Module 03 Quality Control Sterilization and Disinfection Module PDF

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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

Blood Agar

Diffrentiation of hemolytic patterns; Used for fastidious organisms

Bordet-Gengou Agar

Bordetella pertussis

Blood Cystine Dextrose Agar

Francisella tularensis

Buffered Charcoal Yeast Extract (BCYE)

Legionella, spp.

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 Agar (CCFA)

Clostridium difficile

Eosin Methylene Blue (EMB) or Levine’s Medium

Differential media for lactose fermenters (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

MacConkey (MAC) Agar

Differential medium for LF and NLF; 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.

Thayer-Martin Agar

Neisseria gonorrhoeae and Neisseria meningitis

Thioglycollate Broth

Aerobes and Anaerobes

Thiosulfate-Citrate-Bile SaltsSucrose (TCBS) Agar

Vibrio

Xylose Lysine Desoxycholate (XLD) Agar

Salmonella and Shigella

Biochemical Tests A. Triple Sugar Iron (TSI) Test ⦁

Helps in determination of organism through its ability to ferment sugar

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3 Sugars:

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

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Glucose (0.1%)

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Lactose (1%)

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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 Organism

Slant

Butt

Gas

H 2S

Escherichia, Klebsiella, Enterobacter

Acid (A)

Acid (A)

Pos (+)

Neg (-)

Shigella, Serratia

Alkaline (K)

Acid (A)

Salmonella, Proteus

Alkaline (K)

Pseudomonas

Alkaline (K)

Neg (-)

Neg (-)

Acid (A)

Pos (+)

Pos (+)

Alkaline (K)

Neg (-)

Neg (-)

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.

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The positive result is indicated by the red layer at the top of the tube after the addition of Kovac’s reagent.

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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.

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The medium contains ferrous ammonium sulfate and sodium thiosulfate, which together serve as indicators for the production of hydrogen sulfide.

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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.

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Organisms possessing the enzyme tryptophanase degrade tryptophan to indole. Indole is detected upon the addition of Kovacs Reagent.

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Indole combines with p-dimethylaminobenzaldehyde and produces a red band at the top of the medium.

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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.

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Motile organisms extend from the stab line and produce turbidity or cloudiness throughout the medium.

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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.

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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

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Positive: Turning of bromthymol blue indicator from green to blue

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Negative: Absence of growth/ No change in color

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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

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Procedure: Pick up colony with an inoculating loop and immerse in a few drops of 3% H2O2 (hydrogen peroxide)

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Interpretation:  Positive: effervescence or presence and formation of bubbles

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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).

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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.

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Most commonly used BSC in a clinical microbiology laboratory (class IIA).

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Sterilizes the air using HEPA filter that flows over the infectious material and the air to be exhausted.

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Used for BSL 2 and 3 agents.

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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.

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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.

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Biological indicator is Bacillus stearothermophilus.

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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.

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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).

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Lyophilization destroys bacteria through changes in proteins and decrease in chemical reactions.

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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

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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.