History of Microbiology
Discovery of Microorganisms
◉ Microbiology - defined as the study of organisms
that are too small to be seen by the naked eye.
Francesco Stelluti (1577 – 1652)
- He made the earliest observation on bees and
weevils using a microscope supposedly
supplied by Galileo
Anton Van Leeuwenhoek (1632 – 1723)
- He was considered as the "First true
microbiologist."
- He was the first person to observe and
accurately describe living microorganisms,
such as bacteria and protozoa, thus, he was
regarded as the "Father of Bacteriology and
Protozoology.
- He used the term "animalcules," or the tiny
living and moving cells seen under the
microscope, to describe microorganisms.
- He used his self-made single lens microscope
with 50 to 300x magnification to study bacteria
and protozoa
Spontaneous Generation
➢ States that life arises from non-living matter
Aristotle (384 – 322 B.C.)
- He mentioned that simple invertebrates could
come from spontaneous generation
Francesco Redi (1626 – 1697)
- In 1668, he demonstrated that maggots could
not arise spontaneously from decaying meat
- The results of his investigation invalidated the
long-held belief that life forms could arise from
non-living things
John Needham (1731 – 1781)
- He observed that the sealed flask with boiled
mutton broth became cloudy after standing
- He asserted that organic matter possessed a
"vital force" that could give rise to life
Lazzaro Spallanzani (1729 – 1799)
- He improved the previous experiments of
Needham by heating the broth that was
transferred into a sealed jar
- He observed that no growth took place as long
as the flasks remained sealed
- He proposed that air transports microorganisms
into the culture medium
- He concluded that microorganisms from the air
probably had entered Needham's concoction
after they were boiled
Biogenesis
➢ It states that living cells could only arise from pre-
existing living cells
Rudolf Virchow (1821 – 1902)
- He challenged the concept of spontaneous
generation with biogenesis
MICR_111: CLINICAL MICROBIOLOGY | CHAP 1 LECTURE
Theodor Schwann (1810 – 1882)
- He observed that no growth occurred in a flask
that contained a nutrient solution after allowing
the air to pass through a heated tube
Heinrich Schröder (1810 – 1885) & Theodore
Von Dusch (1824 – 1890)
- They noticed that no growth took place after
allowing the air to pass through a sterile cotton
wool placed on a flask with heat-sterilize culture
medium
Louis Pasteur (1822 – 1895)
- He disproved the theory of spontaneous
generation
- He proved that while the air does not generate
microbes, microorganisms are, indeed, present
and can contaminate a sterile solution
- He proposed the use of heat in killing
microorganisms, which is now called the
aseptic technique, or a method utilized in
preventing contamination by unwanted
microorganisms
- He provided evidences that microorganisms
could not originate from "mystical forces"
present in non-living materials
- He developed the vaccine against anthrax
(1881) and rabies (1885)
- He improved the wine-making processes by
introducing the concept of fermentation and
"pasteurization"
John Tyndall (1820 – 1893)
- He showed that dust carry germs that could
contaminate a sterile broth
- Tyndallization is a form of sterilization in the
19th century that uses moist heat for three
consecutive days to eradicate vegetative cells
and endospores
Ferdinand Cohn (1828 – 1898)
- He discovered that there are bacteria that could
withstand a series of boiling because of heat-
resistant structures known as endospores
Antoine-Laurent Lavoisier (1743 – 1794)
- He showed the importance of oxygen to life
Fermentation & Pasteurization
➢ Theodor Schwann explained that yeast cells are
responsible for the conversion of sugars to alcohol.
➢ Louis Pasteur described that certain
microorganisms known as yeasts convert sugar to
alcohol in the absence of air, a process known as
fermentation.
➢ Pasteur stated that the souring and spoilage of
wine are caused by different bacteria. He also
proved that in the presence of air, bacteria convert
the alcohol in the beverage into vinegar or acetic
acid.
MASAKAYAN, J.E.
 History of Microbiology
➢ To resolve the problem in the wine industry, Pasteur
suggested the minimal heating of beers and wines
that is sufficient to kill most of the bacteria also
known as pasteurization
Theory of Antisepsis
Ignaz Semmelweis (1816 – 1865)
- He demonstrated that routine handwashing can
prevent the spread of diseases
John Lister (1827 – 1912)
- He introduced the system of antiseptic surgery.
- He pioneered in promoting among surgeons the
handwashing before and after an operation, the
wearing of gloves, sterilizing of surgical
instruments, and the use of phenol as an
antimicrobial agent for surgical wound dressing
Germ Theory of Disease
➢ It is based on the concept that microorganisms can
cause diseases
Robert Koch (1843 – 1910)
- He was first to show irrefutable proof that
bacteria indeed cause diseases.
- He discovered Bacillus anthracis, the causative
agent of anthrax, in 1876
- He discovered Mycobacterium tuberculosis,
which is the causative agent of pulmonary
tuberculosis in 1882
- He was the first to cultivate bacteria on boiled
potatoes, gelatin, meat extracts, and protein
- He developed a culture medium for observing
bacterial growth isolated from the human body
Koch’s Postulates
1. The microorganism must be present in every case
of the disease but absent from a healthy host.
2. The suspected microorganism must be isolated
from a diseased host and grown in a pure culture.
3. The same disease must be present when the
isolated microorganism is inoculated into a healthy
host.
4. The same organism must be isolated again from the
diseased host
Collaborators of Koch
1. Walther Hesse (1846-1911) – he introduced the
use of culture media
2. Fanny Hesse (1850 – 1934) - She suggested the
use of agar, a solidifying agent, in the preparation of
the culture media.
3. Julius Richard Petri (1852 – 1921) – he developed
the Petri Dish, which is a circular glass or plastic
plate for holding the culture media
4. Martinus Beijerinick (1751 – 1931) & Sergei
Winogradsky (1856 – 1953) – they developed the
enrichment-culture technique and use of selective
media
MICR_111: CLINICAL MICROBIOLOGY | CHAP 1 LECTURE
Advent of Immunology
Edward Jenner (1749 – 1823)
- He introduced the concept of vaccination
- He collected scrapings from cow pox blisters and
inoculated a healthy volunteer by scratching the
person's arm with a pox-contaminated needle
Louis Pasteur (1822-1895) and Pierre Paul Émile
Roux (1853-1933)
- Pasteur used the term "vaccine" for an
attenuated culture
- Pasteur and Roux made a series of
experiments to produce attenuated strains of
bacteria
- They were able to prove that when attenuated
strains are introduced into a healthy host, the
