Asepsis Sterilization – Classification and Significance

The concept of asepsis and its role in the prevention of infection was put forward nearly  2 centuries ago. The general principles for asepsis were laid down by Hungarian abstetrician, Ignaz semmelweiss in europe in early 1850’s and Oliver Holmes in USA. These principles were accepted after Joseph Lister’s studies on prevention of wound infection carried out between 1865-91. Lister, working on antisepsis, initially used phenol (dilute carbolic acid) for contaminated wounds, later applied it in all surgical wounds, also in operating room by nebulization of the solution. Further developments occurred with the introduction of steam sterilization surgical masks, sterile gloves, sterile gowns and drapes etc.

In present days certain guidelines and regulations are recommended by accepted bodies, which have to be followed in dental practice and up graded in every general body meeting.



  1. Sun Light
  2. Drying
  3. Heat
    1. Dry
    2. Moist
  4. Gas
  5. Filtration
  6. Irradiation
  7. Ultra sonic cleaning
  8. Oil


  1. Phenol Derivatives :  Phenol, Cresol, resorcinol, chloroxylenol
  2. Oxidizing agents :  Pot.  Permanganate,  Hydrogen Peroxide, Benzoyol  Peroxide
  3. Halogens : Iodine, chlorine
  4. Biguanide :   Chlorhexidine
  5. Quarternary Ammonium (Cationic) : Cetrimide,  Zephiran
  6. Soaps  : of Sodium  &  Potassious
  7. Alcohols :  Ethanol, Isopropanol.
  8. Aldehydes  : Formaldehyde, Glutaraldehyde
  9. Acids : Boric acid, acetic acid
  10. Metallic salts ;   Silver  Nitrate, Zince Sulfate, Zinc Oxide, calamine, Ammoniated mercury.
  11. Dyes : Gentian violet, proflamine, Acriflamine
  12. Furan derivatives : Nitro flurazone


  1. Sunlight :  Most old & still effective.  It possesses appreciable bactericidal activity.  The action is due to ultra violet rays.  This is one of the natural methods of sterilization in case of water in tanks, rivers and lakes.STERLIZATION BY SUNLIGHTWATER PURIFICATION BY SUNLIGHT
  2. Drying :  It air has deleterious effect on many bacteria.  Spores are unaffected by drying.  Hence it is very unreliable method.
  3. Heat :     Is the most common and one of the most effective methods of sterilization.  HEAT STERILIZATION

Factors influencing sterilization by heat are : –

      1. Nature of heat
        1. Dry
        2. Moist
      2. Temperature & time
      3. No of organism present
      4. Whether organism has sparing capacity
      5. Type of material from which organism is to be eradicated



Killing is due to :

  1. Protein denaturation
  2. Toxic effects of elevated levels of electrolytes

a.   Red Heat :   It is used to sterilize metallic objects by holding them in flame and heated red hot.  heat sterilization

Example : inoculating wires, needles, forceps etc.

b.   Flaming :   The article is passed over flame without allowing it to become red hot.

Example : Glass plates,  Cotton wool plays and glass slides.

c.   Hot Air Oven: It is used to sterilize items, which do not get damaged by high temp. such as laboratory glass wave, flasks, scissors, impression trays (metal), all stainless steel instruments with sharp cutting edges, (preferred) B.P. handles, Dapen dishes, mouth mirrors and poles.  Hot air is poor conductor of heat and poor penetrating capacity.  So grease, oils, powders plastics, rubber-containing substances should be sterilized by other methods.  High temp. can damage fabrics or melt them.

hot air oven

Temp.  &  Time:   The sterilization is complete if these two factors are achieved throughout the load.

  • 160oC  to 320oF –  120/60 min
  • 170oC  to 340oF –  60 min
  • 150oC  to 300oF –  150 min
  • 140oC to  280oF –  180 min


  1. The heat should be uniformly distributed in side the oven.
  2. All the instruments must be clean of dry prior to wrapping.
  3. It should not be over loaded.
  4. Oven must be allowed to cool for about 2 hours before opening other wire glass will crack.

Sterilization Control of Hot Air Oven 

  1. Detectors as spores of non-pathogenous strains of clostridium tetani are used to test dry heat efficiency.
  2. Browne’s tube (green spot) is available for checking sterilization by dry heat.   A green color is produced after 60 min. at 160oC.
  3. Thermocouples may be used.


