Factors Influencing Efficacy of Sterilization and Disinfection antimicrobial agent is affected by at following factors


Sterilization: It is the process by which all living microorganisms including viable spores, are destroyed with reduction of at least 106 log colony forming units (CFU) of microorganisms and their spores. Disinfection: It refers to a process that destroys or removes most if not all pathogenic organisms but not bacterial spores with reduction of at least 103 log CFU of microorganism but not spores. Asepsis: It is a process where the chemical agents (called antiseptics) applied to body surfaces (skin) will kill or inhibit the pathogenic microorganisms (and also commensals) present on skin. 

Decontamination (or Sanitization): It refers to the reduction of pathogenic microbes to a level at which items are considered as safe to handle without protective attire with reduction of at least 1 log CFU of microorganism but not spores.
Decontamination (or Sanitization): It refers to the reduction of pathogenic microbes to a level at which items are considered as safe to handle without protective attire with reduction of at least 1 log CFU of microorganism but not spores.

Factors Influencing Efficacy of Sterilant/Disinfectant

The efficiency of a sterilant/disinfectant antimicrobial agent is affected by at following factors:

1.Organism load: Larger microbial population requires a longer time to die than a smaller one.
2. Nature of organisms: It greatly influences the efficacy of the disinfectants
3. Concentration of the chemical agent and temperature of the physical agent
4. Nature of the sterilant/disinfectant:
  • Microbicidal ability, Rapidity of action, Residual activity.
  • Ability to act in presence of organic matter such as pus, blood, and stool.

5. Duration of exposure: More is the exposure time, better is the efficacy.
6. pH: Heat kills more readily at an acidic pH
7. Biofilm formation: Prevents the entry of disinfectants.

Table Classification of sterilization/disinfection methods.

A. Physical methods
1. Heat
Dry heat: Flaming, Incineration and Hot air oven
Moist heat:

a. Temperature < 100°C, e.g. pasteurization, water bath and inspissation
 b.  Temperature at 100°C, e.g. boiling, steaming and tyndallization
 c.  Temperature > 100°C, e.g. autoclave

2. Filtration: Depth filters and membrane filters

3. Radiation
Ionizing radiation: Y-rays, X-rays and cosmic rays
Non-ionizing radiation: Ultraviolet (UV) and infrared rays

4. Ultrasonic vibration
B. Chemical methods
1. Alcohols: Ethyl alcohol, isopropyl alcohol
2. Aldehydes: Formaldehyde, glutaraldehyde, Ortho-phthaladehyde
3. Phenolic compounds: Cresol, lysol, chlorhexidine, chloroxylenol, hexachlorophene
4. Halogens: Chlorine, iodine, iodophors
5. Oxidising agents: Hydrogen peroxide, Peracetic acid
6. Salts: Mercuric chloride, copper salts
7. Surface active agents: Quaternary ammonium compounds and soaps
8. Dyes: Aniline dyes and acridine dyes
9. Gas sterilization: Low temperature steam formaldehyde, Ethylene oxide (ETO) and Beta-propiolactone (BPL)


Heat Sterilization/Disinfection

Mechanism of Action

  • Dry heat kills the organisms by Charring, Oxidative damage, Denaturation of bacterial protein and Elevated levels of electrolytes (CODE).
  • Moist heat kills the microorganisms by denaturation and coagulation of proteins. Dry Heat (Hot air oven)
  • Holding temperature required: 160°C for 2 hours
  • Materials sterilized: Hot air oven is best method for sterilization of:
  1.  Glassware like glass syringes, petri dishes, flasks, pipettes and test tubes.
  2. Surgical instruments like scalpels, scissors, forceps, etc.
  3. Chemicals such as liquid paraffin, fats, glycerol, and glove dust powder, etc.
  • Sterilization control:
  1. Spores (106) of nontoxigenic strains of Clostridium tetani or Bacillus subtilis subsp. niger
  2. Thermocouples and Browne’s tube.