latter remains protected against the virulent
agent
Charles Chamberland (1851 – 1908)
- He created a porcelain bacterial filter and
developed the anthrax vaccine together with
Pasteur
Emil von Behring (1854-1917)
- He prepared antitoxins for diphtheria and
tetanus
Élie Metchnikoff (1845-1916)
- He was the first to describe the cells of the
immune system and the process of
phagocytosis.
Modern Therapy: “Magic Bullet”
Selman Waksman (1888-1973)
- He discovered the streptomycin and neomycin
antibiotics
- He was regarded as the "Father of Antibiotics"
by some historians because he discovered
antimicrobials before the hype of penicillin
Alexander Fleming (1881-1955)
- He accidentally discovered the antibiotic
penicillin (Penicillium notatum)
- He discovered the lysozyme
Howard Florey (1898-1968) & Ernst Chain
(1906-1979)
- They made the purification process for penicillin
and the clinical trials to humans
Edward Abraham (1913-1999)
- He was the first to propose the correct
biochemical structure of penicillin
Paul Ehrlich (1854-1915)
- He discovered salvarsan (arsphenamine) for
the treatment of syphilis
◉ Chemotherapy
MASAKAYAN, J.E.
 History of Microbiology
- It is the use of chemical substances in the
treatment of diseases
- It also refers to the chemical treatment of non-
infectious diseases, such as cancer
IMPORTANT EVENTS IN THE DEVELOPMENT OF
MICROBIOLOGY
Hans Jansen developed the first
1590-1595
compound microscope
Antonie van Leeuwenhoek discovered
1676
animalcules
Otto Friedrich Muller produced the
1786
first classification of bacteria
Edward Jenner introduced the
1798 smallpox immunization through cow
pox inoculation
Theodor Schwann and Matthias
1838-1839 Jakob Schleiden introduced the cell
theory
Rudolf Virchow proposed the theory
1858
of biogenesis
Louis Pasteur used the aerobic and
1861 anaerobic terminologies in describing
the metabolism of yeast.
Joseph Lister published his work on
1867
antiseptic surgery.
Ferdinand Cohn used the genus
1875
name Bacillus in classifying bacteria
Louis Pasteur described the weak
1880 strain of organisms as attenuated in
developing vaccines.
Robert Koch cultured bacteria on
1881 gelatin. Louis Pasteur developed the
anthrax vaccine
Robert Koch discovered the tubercle
1882
bacilli.
- Koch's postulates were first
published.
- Elie Metchnikoff described
1884 phagocytosis.
- Autoclave was developed.
- Hans Christian Gram introduced
Gram stain
Louis Pasteur produced the rabies
1885
vaccine
Julius Richard Petri introduced the
1887
use of the Petri dish.
Emil von Behring prepared antitoxins
1890
for diphtheria and tetanus.
Jules Bordet discovered the
1895
complement
Ronald Ross showed that malarial
1896
parasites are carried by mosquitoes
Walter Reed proved that yellow fever
1900
is transmitted by mosquitoes
MICR_111: CLINICAL MICROBIOLOGY | CHAP 1 LECTURE
Karl Landsteiner discovered the blood
1901 group system.
Fritz Schaudinn and Erich Hoffman
1906 showed that the Treponema pallidum
causes syphilis.
Recognition of the role of protozoa in
1907
the disease process
Sigurd Orla-Jensen established the
importance of including the
1909
physiologic characteristics of bacteria
in their classification
Paul Ehrlich developed a
1910
chemotherapeutic agent for syphilis.
First edition of Bergey's Manual of
Determinative Bacteriology was
1923 published Subsequent editions were
titled Bergey's Manual of Systematic
Bacteriology
Emnst Ruska developed the first
1933
transmission electron microscope
Edouard Chatton introduced living
1937 organisms as either prokaryotes or
eukaryotes
Selman Waksman discovered
1944
streptomycin
Alexander Fleming discovered
1945
penicillin.
Max Theiler developed the yellow
1951
fever vaccine
Francis Crick and James Watson
1953
discovered the structure of DNA
Rosalyn Yalow and Solomon Berson
1954
developed the radioimmunoassay.
Cornelius van Niel and Roger Stanier
1962
described prokaryotes.
Recognition of archaebacteria as a
1976
distinct microbial group
Development of scanning tunneling
1980
microscope (STM)
- Robert Gallo and Luc Montagnier
identified and isolated HIV.
1983-1984 - Kary Mullis developed
Polymerase Chain Reaction
(PCR)
The first hepatitis B vaccine created
1986 through genetic engineering was
approved for human use
- Chickenpox vaccine was
approved.
- Craig Venter, Hamilton Smith, and
1995 Claire Fraser released the first
complete genome sequence of a
microorganism utilizing
Haemophilus influenzae
MASAKAYAN, J.E.
 Sterilization and Disinfection
Microbial control involves physical and chemical
agents that destroy microorganisms and potential
pathogens or inhibit their growth and prevent their
transmission
Sterilization
➢ Sterilization refers to the removal or destruction of
all forms of life, including bacterial spores
Physical Methods of Sterilization
Heat is the most commonly used method for the
removal of microorganisms
Moist Heat Procedure
• It destroys microorganisms through the coagulation
of enzymes and structural proteins and degradation
of nucleic acids
Boiling
- It destroys vegetative bacteria (non-
sporulating)
- Temperature and time of exposure: 100°C for
10 minutes to 15 minutes
Autoclaving
- It is the fastest and simplest method of
sterilization through which all organism (except
for prions), including those that contain spores,
are killed within 15 minutes
- It is used to sterilize biohazardous trash and
heat-stable objects.
▪ Principle: Steam under pressure
▪ Biological indicator: Geobacillus
stearothermophilus (formerly Bacillus
stearothermophilus)
- Autoclave is a chamber which is filled with hot
steam under pressure.
Pasteurization/Partial Sterilization
- It is used to sterilize milk, dairy products, and
alcoholic beverages
- It eliminates food-borne beverages pathogens
and organisms responsible for food spoilage
- This method cannot bacterial endospores
Types of Pasteurization
Low-Temperature Holding (LTH) / Batch Method
• It destroys milk-borne pathogens.
• Treatment at this temperature reduces spoilage
of food without affecting its taste.
▪ Temperature and time of exposure: 63°C for 30
minutes
High-Temperature Short-Time (HTST)/Flash
Pasteurization
▪ Principle: Quick heating then immediate cooling
▪ Temperature and time of exposure: 72°C for 15
seconds
Ultra-High Temperature (UHT)
• It is ideal for milkproducts and beverage additives
like coffee creamer.
▪ Temperature and time of exposure: 140°C for 3
seconds (cooled very quickly in a vacuum
chamber)
• Advantage: Milk can be stored at room
temperature for two months without affecting its
flavor.
Dry Heat Procedure
• This is a sterilization method that does not require
water.
• It kills microorganisms by denaturing proteins.
• It is recommended for the sterilization of
glassware, oil products, and powder.
• Biological indicator: Bacillus atrophaeus (ATCC
9372)

