non-lactose
fermenters) 2. Temperature and pH
D.8. Antibiotic Medium
✓ It is a medium that contains antibiotics as inhibitors 3. Microbial load (number of organisms present)
✓ Example: Thayer-Martin, Modified Thayer-Martin ✓ The time necessary to kill microorganisms is
D.9. Medium for Biochemical Testing directly proportional to the number of
✓ Used to test biochemical activities and detects microorganisms present
chemicals or metabolites produced by the
organism 4. Concentration of disinfectant
✓ Examples: Russell’s Double Sugar (RDS) Agar,
Kligler Iron Agar (KIA), Triple Sugar Iron (TSI) 5. Amount of organics present (presence of blood, mucus,
Agar, Sulfide-Indole-Motility (SIM) medium, Lysine vomitus, pus)
Iron Agar (LIA) ✓ The time necessary to kill microorganisms
increases in direct proportion to the amount of
INHIBITING THE GROWTH OF MICROBES IN VITRO organics present; thus, organic material should be
Definition of Terms: removed first before chemical sterilization
1. Sterilization
✓ Is the destruction or elimination of all microbes, 6. Nature of surface to be disinfected
including cells, spores, and viruses
✓ STERILE = devoid of microbial life 7. Length of contact time
2. Disinfection
✓ Elimination of most or all pathogens except 8. Type of water available
bacterial spores, from nonliving objects ✓ Hard water may reduce the rate of killing
✓ Chemicals used to disinfect inanimate objects are microorganisms
called DISINFECTANTS ✓ 70% alcohol is more effective than 95% alcohol
✓ Chemicals used to disinfect skin and other living due to its higher water content
tissues are called ANTISEPTICS
3. Pasteurization
✓ Is a method of disinfecting liquids
✓ Used to disinfect milk, wine, and other beverages
4. Sanitization
✓ Reduction of bacterial populations to levels
considered safe by Public Health Standards
5. Bactericidal agents
✓ “cide” or “cidal” refers to killing. Ex. homicide,
suicide, genocide
✓ Drugs or chemicals that kill bacteria but not
necessarily those with bacterial spores
6. Sporicidal agents
✓ Are used to kill bacterial endospores
7. Bacteriostatic agents
✓ “static” means to stop or steady
✓ Are drugs or chemicals that can inhibit the growth
and multiplication of bacteria, but not necessarily
killing them

