This document discusses sterilization and disinfection in microbiology labs. It defines key terms like sterilization, disinfection, antiseptic, and provides examples of physical sterilization methods like heat, UV light, radiation and filtration. It also discusses chemical sterilization methods like ethylene oxide and glutaraldehyde. Specific sterilization techniques are described in detail, such as using autoclaves for moist heat sterilization and hot air ovens for dry heat sterilization. The importance of monitoring autoclaves with indicators is also highlighted.
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.
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.
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