Autoclaving Conditions and Purpose Flaming or Direct Heating

121°C, 15 psi for 15 132°C, 15 psi for 30 - It is the procedure for sterilization of
minutes to 20 minutes: minutes to 60 minutes inoculating loops and needles.
- Culture media
Decontamination of Oven-heating
- Liquids
medical waste - It is used for glassware, oil, petrolatum or
- Glass Pipettes
- Laboratory Utensils powders.=
▪ Temperature and time of exposure: 160°C -
170°C for 1.5 hours - 2 hours
Fractional or Intermittent Sterilization
(Tyndallization)
Incineration
- It destroys vegetative cells and spores after
- It is the most common method of treating
three consecutive days of sterilization.
infectious waste and infected laboratoryanimals
▪ Temperature and time of exposure: 100°C for 30
▪ Principle: Burning of materials into ashes at 300°C
minutes
to 400°C
▪ Instrument: Arnold's sterilizer (free-flowing steam)
▪ Temperature for hazardous materials: 870°C to
980°C
Inspissation
- It is utilized to sterilize protein-rich medium
Cremation
such as the Lowenstein-Jensen medium.
- It is utilized to control the spread of
▪ Principle: Thickening of culture media through
communicable diseases
evaporation
▪ Temperature and time of exposure: 70°C to 80°C
days)

MICR_111: CLINICAL BACTERIOLOGY | LESSON 8: LECTURE MASAKAYAN, J.E.