This method employs a heat transfer device.  The media used are glass beads, molten metal and salt.  The temperature achieved is of 220oC.  The method employs submersion of small instruments such as endodontic files and burs, into the beads; and are sterilized in 10 seconds provided they are clean. A warm-up time of at least 20 minutes is recommended to ensure uniform temperatures in these sterilizers.

glass bead sterilizer

Some hand piece can be sterilized by dry heat.  The hand pieces should be carefully cleaned and lubricated with special heat resistant oils.


Effective by denaturation and coagulation of proteins.

a. Temperature below 100oC.

    1. Pasteurization – milk by Hold Method and  Flash Method.
    2. Vaccine bath – for  vaccines
    3. Inspissation

b. Temperature at 100oC 

  1. Tyndallization
  2. Boiling
  3. Steam Baths

c.  Temperature above 100oC



These are three major factors required for effective autoclave :

  1. Pressure:   It is expressed in pressure (Pounds) pre square inch and it is 15-PSI pressure.
  2.   Temperature:   To achieve required pressure, the temp. must be reached and maintained at 121oC with the increase in temperature and pressure super heated steam is formed and removed Air from chamber and this brings about sterilization.
  3.   Time:  Wrapped loads require a minimum of 20 min. After reaching full temperature and time cycle, a wide variety of materials can be sterilized by this method.

AUTOCLAVEExample: Diagnostic and prognostic Instruments, plastic filling Instrument, impression trays, laboratory equipments, surgical instruments etc.  Higher temperature and greater pressure shorter the time required for sterilization.

             Pressure            Temperature           Time (Min.)
              15  psi               121oC                   15
              20 psi               126oC                   10
              20 psi               134oC                    3

Time required to sterilize for a particular item also varies with the amount of material for the thickness of the wrap.


Radiation used for sterilization is of two types

  1. Ionizing radiation, e.g., X-rays, gamma rays, and high speed electrons and
  2. Non-ionizing radiation, e.g. ultraviolet light, and infrared light.   These forms of radiation can be used to kill or inactivate microorganisms.

Ionizing Radiation 

It is effective for heat labile items.   Bellamy (1959) reported that it has great penetrating properties.  It is commonly used by the industry to sterilize disposable materials such as needles, syringes and swabs.

The lethal action of this radiation is believed to be due to its effect on the DNA of nucleus and on the other vital cell components.  There is no appreciable rise in temperature.   High energy gamma rays from cobalt-60 are used to sterilize such articles.

ionizing radiation

Non-ionizing radiation 

Two types of non-ionizing radiations are used for sterilization:-


It is absorbed by proteins and nucleic acids and kills microorganisms by the chemical reactions it sets up in the bacterial cell.  It has low penetrating capacity and its main application is purification of air in operating rooms; viz, to reduce the bacteria in air, water and on the contaminated surfaces.  All forms of bacteria and viruses are vulnerable to ultraviolet rays below 3000 atmospheric pressure.   Excessive exposure of skin can produce serious burns.  Care must be taken to protect the eyes while using U-V radiation for sterilization.



It is another form of dry heat sterilization.   It is most commonly used to purify air, such as in the operating room.  Infrared is effective, however, it has no penetrating ability.



Several studies have shown that, when performed correctly, ultrasonic cleaning will remove dried serum, whole blood, plaque, zinc phosphate cement, and polycarboxylate cement from instruments, metal surfaces and dentures. It has been found to be more effective than manual cleaning.

Ultrasonic cleaning minimizes the handling of contaminated instruments by the nurse and reduces the chance of injuries from sharp, contaminated instruments.

Instruments are loaded into a metal basket, which is then placed into the ultrasonic bath. The unit is activated for the time recommended by the manufacturer (usually about 6 minutes). Instruments, which are contained in cassettes, are cleaned for 12 minutes.


After the cleaning cycle is complete, the basket is taken to the sink and the instruments are carefully and thoroughly rinsed under tap water. The instruments are checked for residual debris, which may be safely removed manually.

Instruments are taken to the packaging area, where they are unloaded from the baskets onto a thick disposable paper towel. The instruments are thoroughly ‘pat’ dried using strong paper towels. Drying is important.