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PHYSICAL METHODS OF MICROBIAL CONTROL high protein content since high temperatures can denature
proteins; achieved at 76-80°C for 2 hours, 3 consecutive
days
HEAT C. Autoclaving
✓ Is the most common type of sterilization for inanimate ̶ A sterilization technique that uses steam under
objects able to withstand high temperatures pressure to completely destroy all microbial life
✓ Practical, efficient, and inexpensive (except, prions)
✓ Two factors that determine the effectiveness: TIME ̶ The most effective method of sterilizing culture
and TEMPERATURE media and surgical supplies
✓ Thermal Death Point (TDP) – the lowest temperature ̶ Autoclaves should be set to run 20 minutes at a
that will kill a standard pure culture of organisms in a pressure of 15 psi and a temperature of 121.5°C
specific period of time (for culture media, liquids, and instruments)
✓ Thermal Death Time (TDT) – the length necessary to ̶ 132°C for 30-60minutes at 15 psi for infectious
sterilize a pure culture at a specific temperature medical waste
̶ Bacillus stearothermophilus: biological
DRY HEAT (STERILIZATION TECHNIQUE) indicator for the effectiveness of autoclaving
A. Hot air (Oven)
Fig 4. Biological indicator of the
̶ uses a thermostatically controlled oven
̶ Effective sterilization of metals, glassware, some effectiveness of autoclaving
Sealed ampules containing bacterial
powders, oils, and waxes spores suspended in a growth medium
̶ The items are baked at 160-165°C for 2 hours, or are placed within the load to be
170-180°C for 1 hour sterilized. Following sterilization, the
B. Incineration (burning) ampules are incubated at 35°C. If the
̶ Effective means of destroying contaminated spores were killed, there will be no
disposable materials change in the color of the medium; it
̶ Most common method of treating infectious will remain purple. If the spores were
waste not killed, germination will occur, and
acid production by the bacteria will
̶ Contaminated materials are burned to ashes, cause the pH indicator in the medium
temperature is 870- 980°C to changed from purple to yellow.
C. Flaming
̶ Used for sterilizing wire loops and forceps
̶ Electrical heating devices are now used instead of
an open flame from alcohol lamps or Bunsen PASTEURIZATION
burners ✓ A method to achieve disinfection (but not sterilization)
MOIST HEAT of liquids such as milk, and wine
✓ Advantage: reduces spoilage of food without affecting
✓ Heat is applied in the presence of moisture (ex. steaming &
its taste
boiling)
✓ Faster, and more effective than dry heat
A. Flash Pasteurization or High Temperature Short-Time
✓ Can be accomplished at a lower temperature; thus, it is less
(HTST) Pasteurization
destructive to many materials that otherwise would be
✓ Disinfection is accomplished at 72°C (161°F) for 15
damaged at higher temperatures
seconds (or 74°C for 4-5 seconds)
✓ Moist heat causes proteins to coagulate (as occurs when
B. Batch Pasteurization
eggs are hard boiled).
✓ Disinfection is accomplished at 62°C for 30 minutes
✓ Because cellular enzymes are proteins, they are inactivated
(63°C in some references)
by moist, leading to cell death.
A. Boiling
̶ Disinfection is achieved by boiling at 100°C for • Ultra-High-Temperature Treatments (UHTT): a
10 minutes sterilization method, not a pasteurization method) may be
̶ Vegetative forms of most pathogens are easily used to treat milk; done at 140°C for 4 seconds
destroyed by boiling for 30 minutes
̶ Water should always be boiled for longer times at FILTRATION
high altitudes ✓ May be used for both liquid and air
̶ Boiling is not always effective, however, because
heat-resistant bacterial endospores, A. Liquid Filtration
mycobacteria, and viruses may be present ✓ Accomplished through the use of thin membrane
̶ Microorganisms resistant to boiling: Bacillus, filters composed of plastic polymers or cellulose
anthracis, Clostridium tetani, Clostridium esters containing pores of a certain size
botulinum, Hepatitis viruses, and all thermophiles ✓ Most bacteria, yeasts, and molds are retained by
B. Fractional/Intermittent/Discontinuous Sterilization pore sizes of 0.45 and 0.80 μm; however, this
̶ Involves alternate heating, incubation, and heating pore size may allow passage of Pseudomonas-
methods like organisms, and therefore a 0.22-μm size is
̶ Used to sterilize media containing milk and serum available for critical sterilizing (e.g., parenteral
̶ Day 1: destroys vegetative bacteria solutions)
̶ Day 2: destroys spores ✓ Membranes with pore sizes of 0.01 μm are
̶ Day 3: destroys remaining spores capable of retaining small viruses
B.1. Tyndallization – uses Arnold’s sterilizer; uses ✓ The most common application of filtration is in the
flowing steam for 30 minutes using temperature of 100°C sterilization of heat-sensitive solution, such as
at atmospheric pressure for 3 consecutive days parenteral solutions, vaccines, and antibiotic
B.2. Inspissation – thickening through evaporation; used solutions
for sterilization of culture media that are egg-based and

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B. Filtration of air ✓ Inactivate microorganisms by denaturing proteins,

✓ Accomplished with the use of high-efficiency dehydrating cells, and dissolving lipids
particulate air (HEPA) filter ✓ Should be used in concentrations between 60-
✓ HEPA filters are able to remove microorganisms 90%, with 70% as the most effective
larger than 0.3 μm and are used in laboratory ✓ They have effective bactericidal activity against
hoods and in rooms of immunocompromised Gram positive & Gram-negative bacteria
patients ✓ Also effective in killing Mycobacterium
tuberculosis and various fungi as well as some
COLD enveloped viruses
✓ Rapid freezing, using liquid nitrogen, is a good way ✓ Not effective in killing bacterial spores, and some
to preserve foods, biologic specimens, and bacterial non-enveloped viruses
cultures ✓ Filter the alcohol using a 0.22 μm filter to remove
✓ Refrigeration cannot be relied upon to kill bacterial spores (Alcohols may be contaminated
microorganisms; it merely slows their metabolism and with bacterial spores because it is not sporicidal)
their rate of growth
✓ Thawing foods allow bacterial spores in the foods to 2. HALOGENS
germinate and microorganisms to resume growth A. Iodine
✓ Repeated thawing and freezing are also not ✓ 70% Ethyl alcohol followed by an Iodophor and
recommended because it preserves the microbes that another round of alcohol is the most common
might be present leading to deterioration of food when compound used as skin antiseptic before drawing
re-thawed. blood for cultures and before surgery
✓ Prepared as: 2% Iodine in 50% alcohol
DESSICATION ✓ Can be prepared as an Iodophor = Iodine +
✓ Lyophilization – the combined use of freezing and Detergent/neutral polymer (ex. Povidone); best
drying example is Betadine
✓ In a hospital setting, dried clinical specimens and dust B. Sodium hypochlorite
may contain viable microorganisms ✓ Also known as household bleach
✓ Recommended dilution is 1:10
RADIATION ✓ Use 1:10 dilution for porous surfaces; 1:100
A. Ionizing (Gamma) radiation smooth, hard surfaces; 1:5 for large spills
✓ Used for sterilizing disposables (plastic syringe, ✓ Inactivates Hepatitis B virus (HBV) within 10
catheters, gloves) minutes and HIV within 2 minutes
✓ Uses short wavelength but high gamma rays
✓ Biological indicator: Bacillus pumilis 3. QUATERNARY AMMONIUM COMPOUNDS
B. Non-ionizing radiation (UV light) ✓ Example: Zephiran (Winthrop) and Benzalkonium
✓ Uses Mercury arc lamps with wavelength of 240- chloride are used to disinfect bench-tops and floors
280 nm ✓ Non-sporicidal and non-tuberculocidal
✓ For room disinfection to reduce cross-infection ✓ Pseudomonas aeruginosa is resistant to QUATS
among personnel
✓ Does not penetrate well, the organism must have 4. ALDEHYDES
direct surface exposure such as the working table, ✓ 1-8% Formaldehyde or Glutaraldehyde (variable
surface of a biological safety cabinet, cellphones, strength) – not used as surface disinfectants due to its
ballpens irritating fumes