 Sterilization and Disinfection

TERMINOLOGIES Low/Cold Temperature

refers to the lowest temperature - It is considered bacteriostatic because it
Thermal Death at which a suspension of educes the rate of metabolism.
Point (TDP) bacteria - It is important in food microbiology.
is killed in 10 minutes - Exposure to 2°C to 8°C for 72 hours kills the
refers to the shortest period of agents of syphilis
Thermal Death time needed to kill at a
Time (TDT) prescribed temperature and Desiccation and Lyophilization
under specific conditions - Desiccation destroys bacteria through the
refers to the time in minutes to disruption of metabolism that involves removing
reduce the water from microbes, and that is considered as
Decimal bacterial population or spores bacteriostatic.
Reduction Time by 90% at a specified - Lyophilization destroys bacteria through
(DRT/D/D Value) temperature; this is widely used changes in proteins and decrease in chemical
or observed reactions
in the food industry
Examples of bacteria which remain active in a
Filtration dry environment
A. Neisseria gonorrhoeae – viable for one hour
• It is the method of choice for the sterilization of B. Mycobacterium tuberculosis – viable for several
antibiotic solutions, toxic chemicals, months
radioisotopes, vaccines, and carbohydrates C. Bacillus and Clostridium – viable for ten years
(heat-sensitive solutions)
• It may be used with both liquid and air Osmotic Pressure
substances - It is the use of high concentrations of salts and
sugars in food to create a hypertonic
Type of Filters environment.
- In plasmolysis, the plasma membrane shrinks
Depth Filters away from the cell wall (cell may not die, but
- These are made up of fibrous or granular usually stops growing) as water leaves the cell
materials.
• Example: Berkefield filter (diatomaceous earth), Radiation
Chamberland filter (unglazed porcelain),and - The underlying principle is that when the
asbestos radiation passes through the cells, free
hydrogen and hydroxyl radicals and some
Membrane Filters (Circular filters) peroxidases are created. These free radicals, in
- These are porous membranes (almost 0.1 um turn, cause different intracellular damage.
thick) - Biological indicator: Bacillus pumilus
- They are composed of cellulose acetate or
polycarbonate. 1. Ionizing radiation (Cold sterilization)
- They are designed to sterilize pharmaceuticals, - It causes mutation in the DNA and generates
ophthalmic solutions, culture media, antibiotics, peroxides
and oil products. - It destroys vegetative cells and endospores of
both prokaryotes and eukaryotes.
a. Liquid filtration - It has a shorter wavelength.
- It uses cellulose acetate or cellulose nitrate - It is usually designed to sterilize plastic
membrane with a vacuum. syringes, sutures, catheters, gloves, hormone
b. Air filtration solutions, and antibiotics.
- It utilizes high-efficiency particulate air (HEPA) - It is also used to "pasteurize" meat products
filters. - The types of radiation rays used are gamma
- HEPA filters remove organisms larger than 0.3 rays (1500 to 2500 radiation) and x-rays.
um from isolation rooms, operating
c. Filtration of bacteria, yeasts, and molds 2. Non-ionizing radiation
- It utilizes membrane filters with 0.45-um pores. - It causes damage to cellular DNA by producing
- Membrane filters with pores ranging in size thymine dimers
from 0.2um to 0.45um in diameter from isolation - It has a longer wavelength (> 1um) and low
rooms, operating rooms, and biological safety energy.
cabinets (BSCs) - It is used to sterilize exposed surfaces,
- most bacteria as well as fungi but not viruses. operating rooms, nursery rooms, and
d. Critical sterilizing cafeterias.
- It uses membrane filters with 0.22-pm pores - The types of radiation rays utilized are
ideal for parenteral solutions and alcohol ultraviolet rays (10um to 400um) in which
- Membrane filters with 0.22 um pores are used 260um is the most lethal
to eradicate vegetative cells but not viral agents - Microorganisms in water are destroyed when
passed under UV lamps
MICR_111: CLINICAL BACTERIOLOGY | LESSON 8: LECTURE MASAKAYAN, J.E.
 Sterilization and Disinfection
Chemical Methods of Sterilization
Disinfection refers to the removal, inhibition, or killing
of microorganisms which include potential although it
does not remove pathogens by utilizing chemical agents
usually on inanimate objects all bacterial spores
Mechanisms by Which Chemical Disinfectants
Destroy Microorganisms
1. Reaction with components of the cytoplasmic
membrane
2. Denaturation of cellular proteins
3. Reaction with the thiol groups of enzymes
4. Damage to DNA and RNA
Factors That Influence the Activity of Disinfectants
1. Types of organism present
2. Number of organisms present (microbial load)
3. Temperature and pH level of the process
4. Concentration of disinfectants
5. Amount of organics and biofilms
6. Length of contact time between the chemical
agent and the microorganism
7. Type of water used for disinfection
◉ Antiseptic
- It is applied topically on the skin.
- It inhibits sepsis formation.
- Examples: Hexachlorophene and tincture of
iodine/povidone alcohol (iodophor)
◉ Disinfectant
- It is usually applied to inanimate objects.
- Sodium hypochlorite (NaOCI) in 1:10 dilution is
an effective disinfectant.
- Examples: Cresols, chlorine, and sodium
hypochlorite
◉ Bactericidal
- It precipitates bacterial protein and kills all
bacteria in the specimen.
- Some examples are strong acids such as
sulfuric acid and hydrochloric acid
◉ Bacteriostatic
- It inhibits the growth of organisms
Common Chemical Agents Used in Microbial
Control
Acid and Alkaline Solutions
- It hydrolyzes and coagulates proteins.
Phenol (Tuberculocidal)
- The first widely used antiseptic and disinfectant.
- It destroys plasma membranes and denatures
cell proteins
- It is effective even in the presence of organics.
- 5% phenol with 10 to 30 minutes contact time is
effective against mycobacteria
- Examples: Cresols, xylenols,
hexachlorophenes, amphyls, and
orthophenylphenols
MICR_111: CLINICAL BACTERIOLOGY | LESSON 8: LECTURE
Alcohol (Non-sporicidal)
- It causes denaturation of proteins and
dissolution of lipid membranes
- It is used as both an antiseptic and a
disinfectant (bactericidal and fungicidal).
- It should be used in 60% to 90% concentration
to achieve its antimicrobial property.
- It should be allowed to evaporate from the
surface to achieve complete antisepsis.
- Examples: Isopropanol (rubbing alcohol) and
ethanol
- Disadvantage: Poor activity against non-
enveloped viruses and ineffective in the
presence of organic material
Halogen
- It destroys microorganisms through the
oxidation process.
- Examples: Chlorine, iodine, fluorine, bromine,
and astatine
- NaOCl should be freshly prepared every day
with 10 minutes to 30 minutes of contact time to
make it an effective tuberculocide - 1:10 dilution
is an effective bleach
- Halozone (parasulfone dichloraminobenzoic
acid) contains chloride and is used to disinfect
drinking water.
Preparations of lodine: Tincture and Iodophor
• Tincture of iodine contains 2% iodine in 70%
alcohol and is used as an antiseptic.
• Iodophor is composed of iodine and a neutral
polymer carrier and acts as either an antiseptic or
a disinfectant
• Before drawing blood for culture, iodophor is
applied after 70% alcohol.
• Improperly diluted iodophors may not kill
microorganisms due to the lack of free iodine in
the solution.
• The antibacterial effect of iodophors is oxidative
effects of molecular iodine and hydroiodic acid.
• Contact time for lodine: 30 to 60 seconds onto the
skin prior to blood collection
• Disadvantage: Non-sporicidal and non
tuberculocidal
Chlorine
• It is used in the form of hypochlorite.
• Its antibacterial activity is manifested by the
oxidative and disinfecting effects of hypochlorous
acid (formed when chloride ions are dissolved in
water).
• Concentrated solutions (corrosive) should not be
used for disinfection.
• The CDC recommendation for this chemical agent
is 1:10 dilution of 5.25% solution of
• sodium hypochlorite, especially for disinfecting
tabletops and floors with blood spills.
• Contact time: 3 minutes for organic materials and
10 minutes to 30 minutes for mycobacteria
• Disadvantage: Ineffective in the presence of large
amounts of proteins
MASAKAYAN, J.E.
 Sterilization and Disinfection
Salts of Heavy Metals
• It destroys microorganisms by inactivating and
precipitating cell proteins.
• Some examples are copper, arsenic, mercury,
silver, and zinc (bacteriostatic).
• AgNO3 is an eye drop solution used historically
against N. gonorrhoeae while mercuric
• chloride is an antiseptic
Quaternary Ammonium Compounds (Non-
tuberculocidal and Non-sporicidal)
• It is widely used as surface-active agents.
• Its antibacterial effect is the disruption of the cell
membrane contents that results in the leakage of
cell
• It is used on laboratory bench-tops and floors, in
• food outlet facilities. cleaning dairy utensils, and in
disinfecting
• Some examples are zephiran (benzalkonium
chloride and cetylpyridinium chloride
• Culture of Pseudomonas aeruginosa in an
ammonium acetate medium is resistant to quats.
Phenolic
• It is a derivative of phenol withreduced toxicity
• It is a stabe surface disinfectant and effective in
the presence of organics.
• It disrupts microbial cell walls and membranes and
precipitates proteins
• Example: Chlorhexidine gluconate (CHG),
chloroxylenol (PCMX) and triclosan
• CHG inactivates certain lipid-enveloped viruses,
such as HIV, respiratory virus, and
cytomegalovirus.
• CHG binds to the skin and remains active for at
least 6 hours.
• CHG is an effective body disinfectant used in the
hospital prior to surgery.
• PCMX is effective for surface cleansing
contaminated with body fluids, such as blood and
sputum; it is also used as a skin antisepsis.
• Triclosan is more effective compared to CHG,
and it is utilized for clothing and furniture
decontamination and, as an additive to dental care
liquid.
• Disadvantage: Nonsporocidal
Aldehyde- Cold/Chemical Sterilant
• Its antibacterial effect is the inactivation of proteins
and nucleic acids.
• It is recommended for sterilizing medical
instruments.
• Example: Formaldehyde and 2% glutaraldehyde
Formaldehyde (HCHO)
- It is generally used as formalin which consists
of 37% aqueous solution or HCHO gas.
- It is best for sterilizing HEPA filters in a
biological safety cabinet (formaldehyde vapor)
- Its usefulness, however, is limited because it
has an irritability factor, and it is a known
carcinogen.
- Recommended concentration: 8% HCHO
MICR_111: CLINICAL BACTERIOLOGY | LESSON 8: LECTURE
- For mycobacteria, 3% to 8% HCHO is used with
a contact time of at least 30 minutes
Glutaraldehyde (Pseudomonacidal,
Tuberculocidal, Fungicidal, and Virucidal)
- It is commonly utilized on medical instruments
(heat-labile) that are made of plastic and rubber
materials.
- It is effective against human immunodeficiency
virus (HIV) and hepatitis B (HBV) when
organisms are exposed for 10 minutes.
- It has a rapid killing action but does not
penetrate organic materials well when used as
a sterilant.
- Recommended concentration: 2%
Glutaraldehyde
- 2% glutaraldehyde is bactericidal (including
tuberculocidal) in 10 minutes and sporicidal in 3
hours to 10 hours
Gas Sterilant
a. Ethylene oxide (EtO)
- It is the most commonly used gas for
sterilization
It is utilized to sterilize plastic Petri dishes,
sutures, catheters, heat-sensitive equipment
such as the heart-lung machines. nitrogen or
carbon dioxide before
It has an explosive property so it should be
mixed with use.
- Its antibacterial effect is the alkylation of nucleic
acids in spores and vegetative cells
- The concentration used is from 450 mg/L to 700
mg/L of chamber space at 55oC to 60oC for two
hours
- Biological indicator: Bacillus species
b. Periacetic Acid
- It is active against all vegetative
microorganisms and fungal spores.
- It is used to sterilize pieces/parts of medical
equipment.
Disinfectant Screening Test - Phenol Coefficient
(PC)
- It is the highest dilution that kills the bacteria
after 10-minute exposure.
- The potency of a disinfectant is compared with
phenol.
- Test organisms: Staphylococcus aureus and
Salmonella serotype Typhi(20°C or 37°C)
In solving for the PC, the following formula is used:
𝑃𝐶
highest dilution of disinfectant that will kill organisms at a given time
=
ℎ𝑖𝑔ℎ𝑒𝑠𝑡 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝ℎ𝑒𝑛𝑜𝑙 𝑡ℎ𝑎𝑡 𝑤𝑖𝑙𝑙 𝑘𝑖𝑙𝑙 𝑜𝑔𝑟𝑛𝑖𝑠𝑚 𝑎𝑡 𝑎 𝑔𝑖𝑣𝑒𝑛 𝑡𝑚𝑒
• Result: PC > 1, which indicates that the disinfectant
is more effective than phenol
• Interpretation: The higher the PC value, the more
effective the disinfectant.
MASAKAYAN, J.E.
 Lesson 3: Bacterial Taxonomy and Physiology
o Ex: Staphylococcus aureus or Staphylococcus
aureus

