sterilization techniques in microbiology

1. Sterilization techniques
Ria

2. What is sterilization?
• Removal or destruction of microorganisms ,including viruses and bacteria
• Doesnot apply to prions , cant be destroyed by standard techniques
• Aseptic- area free of contaminants.
• Disinfection- can be both chemical and physical agents classified as
disinfectants. Eg., UV rays, heat ,alcohol,bleach …..
• Antisepsis- chemical used on skin, called antiseptic
• Degerming- use of soaps or alcohol on a surface by scrubbing.
• Sanitation-disinfecting places and utensils, for public care ..
• Pasteurization- heat to kill pathogens especially in milk ,fruit juices ,
beverages…

3. Microbial death rates
• Permanent loss of reproductive ability under environmental
conditions.
• Constant overtime for particular microorganisms.

4. Action of microbial agents
• Alteration of cell walls and membranes- integrity of cells are
maintained by cell walls.da,age to cell wall can rupture the
cell.,leaking out of cell components, causes death of the cell.
in case of viruses, enveloped viruses use envelope to attach to target .
Damage to this cell envelope can affect this attachment.
• Damage to proteins and nucleic acids-Metabolic pathway affected,
proteins have definite structure which when altered can affect the
whole process.
• Destruction of nucleic acids can cause mutations

5. Selection of microbial control methods
• It should act fast
• Should not be expensive
• Stable during storage
• Available easily
• Harmless to humans, animals and objects
Eventhough every agents have limitations and disadvantages.

6. Factors affecting efficacy of antimicrobial
methods
• Site to be treated –eg, harsh chemicals cannot be used on humans.
increased amount of infection occurs if needles or scalpels are not
sterilized.
Relative susceptibility of microorganisms- death rate is different for
different organisms
Easy to affect enveloped viruses than non enveloped ones.
Some are resistant- bacterial endospores, species of mycobacterium,cysts
of protozoa
Prions- infectious protein ,causes degenerative diseases of brain,are more
resistant than any living thing.(refer- JungM J ,et.al 2003 , Prion ,prion
diseases and decontamination)

7. Environmental conditions
Temperature and pH affect microbial death rate.
Warm disinfectants usually work better than cooler ones.
Chlorine acts better at low pH.
Biosafety Levels- Center for disease control and prevention
Biosafety Level-1 : suitable for handling microbes
not known to cause disease
precautions include handwashing, use of
disinfectants

8. Biosafety level-2
• Moderately hazardous agents
• Examples- Staphylococcus aureus ,influenza virus.
• Extreme precautions taken while working with contaminated sharp objects, use
of safety cabinets.
Biosafety level-3
• more strict
• Works done only in safety cabinets
• Air enters through one door but leaves through HEPA( High efficiency particulate
air filters) to outside of the room.
• Usually for tuberculosis,anthrax bacteria,viruses of yellow fever and rocky
mountain spotted fever.

9. Biosafety level-4
• For working with dangerous microbes that causes ebola, smallpox…
• separate buildings
• Entry and exit through electronically sealed airlocks with multiple
showers, a vacuum room, a UV light room– strictly controlled
• Air and water are filtered – entry and exit
• Special suits with air hoses

10. Physical Methods
• Heat, Cold, Dessication , filtration ,osmotic pressure, radiation
• Heat-
Older means of microbial control
High temp can denature proteins
Thermal death point- microbes differ in susceptibility to heat
The lowest temperature that kills all cells in a broth in 10 min.
Thermal death time- Time taken to completely sterilize a particulare
volume of liquid at a set temperature.

12. Autoclaving
• Consists of a pressure chamber, pipes to introduce and evacuate steam ,
valves to remove air and control pressure
• 121ᵒ C for 15 min
• Temp above than boiling
• Within 10 min , all microbes gets destroyed from small quantities.
• Larger volumes requires more time.
• Autoclaving solid substances also require more time , as heat should
penetrate the centers.
• Sterilization requires entry of steam to the contents,so should be
wrapped loosely with cloth or paper.
• To ensure proper sterilization, use of plastic beads or color indicators
impressed on tape is done.

13. Pasteurization
• Older times, 63ᵒ for 30 min
• Flash pasteurization- 72ᵒ for 15 sec through heated tubes
• Ultra high temperature pasteurization- 135ᵒ for 1 sec (may affect the
taste)
• Louis Pasteur – developed method of heating beer and wine to
destroy microorganisms
• Heat tolerant and heat loving organisms survive pathogens,
eventhough willnot cause spoilage and consumption.

14. Dry heat
• Some substances like powder or oils cannot use moist heat
• Requires higher temp fro more hours, penetrates slowly
• 171ᵒ for 1 hour or 160ᵒ for 2 hours- Hot air oven
• Incineration- complete destruction of material, direct heat given
• In case of loops , contaminated dressings..

15. Refrigeration and Freezing
• Refrigeration- 0 to 7 C
• Freezing- below 0 C
• Chemical reactions slow down at lower temperatures,so growth
reduces.
• Cold Loving microbes ( psychrophils) can multiply and affect the food
kept .
• Slow freezing is most effective as ice crystals have time to puncture
the cell wall
• Some can survive these temperatures, like endospores and viruses.

16. Dessication and Lyophilization
• Dessication or drying- to preserve foods like peas ,beans,
fruits,grain,nuts...
• Inhibits metabolism which requires water.,so microbial growth is
affected.
• Molds can grow on dried raisins.
• Lyophilization- technique combining freezing and drying,done to
preserve microbes.
• Freeze the culture in liquid nitrogen or frozen carbon dioxide and then
subject it to vacuum that remove frozen water to gas(sublimation).