CHEMICAL METHODS OF MICROBIAL CONTROL 5. OXIDIZING AGENTS

✓ 3-30% Hydrogen peroxide (Agua Oxinada) and
CHEMICAL METHODS OF STERILIZATION Potassium permanganate
1. Ethylene Oxide (EtO)
✓ Used in gaseous form for heat-sensitive objects
6. HEAVY METALS
2. Formaldehyde vapor and Vapor-phase Hydrogen
Peroxide (oxidizing agent) ✓ Mercury – active component of Merthiolate; toxic to
✓ Used to sterilize HEPA filters in Biological Safety humans and environment
Cabinets (BSCs) ✓ Copper – algaecide
3. Glutaraldehyde or Peracetic acid (Cold sterilization) ✓ Silver - 1% AgNO₃; used in Crede’s prophylaxis for
✓ Glutaraldehyde is sporicidal in 3-10 hours and infants with eye infection known as Opthalmia
may be used for medical equipment such as neonatorum caused by Neisseria gonorrhoeae
bronchoscope since it does not corrode lenses, ✓ Manganese – present in Potassium permanganate
metal, or rubber
✓ Peracetic acid is effective in the presence of 7. ACIDS
organic material; used in the sterilization of ✓ Nitric acid
surgical instruments ✓ Sulfuric acid

CHEMICAL METHODS OF DISINFECTION 8. ALKALIS

✓ Potassium hydroxide
1. ALCOHOLS ✓ Sodium hydroxide
✓ The most effective alcohols used in the hospitals
are ETHYL and ISOPROPYL

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9. PHENOLS AND PHENOLICS

A. Chlorhexidine gluconate (CHG)
✓ Disrupts the microbial cell membrane and precipitates
the cell contents
✓ CHG (0.5% to 4%) is more effective against Gram-
positive than Gram-negative bacteria and has less
activity against fungi and tubercle bacilli
✓ Lipid-enveloped viruses (e.g., herpes virus, HIV,
respiratory viruses, influenza virus, cytomegalovirus)
are rapidly inactivated
✓ CHG is safe and nontoxic, but skin reactions are seen
in infants
✓ CHG should not come into contact with the eyes, the
middle ear, or meninges
✓ ADVANTAGE: remains active in skin for 6 hours
B. Hexachlorophene
✓ Effective against gram-positive bacteria
✓ It is a chlorinated bisphenol that interrupts bacterial
electron transport, inhibits membrane-bound
enzymes at low concentrations, and ruptures
bacterial membranes at high concentrations
✓ Gram-positive bacteria are killed by 3%
hexachlorophene within 15 to 30 seconds, but a
longer time is needed for gram-negative bacteria
✓ Associated with severe toxic effects, including
deaths
✓ 3% hexachlorophene to be available only by
prescription and designated
✓ Indicated to control outbreaks of gram-positive
infections when other infection control procedures
have been unsuccessful
C. Chloroxylenol [parachlorometaxylenol (PCMX)]
✓ Halogen-substituted phenolic compound PCMX at
concentrations of 0.5% to 4%
✓ Acts by microbial cell wall disruption and enzyme
inactivation
✓ PCMX has good activity against gram-positive bacteria