Subspecies and Serovar Nomenclature

Subspecies
➢ Capital first letter of genus → Small letter species →
followed by the word subsp. → small first letter of the
subspecies of the organism
➢ For organism with (serovar/serotype): write ser.
→ followed by capital first letter of serotype
❖ Salmonella enterica subsp. arizonae
❖ Salmonella enterica subsp. enterica ser. Typhimurium

Common name Organism

Anthrax Bacillus Bacillus anthracis
Bang’s Bacillus Brucella spp.
Battey Bacillus Mycobacterium intracellulare
Bordet-Gengou Bacillus Bordetella pertussis
Canned food Bacillus Clostridium botulinum
Colon Bacillus Escherichia coli
Comma-shaped / Curved
FAMILY -aceae Vibrio spp.
Bacillus
GENUS -ccus Corroding Bacillus Eikenella corrodens
SPECIES aureus Foam-loving Bacillus Aggregibacter aphrophilus
SUB SPECIES anarobius Friedlander’s Bacillus Klebsiella pneumonia
Fried rice Bacillus Bacillus cereus
Taxonomy – area of biological science comprising of Gas gangrene Bacillus Clostridium perfringes
three distinct disciplines:
Hansen’s Bacillus Mycobacterium leprae
1. Classification
Hay Bacillus Bacillus subtilis
2. Nomenclature
Corynebacterium
3. Identification of Organism Hoffman Bacillus
pseudodiptheriticum
Classification Kleb-Loeffler Bacillus Cornyebacterium diphtheriae
• Method of organizing microorganisms into groups or taxa Koch’s Bacillus Mycobacterium tuberculosis
based of similar morphologic, physiologic, and genetic Koch-Weeks Bacillus Haemophilus aegypticus
traits Morax-Axenfeld Bacillus Moraxella lacunata
Pfeiffer’s Bacillus Hemophilus influenza
Microbial Taxonomy Plague Bacillus Yersina pestis
FAMILY Tackhead Bacillus Clostridium tetani
➢ Group of organisms that may contain multiple genera Tap Water Bacillus Mycobacterium gordonae
and consists of organisms with common attribute Whitmore Bacillus /
Burkholderia pseudomallei
➢ Naming: addition of -aceae to the root name of the Vietnamese Time Bomb
type genus (ex: Streptococcaceae, Mycobacterium avium
Wood pigeon Bacillus
Enterobacteriaceae) subsp.Silvaticum
GENUS Yellow Bacillus Mycobacterium kansasii
➢ Different species that have several important
features in common Microbial Identification
SPECIES • Process by which a microorganism’s key features are
➢ sp., spp. delineated
➢ Collection of bacterial strains that share common • Two general categories:
physiologic and genetic features and differ from other (1) Genotypic Characteristics – refers to organism’s
microbial species genetic makeup: nature of the organism’s genes
➢ Subspecies – group within a species and constituent nucleic acid (DNA,RNA)
▪ Biotype, Serotype, Genotype – groups below (2) Phenotypic Characteristics – based on features
the subspecies level but relatively minor beyond the genetic level and include observable
characteristics characteristics that may require extensive analytic
▪ Serotype – same species but different procedures to be detected (ex: colony morphology,
serological feature staining, biochemical and susceptibility test)
▪ Biotype – same species but different
physiological characteristics Terminologies
▪ Epithet – the proper word for the name of the 1. Biogroup – is a population of species that share the
species same biochemical properties
2. Epithet – is the proper word for the name of the
Nomenclature species
• Naming of microorganisms according to established rules 3. Genotype – is the collection of genes that describes
and guidelines set forth in the International Code of the characteristics of an organism
Nomenclature of Bacteria (ICNB) or the 4. Morpho Var - also known as a morphotype, is a
Bacteriological Code (BC) prokaryotic strain which differs morphologically from
• Binomial name – genus and species other strains
→ Genus – capitalized and italicized/underlined 5. Phenotype – is the observable physical and
→ Species – first name is lower case and biochemical properties of the organism
italicized/underlined 6. Polyphasic Taxonomy – is a modern system of
bacterial classification and identification combining

