• Sterilization: Object, surface or medium is freed of all living

microorganisms either in the vegetative or in the spore state.

• Disinfection: Destruction of microorganisms, especially potential
pathogens on the surfaces of inanimate objects or in the
environment.
• Reduce the microbial population not the bacterial endospores on an
inanimate surfaces or in the organic materials.
• Antisepsis: Destruction or inhibition of miroorganisms on living
tissues by chemicals (non toxic and non irritating).
• Antiseptics: Chemical agent
• Sepsis: Growth of microorganisms in the body or the presence of
microbial toxins in the blood and other tissues.
• Asepsis: Practice of preventing entry of infectious agents into sterile
tissues and thus preventing the infections.
• Sanitization: Cleansing technique that mechanically removes
microorganisms to reduce the level of contaminants. (Sanitizer, soap
or detergent)
Uses of sterilization
• Sterilization of surgical procedures or medicines: Gloves, aprons,
surgical instruments, syringes.

• Sterilization in microbiological works: Preparation of culture media,

reagents and equipment.
Methods of sterilization
• 1. Physical method
• 2. Chemical method.
Physical Methods:
a) Heat:
I) Dry heat II) Moist Heat III) Sunlight
b) Radiation:
I) UV Radiation ii) X ray, gamma ray
c) Filtration: cellulose membrane filter.
Sunlight

• Direct sunlight is a natural method of sterilization of water in tanks,

rivers, and lakes.
• Direct sunlight has an active germicidal effect due to its content of
ultraviolet and heat rays.
• Bacteria present in natural water sources are rapidly destroyed by
exposure to sunlight.
Heat
• Heat is the most dependable method of sterilization

• As a rule, higher temperatures (exceeding the maximum) are

microbicidal, whereas lower temperatures (below the minimum)
tend to have inhibitory or microbistatic effects.
Heat

Two major methods:

Dry heat and moist heat

•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
Moist Heat:
• Kills the microorganisms by denaturation and coagulation of proteins
• Temperature required to kill microbe by dry heat is more than moist
heat

• Thermal death time-

• Minimum time required to kill a suspension of organisms at a

predetermined temperature in a specified environment.
Heat
• In the form of
• Hot water, Boiling Water, Steam (Vaporized Water)
• Temperature ranges 60-135°C.
• Adjustment of pressure in a closed container regulates the
temperature and steam.
• Sterilization by moist heat:
temperature <100°C (Hot water- Pasteurization)
temperature of 100°C (Boiling water- Water Bath)
temperature >100°C (Steam – Autoclaving)
Pasteurization: Pasteurization is a technique in which heat is applied to
liquids to kill potential agents of infection and spoilage, while at the same
time retaining the liquid’s flavor and food value.
• originally employed by Louis Pasteur and is used for in food and dairy
industry
Two methods of pasteurization
• holder method (heated at 63oC for 30 minutes)
• flash method (heated at 72oC for 15 seconds)
• Boiling: Water Bath
• Boiling water (100oC) for 10–30 minutes kills most vegetative bacteria and
viruses.
Autoclaving
Sterilization by steam under pressure.
• Pressure–temperature combinations achieved
with special device “Autoclave”
• At 15 lb per sq. inch pressure, 121°C
temperatures is obtained, kept for 15 minutes for
sterilization.
• Culture media, certain heat resistant equipment,
liquids etc.
• In effective for substances that repel moisture.
Wax, oil, powders.
• Kills all the vegetative and spore form of
bacteria.
Intermittent sterilization:

