steres

1. Sterilisation and
disinfection
Dr Sushil Adhikari
First year resident
Department of Orthopedics
NAIHS

2. Objectives:
• Introduction
• Sterilising agents
- Physical agent
- Chemical agents
• Spaulding classification
• Levels of disinfectants

3. INTRODUCTION
• Sterilisation: process by which an article, surface or
medium is freed from all living microorganisms either in
vegetative or spore state
• Disinfection: destruction of all pathogenic organism
• Asepsis: state of complete absence of viable pathogenic
microorganisms in any environment
• Antiseptics: agents that can be applied on the skin or
mucus membrane to prevent infection by inhibiting the
growth of bacteria

4. • Bactericidal agents: substance that kill bacteria
• Bacteriostatic agents: prevent multiplication of bacteria
which may, however, remain alive
• Decontamination: process of rendering an article or area
free of contaminants, including microbial, chemical,
radioactive and other hazardous materials from an area,
object or body surface

5. Sterilising agents
• Physical agents:
- Dry heat: by flaming, incineration or using hot air
- Moist heat: by boiling, steam at atmospheric pressure, steam
above atmospheric pressure
- Filtration: using candles, asbestos pads, membranes
- radiation

6. • Chemical agents:
- alcohols: ethyl, propyl, trichlorobutanol
- Aldehydes: formaldehyde, glutaraldehyde, orthophthalaldehyde
- Acids: peracetic acids, hypochlorous acid
- Dyes: aniline, acridine
- Halogens: iodine, chlorine
- Phenols
- Gases: ethylene oxide, formaldehyde, hydrogen peroxide fogging
- Surface reactive agents
- Metallic salts

7. Physical agents
Heat:
• Most reliable method of sterilization
• Factors influencing sterilization by heat:
- Nature of heat: dry or moist
- Temperature and time
- Number of microorganisms present
- Characteristics of microorganisms, such as species, strain,
presence of spores
- Types of material from which the organisms must be eradicated

8. • Mechanism of action:
- dry heat: protein denaturation, damage by oxidizing molecules,
destroying cell constituents, toxic effect of elevated levels of electrolytes
- Moist heat: protein denaturation and coagulation
• Dry heat vs moist heat
- Dry heat requires more temperature
• Thermal death time: minimum time required to kill a suspension
of organisms at a predetermined temperature in a specified
environment
- Presence of organic substances, proteins, nucleic acids, starch, sugar,
fats and oils increase thermal death time
- Presence of disinfectants and high acid or alkaline pH hastens bacterial
killing

9. • Dry heat:
- flaming:
• inoculating loop or wire, tip of forceps and searing spatulas
• Inoculating loops carrying infective materials may be dipped in
disinfectant before flaming to prevent spattering
- Incineration:
• excellent method
• Burning microbiological and anatomical waste at very high
temperature (870-1200C)

10. - Hot air oven:
• most widely used method of dry heat sterilization
• Conduction of heat
• Holding period of 160 C for 2 hours
• Items sterilized: glassware (petridish, test tubes,
syringe, flask, beakers), sharp surgical instruments
(scalpels, scissors), chemicals (liquid paraffin, jellies, oil,
glycerol, grease, fats)
• Free circulation of air should be ensured

11. • Precautions:
- Even distribution of air should be ensured
- Avoid overloading
- Do not use inflammable items such as rubber
- Ensure all items are dry
- Test tubes and flask should be wrapped in craft
paper
• Allow to cool for 2 hours after holding period
• Sterilisation control
- physical: temperature monitoring by
thermocouple
- Chemical: browne tube
- Biological: heat resistant spore of non-toxic
clostridiums tetani or bacillus subtilis subsp
niger

12. Moist heat
• Temperature below 100 C
- Pasteurisation:
- Milk and milk products
- holder method or flash process, followed by cooling to 13 C or
lower
- All non-sporing pathogens such as mycobacteria, brucella and
salmonella are destroyed
- Coxiella is heat resistant and may survive by holder method
- Methods to assess efficacy: phosphatase test, coliform count,
standard plate count

13. - Inspissation:
• Media such as LJ and Loeffler serum: Heating at 80-85C for 30
min on 3 successive days in an inspissator
• Day 1: vegetative forms are killed
• Day 2: vegetative form produced by spores are killed
• Day 3: germinated spores are killed
- Water bath
• Bacterial vaccines are heat
inactivated at 60 C for 1 hour
• Serum or body fluids containing
coagulable proteins: heating for
1 hr at 56C

