micro

1. Microbiology Lab
Sterilization & Disinfection
Prepared by: Kazhaleh Mohammadi
Knowledge University
2022-2023

2. Terms
• Sterilization: Complete killing of all forms of microorganisms, including
bacterial spores
• Disinfection: Killing or removing of harmful vegetative microorganisms.
• Disinfectant: Chemical substance used on inanimate objects . Toxic to human .
• Antiseptic: Disinfectant that can be safely used on living tissues
• Germicide/microbicide: Chemical agent that kills pathogenic microorganisms

3. • Sepsis: Growth of microorganisms in the body or the presence of
microbial toxins in blood and other tissues
• Asepsis: Practice to prevent entry of infectious agents into sterile tissues
and thus prevents infection
• Sanitization: Cleansing technique that mechanically removes
microorganisms to reduce the level of contaminants
• Sanitizer- Compound (e.g., soap or detergent)

4. Uses of Sterilization
1. Sterilization in Microbiological works:
• Preparation of culture media, reagents and equipments
2. Sterilization for Surgical Procedures and medicines:
• Gloves, aprons, surgical instruments, syringes, drugs and other
supplies etc.

5. Methods of Sterilization
1. Physical Methods:
• Heat (moist heat & dry heat)
• U.V.( ultra violet) Light
• Ionizing Radiation
• Filtration
2. Chemical Methods: (used for heat sensitive equipment)
• Ethylene oxide , Gluteraldehyde

6. Physical Methods
• Heat : Most important ,should be used whenever possible, can be :
A- Dry heat induces:
• Denaturation of protein, oxidative damage and toxic effect due to the high level of
electrolytes
• Also damage the DNA of the microorganism
• As a result, the microorganism got killed
B- Moist heat : kills the microorganisms by denaturation and coagulation of
proteins, eg. Autoclaves
Temperature required to kill microbe by dry heat more than moist heat

7. Dry Heat: Hot air oven
• Introduced by Louis Pasteur
• Metallic instruments (like forceps, scalpels, scissors)
• Glasswares (such as petridishes, pipettes, flasks, all-glass syringes)
• Swabs, oils, grease, petroleum jelly and some pharmaceutical products
• Unsuitable for rubber and plastics
• 160οC for two hours, 170οC for 1 hour and 180οC for 30 minutes
• The hot air oven must not be opened until the temperature inside has
fallen below 60o C to prevent breakage of glassware

8. Moist Heat: Autoclave
• Uses hot water
• Autoclaving – the standard sterilization method in hospitals.
• Autoclave works under the same principle as the pressure cooker where
water boils at increased atmospheric pressure, because of the increased
pressure the boiling point of water is >100 °C.
• The autoclave is a tough double-walled chamber in which air is replaced by
pure saturated steam under pressure.

9. Monitoring of Autoclaves
1. Physical- Use of thermocouple to measure accurately the temperature.
2. Chemical- It consists of heat sensitive chemical that changes color at the right
temperature and exposure time. e.g.
 Autoclave tape ( commonly used)
3. Biological – The biological indicator is used for monitoring sterilization. This tube
contains bacterial spores on a filter-paper carrier packaged within a small,
thermoplastic culture tube. Inside the thermoplastic culture's tube is a sealed-glass
ampoule containing a color indicator that turns a dramatic yellow when spores
grow.

10. Autoclave
Autoclave tape Biological indicator
(Spore test)

11. Physical methods : U.V. light
• U.V. light
• Limited sterilizing power because of poor penetration into most materials.
• Disinfection of closed areas in the microbiology laboratory, inoculation
hoods, and laminar flow
• Harmful to skin and eyes
• Doesn't penetrate glass, paper, or plastic
• Of use in surface disinfection

12. Physical methods : Radiation
• Ionizing radiation
• High-energy rays, good penetrative power
• Radiation does not generate heat- "cold sterilization“
• (a) X-rays, (b) gamma rays
• Gamma radiation: has greater energy than U.V. light, therefore more effective.
Used mainly in industrial facilities
• e.g. sterilization of disposable plastic syringes, gloves, specimens, containers,
and Petri dishes

13. Physical Methods : Filtration
• Use of membrane filter made of cellulose acetate or other material.
• Membrane filters with pore sizes between 0.2-0.45 μm
• Does not kill microbes, it separates them out
• Used to remove microbes from heat-labile liquids such as serum,
antibiotic solutions, sugar solutions, urea solution
• For removing bacteria from ingredients of culture media
• Thus filtration does not technically sterilize the items but is
adequate for circumstances under which is used.

14. Chemical Methods
• Using Disinfectants /Antiseptics
• eg. phenolics, chlorhexidine, alcohol, iodine, etc.
• Some strong chemical substances may be used to achieve
sterilization (kill spores) such as glutaraldehyde & ethylene oxide

15. ALCOHOLS
• Alcohols dehydrate cells, disrupt membranes, and cause coagulation of protein
• Examples: Ethyl alcohol, isopropyl alcohol, and methyl alcohol
• Application:
• 70% ethyl alcohol (spirit) is used as antiseptic on the skin
• Isopropyl alcohol is preferred to ethanol
• Also used to disinfect surfaces
• Used to disinfect clinical thermometers
• Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods
• Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

16. Action of Disinfectants
• Damage to the cell wall and alter the permeability of the cell
membrane, resulting in exposure, damage, or loss of the cellular
contents
• Alter proteins and form protein salts or cause coagulation of proteins
• Inhibit enzyme action, nucleic acid synthesis, or alter nucleic acid
molecules
• Cause oxidation or hydrolysis

17. Important to remember
• Any instrument or item used for sterile body site
should be sterile.
• Any instrument or item used for non-sterile body site
can be disinfected