LESSON 3: QUALITY CONTROL: STERILIZATION AND DISINFECTION

I. Learning Objectives
At the end of the lesson, students should be able to:
A. Identify basic culture medium; and
B. Differentiate their purposes.
C. Identify the different biochemical tests;
D. Explain the procedures for each biochemical tests; and
E. Interpret the results of the tests.
F. Explain the importance of stringent biosafety measures and the objectives of quality
control;
G. Differentiate the types of biological safety cabinet; and
H. Enumerate and describe the biological agents
I. Discuss the sterilization techniques through physical and chemical methods; and
J. Describe the chemical agents utilized in disinfection.

II. Concepts and Information from the PowerPoint

Common Culture Media and Their Purposes

Culture Medium Purpose

Alkaline Peptone Broth Vibrio

Diffrentiation of hemolytic patterns; Used for

Blood Agar
fastidious organisms

Bordet-Gengou Agar Bordetella pertussis

Blood Cystine Dextrose Agar Francisella tularensis

Buffered Charcoal Yeast Extract
Legionella, spp.
(BCYE)

Campy-Blood Agar Campylobacter spp.

Cetrimide Agar Pseudomonas aeruginosa

Chocolate Agar Nisseria; for fastidious bacteria

Cystine Tellurite Blood Agar (CTBA) Corynebacterium diphtheriae

Cycloserine Cefoxitine Fructose

Clostridium difficile
Agar (CCFA)

Eosin Methylene Blue (EMB) or Differential media for lactose fermenters

Levine’s Medium (LF) and non-lactose fermenters (NLF).

Fletcher’s Semi-solid Medium Leptospira

Hektoen Enteric (HE) Agar Differentiation of Salmonella and Shigella

Löeffler’s Blood Serum Medium Corynebacterium diphtheriae

Löwenstein-Jensen (LJ) Agar Mycobacterium

Differential medium for LF and NLF;

MacConkey (MAC) Agar
Selective for Gram-negative bacteria.

Mannitol Salt Agar Staphylococcus aureus

Middlebrook 7H10 Agar Mycobacteria

Martin-Lewis Agar Neisseria gonorrhoeae

Mueller-Hinton Agar Susceptibility Test (Antimicrobial Testing)

New York City (NYC) Agar Neisseria gonorrhoeae

Phenylethyl Alcohol (PEA) Medium Gram-positive bacteria

Regan-Lowe Agar Bordetella pertussis

Salmonella-Shigella Agar (SSA) Salmonella and Shigella

Selenite Broth (Enrichment Medium) Salmonella spp.

Skirrow Agar Campylobacter spp.

Tetrathionate Broth Salmonella and Shigella spp.

Neisseria gonorrhoeae and Neisseria

Thayer-Martin Agar
meningitis

Thioglycollate Broth Aerobes and Anaerobes

Thiosulfate-Citrate-Bile Salts-
Vibrio
Sucrose (TCBS) Agar
 Xylose Lysine Desoxycholate (XLD)
Salmonella and Shigella
Agar

Biochemical Tests
A. Triple Sugar Iron (TSI) Test
⦁ Helps in determination of organism through its ability to ferment sugar
⦁ 3 Sugars:
⦁ Glucose (0.1%)
⦁ Lactose (1%)
⦁ Sucrose (1%)
⦁ Procedure:
⦁ Inoculate TSI agar by first stabbing through the center of the medium and
then streaking on the surface of the agar slant.
⦁ Leave the cap on loosely and incubate the tube at 35°C for 18 to 24 hours.
 Expected Results Triple Sugar Iron Agar
o Alkaline slant/Alkaline butt (K/K) i.e Red/Red = glucose, lactose and
sucrose non-fermenter
o Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation only,
gas (+ or-) , H2S (+ or -)
o Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or
sucrose fermenter gas (+ or -), H2S (+ or -).
o If H2S is produced, the black color of ferrous sulfide is seen.
 Some Examples of TSI Agar Reactions