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 Lesson 3: Bacterial Taxonomy and Physiology
phylogenetic, phenotypic, and genotypic
characterizations, likewise utilizing molecular
sequences and epigenetic factors
7. Serogroup - is a serovar having similar antigens
8. Strain – an altered or a variant microorganism within
the same species
Bacterial Physiology
Prokaryotic Cell – Unicellular, without nucleus and other
organelles
Eukaryotic Cell – multicellular, with nucleus and other
organelles
❖ Archaea – closely related to eukaryotic cell
- Different cellular structure
- Cell envelope and enzymes allows them to
thrive under harsh condition
- Halophiles (Salt loving) Thermophiles ( Heat-
loving) Cryophiles (cold loving)
Bacterial Cell Structures
Cell Envelope
• Outermost layer consisting of membrane and structures
surrounding the cytoplasm (capsule and slime layers)
Cell Wall
• Rigid structure that protects and maintains the shape of
the cell
❖ Gram positive – Staphylococcus spp.
❖ Gram negative – Salmonella spp.
❖ Acid fast – Mycobacterium spp.
➢ Mycoplasma / Urea plasma – cell wall less, no cell wall
but with sterol
Types of Cell Wall
Gram-positive Cell Wall
1. With thick peptidoglycan layer (murein layer)
• Peptidoglycan/ Murein layer – cross linked glycan
chains of N-acetyl-D-glucosamine (NAG) and N-
acetyl-D-muramic acid (NAM)
• Penicillin binding protein (PBS) – enzymes that
promotes cross-link of NAG-NAM
2. Teichoic acid and Lipoteichoic Acid
- Made up of alcohol (glycerol or ribitol) and phosphate
- Makes the cell wall negatively charged
MICR_111: CLINICAL MICROBIOLOGY | TAXONOMY
Gram Negative Cell Wall
1. Thick outer membrane – proteins (porins channel),
Phospholipid, and lipopolysaccharide
2. Lipopolysaccharide (LPS) – makes the cell wall (-)
- Lipid A (endotoxin) – fever and shock
- Core polysaccharide
- Antigenic O specific polysaccharide
3. Periplasmic space – space between OM and PL
- Nutrient binding proteins and enzymes
4. Thin inner peptidoglycan layer
Gram Staining Procedure (VIAS)
1. Crystal Violet – primary stain (60 secs)
2. Grams Iodine – mordant makes dye less soluble;
increases binding (60 secs)
3. Acetone Alcohol – decolorizer (until colorless)
4. Safranin Red – secondary stain; counterstain (30 secs)
Acid Fast Cell Wall
• May resemble gram + bacteria (gram staining)
• Contains Methoxy mycolic acid
• IUPAC: 2-[1-hydroxy-18-[2-(17-methoxy-18-
methylhexatriacontyl)cyclopropyl]octadecyl]tetracos
anoic acid]
• Makes the organism difficult to visualize using gram
stain
• Provides a strong hydrophobic structure
• Ex: Mycobacterium and Nocardia (partially acid-
fast) and some protozoan parasites (Coccidian)
Acid Fast Staining
1. Zeihl-Neelsen – hot method (heat)
2. Modified Kinyoun – cold method (tergitol)
Reagents:
1. Carbol Fuchsin – primary stain
2. Acid-alcohol – decolorizer
3. Methylene blue – secondary stain
Differential Stain
Gram Stain Acid Fast Stain
Positive Violet Red
Negative Red Blue
Plasma Membrane
• Phospholipid bilayer embedded with proteins
- Prokaryotes – without sterol except (Mycoplasma)
- Eukaryotes – with sterol
• Act as osmotic barrier
• Site for electron transport chain
MASAKAYAN, J.E.
 Lesson 3: Bacterial Taxonomy and Physiology
Bacterial Cytoplasmic Structures and Appendages
1. Bacterial Ribosomes
- Site of protein synthesis
- 70s divided into 50s and 30s
- Important structure for genotypic identification of
microorganisms
• Svedberg unit – sedimentation rate during centrifugation
(Theodor Svedberg)
2. Inclusion Bodies
- energy and food storage
3. Genetic Material Bacterial Morphology
- single circular chromosome
Type Shape Example
4. Plasmid
Round/spherical Staphylococcus,
- extrachromosomal DNA structure capable of
Coccus (Cocci) Coffee-bean/ Streptococcus,
horizontal gene transfer (antibiotic resistance)
lancet shaped Gonococcus
5. Capsule (India Ink/Nigrosin)
Rod-shaped,
- enables the bacteria to evade host immune system
club shaped, Escherichia coli,
- negative stain; black background
Bacillus (Bacilli) Comma Shigella, Vibrio,
6. Flagella (Leifson Stain)
shaped, Mycobacterium
- locomotion/ motility
Filamentous
• (Atrichous, Monotrichous, Ampitrichous,
Treponema
Lopotrichous, and Peritrichous)
(Fine regular
7. Endospores (Clastridium and B. anthracis)
coils)
(Schaeffer-Fulton Stain)
Spirillum Leptospira (very
• enables the bacteria to thrive under hash Spiral or coiled
(Spirilli/Spirochetes) fine regular coils
environment
with hook on
8. Pili/Fimbriae (Somatic or Sex)
one or both
• hairlike appendages that serves as protein tubes ends)
allowing exchange of DNA through conjugation
Organism Flagella Motility Pattern
Twitching in wet
Bartonella spp. Atrichous
mounts
Capnocytophaga Atrichous Gliding motility
Chromobacterium
Polar Flagella -
violaceum
Pseudomonas spp. Polar Flagella -
Burkholderia spp.
(except B. mallei = Polar Flagella -
non motile)
Burkholderia Polar tuft of
-
pseudomallei Flagella Morphological Arrangement of Bacteria
Aeromonas Bacteria Arrangement and Examples
(mesophilic grp: Single or polar Shape
-
A.hydrophilia, A. flagellum 1. Singly or in pairs – Pneumococcus,
Veronii, A. Caviae Gonococcus, Meningococcus
Campylobacter Single or polar 2. Tetrads – Geffyka tetragena
Darting motility Cocci
spp. flagellum 3. Sarcinate – Sarcinna lute
Monopolar or 4. Clusters – Staphylococcus
Helicobacter spp. - 5. Chains – Streptococcus
multipolar flagella
Monotrichous 1. Singly or in pairs – Klebsiella
➢ broth = pneumoniae
polar, 2. Chains – Bacillus subtilis, Bacillus
Shooting star Bacilli
Vibrio spp. sheathed anthracis
motility 3. Palisade – Mycobacterium leprae
➢ solid media
= 4. Groups – Mycobacterium tuberculosis
unsheathed 1. Singly or in pairs
Peritrichous Spirillum 2. Groups
Acaligenes faecalis - 3. No typical arrangement
flagella
Bacillus spp.
(except B. Peritrichous Gram Stain Reaction
-
anthracis & B. flagella • Neisseria
mycoides) All Cocci are Gram Positive
• Veilonella
except:
Enterobacteriaceae Peritrichous - • Moraxella
(except Klebsiella, flagella • Bacillus
Yersinia, Shigella) • Listeria
Kurthia spp. Peritrichous - • Erysipelothrix
flagella All Bacilli are Gram Negative • Corynebacterium
Listeria Peritrichous Tumbling except: • Mycobacterium
monocytogenes flagella (hanging drop)
• Lactobacillus
Umbrella/
• Priopionibacterium
Inverted
• Eubacterium
Christmas Tree)

MICR_111: CLINICAL MICROBIOLOGY | TAXONOMY MASAKAYAN, J.E.

