2. Lecture conducted for I B.Sc,biotechnology
(2013-2014 session)
• The following lecture is complied from
different textbooks and is for teaching learning use only
• This lecture is not a substitute for
classroom lecture but an adjunct for the
same
• This lecture may be useful for BSc
Nursing, BMLT & DMLT students too
3. DEFINITION
• The process of freeing an article or a
surface from all living microorganisms
including viruses & bacterial spores
• Various methods
– Heat
– Filtration
– Radiation
– Sterilant gases
4. HEAT
• Factors affecting
– Nature of heat – dry or moist
– Temperature & time
– No of organisms present
– Characteristics of the organism
– Type of material
• Two methods
– Dry heat
– Moist heat
5. DRY HEAT
• Kills by oxidation, protein denaturation &
toxic effect of elevated levels of electrolyte
• Types of processes
– Flaming
– Incineration
– Hot air oven
6. Dry heat - FLAMING
• 2500C – 3000C
• Points of forceps & Inoculation loops –
heat in bunsen flame till red hot
• Slow passage through flame to destroy
vegetative bacteria on surface of scalpel
blade, glass slides, mouths of test tubes
10. Dry heat - HOT AIR OVEN
• Holding temp & time: 1600C for 1 hr
• Used for glassware, forceps, swabs, water
impermeable oils, waxes & powders
• Before placing in hot air oven
– Dry glassware completely
– Plug test tubes with cotton wool
– Wrap glassware in Kraft papers
• Don’t over load the oven
• Allow free circulation of air between the material
11. Dry heat - HOT AIR OVEN
• Sterilization controls: to check whether the
equipment is working properly
– Chemical controls: Browne’s tubes
• Color change from red to green
– Thermocouples
– Biological controls: paper strips containing106 spores
of Clostridium tetani
• Place strips in oven along with other material for the
sterilization
• Later culture the strips in thioglycollate broth or RCM at 37 0C
for 5 days
• Growth in medium indicates failure of sterilization
16. Moist Heat - Temp at 100 C
0
• Boiling - 1000C for 10 min
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Kills all vegetative bacteria
Water should be soft, deionized or distilled
2% sodium bicarbonate promotes the process
Kills vegetative bacteria, hepatitis virus & some spores
• Steaming (free steam) – 30-60 min in Arnold
/Koch steamer
– For heat labile media – DCA, TCBS
– Tyndallisation (intermittent sterilization) - 1000C, 30
min, 3 days
• Nutrient media & media containing sugars or gelatin
• I day all vegetative bacteria are killed. On II & III day spores
that germinate are killed
17. Moist Heat - Temp above 100 C
0
• Autoclave (steam under pressure) - 1210C, 15
min, 15 lbs
– Used for rubber articles, dressings, sharp
instruments, infectious medical waste, culture media
– Principle – (refer Ananthanarayan & Paniker 7 edn Page 27)
th
• Sterilization control
– Thermocouples
– Browne’s tube (red-green), Bowie & Dick tape (white-brown)
– 106 spore of B stearothermophilus. Incubate at 550C for 5 days
22. FILTRATION
• Aqueous liquids may be sterilized by forced
passage through a filter of porosity small
enough to retain any microorganisms present
in them
• Used to sterilize serum, carbohydrates soln,
filtrates of toxins & bacteriophages, in water
bacteriology, in examination of Schistosoma
eggs
23. FILTRATION
• Types of filters
– Earthenware candles
• Unglazed ceramic & diatomaceous earth filters
• Eg. Chamberland filters, Doulton filters
– Asbestos filter – Seitz, Carlson, Sterimat
– Sintered glass filter
– Membrane filters – cellulose nitrate, cellulose acetate,
polycarbonate, polyester filters
– Pore size: 0.015 – 12 μm
• HEPA filters – for large volumes of air
Sterilization control – bubble pressure test
29. DISINFECTION
• The process of freeing an article or a
surface from all or some of the living
microorganisms but not necessarily
bacterial spores
• Strong disinfectants – for inanimate object
• Mild disinfectant (antiseptic) – superficial
application on living tissue
30. Factors affecting DISINFECTION
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Conc of disinfectant
Time of action
pH of the medium
Temperature
Nature & number of organisms
Presence of extraneous material
Others – hardness of water, relative
humidity
32. ALCOHOL
• Ethanol, isopropyl alcohol
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Skin antiseptics at 70%
Less sporicidal & virucidal activity
Denature bacterial proteins
Isopropyl alcohol better fat solvent, more bactericidal
and less volatile