Name of the
Slant Butt Gas H2S
Organism

Escherichia,
Klebsiella, Acid (A) Acid (A) Pos (+) Neg (-)
Enterobacter
 Shigella, Alkaline
Neg (-) Neg (-)
Serratia (K) Acid (A)

Salmonella, Alkaline
Proteus (K) Acid (A) Pos (+) Pos (+)

Alkaline Alkaline
Pseudomonas Neg (-) Neg (-)
(K) (K)

B. IMViC Tests
1. Indole Test
⦁ Principle: Used to determine the ability of an organism to split tryptophan to form
the compound indole.
⦁ It is performed on sulfide-indole-motility (SIM) medium or in tryptophan broth.
Result is read after adding Kovac’s reagent.
⦁ The positive result is indicated by the red layer at the top of the tube after the
addition of Kovac’s reagent.
⦁ A negative result is indicated by the lack of color change at the top of the tube
after the addition of Kovac’s reagent.

SIM Medium (Hydrogen Sulfide Production)

⦁ The formulation of SIM Medium is designed to allow the detection of sulfide
production, indole formation and motility.
⦁ The medium contains ferrous ammonium sulfate and sodium thiosulfate,
which together serve as indicators for the production of hydrogen sulfide.
⦁ Hydrogen sulfide production is detected when ferrous sulfide, a black
precipitate, is produced as a result of ferrous ammonium sulfate reacting with
H2 S gas.
SIM Medium (Indole Test)
⦁ Casein peptone, another component of SIM Medium, is rich in tryptophan.
 ⦁ Organisms possessing the enzyme tryptophanase degrade tryptophan to
indole. Indole is detected upon the addition of Kovacs Reagent.
⦁ Indole combines with p-dimethylaminobenzaldehyde and produces a red
band at the top of the medium.
⦁ A negative indole test produces no color change upon the addition of Kovacs
Reagent.
SIM Medium (Motility Test)
⦁ The small amount of agar added to the medium provides a semi-solid
structure allowing for the detection of bacterial motility.
⦁ Motile organisms extend from the stab line and produce turbidity or
cloudiness throughout the medium.
⦁ Non-motile organisms grow only along the stab line and leave the
surrounding medium clear.

2. Methyl Red Test

 Principle: Methyl red test, commonly known as MR test is used to determine the
ability of an organism to produce and maintain stable acid end products from
glucose fermentation.
 MR test along with VP test is performed simultaneously because they are
physiologically related and are performed on MRVP broth.
 Procedure of Methyl Red (MR) Test:
o Inoculate MRVP broth with a pure culture of the organism.
o Incubate at 35°-37°C for a minimum of 48 hours in ambient air.
o Add 5 or 6 drops of methyl red reagent per 5 mL of broth.
o Observe for the color change in the broth medium.
 Positive: Bright red color
 Negative: Yellow color

3. Voges- Proskauer Test

 Principle: The Voges-Proskauer (VP) test is used to determine if an organism
produces acetylmethyl carbinol from glucose fermentation
 Procedure:
 i. Using organisms taken from an 18-24-hour pure culture, lightly inoculate
the medium.
ii. Incubate at 37 degrees C. for 24 hours.
iii. Add 6 drops of 5% alpha-naphthol (Barritt’s A), and mix well.
iv. Add 2 drops of 40% potassium hydroxide (Barritt’s B), and mix well.
v. Observe for a pink-red color at the surface within 30 min. Shake the
tube vigorously during the 30-min period.

4. Citrate Utilization Test

 Principle: Used to determine the ability of an organism to utilize sodium citrate as
its only carbon source.
 The test is performed on Simmons citrate agar
 Positive: Turning of bromthymol blue indicator from green to blue
 Negative: Absence of growth/ No change in color
 Procedure:
i. Streak the slant back and forth with a light inoculum picked from the
center of a well-isolated colony.
ii. Incubate aerobically at 35 to 37C for up to 4-7 days.
iii. Observe a color change from green to blue along the slant.