 Lesson 4: Bacterial Genetics, Growth, and Metabolism
Common Stains used for Microscopic Visualization Genetic Recombination
▪ Purple (+) • Refers to transfer or exchange between two
Gram Stain (VIAS)
▪ Red (-) homologous regions of DNA
Acid Fast Chain ▪ Red (+) • Bacterial Cell
(CH/DAM) ▪ Blue - Donor
▪ Auramine-rhodamine (Cell - Recipient (competent)
Wall – yellow or orange) • Plays a major role in bacterial survival
▪ Acidirine orange (Nucleus-
Fluorochrome Stain
bright orange) Restriction Enzyme (RE)
▪ Calcoflour White (Chitin • Allows cutting of incoming foreign DNA
(fungi) – bright apple green) • Mechanism of bacteria to preventing cutting of their own
C. diphtheriae metachrom DNA
Methylene Blue
granule
Lactophenol Blue Fungal cell wall Example:
Indi ink and Nigrosin Negative stain ▪ EcoRI – Escherichia coli
Endospore stain (M-H-S) ▪ HindIII – Haemophilus influenzae

Bacterial Genetics Mechanism of Gene Transfer

1. DNA (Deoxyribonucleic Acid) Transformation / DNA cloning

• Double stranded • Involve uptake of free/naked DNA
• Capable of replication • When bacteri5al cell (donor) dies, the recipient uptakes
• Contains nitrogenous bases (CGAT) the free DNA

2. RNA (Ribonucleic Acid)

• Single stranded
• Incapable of replication (needs to convert)
• Contains nitrogenous bases (CGAU)

Naturally Competent Bacteria

➢ Streptococcus pneumoniae (gram positive
➢ Neisseria gonorrhea
➢ Haemophilus influenzae
➢ Escherichia coli – most commonly used for cloning

Purines: Cytosine, Thymine, Uracil (CTU)
Pyrimidines: Adenosine, Guanine (AG)
Central Dogma of Molecular Biology
Transduction
• (Generalized and Specialized)
▪ Generalized transduction – bacterial DNA is
incorporated into another bacterium specially during
the lysis of the virulent bacteriophage
▪ Specialized transduction – part of a fragment
bacterial DNA with viral nucleic acid is transferred to
another bacterium by the temperate bacteriophage
during lysogenic process
• Utilize bacteriophage
• Bacteriophage – virus that infects bacteria
• Lysogenic cycle – bacteriophage will die and leave the
genetic material, and transferred to the second bacteria
being infected = tempering

Conjugation
• Use sex pili
• Occurs between two living cells (requires cell to cell
contact)
Mutation • E. coli F factor (both plasmid and X genes)
• Result of alteration of the original nucleotide sequence
during the transcription-translation process.
• 3 types:
(1) Insertion
(2) Deletion
(3) Substitution
• Transposons – mobile elements that often carry drug
resistant genes (plasmid)
• AMR – one of the result of mutation

MICR_111: CLINICAL MICROBIOLOGY | BACTERIAL GENETICS MASAKAYAN, J.E.

 Lesson 4: Bacterial Genetics, Growth, and Metabolism
Bacterial Growth

3 major nutritional needs for growth

50% Carbon Cellular constituents
14% Nitrogen Proteins and Nucleic acid
ATP Phosphate (PO4-), sulfate (SO4-)
Electrolytes Na+, K+, Cl- , Ca+2
Nutritional Requirements for Growth
Autotrophs/Lithotrophs Utilize CO2
Phototrophs Photosynthesis
Chemolithotrophs Oxidation
Heterotrophs Utilize glucose (OF Pathway)

Physiologic Requirements of Bacteria

According to Oxygen Requirement
Aerobes ▪ BBMP ( Bordetella,
▪ 15 – 21% Oxygen Brucella, Mycobacteria, and
▪ 1% Carbon Pseudomonas)
Dioxide
Superoxide dismutase and (A) Gaspak Jar
catalase
Obligate – Does not require
Oxygen
▪ Clostridium
▪ Bacteroides
Anaerobes Facultative – may or may not
▪ Enzymes require oxygen
▪ Enterobacteriaceae
Aerotolerant – Exposure leads
to absence of metabolic
activity
▪ Lactobacillus
▪ Cutibacterium
Microaerophile ▪ 2-10%
According to Carbon Dioxide (CO2)
Aerobic and Facultative (B) Candle Jar (C) Incubator
▪ Capnophiles (5 – 10%)
aerobic
▪ 0.03% CO2
According to Temperature
Psycrophiles/ 0 – 20oC
Cryophiles
Mesophiles 20 – 45 oC
Thermophiles 50 – 60 oC
Extremophiles Below earth surface
According to pH (6.5 – 7.5)
Acidophile 5.5
Neutrophile 5.5 – 8.0 (D) CO2 generating packet
Alkalophile 8.5 – 11.5
Other Physiologic Requirements of Bacteria
Halophiles Requires salt
Barophiles Requires pressure
Fastidious Requires growth factors
▪ Haemophilus
influeanzae

Bacterial Incubators

• It is an insulated and enclosed device that provides an

optimal condition of temperature, humidity, and other
environmental conditions required for the growth of
organisms.

Uses of Bacteriological Incubator

➢ Cultivation of bacteria
➢ Maintenance of bacterial cultures
➢ Microscopic examination (E) Blood Culture Incubator
➢ Biochemical studies
➢ Growth pattern of bacteria

MICR_111: CLINICAL MICROBIOLOGY | BACTERIAL GENETICS MASAKAYAN, J.E.

 Lesson 4: Bacterial Genetics, Growth, and Metabolism
Rate of Bacterial Growth

⦿ Length of generation time – measure of the growth rate

of an organism
⦿ Growth Pattern – exponential

Organism Generation time

Bacillus cereus 28 minutes
Escherichia coli 12.5 minutes
Staphylococcus aureus 27-30 minutes
Mycobacterium 18-24 minutes
tuberculosis
Treponema pallidum subsp. 30 hours
pallidum

Bacterial curve

LaLoStD

Bacterial Growth Curve

▪ No cell division
Lag Phase ▪ No increase in cell mass
▪ Adjustment phase
▪ Logarithm cell division
Log (exponential ▪ Biochemical testing
case) ▪ Antimicrobial susceptibility
testing
▪ Sporulation and antibiotic
synthesis
Stationary Phase ▪ Slow metabolic activity
▪ Division = death
▪ Growth cessation
▪ Death cell > live cell
Death Phase
▪ Toxic waste generation

MICR_111: CLINICAL MICROBIOLOGY | BACTERIAL GENETICS MASAKAYAN, J.E.