14. • Temperature at 100C
- boiling:
• Vegetative bacteria are killed almost immediately at 90-100 C
but spares spores
• Material should be immersed in water and boiled for 10-30 min
• Lid of the sterilizer should not be open
• Not recommended for sterilization of instruments used for
surgical procedures
- steaming:
• Steam condenses onto the surface of object and gives latent
heat to the surface which acts as antimicrobial
• Koch or Arnold steamer is usually used
• Used for sugar solutions

15. - tyndallisation or intermittent sterilisation
• Heating an article for 100C for 20minutes on 3 successive days
• Principle same as inspissation
• Used for media containing sugars or gelatin
• This method may fail to kill spores of certain anaerobes and
thermophiles

16. • Temperature above 100C
- Autoclave
• Most commonly: 121C
temperature for 15 minutes at
15lbs pressure
• Article sterilized: all heat stable
media, gloves, gowns, dressing
material, surgical instruments,
water solutions, biohazardous
waste
• Sterilisation control: spores of
Geobacillus stearothermophilus

17. • Principle: water boils when its vapor pressure is equal
to atmospheric pressure (ie at 100C), increase in
pressure at a closed vessel increases the temperature
at which water boils
• Precautions:
• Polyethylene tray not to be kept
• Do not keep dry items or water proof items
• Arrange materials such that efficient steam penetration is
allowed
• Materials should not touch sides or top
• Clean items and waste should be autoclaved separately

18. Radiation:
• 2 types:
- Ionising radiation: Xrays, gamma rays and cosmic rays
• Highly lethal to DNA and other vital constituents
• Very high penetrative power
• Cold sterilisation
• Used for disinfectant and sterilisation of disposable platics,
adhesive dressing, bone and tissue graft, catgut sutures
• Biological indicator: Bacillus pumilus

19. - non- ionizing radiations: UV rays, infrared rays
• Electromagnetic rays with wavelength longer than those of
visible lights
• Poor penetrating power
• Disinfectant
• Infrared radiation: rapid mass sterilisation of prepacked items
such as syringes and catheters
• UV rays: disinfecting enclosed areas such as biosafety cabinets
in laboratories, entryways, operation theatre

20. Filtration:
• Uses:
- For heat labile fluids such as sera and solutions of sugars or
antibiotics
- Separation of bacteriophage and toxins from bacteria
- Viral isolation
- Purification of water
• Types
- Candle filters: used for purification of water for industrial and
drinking purpose
- Sintered glass filters: made up of crushed glass particles, not
used nowadays as it is brittle

21. - Membrane filter: made up of
cellulose esters or other polymers,
used in water purification and
analysis, sterilisation and sterility
testing and for preparation of
solutions for parenteral use
• Wide range of sizes, 0.22mm (most
commonly used)
• Biological indicators: Brevundimonas
diminuta, Serratia

22. Filtration of air:
- Ultraclean air needed in OT, ICUs, transplantation units
- Methods:
• Surgical masks
• HEPA filter: removes 99.97% of particle of 0.3 microns
• ULPA filter: removes 99.999% of particle of 0.12 microns

23. Chemical agents
• Ideal antiseptic or disinfectant should
- Have a wide spectrum of activity and be effective against all
microorganism
- Be active in presence of organic matter
- Be effective in acid as well as alkaline media
- Have a speedy action
- Have ahigh penetrating power
- Be stable

24. • Mode of action of chemical agents
- By protein coagulation
- By disruption of cell membrane resulting in exposure, damage or
loss of contents
- By removal of free suphydryl groups essential for functioning of
enzymes
- By substrate competition of enzymes necessary for metabolism of
cell

25. • Alcohol: disinfection and antisepsis
- Ethyl alcohol and isopropylalcohol
- Mainly used as a skin antiseptics at a concentration of 60-90%
- Acts by denaturing bacterial proteins,
- No action on spores
- Protein slows its action while 1% mineral acid or alkali enhances it
- Isopropyl alcohol is preferred as it is fat solvent, is more
bactericidal and less volatile
- Used for disinfection of clinical thermometers
- Methyl alcohol: against fungal spores, used in cleaning cabinets
and incubators

26. • Aldehydes:
- MOA: alkylation of nucleic acids and proteins
- Disinfection and sterilisation
- Formaldehyde:
• Active against amino group in protein molecule
• in aqueous solutions: bactericidal, sporicidal, virucidal
• preserve anatomical specimens, destroy antrax spores in hair
and wool;
• Gas: sterilizing instruments, heat sensitive catheters and for
fumigating wards, isolation rooms and laboratories
• Gas is an irritant and toxic when inhaled