IMViC Test Results

1. IMViC tests of Escherichia coli
1. Indole: Positive
2. Methyl-Red: Positive
3. Voges-Proskauer test: Negative
4. Citrate test: Negative
2. IMViC tests of Klebsiella (formerly Enterobacter) aerogenes
1. Indole: Negative
2. Methyl-Red: Negative
 3. Voges-Proskauer test: Positive
4. Citrate test: Positive
3. IMViC tests of Proteus vulgaris
1. Indole: Positive
2. Methyl-Red: Positive
3. Voges-Proskauer test: Negative
4. Citrate test: Negative
4. IMViC tests of Citrobacter freundii
1. Indole: Negative
2. Methyl-Red: Positive
3. Voges-Proskauer test: Negative
4. Citrate test: Positive

C. Catalase Test
⦁ Principle: The enzyme catalase mediates the breakdown of hydrogen peroxide
into oxygen and water
⦁ Procedure: Pick up colony with an inoculating loop and immerse in a few drops of
3% H2O2 (hydrogen peroxide)
⦁ Interpretation:
 Positive: effervescence or presence and formation of bubbles
⦁ This test is used to differentiate between staphylococci (+ve) and streptococci (-
ve)

D. Coagulase Test
⦁ Principle: Coagulase is an enzyme that coverts soluble fibrinogen into insoluble
fibrin
⦁ This test is used to differentiate Staphylococcus aureus (+ve) from coagulase
negative staphylococci
a. Bound coagulase (clumping factor)
• Detected in tube coagulase test
 • Mix 0.1mL of culture + 0.5 mL of plasma
• Incubate at 37C for 4 hours
• Observe the tube for clot formation
• Any degree of clotting constitutes a positive test
• More accurate. Time-consuming
b. Free coagulase
• Detected in slide coagulase test
• Add one drop of plasma on slide
• Mix well and observe clumping within 10 seconds
• Rapid diagnosis. Less accurate

Biosafety and Quality Control in a Laboratory

 Clinical laboratory specimens are potential hazards since they may contain
infectious agents.
 Universal precautions are recommended by the Center for Disease Control
(CDC). These measures must be observed and strictly implemented.

Biological Safety Cabinet (BSC)

⦁ A device that encloses a working area to protect workers from aerosol exposure
and infectious disease agents.
⦁ The air that contains infectious material is sterilized either by heat or UV light, or
by passage through a high-efficiency particulate (HEPA) resistance filter.
A. Class I Cabinet
⦁ Open-fronted type of cabinet with negative pressure (ventilated).
⦁ Allows room (unsterilized) air to enter the cabinet, circulate around the area,
and expose the material within; only the air to be exhausted is sterilized using
HEPA filter.

B. Class II Cabinet
⦁ Known as laminar flow BSC.
⦁ Most commonly used BSC in a clinical microbiology laboratory (class IIA).
 ⦁ Sterilizes the air using HEPA filter that flows over the infectious material and
the air to be exhausted.
⦁ Used for BSL 2 and 3 agents.
⦁ There are 2 types of Class II cabinets:
 Class IIA – has fixed opening; 70% of the air is recirculated.
 Class IIB – variable sash opening; used for chemicals, radioisotopes
and carcinogens.

C. Class III Cabinet

⦁ Provides highest level of safety to the worker.
⦁ Air coming in and out of the cabinet is sterilized using a HEPA filter and the
infectious material within is handled with rubber gloves that are attached and
sealed to the cabinet.
⦁ Used for biosafety levels (BSL) 4 agents

Classification of Biologic Agents Based on Hazard

⦁ BSC is composed of different biosafety levels that range from BSL 1 to 4
depending on the level of bio-containment precaution required for the specimen
being studied.

A. Biosafety Level I Agents

⦁ Have no known potential for infecting healthy people.
⦁ Containment level is used in laboratory activities of students.
⦁ Some examples of pathogens that requires this containment level: Bacillus
subtilis and Naegleria gruberi

B. Biosafety Level II Agents

⦁ Acquired by ingestion or exposure to percutaneous or mucous membrane.
⦁ Include all the common agents of infectious diseases.
⦁ Access to laboratory is limited. It requires the personnel to change their
clothes with the recommended laboratory clothing before going to their
stations.
⦁ Personnel handling these agents should receive immunization.
⦁ Some examples of pathogens that requires this containment level: HIV,
Bacillus anthracis, Yersinia pestis, Salmonella, and Shigella.