REFERENCES

Notes from the audiobook transcript on Controlling Microbial

Growth In Vitro by Ms. Rolaine Ann A. Polo, RMT, MPH

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 06
[TRANS] TOPIC 6: SPECIMEN COLLECTION & PROCESSING IN MICROBIOLOGY
6. For specimens collected via swabs, Calcium alginate
Dacron or Rayon swabs may be used for bacterial culture,
OUTLINE because cotton swabs may release fatty acids that are toxic to
I General Guidelines for Specimen Collection the bacteria.
II Fundamentals of Specimen Collection • However, if we need to collect specimens for viral
III Specimen Labels
culture or determination, Calcium alginate is NOT
IV Specimen Requisition
V Grounds for Specimen Rejection recommended because it is toxic to viruses.
VI Most Common Specimens Received in the Laboratory 7. The quantity of the specimen must be adequate because
A Abscess (also lesion, wound, pustule, ulcer) the greater the quantity of the specimen, the higher the chance
B Blood or Bone Marrow Aspirate/ Specimens of isolating the organism.
C Cerebrospinal Fluid 8. Specimens for microbiologic analysis must be taken to the
D GI Tract Specimens laboratory and examine promptly. (Preferably within 2 hours)
E Female Genital Tract 9. Specimens must be labeled accurately with the correct
F Male Genital Tract
patient’s information such as:
G Lower Respiratory Tract
H Upper Respiratory Tract • Patient’s complete name
I Urine Specimens • Patient’s unique identifying number/hospital/sample
VII Other Specimens Received in the Laboratory number
• Patient’s birthdate
• Also include the specimen’s date and time of collection
GENERAL GUIDELINES FOR SPECIMEN • Source of the specimen
COLLECTION • Initials of the individual collecting the sample
10. All appropriate specimens should have a direct
10. Specimen should be collected at the correct microscopic examination.
anatomic site and should be representative of • A direct microscopic examination gives the
infection. microbiologist and the clinician an early indication as to
• That’s why in the specimen label, the source is the possible condition of the patient. Although usually,
important. the microscopic exam doesn’t give the organism in
2. Specimen should be collected in the acute phase of question but it will give an idea as to the morphology of
illness/infection and as much as possible, before the initiation the cell, and the gram-staining characteristics.
of antibiotics or chemotherapeutic agents. • It takes a lot of time to grow, isolate, and detect the
• This is to ensure a greater chance of microbial bacteria in question. Usually, it takes about 18-24
recovery, meaning to say, we have to make sure that hours incubation; some bacteria require 7 days (for
the bacteria yield is high. blood culture); Brucella spp. needs 21 days
• If patient is on anti-microbial therapy, you may use • Direct microscopic examination is also important
anti-microbial removal device in the specimen because it is used to assess the quality of the specimen
container. collected
3. For specimen preservation, preservatives must be used if o Used to serve as a guide during the work of
ever delay is anticipated. the specimen
• Use boric acid to preserve urine 11. The microbiologist or medical technologist should talk
• Polyvinyl alcohol to preserve stool specimen to the requesting physician or another member of healthcare
• Use 0.025% (w/v) Sodium Polyanethole Sulfonate team before discarding any unacceptable specimens.
(SPS) for blood specimens; this is found in yellow top 12. If specimens are not processed as soon as they are
tube or standard blood culture tubes with SPS received, they must be stored properly. Storage methods
o SPS is not just an anticoagulant, it is also an include:
anti-phagocytic and anti-opsonization agent - refrigerator temperature – 4°C
4. Timing of collection must be considered because there are - ambient room temperature
bacteria that can be found in a certain specimen, and over the - body temperature
duration of the specimen, these bacteria will disappear and will - freezer temperature (-20°C to -70°C)
now be more detected in another specimen. • Depending on the organism and the culture media
• For instance, Salmonella infection in its early week (1st used
week) is detect in blood. • Anaerobic bacteria should never be stored in the
• For 2nd & 3rd week, the best specimen is stool refrigerator
specimen • CSF specimen should always be kept at body
• In 4th & 5th week, the best specimen is serum for temperature (37°C)
antibody titer. • Urine, stool, viral specimens, sputum, swabs, and
5. Specimens should be collected under sterile, aseptic foreign devices such as catheters, should be stored at
conditions to avoid contamination with normal flora and refrigerator temperature which is at 4°C
organisms from adjacent tissues. • Serum for serologic studies may be frozen at -20°C
• Because we only need the organism in question or • Specimens for long-term storage such as tissue
the pathogenic microorganism to grow. specimens should be frozen at -70°C