Moist Heat at a Temperature below 100ºC

  • Pasteurization: Used for perishable beverages like fruit and vegetable juices, beer, and dairy products such as milk.
  1. Two methods: Holder method (63ºC for 30 min) and Flash method (72ºC for 20 sec followed by cooling to 13ºC). 
  2. All nonsporing pathogens are killed except Coxiella burnetii which may survive in holder method.
  • Water bath: Used for disinfection of serum, body fluids, and vaccines (60°C for one hour)
  • Inspissation (Fractional sterilization):
  1.  It is a process of heating an article on 3 successive days at 80–85ºC for 30 min
  2. Used for sterilization of egg based (LJ and Dorset’s egg medium) and serum based media (Loeffler’s serum slope).

Moist Heat at a Temperature of 100ºC

  • Boiling: Boiling of the items in water for 15 minutes may kill most of the vegetative forms but not the spores.
  • Steaming: Koch’s or Arnold’s steam sterilizers are used to provide temperature of 100°C for 90 minutes. It is useful for those media which are decomposed at high temperature of autoclave. It kills most of the vegetative forms but not the spores.
  • Tyndallization or intermittent sterilization: Involves steaming at 100°C for 20 min for 3 consecutive days. It is used for sterilization of gelatin and egg, serum or sugar containing media. It kills most of the vegetative forms including spores.

Moist Heat at a Temperature above 100ºC (Autoclave)

  • Principle: Autoclave functions similar to a pressure cooker. At normal pressure, water boils at 100°C but when pressure inside a closed vessel increases, the temperature at which water boils also increases.
  • Sterilization conditions: 121°C for 15 min at pressure of 15 psi (most commonly used).
  • Uses of autoclave: Autoclave is useful for surgical instruments and culture media and those materials which cannot withstand the higher temperature of hot air oven or media containing water that cannot be sterilized by dry heat. 
  • Sterilization control:
  1.  Biological indicator-Spores of Geobacillus stearothermophilus (best indicator)
  2. Thermocouple and indicators like Browne’s tube, Autoclave tapes:


Filtration is an excellent way to remove the microbial population in solutions of heat-labile materials like vaccine, antibiotics, toxin, serum and sugar solution as well as for purification of air in laminar air flow systems. There are two types of filters; depth and membrane filters.

Depth filters: They are porous filters that retain particles throughout the depth of the filter, rather than just on the surface. They are used for industrial applications such as filtration of food and beverage, and chemicals, but are not to filter bacteria. Examples include:
  • Candle filters made up of diatomaceous earth (Berkefeld filters), unglazed porcelain (Chamberland filters)
  • Asbestos filters (Seitz and Sterimat filters)
  • Sintered glass filters
Membrane filters: They are widely used filters for bacterial filtration. They are porous; retain all the particles on the surface that are smaller than their pore size.
  • Made up of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride
  • Pore size: Membrane filters have an average pore diameter of 0.22 µm (MC used)
  • Filtration of air: Air filters are membrane filters used to deliver bacteria-free air. Examples:
  1.  Surgical masks (that let air in but keep microorganisms out).
  2. In biological safety cabinets and laminar airflow systems; two filters are used
  •  HEPA filters (High-efficiency particulate air filters): HEPA filter removes 99.97% of particles of size ≥ 0.3 µm.
  • ULPA filters (Ultra-low particulate/penetration air filters): Removes from the air 99.999% of particles of size ≥ 0.12 µm.
  • Sterilization control includes Brevundimonas diminuta and Serratia marcescens.


Ionising radiations:
  • Examples include, X-rays, gamma rays (from Cobalt 60 source), and cosmic rays.
  • Mechanism: It causes breakage of DNA without temperature rise (hence called as cold sterilization). 
  • It destroys spores and vegetative cells, but not effective against viruses. It is used for:
  1.  Disposable plastics, e.g. rubber or plastic syringes, infusion sets and catheters.
  2. Catgut sutures, bone and tissue grafts and adhesive dressings, antibiotics and hormones.
  • Advantages of Ionizing radiation:
  1.  High penetrating power,
  2. Rapidity of action and
  3. Temperature is not raised
  • Sterilization control: Efficacy of ionising radiation is tested by using Bacillus pumilus.
Nonionizing radiation:
  • Examples of nonionizing radiation include infrared and ultraviolet radiations.
  • They are quite lethal but do not penetrate glass, dirt films, water.
  • Dose: 250–300 nm wavelength for 30 min
  • Used for sterilization of clean surfaces in operation theatres, laminar flow hoods as well as for water treatment.