C. Biosafety Level III Agents

⦁ Potential agents for aerosol transmission
 ⦁ Air movement in the laboratory must be controlled to contain the infectious
materials.
⦁ Some examples of pathogens that requires this containment level:
Mycobacterium tuberculosis, Francisella tularensis, Brucella spp., Coxiella
burnetti, St. Louis encephalitis virus, and systemic fungi.

D. Biosafety Level IV Agents

⦁ Cause life-threatening infections.
⦁ Maximum containment and decontamination of all personnel and materials
before leaving the area are observed.
⦁ Aerosol transmission with pressure is possible.
⦁ Some examples of pathogens that requires this containment level: arbovirus,
arenavirus, filovirus, and smallpox virus.

Notes to remember:
 All clinical laboratrories must adhere to biosafety level 2 guidelines.
 The agents that pose the greatest risk are those that are transmitted by
aerosols.
 The 5 most frequently acquired laboratory infections are: shigellosis,
salmonellosis, tuberculosis, brucellosis, and hepatitis.
 The laboratory procedures that create aerosol are pipetting, flaming loops,
agar plates streaking, and centrifugation.

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
o Refers to the removal or destruction of all forms of life, including bacterial
spores.
Physical Methods of Sterlization
A. Application of Heat
o Heat is the most commonly used method for the removal of
microorganisms.
1. Moist Heat Procedure
 It destroys microorganisms through the coagulation of enzymes,
structural proteins and degradation of nucleic acids.
 a. Tyndallization

 Flowing steam, 100 degrees Celsius for 30 minutes for 3

successive days
b. Inspissation
 75 degrees Celsius for 2 hours for 3 consecutive days
c. Autoclaving
 Fastest and simplest method of sterilization through which all
organisms (except prions), including those that contains spores,
are killed within 15 minutes.
 Sterilizes bio hazardous trash and heat-stable objects.
 Principle: steam under pressure.
 Biological indicator is Bacillus stearothermophilus.
 Autoclave is a chamber which is filled with hot steam under
pressure.
o 121 degrees Celsius, 15 psi for 15 minutes: for media,
liquids, utensils, glass, pipettes, and instruments for
assay.
o 132 degrees Celsius, 15 psi for 30-60 minutes: for
decontaminating medical wastes.
2. Dry Heat Procedure
 Does not require water.
 Kills microorganisms by denaturing proteins.

 Utilized for the sterilization of glassware, oil products, and powder.

 Biological indicator is Bacillus atropheus (Bacillus subtilis var. niger).
a. Flaming/Direct Heating
b. Oven-heating

 Used for glassware, oil, petroleum or powders.

 Temp. and time exposure: 160-170 degrees Celsius for 1.5-2
hours.
c. Incineration
  Most common method of treating infectious waste and infected
laboratory animals.
 Principle: Burning of materials into ashes at 300-400 degrees
Celsius
 Temp used for the hazardous material: 870-980 degrees
Celsius.

d. Cremation

 Used to control spread of communicable diseases.

B. Filtration
 Method of choice for the sterilization of antibiotic solutions, toxic chemicals,
radioisotopes, vaccines, and carbohydrates (heat-sensitive solutions).
 May be used with both liquid and air substances.
 Types of Filters:
1. Depth Filters
2. Made of fibrous or granular materials.
3. Examples: Berkefield filter, Chamberland filter, HEPA filters and
asbestos.
4. Membrane Filters (Circular Filters)
5. Porous membranes (almost 0.1µm thick)
6. Composed of cellulose acetate or polycarbonate.
7. Sterilizes pharmaceuticals, ophthalmic solutions, culture media,
antibiotics, and oil products.