Certain heat-labile substances (e.g., serum, sugar, egg, etc.) that

cannot withstand the high temperature of the autoclave can be
sterilized by a process of intermittent sterilization, known as
tyndallization.
• Tyndallization is carried out over a period of 3 days and requires a
chamber to hold the materials and a reservoir for boiling water. Items
to be sterilized are kept in the chamber and are exposed to free-
flowing steam at 100°C for 20 minutes, for each of the three
consecutive days.
• On the first day, the temperature is adequate to kill all the vegetative
forms of the bacteria, yeasts, and molds but not sufficient to kill
spores.
• The surviving spores are allowed to germinate to vegetative forms on
the second day and are killed on re-exposure to steam.
• The third day re-ensures killing of all the spores by their germination
to vegetative forms.
DRY HEAT:
Red heat:
Sterilized by holding them in Bunsen flame till
they become red hot.
bacteriological loops, straight wires, tips of
forceps and spatulas
Limited to those articles that can be heated to
redness in flame.

Flaming:
Method of passing the object over a Bunsen
flame but not heating it to redness.
Scalpels, mouth of test tube, flask, glass slides
and covers.
Incineration
• Method of destroying contaminated material
by burning them in the incinerator.
• E.g Pathological materials and beddings.
• Suitable only for those objects that are
needed to be disposed off.
Hot Air oven:

• Introduced by Louis Pasteur

• Metallic instruments (like forceps, scalpels, scissors)
• Glass wares (such as petri-dishes, pipettes, flasks, all-glass syringes)
• Swabs, oils, grease, and some pharmaceutical products
• Unsuitable for rubber and plastics.
• 160°C. for two hours, 170°C for 1hour and 180°C. for 30 minutes.
• The hot air oven must not be opened until the temperature inside has
fallen below 60°C to prevent breakage of glass ware.
Radiation
UV rays: Non ionizing
• low energy rays, poor penetrative power,
Microbicidal- inhibits DNA replication,
readily induces mutation in cell, don’t kill
spores.
• Disinfection of closed areas in microbiology
laboratory, inoculation hoods, and operating
theaters.
• Ionizing rays:
• High-energy rays, good penetrative power, Radiation does not
generate heat-"cold sterilization“ e.g.
• Gamma radiation: Sterilization of antibiotics, hormones, vitamins,
catheters, animal feeds, metal foils, and plastic disposables, such as
syringes, petri dishes.
• Damage the nucleic acid of the microorganism
Filtration
Does not kill microbes, it separates them out.
Membrane filters with pore sizes between 0.2-0.45μm.
• Used to remove microbes from heat labile liquids such as serum,
antibiotic solutions, sugar solutions.
• Eg. Membrane filters
Membrane filters
• Widely used, circular porous membranes,
usually 0.1 mm thick, Variety of pore sizes
(0.015–12 μm) Membranes with pores about
0.2μm are used, smaller than the size of
bacteria.
• Sterilize pharmaceutical substances,
ophthalmic solutions, liquid culture media,
oils, antibiotics, and other heat- sensitive
solutions.
• To obtain bacterial free filtrates of clinical
specimens for virus isolation.
Chemical method of sterilization
• Several chemical agents, Antiseptics and disinfectants.
Properties of Ideal Disinfectant
• Wide spectrum of antimicrobial activity
• Non-toxic, non-corrosive
• Stable upon storage, no chemical change.
• Soluble in water, lipids for penetration into microorganisms
• Effective in acidic as well as in alkaline media
• Speedy action, relatively inexpensive.
Types of Disinfectants

• Phenolic compounds (Dettol)

• Alcohols (Spirit)
• Gases (Chlorine dioxide)
• Aldehydes (Formaldehyde)
• Oxidizing agents (Chlorine)
• Dyes (Malachite green)
• Aids and Alkalis (HCL, chlorine bleach)
• Heavy metals (Zinc, mercury)
List of Instruments used in Microbiology Lab

• Analytical Balance Incubator

• Autoclave Laminar Hood
• Bunsen burner Magnetic Stirrer
• Centrifuge Microscope
• Colony Counter PH meter
• Deep Freezer Spectrophotometer
• Hot air oven Vortex
• Water Bath Water distiller
• Difference between antiseptics and disinfectants?
• Difference between bactericidal and bacteriostatic agents?