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13. Transport media may be used in order to maintain the 6. The specimen transport time exceeds 2 hours post-collection
viability of the organisms and to avoid hazard that result from and the specimen is unpreserved.
leakage. 7. The specimen is received in formalin which can kill any
• Transport media such as Stuart’s, Buffered Semi- microorganisms present.
solid Agar, Amie’s, and Cary-Blair medium may be 8. The specimen has been received for anaerobic culture but
used from the site known to have anaerobes as part of the normal
14. The contents of the specimen request form should flora such as the vagina and mouth.
include the following: 9. The specimen has dried up.
• Patient’s name 10 Processing a specimen that would produce a formation of
• Hospital identification number questionable medical value, such as specimens from Foley
• Age and birthdate catheter tip.
• Sex
• Collection date and time MOST COMMON SPECIMENS RECEIVED IN THE
• Ordering physician LABORATORY
• Exact nature and source of the specimen
• Current antimicrobial therapy ABSCESS
• Diagnosis (if needed) (ALSO LESION, WOUND, PUSTULE, ULCER)
SUPERFICIAL
FUNDAMENTALS OF SPECIMEN COLLECTION • CONTAINER: Aerobic swab moistened with Stuart’s or
1. If possible, collect the specimen in the acute phase of the Amie’s medium
infection and before antibiotics are administered. • PATIENT PREP.: Wipe area with sterile saline or 70%
2. Select the correct anatomic site for collection of the specimen. alcohol
3. Collect the specimen using the proper technique and supplies • Aspirate, if possible, swab along leading edge of
with minimal contamination from normal biota (normal flora). wound.
4. Collect the appropriate quantity of specimen. • TRANSPO TO LAB: less than 2 hours
5. Package the specimen in a container or transport medium • STORAGE BEFORE PROCESSING (if delayed): 24
designed to maintain the viability of the organisms and avoid hours in room temperature
hazards that result from leakage.
• PRIMARY PLATING MEDIA: Blood Agar, Chocolate
6. Label the specimen accurately with the specific anatomic site
Agar, MacConkey Agar; optional: Colistin-Nalidixic
and the patient information – patient’s name and a unique
acid agar
identification number, as well as date and time of collection.
• DIRECT EXAM: Gram-staining is important
7. Transport the specimen to the laboratory promptly or make
• Add Colistin-Nalidixic acid agar if smear suggest mixed
provisions to store the specimen in an environment that will not
degrade the suspected organism(s). gram-positive and gram-negative flora.
8. Notify the laboratory in advance if unusual pathogens or DEEP
agents of bioterrorism are suspected. • CONTAINER: Anaerobic transporter
• PATIENT PREP: Wipe area with sterile saline or 70%
SPECIMEN LABELS alcohol
1. Patient’s name • Aspirate material from wall or excise tissue.
2. Identifying number (hospital or sample number) • TRANSPO TO LAB: less than 2 hours
3. Birth date • STORAGE BEFORE PROCESSING: 24 hours in room
4. Date and time of collection temperature
5. Source • PRIMARY PLATING MEDIA: Blood agar, Chocolate
6. Initials of the individual that collected the sample agar, MacConkey agar, CNA, anaerobic Brucella
Blood agar (BBA), Laked Brucella Blood agar with
SPECIMEN REQUISITION Kanamycin & Vancomycin (LKV), Bacteroides Bile
A complete requisition should include the following: Esculin agar (BBE)
1. The patient’s name • DIRECT EXAM: Gram-staining is important
2. Hospital identification number • Wash any granules and “emulsify” in Saline.
3. Age and date of birth
4. Sex BLOOD OR BONE MARROW ASPIRATE/SPECIMENS
5. Collection date and time • Blood culture is necessary to evaluate bacteremia,
6. Ordering physician septicemia, and fever of unknown origin.
7. Exact nature and source of the specimen • Blood is normally sterile
8. Diagnosis (may be ICD-10-CM code)
• The presence of bacteria in the blood accompanied by
9. Current antimicrobial therapy
fever, chills, increased pulse rate, decreased blood
pressure is known as septicemia, which is deadly
GROUNDS FOR SPECIMEN REJECTION • Blood for culture must be collected 30-45 minutes
Specimens in the Bacteriology laboratory must be before fever spikes.
rejected if: o Example, if patient is taking paracetamol, it is
imperative to collect blood for culture 30-45
1. The information on the label does not match the information minutes after the 4th hour of paracetamol use
on the request form. because usually, paracetamol lasts for 4-6
2. The specimen has been transported at the improper hours
temperature.
• Blood culture bottles may contain anticoagulant such
3. The specimen has not been transported in a proper medium.
as 0.025-0.05% SPS (Sodium polyanethole sulfonate)
4. The quantity of the specimen is insufficient for testing.
o Heparin may be used; however, it may inhibit
(QNS = Quantity Not Sufficient)
the growth of gram-positive bacteria in yeast.
5. The specimen is leaking.