C. Low/Cold Temperature
 Considered bacteriostatic because it reduces the rate of metabolism.
 Important in food microbiology.
 Exposure to 2-8 degrees Celsius for 72 hours kills the agents of syphilis.
 D. Desiccation and Lyophilization
⦁ Desiccation destroys bacteria through the disruption of metabolism that
involves removing water from microbes (bacteriostatic).
⦁ Lyophilization destroys bacteria through changes in proteins and decrease in
chemical reactions.
⦁ Examples of bacteria which remain active in a dry environment are as follows:
 Neisseria gonorrheae –viable for one hour
 Mycobacterium tuberculosis – viable for several months
 Bacillus and Clostridium – viable for ten years

Chemical Method of Sterilization

A. Ethylene Glycol
⦁ Most common chemical sterilant
⦁ Used for materials that cannot be autoclaved
⦁ Quality control: Bacillus subtillis
B. Formaldehyde
⦁ Sterilize HEPA Filters in BSCs
C. Glutaraldehyde
⦁ Sporicidal (kills spores) in 3-10 hours
⦁ Used for bronchoscopes

Disinfection

 Refers to the removal, inhibition, or killing of microorganisms which includes

potential pathogens, by using chemical and physical agents usually on inanimate
objects, although it does not remove all bacterial spores.
Terminologies to Remember:
1. Antiseptic
⦁ Applied topically on the skin.
⦁ Inhibits sepsis formation.
 ⦁ Examples: phisohex, hexachlorophene, and tincture of iodine/povidine
alcohol (iodophor).
2. Disinfectant
⦁ Applied to inanimate objects.
⦁ Examples: Lysol (cresols), chlorine, and sodium hypochlorite.
⦁ Sodium hypochlorite in a 1:10 dilution is an effective disinfectant.
3. Bactericidal
⦁ Precipitates bacterial protein (H2SO4, HCl) and kills all bacteria in the
specimen.
⦁ Example: strong acids
4. Bacteriostatic
⦁ Inhibits the growth of organisms.

Physical Methods of Disinfection

A. Boiling
⦁ Destroys vegetative bacteria (non-sporulating).
⦁ Temperature and time of exposure used are 100 degrees Celsius for 10-15
minutes
B. Pasteurization/ Partial Sterilization
⦁ Used to sterilize milk, dairy products, and alcoholic beverages.
⦁ Eliminates food-borne pathogens and organisms responsible for food
spoilage.
⦁ Cannot eliminate bacterial endospores.

Chemical Methods of Disinfection

A. Alcohol
⦁ Ethyl or Isopropyl alcohol is nonsporicidal (does not kill spores) and
evaporates quickly
⦁ Its use is limited on the skin as an antiseptic or on thermometers as a
disinfectant
 B. Quarternary Ammonium Compounds (QUATS)
⦁ Used to disinfect bench tops or other surfaces in the laboratory
⦁ Low toxicity
⦁ Examples: Benzalkonium chloride (Zephiran)
C. Halogens
⦁ Halogens especially iodine and chlorine are frequently used as disinfectant
⦁ Examples: Povidone-Iodine and Sodium Hypochlorite (household bleach)
D. Phenolics
⦁ Denature proteins; disrupts cell membranes
⦁ Disinfectants at high concentrations; used in soaps at low concentrations
⦁ Examples: Phenol, Carbolic acid, Lysol

III. Assignment
Crossword Puzzle
1. Puzzle must include 10 word entries, in a combination of across and down using any
of the concepts in this lesson.
2. Color block the boxes that are not used.
3. Must include clear but concise clues.
4. All entries must intersect at least one other entry.
5. Use only the terms and concepts discussed in this lesson.
6. Submission must include an unsolved puzzle, a number clue list (divided into
“across” and “down” lists), and a solved version of the puzzle.
TIP:
⦁ Start by writing a list of possible words, including short, medium, and long words.
⦁ When designing the puzzle, start with one long word in the middle and work out from
there instead of starting in the corner.