Table: Classification of chemical disinfectants based on their efficacy

Level of disinfectant
Bacterial spores
Tubercle bacilli
Non enveloped viruses
Enveloped viruses
Vegetative bacteria
Low level disinfectant
Intermediate level disinfectant
High level disinfectant
May be
Chemical sterilants


  • They act on bacteria, fungi, some enveloped viru (e.g. HIV); but not spores.
  • They act by denaturing proteins and possibly by dissolving membrane lipids. 
  • Ethyl alcohol is used as surgical spirit (70%) in hand rubs as antiseptics.
  • Isopropyl alcohol: Used for clinical thermometers.


They combine with nucleic acids and proteins and inactivate them, probably by crosslinking and alkylating the molecules. They are sporicidal and can be used as chemical sterilants.

1. Formaldehyde: It is best used for:

  • Preservation of anatomical specimen
  • Formaldehyde gas is used for fumigation of closed areas such as operation theaters
  • Preparation of toxoid from toxin. It is toxic, irritant and corrosive to metals.

2. Glutaraldehyde is less toxic, less irritant and less corrosive, hence is best used to sterilize endoscopes and cystoscopes:

  • It is used as 2% concentration (2% cidex) for 20 min.
  • It has to be activated by alkalinization before use. Once activated, it remains active only for 14 days.

3. Ortho-Phthalaldehyde (0.55%): It can also be used for sterilization of endoscopes and cystoscopes and has many advantages over glutaraldehyde:

  • It does not require activation
  • Low vapor property
  • Better odor
  • More stable during storage
  • ↑ mycobactericidal activity.

Phenolic Compounds

Phenolics as disinfectants: Cresol, xylenol, Lysol and ortho-phenylphenol are used as disinfectants in laboratories and hospitals.

  • All have the ability to retain activity in presence of organic matter.
  • They are toxic and irritant to skin, hence used as disinfectants but not as antiseptics.

Phenolics as antiseptics: Certain phenolics are less irritant to skin, persist in skin for longer period and are widely used as antiseptics. In general they are more active against grampositive than gram-negative bacteria.

  • Chlorhexidine: It is an active ingredient of savlon (chlorhexidine and cetrimide)
  • Chloroxylenol: It is an active ingredient of dettol.
  • Hexachlorophane: As it can cause brain damage, hence its use as antiseptic is restricted only to a staphylococcal outbreak.


Iodine: It is used as a skin antiseptic and kills microorganisms by oxidizing cell constituents and iodinating cell proteins, e.g. Tincture of iodine (2%) and Iodophor (iodine complexed with an organic carrier) e.g. Betadine.

Chlorine: It is the most commonly used disinfectant:

  • For municipal water supplies and swimming pools and is also employed in the dairy and food industries
  • As laboratory disinfectant
  • As bleaching agent: to remove the stain from clothes. (Common uses of chlorine are given in table below).
  • Preparations: It may be available as: (i) chlorine gas, (ii) sodium hypochlorite (household bleach, 5.25%), or (iii) calcium hypochlorite (bleaching powder)
  • Mechanisms: All preparations yield hypochlorous acid (HClO) which causes oxidative destruction of vegetative bacteria and fungi, but not spores.
  • Disadvantages: 

  1. Organic matter interferes with its action, hence excess chlorine always is added to water to ensure microbial destruction
  2. Carcinogenic 
  3. Need daily preparation 
  4. They are not active against Giardia and Cryptosporidium,
  5. Sodium hypochlorite is corrosive and should be handled cautiously.

Oxidising Agents

Hydrogen Peroxide (H2O2)

  • Mode of action: It is a chemical sterilant, acts by liberating toxic free hydroxyl radicals which attack membrane, lipid, DNA, and other cellular components.
  • Concentration of H2O2  3–6% is ideal, except for catalase producing organisms and spores which require 10% of H2O2.
  • Used to disinfect ventilator, soft contact lenses, and tonometer biprisms. Vaporized H2O2 is used for plasma sterilization.
  • Advantage: 1. It acts perfect even in presence of organic matter 2. Low toxicity 3. Environmentally safe.

Peracetic Acid

It is a chemical sterilant; often used in conjunction with H2O2, to disinfect hemodialyzers and in plasma sterilization. It is also used for sterilizing endoscopes. However, it may corrode steel, iron, copper, brass and bronze.