17. Filtration
• Passage of fluid( liquid or gas) through a sieve.
• Different pore sizes for different organisms.
• Used to sterilize ophthalmic solution, antibiotics,cacci, liquid vitamins,
culture media and enzymes..
• Made of glass, porcelain,cotton, asbestos and diatomaceous earth
• Membrane filters made of plastic or nitrocellulose ,pore sizes range from
25 micrometre to 0.01 micrometre diameter
• HEPA filters- high efficiency particulate air filter attached to air ducts.

18. Osmotic pressure
• Use of high concentrations of sugar and salt in order to preserve
foods.
• Osmosis- movement of water across a semipermeable membrane
from high water concentration to lower water concentration.
• Cell shrinks in hypertonic ,removal of water prevents the metabolic
activities of microbes.
• Examples.,jams,salted fish, jellies, pickles
• Fungus can grow in hypertonic than bacteria

19. Radiation
• Two types of radiation- particulate radiation and electromagnetic
radiation.
• Ionizing radiation-electron beams ,gamma beams, some X rays of
wavelength shorter than 1 mm
• Such ions disrupt hydrogen bonding, form hydroxyl radicals and can
cause mutations and cell death
• These beams are produced by cathode ray machines
• Used to sterilize spices ,meats,dental and medical
supplies,gloves,syringes....
• Gamma rays require more hours ,used to inhibit microbial growth,larva
and eggs
• X rays travel farthest but have less energy than gamma rays

20. Chemical methods of microbial control
• Phenols
• Alcohols
• Halogens
• Oxidizing agents
• Surfactants
• Heavy metals
• Aldehydes
• Gaseous agents
• Enzymes
• Antimicrobials

21. Phenols and phenolics
• In 1867, Lister used phenol for reducing infection during surgery.
• Phenolics- derived from phenol molecules ,which are chemically
modified by addition of halogens or organic functional groups
• Examples- chlorinated phenol- contain more chlorine atoms
natural oils – such as pine and clove oils
• Bisphenolics – composed of two covalently linked phenolics.
examples- orthophenylphenol( active ingredient: Lysol and triclosan)
this is incorporated into cutting boards, garbage bags..

22. .
• PHENOLS AND PHENOLICS – denature proteins and remain active for prolonged
time
• Negative effects- strong odor and possible side effects; bisphenolics can damage
brains of infants.
Alcohols-
• not effective against fungal spores
• Affects bacteria,virus,fungus
• Commonly used ones- rubbing alcohol( isopropanol) and consuming
alcohol(ethanol).
• Denature proteins, eventhough pure alcohol is not effective
• Need water content, so used as 70% to 90% solutions.
• Evaporate easily- so no residue remains
• Disadvantage- may not contact microbes for long enough to be effective.
• Alcohol based antiseptics- more effective against bacteria and not on viruses
• Swabbing skin with alcohol remove microbes by physical action(degerming)

23. Halogens
• Four very reactive , nonmetallic chemical elements:
iodine,chlorine,bromine,fluorine
• Intermediate level antimicrobial chemicals- effective against fungal
spores, fungus, bacterial endospores, some viruses.
• Denature essential proteins.
• Iodine is effective. But protozoan cysts can remain
• Example- betadine- iodophor (long lasting and non irritating)
• Chlorine is used to treat drinking water, swimming pools..
• Bleaching done to clean utensils and floors

24. Oxidizing agents
• High level disinfectants- oxidize the enzymes, prevent metabolism
• Effective against anaerobic microorganisms
• Hydrogen peroxide – household chemical, sterilize inanimate objects
is not good for open wounds- catalase from our body
neutralizes hydrogen peroxide.
• Ozone – reactive form of oxygen,generated when molecular oxygen to
electrical discharge. Fresh smell is given to air after a thunderstorm by
ozone.
• In some countries, ozone is used instead of chlorine
• Peracetic acid- sterilize equipments and will not affect organic
contaminants, it leaves no toxic residue.

25. Surfactants
• Surface active chemicals
• Soaps and detergents- soaps have fatty acid at one end and at other
end is hydrophilic & negatively charged.
• Can remove the tiny droplets of oils and any bacteria they harbor
• Synthetic detergents- positively charged organic surfactants.

26. Heavy metals
• Ions of arsenic,zinc , mercury, silver,copper are antimicrobial,they
combine with the sulphur atoms in molecules of cysteine
• Low-level bacteriostatic and fungistatic agents with few exceptions.
• Examples- use of silver nitrate against Neisseria gonorrhoea which
affect eyes of babies.
• Silver is still used in burn cream , surgical dressings, catheters..
• Copper interferes with chorophyll control algal growth in fish tanks,
swimming pools...

27. Gaseous agents
• Used to treat pillow,dried and powdered foods,heart -lung machine
components,catheters...
• Ethylene oxide,propylene oxide and beta propiolactone are examples
and can penetrate through paper and plastic wraps
• Ethylene oxide is used by NASA to sterilize spacecraft, hospitals to
sterilize instruments sensitive to heat
• Disadvantages- they are toxic.betapropiolactone is potentially
carcinogenic.

28. Aldehydes
• Compounds containing terminal CHO group.
• Glutaraldehyde,which is liquid and formaldehyde which is gas are
highly reactive.
• Denature proteins and inactivate nucleic acids
• 2%solutions are used at hospitals.
• Formaldehyde can cause irritation in mucous membranes and is
carcinogenic whereas glutaraldehyde is less irritating and effective.

29. Methods to evaluate disinfectant and
antiseptics
• Phenol coefficient test
• Use dilution test
• Kesley-Sykes capacity test
• In use test