– Methyl alcohol – to treat cabinets / incubators affected
by fungal spores
– Others – benzyl alcohol, chlorbutol, phenylethanol
33. ALDEHYDE
• Formaldehyde - 10% used
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In aq. soln is virucidal, bactericidal, sporicidal
Used to fumigate wards, sick rooms, labs
Expose to ammonia to remove residual formaldehyde
Has pungent smell, irritant to skin, eyes, mucus memb & toxic
when inhaled
• Glutaraldehyde – less toxic, less irritant
– Endotracheal tubes, metal instruments, polythene tubing
• Β propiolactone (BPL) – condensation product of ketane
& formaldehyde
– More efficient for fumigation but is carcinogenic
– 0.2% generally used
34. ETHYLENE OXIDE
• Highly inflammable, mixed with inert gases
– CO2, N
• Especially for heart lung machines,
respirators, sutures, syringes, dental
equipments
35. DYES
• Combine with nucleic acids
• Aniline dyes
– Brilliant green, malachite green, crystal violet
• Acridine dyes
– Proflavine, acriflavine, euflavine, aminacrine
• Skin & wound antiseptics
• Bacteriostatic, more active against GP
bacteria
36. HALOGENS
• Kills by oxidation
• Iodine – 2.5% in 70% alcohol, Skin antiseptic
• Iodophores (iodine + non-ionic surface active
agent) – betadine – non staining, less irritant,
less toxic
• Chlorine – disinfect water supplies, swimming
pools
• Sodium hypochlorite – 1% for HIV
• Organic chloramines – antiseptic for wound
dressings
37. PHENOLICS
• Carbolic acid – 2-5%
– Powerful microbicidal, very corrosive
– General purpose disinfectant in hospital
• Cresol, lysol
• Chloroxylenol, chlorophenol,
hexachlorophane – less toxic, less irritant,
less active, more readily inactivated by
organic matter
38. SURFACE ACTIVE AGENTS
• Disrupt cell memb, 4 main groups
• Anionic surfactants – strong detergent
action, weak antimicrobial action
• Non-ionic surfactants
• Cationic surfactants – quaternary
ammonium compounds – cetrimide,
benzalkonium chloride - bacteriostatic
• Amphoteric surfactants – both detergent &
antimicrobial properties – Tego comps
42. TESTS FOR DISINFECTANTS
• To determine efficacy of disinfectants
– Phenol Co-efficient method
• Rideal Walker test
• Chick Martin test
– Compares disinfectant with phenol
– Kelsey & Sykes Capacity test
– Determines dilution of disinfectant to be used
– Kelsey & Mauer In-use test (stability test)
– Checks end result of disinfection
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PRIONS
BACTERIAL SPORES
Bacillus subtilis
Clostridium sporogenes
MYCOBACTERIA
Sterilisation
High Level Disinfection
Intermediate Level disinfection
M. tuberculosis var bovis
NON LIPID OR SMALL VIRUSES
Polio virus, Rhino virus
FUNGI
Trichophyton, Candida, Cryptococcus
VEGETATIVE BACTERIA
Pseudomonas, Staphylococcus, VRE
LIPID OR MEDIUM SIZED VIRUSES
HBV, HIV, HSV, HCV, EBOLA, CMV
Low Level Disinfection
44. Categories
• Sterilisation
• High level disinfection (HLD) kills all microorganisms except
high number of bacterial spores - aldehydes, hydrogen
peroxide, ortho-phthaldehyde
• Intermediate level disinfection (ILD) kills all vegetative
bacteria including M. tuberculosis var bovis, all fungi and most
viruses - phenolics, iodophores, chlorine compounds,
alcohols
• Low level disinfection (LLD) kills most vegetative bacteria but
not M. tuberculosis var bovis, some fungi and some viruses Hospital type germicides- quaternary ammonium
compounds
45. Plasma technology
• Glow discharge or low temperature
plasmas
• Plasma has sufficient energy to disrupt
molecular bonds
• Direct current, radiofrequency, microwave
power is used to produce the plasma
• Used in disinfection of OTs
46. A known HIV positive patient is admitted in an
isolation ward after an abdominal surgery following
an accident. The resident doctor who changed his
dressing the next day found it to be soaked in
blood. Which of the following would be the right
method of choice of discarding the dressings :
a) Pour 1% hypochlorite on the dressing material and send it for
incineration in a appropriate bag
b) Pour 5% hypochlorite on the dressing material and send it for
incineration in a appropriate bag
c) Put the dressing material directly in an appropriate bag and send
for incineration
d) Pour 2% Lysol on the dressing material and send it for
incineration in a appropriate bag
47. Heat labile instruments for
use in surgical procedures
can be best sterilized by
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Absolute alcohol
Ultra violet rays
Cholorine releasing compound
Ethylene oxide gas