Plasma Sterilization

This is recently introduced sterilization device (e.g. Sterrad and Plazlyte) used for creating plasma state, so as to maintain a uniform vacuum inside the chamber.

  • Chemical sterilants such as H2O2 alone or a mixture of H2O2 and peracetic acid
  • Active agent is Ultraviolet (UV) photons and radicals (e.g., O and OH): Kill microorganisms and spores.
  • Low temperature is maintained (< 50°C), So best for heat labile surgical instruments.
  • Sterilization control: Geobacillus stearothermophilus, Bacillus subtilis subsp. niger.

Heavy Metal Salts

Heavy metallic salts are of limited use in certain areas:

  • Silver sulfadiazine is used on burns surfaces. 
  • Silver nitrate (1%) solution used for eyes of infants to prevent ophthalmia neonatorum.
  • Copper sulfate is an effective fungicide (algicide) in lakes and swimming pools.
  • Mercury salts such as mercuric chloride, thiomersal and mercurochrome were known antiseptics in past. Thiomersal (merthiolate) is used as preservative in vaccines and sera.
  • Mechanism of action: Heavy metals combine with bacterial cell proteins, often with their sulphydryl groups, and inactivate them. They may also precipitate cell proteins. Many heavy metals are more bacteriostatic than bactericidal.

Surface Active Agents

They lower the surface tension between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, and emulsifiers because they have both polar hydrophilic and nonpolar hydrophobic ends.

1. Cationic surfactants (Quaternary ammonium compounds):

  • They disrupt microbial membranes and may also denature proteins.
  • They kill most bacteria (gram-positives are better killed than gram-negatives) but not M. tuberculosis or spores.
  • Nontoxic but are inactivated by acidic pH, organic matter, hard water and soap.
  • Cationic detergents are often used as disinfectants for food utensils and small instruments and as skin antiseptics.
  • Examples include: ○ Acetyl trimethyl ammonium bromide (cetavlon or cetrimide)○ Alkyltrimethylammonium salts ○ Benzalkonium chloride and Cetylpyridinium chloride
2. Anionic surfactants, e.g. soaps, have strong detergent but weak antimicrobial properties. They are active at acidic pH.

3. The amphoteric surfactants: They have both detergent and antimicrobial activity.

  • They are active over a wide range of pH, but is reduced in presence of organic matter.
  • E.g. ‘Tego compounds’: Used as antiseptics in dental practice, but cause allergic reactions.


Aniline and acridine dyes have been used extensively as skin and wound antiseptics.

1. Aniline dyes: E.g. crystal violet, gentian violet, brilliant green and malachite green:

  • They are more active against gram-positive bacteria than gram-negative and have no activity against M. tuberculosis.
  • They are non-toxic and non-irritant to the tissues.
  • Their activity is reduced in presence of organic material such as pus.
  • They interfere with the synthesis of peptidoglycan component of the cell wall.
  • These dyes are used in the laboratory as selective agents in culture media (e.g. malachite green in LJ medium)

2. Acridine dyes: These include acriflavine, euflavine, proflavine and aminacrine:

  • They are affected very little by the presence of organic material. 
  • More active against gram-positive bacteria but are not as selective as the aniline dyes. 
  • They interfere with the synthesis of nucleic acids and proteins in bacterial cells. 

Gaseous Sterilization

Ethylene Oxide (ETO)

Ethylene oxide sterilizer is one of the widely used gaseous chemical sterilants in present days.

  • It has high penetration power, has both microbicidal and sporicidal activity; acts by combining with cell proteins.
  • However, it is highly inflammable, irritant, explosive and carcinogenic. Hence it is usually supplied in a 10 to 20% concentration mixed with inert gases.
  • Sterilization condition: 5 to 8 hours at 38°C or 3 to 4 hours at 54°C.
  • Sterilization control: Bacillus globigii.
  • Use: For sterilization of many heat sensitive items such as disposable plastic petri dishes and syringes, heart-lung machine, sutures, catheters, respirators and dental equipments.