27. - glutaraldehyde:
• Action similar to formaldehyde
• Effective against tubercle bacilli, fungi and viruses
• Has no deleterious effect on cement or lenses of instruments,
so is used to sterilize cystoscope, bronchoscopes, rubber
anesthetic tubes, plastic ET and polythene tubing (2% cidex)
• Less irritant, toxic, corrosive than formaldehyde
- Orthophthalaldehyde (0.55%)
• Has bactericidal activity
• Used to cleanse endoscope between patients as it is quick,
effective and safe

28. • Acids:
- Peracetic acid:
• Good sterilisation effect on bacteria particularly common
antibiotics resistant bacteria (MRSA, VRE and Clostridium difficle)
- Hypochlorous acid
• Generated from reverse reaction of sodium hypochlorite and
hydrogen peroxide
• Bactericidal activity against common pathogenic microorganisms
• Active against biofilms and microorganisms in the biofilms

29. • Gases:
- Ethyle oxide:
• MOA: acts by alkylating the amino, carboxyl, hydroxyl and
suphydryl groups in protein molecules within the microbes and
spores. Causes DNA damage
• It is highly irritant, inflammable, explosive and carcinogenic
• Rapidly penetrates packing materials
• Used for heat sensitive items (disposable plastics, heart lung
machine, respirators, dental equipment, sutures, catheter)

30. • Halogens:
- Chlorine and its compound
• Sodium hypochlorite, calcium hypochlorite and chlorine gas
• Used for disinfection not sterilization and antisepsis
• MOA: oxidizing agents by releasing hypochlorous acid
• Good action on gram positive, gram negative and viruses
• Poor action on spores and mycobacterium tubercle
- Iodinated compound
• Used as skin disinfectant (antiseptics)
• MOA: oxidizing agents
• Effective against most microbes but not spores
• Tincture iodine: 2% iodine in water ethanol solution of potassium iodide
• Iodophores: compound made by complexing iodine with organic carrier
(povidine iodine)

31. • Phenols/ carbolic acid
- MOA: damages the cell membrane ( cell lysis and release of cell
contents) and denaturation proteins
- Active against most gram positive, fairly effective against gram
negative bacteria
- Disinfectant of choice for sputum
- Poor action on spores and viruses
- Commonly used phenols: Lysol, cresols, xylenol
- Activity of phenol decreases in presence of organic mater
- chlorhexidine: widely used antiseptic and disinfectant, bactericidal,
mycobacteristatic, sporistatic

32. • Surface active agents:
- Alters the energy relationship at interfaces, producing a reduction
in surface tension
- Widely used as wetting agents, detergents and emulsifiers
- MOA: acts on phosphate group of cell membrane and also enters
the cell membrane loses its semi permeability and cell proteins are
denatured
- Acts on bacteria but not on spores, tubercle bacilli and most
viruses
- Eg: trimethyl ammonium bromide (cetavlon or cetrimide) and
benzalkonium chloride
- Most active at alkaline pH and acid inactivates them

34. • Metallic salts:
- All salts have certain germicidal actions but salts of heavy metals
have greater action
- Silver, copper and mercury salts are used as disinfectants
- MOA: protein coagulation and combine with free sulphydryl groups
of cell enzymes
- Thiomersal, phenyl mercury nitrate and mercurochrome are less
toxic and used as mild antiseptics and have marked bacteriostatic
but weaker bactericidal and limited fungicidal action
- Copper salts are used as fungides

35. • Dyes:
- Aniline dyes:
• Include brilliant green, malachite green, crystal violet
• Do not act against tubercle bacilli so, is used in LJ media as
selective agent
• Non-irritant and non-toxic to tissue
• Acts by their reaction with acid groups in the cells
- Acridine dyes:
• Not as selective as aniline dyes
• Minimally affected by presence of organic matter
• Impair DNA complexes of organisms and prevent replication

36. • Plasma sterilization:
- 4th
state of matter and contains ions, electrons or neutral particles
- Microbiocidal and sporicidal
- Created from H2O2 alone or H2O2 and peracetic acid combination
- Used in sterilization of surgical instruments, items which cannot
tolerate high temperature and humidity of autoclave
- Low temperature is maintained
- Biological indicator: spores of Geobacillus stearothermophilus

37. Testing of disinfectants
• Phenol coefficient test/ reidel walker test
- Ratio of dilution of any disinfectant that kills a microorganism to
dilution of phenol which kills the microorganism in same time
under identical conditions
- If PC >1 test disinfectant is superior than phenol
- Disadv: does not take account of the presence of organic matter
• Chick martin test:
- Modification of phenol coefficient test
- Acts in presence of organic matter (dried yeast or feces)

38. • Capacity test/ Kelsey-sykes test
- Assess the activity of test disinfectant when microbial load is
increasing
• In use test:
- Test done in hospital
- Tests whether the actively used disinfectant is microbiologically
contaminated or not

44. Thank you