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• CONTAINER: Blood culture media set (aerobic and • Smears from CSF are prepared from CSF sediments,
anaerobic bottle) or Vacutainer tube with SPS thus, specimen should be centrifuged
• PATIENT PREP: Disinfect venipuncture site with 70% • CONTAINER: sterile, screw-cap tube
Alcohol → Povidone iodine → 70% alcohol • PATIENT PREP: disinfect skin with iodine or
• Draw blood at time of febrile episode; draw blood using chlorhexidine before aspirating specimen
2 sets of blood culture specimen bottles (both from left • Consider rapid testing (e.g., Gram stain, cryptococcal
and right arms); do not draw more than 3 sets in a 24- antigen)
hour period; draw 20 mL/set (adults) or 1-20 mL/set • TRANSPO TO LAB: less than 15 minutes
(pediatric) depending on patient’s weight or per • STORAGE BEFORE PROCESSING: less than 24
manufacturer’s instructions hours; Routine incubation at 37°C except for viruses,
o In blood culture, 1 set of bacterial culture is which can be held at 4°C for up to 3 days
composed of 2 bottles; 1 bottle is incubated • PRIMARY PLATING MEDIA: BA, CA (Routine); BA,
aerobically and 1 bottle is incubated CA, thio (Shunt); CA – medium for fastidious
anaerobically organisms
o In a span of 24 hours, may collect up to 2-3 • DIRECT EXAM: Gram staining, best sensitivity for
sets at 1 hour interval per set cytocentrifugation (may also want to do AO if
• TRANSPO TO LAB: within 2 hours at room temp. cytocentrifuge not available)
• STORAGE BEFORE PROCESSING: stored less than • Add thio for CSF collected from shunt; recommended
2 hours at room temp. if delay is anticipated; must be to also collect blood culture.
incubated at 37°C on receipt in laboratory
o Blood culture incubated at 35-37°C for up to GI TRACT SPECIMENS
5-7 days, thus there is no stuck blood culture
GASTRIC ASPIRATE
• For cases of Brucella: culture should be incubated for
3 weeks/ 21 days • CONTAINER: sterile, screw-cap tube
• Positive signs of growth in blood culture include • PATIENT PREP: Collect in early morning before
hemolysis of RBCs, formation of gas bubbles, turbidity, patient eats or gets out of bed
and appearance of small colonies in broth or in surface • Most gastric aspirates are on infants or for Acid-Fast
of RBCs Bacilli
• To process specimen further, if there are signs of • TRANSPO TO LAB: less than 15 minutes at room temp
growth, perform gram-staining followed by subculturing • STORAGE BEFORE PROCESSING: less than 24
the specimen on a blood agar plate and MacConkey hours at 4°C; must be neutralized with sodium
agar plate; Chocolate agar is also included bicarbonate within 1 hour of collection
• PRIMARY PLATING MEDIA: Blood culture bottles may • PRIMARY PLATING MEDIA: BA, CA, MacConkey
be used; BA, CA, anaerobic Brucella Blood Agar (BBA) agar, Hektoen Enteric (HE) Agar, CAN, Enrichment
• DIRECT EXAM: Direct Gram stain from positive blood Broth (EB)
culture bottles • DIRECT EXAM: Gram-staining/AO is performed
• Other considerations for blood culture specimens, • Other considerations: Acid-Fast Bacilli
aside from brucellosis: tularemia, cell wall-deficient GASTRIC BIOPSY
bacteria, leptospirosis, or Acid-Fast Bacilli; blood • CONTAINER: sterile, screw-cap tube (normal saline
cultures should be collected before administration of <2 hrs transport medium recommended)
antibiotics when possible • Rapid urease test or culture for Helicobacter pylori
• TRANSPO TO LAB: less than 1 hour at room temp.
CEREBROSPINAL FLUID • STORAGE BEFORE PROCESSING: less than 24
• Collected through lumbar puncture or spinal tap hours at 4°C
• Lumbar puncture is performed only by physicians, and • PRIMARY PLATING MEDIA: Skirrow, BA, BBA
CSF is collected between 3rd and 4th / 4th and 5th lumbar • DIRECT EXAM: H&E stain; optional: immunostaining
vertebrae • Other considerations: urea breath test, antigen test (H.
• CSF is normally sterile and it is usually used to pylori)
evaluate meningitis RECTAL SWAB
• Bacteria may enter through: the colonization of nasal
pharynx • CONTAINER: Swab placed in enteric transport
• Virus may enter through: the nerve cells medium
• Pathogenic microorganisms in CSF are usually with pili • Insert swab, 1-1.5 cm past anal sphincter; feces should
and capsule; may perform rapid testing for detection of be visible on swab
Cryptococcus • TRANSPO TO LAB: less than 2 hours at room temp.
• Primary isolation medium should include: BA, a • STORAGE BEFORE PROCESSING: less than 24
medium for fastidious organisms such as Chocolate hours at room temp. if there is delay
Agar Plate • PRIMARY PLATING MEDIA: BA, Mac, Xylose Lysine
• It should be examined immediately or less than 15 Deoxycholate (XLD) agar, HE, Campy, EB
minutes • DIRECT EXAM: Perform Methylene blue for fecal
• If ever delay is anticipated: should be placed in an leukocytes
incubator at 37°C for no longer than 1 hour • Other considerations as to the need for rectal swabs:
• usually, physician will collect 3 tubes; 1st tube should Vibrio, Yersinia enterocolitica, Escherichia coli
be transported to Chemistry or Serology dept.; 2nd tube O157:H7, Neisseria gonorrhoeae, Shigella,
is intended for Microbiology dept.; 3rd tube is for Campylobacter, herpes simplex virus, and carriage of
Hematology dept. Group B streptococci
o if there is only 1 tube collected: sent to STOOL (FECES) ROUTINE CULTURE
Microbiology dept first; then to Hematology,
then to Chemistry dept.