 Lesson 4: Specimen Collection, Culture, and Inoculation Technique
Specimen Collection
• Specimens for microbiology cultures should be collected
in sterile containers except for stool specimens
• Swabs
➢ Submit 2 samples – first is for smear; second is for
culture
Alternative for cotton used in swab sample/ swab tips:
Dacron, rayon, or calcium alginate (SARS-Cov2)
Because cotton contains fatty acid that kills your
microorganism
❖ CSF 4 vials:
➢ 1st – probably contaminated with blood; can be use
for in Immunology and Clinical Chemistry section
➢ 2nd – intended for Microbiology section for culture
and sensitivity
➢ 3rd – Hematological analysis
➢ 4th – reserved
❖ Anticoagulant present in Blood Culture bottle: SPS
(sodium polyanethole sulfonate)
Transport Media
• It provides a controlled environment to maintain the
viability of the organism during transport
Example:
➢ Cary Blair medium
➢ Amies medium
➢ Stuart medium
➢ Transgrow
➢ JEMBEC – “John E Martin Biological Environmental
Chamber"
❖ Charcoal – added for N. gonorrhoeae and B. pertussis
Blood Culture Collection
• Aseptic technique (30 seconds)
- 70% alcohol followed by 2% tincture of iodine
- If allergic to iodine: Chlorhexidine gluconate and
Benzalkonium chloride
MICR_111: CLINICAL MICROBIOLOGY | SPECIMEN COLLECTION
• Skin contaminants – CoNS (Coagulase-negative
staphylococci), Micrococcus and Propionibacterium
Blood Volume
Pediatric Sample 5-10ml per set
Adult 20ml per set
Butterfly method: Aerobic → anaerobic
Syringe method: Anaerobic → aerobic
Common Specimens in Microbiology
Urine (UTI)
• Clean catch midstream
• Cleaning of genitalia is required prior to collection
• UroPathogenic Escherichia coli
Stool (GI infection)
• Rectal swab (3 specimen)
• Transport medium (Carry Blair, Stuart, or Amies)
• Helicobacter pylori, Campylobacter jejuni, Vibrio
cholerae, and members of Enterobacteriaceae
Sputum (Pneumonia)
• Rinse the mouth with water
• Expectorated sputum – deep coughing sputum
• Induced sputum – device that help induce sputum
• 3 separate early morning specimen on the same day
(MTB and Fungi)
• Klebsiella pneumoniae, Streptococcus pneumoniae,
Mycobacterium tuberculosis
DSSM (Direct Sputum Smear Microscopy) – Scan and
screen if the sputum is suitable specimen for culture
NOTES:
• Ideally specimen is transported within 30 mins (no
fixative)
• For anaerobic bacteria, transport should not take
more than 10 minutes
• CSF Samples within 15 minutes
Specimen Collection Flowchart
❖ First you have to perform gram stain, followed by primary
plated culture media
❖ Primary plated media: BAP (Blood Agar plate), CAP
(Chocolate Agar plate), MAP (McConkey Agar plate)
MASAKAYAN, J.E.
 Lesson 4: Specimen Collection, Culture, and Inoculation Technique
❖ once you plate it with your primary plated media, you Preservatives and Anticoagulant
perform Biochemical tests to identify the species, the Preservative
genus of your bacteria. - Boric acid
❖ Then you can opt to your serological test or perform - Formalin and Polyvinyl alcohol (Trophozoite and
antimicrobial test Cyst)
❖ Antimicrobial test – use of antibiotics, to determine if the - Refrigeration/ Freezing (ex: C. difficile toxin)
organism is susceptible or resistant to certain antibiotic Anticoagulant
- SPS (sodium polyanethol sulfonate) – 0.025%
Specimen Labelling - Heparin – Mycobacterium tuberculosis
• “Do not ACCEPT unlabeled specimen” - Citrate and EDTA not used in Microbiology –
because it is toxic to microorganism
Specimen Label
➢ Name ❖ Aside from sputum, blood sample can be used in
➢ Identification number detection of Mycobacterium tuberculosis
➢ Room number ❖ TB QuantiFERON – is a blood examination for M.
➢ Physician tuberculosis; and we have what we called TB
➢ Culture site QuantiFERON tubes (3) purple, green, and red. As an
➢ Date and time of collection alternative, Lithium heparinized tube is used (green top)
Requisition form
➢ Patient’s name Specimen Prioritization
➢ Patient’s age (or date of birth) and gender
➢ Patient’s room number and or location Level Description Specimens
➢ Physician’s name, address, and phone number ▪ Amniotic fluid
➢ Specific anatomic site ▪ Blood
➢ Date and time of specimen collection ▪ Brain
1 Critical/Invasive
➢ Clinical diagnosis or relevant patient history ▪ Cerebrospinal fluid
➢ Antimicrobial agents (if patient is receiving any) ▪ Heart halves
➢ Name of individual transcribing order ▪ Pericardial fluid
▪ Body fluids (not listed for lvl 1)
Specimen Storage ▪ Bone
▪ Drainage from wounds
2 Unpreserved
Specimen Storage Guidelines ▪ Feces
Refrigerate Room temperature ▪ Sputum
▪ Catheter tips (IV) ▪ Abscess, lesion, wound ▪ Tissue
▪ CSF for virus ▪ Body fluids ▪ Catheter tip
Quantitation
▪ Ear: outer ▪ CSF for bacteria 3 ▪ Urine
required
▪ Feces (unpreserved ▪ Ear: inner ▪ Tissue for quantitation
▪ Feces for Clostridium ▪ Feces (preserved) ▪ Feces in preservative
difficile toxin (up to 3 ▪ Genital ▪ Urine in preservative
4 Preserved
days: > days store at - ▪ Nasal. N/P, throat ▪ Swabs in holding medium
70oC ▪ Tissue (aerobic and anaerobic)
▪ Sputum ▪ Urine (preserved)
Specimen Rejection
Specimen Transportation
• All specimen must be leak proof • The information on the requisition does not match the
information on the specimen label. If the patient name or
• Triple packaging technique
source does not match, the specimen should be collected
➢ Primary, secondary, tertiary receptacle
again
▪ International Air Transport Association (IATA) and
International Civil Aviation Organization (ICAO) • There is no patient identification on specimen container
- Requirement must be met prior to shipment of • The specimen is not submitted in the appropriate
biological specimen transport container or the container is leaking
• The quantity of the specimen is inadequate to perform all
tests requested
• The specimen transport time is more than 2 hours and the
specimen has not been preserved
• The specimen is received in a fixative such as formalin;
stools for ova and parasite examination are an exception
• An anaerobic culture is requested on a specimen in which
anaerobes are indigenous
• Microbiology processing of a particular specimen results
in questionable data (e.g., Foley catheter tip)
• The specimen is dried up
• More than one specimen from the same source was
submitted from the same patient on the same day; blood
cultures are an exception
• One swab was submitted with multiple requests for
various organisms
• Gram stain of expectorated sputum reveals fewer than 25
white blood cells (WBCs) and more than 10 epithelial
cells per low-power field and mixed bacterial flora

MICR_111: CLINICAL MICROBIOLOGY | SPECIMEN COLLECTION MASAKAYAN, J.E.