The decreasing order of resistance of microorganisms to disinfectant or sterilizing agents is as follows:

Prions (highest resistance) > Cryptosporidium oocysts > Coccidian cyst > Bacterial spores > Mycobacteria > Other parasite cysts (Giardia) > Small non-enveloped viruses > Protozoan Trophozoites > Gram-negative bacteria > Fungi > Large non-enveloped viruses > Gram-positive bacteria > Enveloped viruses.

Sporicidal agents include:

  • EFGH: Ethylene oxide, Formaldehyde, Glutaraldehyde, Hydrogen peroxide. 
  • 3P: Peracetic acid, O-Phthalic acid and Plasma sterilization .
  • Autoclave and Hot air oven.


1. Phenol coefficient (Rideal Walker) test:

  • Determined by the dilution of the disinfectant in question which sterilizes the suspension of Salmonella Typhi in a given time divided by the dilution of phenol which sterilizes the suspension in the same time.
  • Phenol coefficient of > 1 is taken as satisfactory.
  • Drawbacks: ○ Only the phenolic compounds can be assessed ○ It does not assess disinfectant ability to act in presence of organic matter.

2. Chick Martin test: Modified rideal and walker test in which the disinfectants act in the presence of organic matter (e.g. dried yeast, feces, etc.) to simulate the natural conditions.

3. Capacity (Kelsey-Sykes) test: It tests the capacity of a disinfectant to retain its activity when repeatedly used microbiologically.

4. In-use (Kelsey and Maurer) test: It determines whether the chosen disinfectant is effective in actual use in hospital practice.

Table: Biological sterilization indicator

Hot air oven
Clostridium tetani nontoxigenic strain, B. subtilis subsp.niger
Geobacillus stearothermophilus
Brevundimonas diminuta, Serratia
Ionizing radiation
Bacillus pumilus
Ethylene oxide
Bacillus globigi
Plasma sterilization
Geobacillus stearothermophilus, Bacillus subtilis subsp.niger

Table: Methods of sterilization/disinfection used in different clinical situations

Method of sterilization/disinfection
Clinical thermometer
Isopropyl alcohol
Paraffin, glass syringe, flask, slide, oil, grease, fat, glycerol
Hot air oven
OT, entryway, ward, laboratory fumigation,
Formaldehyde gas > UV > BPL
Preservation of anatomical specimen, woolen blanket
Orthophthaldehyde > glutaraldehyde 2% (cidex)
Cystoscope, bronchoscope
Ethylene oxide
Heart lung machine, respirator, dental equipments
Method of sterilization/disinfection
Vaccine, sera, antibiotic, sugar solution, antibiotic and body fluids
Sharp instruments
Plastic syringe, catgut suture, swab, catheter, bone and tissue grafts, adhesive dressing
Ionizing radiation
Culture media, metal instruments, glassware and all suture materials except catgut
Metallic inoculation wire
Red hot by Bunsen burner
Infective material like soiled dressing, bedding, animal carcasses
Burning (incineration)
Metallic surgical instruments
Autoclave, infra-red radiation
Chlorine as hypochlorite 0.2%
Tincture iodine, spirit (70% ethanol), savlon
Contact lenses

Table: Common disinfectants and their spectrum of action

Germicide and their concentrations
Level of disinfectant
Bacteria and enveloped viruses
Unenveloped viruses
M. tuberculosis
Inactivated by organic matter
Glutaraldehyde (2%)
H2O2 (3–25%)
Chlorine (100–1000 ppm)
Isopropyl alcohol (60–95%)
Phenol (0.4–5%)
Iodophor (30–50 ppm of free iodine)
Quaternary–ammonium (0.4–1.6%)

Table: Spaulding’s classification of medical devices according to the degree of risk for infection involved

Medical device
Recommended method
Critical device
Enter a normally sterile site
Surgical instruments, cardiac and urinary catheters, implants, eye and dental instruments
Heat based sterilizations, Chemical sterilant or high level disinfectant
Semicritical device
Come in contact with mucous membranes or minor skin breaches
Respiratory therapy equipments, anesthesia equipments, endoscopes, laryngoscope, rectal/vaginal/esophageal probes
High level disinfectant
Noncritical devices
Comes in contact with intact skin
BP cuff, ECG electrodes, bedpans, crutches, stethoscope, thermometer
Intermediate level or low level disinfectant
Noncritical surfaces
Less direct contact with patient
Surfaces of medical equipments, examination table, computers
Low level disinfectant