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• CONTAINER: clean, leak-proof container; transfer • STORAGE BEFORE PROCESSING: 24 hours at room
feces to enteric transport medium (Cary-Blair) if temp.
transport will exceed 1 hour • PRIMARY PLATING MEDIA: BA, CA, Thayer-Martin
• Routine culture should include Salmonella, Shigella, (TM) agar
and Campylobacter; specify Vibrio, Aeromonas, • DIRECT EXAM: Gram-staining is also important
Plesiomonas, Yersinia, Escherichia coli O157:H7, if • Other considerations: chlamydia, mycoplasma
needed. Follow-up may include Shiga toxin assay as VAGINA
recommended by CDC.
• CONTAINER: Swab moistened with Stuart’s or Amie’s
• TRANSPO TO LAB: within 24 hours at room temp. in
medium or place specimen in JEMBEC transport
holding media (Cary-Blair); if unpreserved, less than 1
system
hour at room temp.
• PATIENT PREP: Remove exudate
• STORAGE BEFORE PROCESSING: 24 hours at 4°C,
• Swab secretions and mucous membrane of vagina. If
or less than 48 hours at room temp. or 4°C
a smear is also required, use a second swab.
• PRIMARY PLATING MEDIA: BA, Mac, XLD, HE,
• TRANSPO TO LAB: less than 2 hours at room temp.
Campy, EB; optional: Mac-S, chromogenic agar
• STORAGE BEFORE PROCESSING: 24 hours at room
• DIRECT EXAM: Methylene blue for fecal leukocytes;
temp.
optional: Shiga toxin testing
• PRIMARY PLATING MEDIA: BA, TM
• Other considerations: (same with rectal swabs)
o Culture is not recommended for diagnosis of
• Do not perform routine stool cultures for patients
bacterial vaginosis
whose length of stay in the hospital exceeds 3 days
o If we are after for group B streptococcus, we
and whose admitting diagnosis was not diarrhea; these
need to inoculate sample into a selective
patients should be tested for Clostridium difficile
medium, usually LIM broth for pregnant
O&P women
• CONTAINER: O&P transporters (e.g., 10% formalin & o LIM Broth – aka Todd Hewitt with Colistin &
PVA) Nalidixic acid
• PATIENT PREP: Collect 3 specimens every other day o Group B streptococcus is harmful to pregnant
at a minimum for outpatients; hospitalized patients women especially Streptococcus agalactiae
(inpatients) should have a daily specimen collected for because it can actually be passed to the baby
3 days; specimens from inpatients hospitalized more during child birth and sometimes, group B
than 3 days should be discouraged streptococcus infection in newborn babies
• Wait 5-10 days minimum (up to 2 weeks) if patient has can cause serious complications that can be
received antiparasitic compounds, barium, iron, life-threatening
Kaopectate, metronidazole, Milk of Magnesia, Pepto- • DIRECT EXAM: Gram
Bismol, or tetracycline • Other considerations of vaginal specimens: bacterial
• TRANSPO TO LAB: Fresh non-preserved liquid vaginosis esp. WBCs, clue cells of Gardnerella
specimens should be examined w/in 30 mins of vaginitis, gram-positive rods indicative of Lactobacillus,
passage; Semi-formed within 1 hour of passage; & curved, gram-negative rods indicative of Mobiluncus
Specimens in fixatives: 24 hours at room temp. spp.
• STORAGE BEFORE PROCESSING: Indefinitely at
room temp MALE GENITAL TRACT
• DIRECT EXAM: Liquid specimen should be examined URETHRA
for the presence of motile organisms • CONTAINER: Swab moistened with Stuart’s or Amie’s
medium or place specimen in JEMBEC transport
FEMALE GENITAL TRACT system for Neisseria gonorrhoeae detection
• These specimens are used to determine the cause of • Insert flexible swab 2-4cm into urethra and rotate for 2
vaginitis, urethritis, and cervicitis as well as child-birth seconds (same with female urethra), or collect
infections. discharge on JEMBEC transport system
• It is also often used to determine sexually transmitted • TRANSPO TO LAB: less than 2 hours at room temp.
infections (STIs) for swab; within 2 hours for JEMBEC system
• Pathogens such as Neisseria gonorrhoeae are • STORAGE FOR PROCESSING: 24 hours per room
susceptible to temperature fluctuations and low levels temp.; put JEMBEC at 37°C immediately on receipt in
of CO₂ laboratory
• Transport medium such as JEMBEC or GonoPack • PRIMARY PLATING MEDIA: BA, CA, TM
must be used • DIRECT EXAM: Gram-staining
• Fastidious organisms mostly grow in the genital-urinary • Other considerations: chlamydia, mycoplasma
specimens and require the use of culture medium of
fastidious org., usually in the name of Chocolate Agar LOWER RESPIRATORY TRACT
FEMALE URETHRAL SPECIMEN SPUTUM, TRACHEAL ASPIRATE (SUCTION)
• CONTAINER: Swab moistened with Stuart’s or Amie’s • CONTAINER: sterile, screw-top container
medium
• PATIENT PREP: have patient brush teeth & then rinse
• PATIENT PREP: Collect 1 hour after patient’s last or gargle with water before collection
urination; remove exudate from urethral opening (if
• Have patient collect from deep cough; specimen
there are visible pus or exudates in urethral opening)
should be examined for suitability for culture by Gram
• Collect urethral discharge by massaging urethra stain; induced sputa on pediatric or uncooperative
against pubic symphysis or insert flexible swab 2-4 cm patients may be watery because of saline nebulization
into urethra and rotate swab for 2 seconds; collect at
• TRANSPO TO LAB: less than 2 hours at room temp.
least 1 hour after patient has urinated
• STORAGE BEFORE PROCESSING: 24 hours at 4°C
• TRANSPO TO LAB: less than 2 hours at room temp.