IV. Generalization/ Summarization

Give a summary or generalization of what you have learned from Lesson 3: Quality
Control: Sterilization and Disinfection. Minimum of 5 sentences.
V. Assessment Quiz with Answers
Choose the best answer among the given choices.
1. All of these media are used for the isolation of Neisseria gonorrhoeae except:

Martin-Lewis Agar

Regan-Lowe Agar

Thayer- Martin Agar

New York City Agar

2. Alkaline slant and Acidic butt result on TSI agar indicates presence of an organism
that is a

glucose, lactose and sucrose fermenter

lactose and sucrose fermenter

glucose, lactose and sucrose nonfermenter

glucose fermenter

3. Which organism has the following IMViC test results? Indole negative, Methyl Red
negative, VP positive and Citrate test positive.

Proteus vulgaris

Escherichia coli

Klebsiella aerogenes

Citrobacter freundii

4. This biologic agent is acquired by ingestion or exposure to percutaneous or mucous

membrane.

Biosafety Level 3 Agents

 Biosafety Level 2 Agents

Biosafety Level 1 Agents

Biosafety Level 4 Agents

5. This chemical method of disinfection denatures proteins and disrupts cell

membranes.

Halogens

Phenolics

Quarternary Ammonium Compounds (QUATS)

Alcohol

6. This physical method of sterilization is used to control communicable diseases.

Autoclaving

Incineration

Cremation

Desiccation

7. This enzyme mediates the breakdown of hydrogen peroxide into oxygen and water.

Coagulase

Catalase

Trytophanase

DNAse

8. A positive citrate utilization test will result in

 effervescence or appearance of bubbles

appearance of red layer on top

change of color from green to blue

appearance of black precipitate

9. Plasma used for bound coagulase test is incubated for

3 hours

1 hour

4 hours

2 hours

10. This IMViC test uses Barritt's A and Barritt's B reagent.

Methyl Red test

Indole test

Voges Proskauer test

Citrate Utilization test

11. Motile organism in SIM medium will result in

turbidity or cloudiness

effervescence or production of bubbles

clot formation
 production of red band at the top of the medium

12. This is an open-fronted type of cabinet with negative pressure.

Class IIA Cabinet

Class IIB Cabinet

Class III Cabinet

Class I Cabinet

13. All of these pathogens are Biosafety Level 3 agents except:

Bacillus anthracis

Coxiella burnetti

Francisella tularensis

Mycobacterium tuberculosis

14. All clinical laboratories must adhere to

biosafety level 2 guidelines

biosafety level 3 guidelines

biosafety level 4 guidelines

biosafety level 1 guidelines

15. What is the biological indicator of Autoclaving?

Bacillus atropheus

Bacillus anthracis
 Bacillus subtilis var. niger

Bacillus stearothermophilus

16. This is used to disinfect bench tops or other surfaces in the laboratory.

Phenolics

Quarternary Ammonium Compounds (QUATS)

Alcohol

Halogens

17. What medium is used for the isolation of Vibrio spp.?

Alkaline Peptone Broth

Lowenstein Jensen Agar

Cetrimide Agar

Hektoen Enteric Agar

18. Which item is utilized as a chemical sterilant for HEPA filters in a biological safety
cabinet?

Halogens

Quarternary Ammonium Compounds (QUATS)

Phenol

Formaldehyde

19. What is the most commonly used biological safety cabinet in a clinical microbiology
laboratory in which the air is sterilized using a HEPA filter?
 Class IIA Cabinet

Class IIB Cabinet

Class III Cabinet

Class I Cabinet

20. Which is the typical mode of transmission for microorganisms that pose the greatest
risk in a microbiology laboratory?

Needle stick

Aerosol

Vector bite

Ingestion

VI. References
▸ Mahon, C.R., Lehman, D.C., Manuselis, G. (2014). Textbook of
Diagnostic Microbiology (5th ed.). New York: Saunders
▸ Bailey, W. R., Scott, E. G., Finegold, S. M., & Baron, E. J. (1986). Bailey and Scott's
Diagnostic microbiology. St. Louis: Mosby.
▸ Rodriguez, M.T. (2018). Review Handbook in Diagnostic Bacteriology. C&E
Publishing Inc.