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• PRIMARY PLATING MEDIA: BA, CA, Mac, • CFU/mL is calculated by dividing the number of
Pseudomonas cepatia (PC) agar, Oxidative colonies counted by the volume capacity of loop in mL
Fermentative Polymyxin B Bacitracin Lactose (OFPBL) o For instance, after incubating BA plate for 24
agar for cystic fibrosis hours, count the number of colonies
• DIRECT EXAM: Gram and other special stains as o
𝐶𝐹𝑈
=
𝑛𝑜.𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑚𝐿 𝑣𝑜𝑙𝑢𝑚𝑒 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 𝑜𝑓 𝑙𝑜𝑜𝑝 𝑖𝑛 𝑚𝐿
requested (e.g., Legionella DFA, acid-fast stain) 𝐶𝐹𝑈 54 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠
• Other considerations: Acid-Fast Bacilli for the presence o =
𝑚𝐿 1 µL →mL
of Nocardia o
𝐶𝐹𝑈
=
54 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠
= 54,000 𝐶𝐹𝑈/𝑚𝐿
𝑚𝐿 0.001 mL
UPPER RESPIRATORY TRACT • A significant bacterial colony count is 1,000 CFU/mL
suspecting for cystitis and 10,000 above CFU/mL
NASOPHARYNX, NOSE, NASOPHARANGEAL SWAB
suspecting for pyelonephritis or kidney infection.
• CONTAINER: Swab moistened with Stuart’s or Amie’s • Lower levels of CFU or number of colonies may
medium indicate contamination only.
• Insert flexible swab through nose into posterior
nasopharynx and rotate for 5 seconds; specimen of
choice for Bordetella pertussis (causes whooping REFERENCES
cough)
Transcript notes and discussion by
• TRANSPO TO LAB: less than 15 minutes at room
temp. if without transport media; less than 2 hours at Ms. Rolaine Ann A. Polo, RMT, MPH
room temp. using transport media
• STORAGE BEFORE PROCESSING: 24 hours at room
temperature if there is delay
• PRIMARY PLATING MEDIA: BA, CA, chromogenic
agar
• Other considerations: add special media for
Corynebacterium diphtheriae, pertussis, chlamydia,
and Mycoplasma
PHARYNX (THROAT)
• CONTAINER: Swab moistened with Stuart’s or Amie’s
medium
• Swab posterior pharynx and tonsils; routine culture for
group A streptococcus (S. pyogenes) only. (causes
tonsilitis or strep throat)
• TRANSPO TO LAB: less than 2 hours at room temp.
• STORAGE BEFORE PROCESSING: 24 hours at room
temp.
• PRIMARY PLATING MEDIA: BA, Group A Selective
Strep Agar (SSA)
• Other considerations: add special media for C.
diphtheriae, Neisseria gonorrhoeae, and Haemophilus
influenzae (epiglottis)

URINE
• Best specimen: 1st morning urine (most concentrated)
• In midstream clean catch: the patient is instructed to
clean the genitals and to only include the middle flow
of urine in the container; the 1st and last flow should be
discarded
• Catheterized urine: may be collected by inserting
catheter into the bladder then allowing passage of the
15 mL of urine which will be discarded and the
remainder should be collected
• Suprapubic aspiration: is in fact the best specimen;
however, it is invasive to the patient; requires
ultrasonography or guided collection
• Urine specimen must be cultured in 1 hour, or
refrigerated, however, never refrigerate urine
specimens for longer than 24 hours
• Inoculate urine specimens in plating media such as
blood agar, MacConkey agar, and chromogenic agar
as an option
• When inoculating specimen, use a calibrated loop
• A calibrated loop is designed to carry or deliver 1 µL of
urine or 10 µL of urine
• Colony count is performed on Blood Agar plate and it
is reported as Colony Forming Unit (CFU) per mL in
urine

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