Small rotary and Endodontic instruments should be held in beakers of ultrasonic cleaning solution which are suspended in the cleaning bath


The Clinical Research Associates (CRA) recommended the following ultrasonic cleaners:

BIOSONIC – Whaledent

T33C – Health Sonics Group

CLOSTER 3 – Provides ultrasonic clean, rinse and dry, but is noisy

Details of these cleaners are given in the report.


  • Ensure bath is 3/4 full as per manufacturer’s instructions.
  • Ensure lid is well fitting to avoid creating aerosols.
  • Instruments should always be placed in a basket within the bath to ensure that they are kept a proper distance from the bottom of the bath. Burs should be placed in the beakers provided.
  • Never overload the basket. Overloading the basket with instruments causes ‘wave shadows’ – inactive zones within the bath.
  • Choose cycles as per manufacturer’s instructions. Currently the counter top ultrasonic baths in use operate at <40 C. At this temperature a cycle of 15 minutes is recommended. Alkazyme is currently considered an appropriate solution to use in this manner. (If the throughput is very large and instruments are heavily contaminated it is currently recommended to change the solution twice daily after the a.m. and p.m. sessions respectively).
  • On removal of instruments rinse thoroughly (in basket) under running water, inspect for any residual cement etc., dry and pack.


  1. Ultrasonic units should be tested on a regular basis. Place a piece of tinfoil in the solution and run the unit. If the foil shows uniform pitting after 5 minutes this indicates that the submerged items received adequate ultrasonic cleaning during the cycle. No holes or an uneven pattern may indicate that the machine is not functioning properly.
  2. Every new solution must be ‘degassed’ by running the ultrasonic bath free of instruments for 15 minutes.
  3. Ultrasonic cleaning is not suitable for hand pieces.
  4. Amalgam carriers should not be placed in the ultrasonic bath


They are used to disinfect the skin of a patient prior to surgery, and to disinfect the hands of the operator.  No available chemical solution will sterilize instruments immersed in it.   Secondly, there is a risk of producing tissue damage if residual solution is carried over into the wound while it is being used.  The chemicals used are


  1. Aqueous solution of formaldehyde (formalin) and
  2. Glutaraldehyde (cidex) are effective disinfectants.

i.   Formaldehyde:    This is a broad-spectrum antimicrobial agent, which is used for disinfection.  It is a hazardous substance, inflammable and irritant to the eye, skin and respiratory tract.  This is used to upto 500c  and has limited sporicidal activity.  It is used for large heat-sensitive equipment such as ventilators and suction pumps excluding rubber and some plastics.


ii.  Glutaraldehyde:   It is toxic, irritant and allergenic.  It is a high level disinfectant.  It is applicable where heat cannot be used.  It is active against most vegetative bacteria (including M. Tuberculosis) and some viruses (including HIV and HBV), fungi and bacterial spores.  It is frequently used for heat sensitive material.  A solution of 2 percent glutaraldehyde (Cidex), requires immersion of 20 minutes for disinfection; and 6 to 10 hours of immersion for sterilization. Stonehill et al (1963) reported that glutaraldehyde kills vegetative bacteria, spores, fungi and virus by alkylation on a 10-hour contact.  The Centre for Disease Control includes it in the list of effective agents against hepatitis viruses.  It is also toxic and irritating, and hence, not used on certain surfaces such as furniture, walls and floors.  It can be safely used on metal instruments (for less than 24 hours), rubber, plastics and porcelain.  It is activated by addition of sodium bicarbonate, but in its activated form in remains potent only for 14 days



Ethanol and isopropyl alcohols are frequently used as antiseptic.  Alcohols possess some antibacterial activity, against some Gram-positive and negative bacteria, and especially against M tuberculosis.   Alcohols act by denaturing proteins.  They are not effective against spores and viruses.

The alcohol must have a 10 minute contact with the organisms.  Solutions of 70 percent alcohol are more effective than higher concentrations, as the presence of water speeds up the process of protein denaturation as reported by Lawrence and Block (1968).   The alcohols do not function as disinfectants when instruments, hand pieces, or other equipment are simply wiped with them, since they evaporate quickly. Alcohols can dissolve cements holding instruments together, and plastics may harden and swell in their presence.

They are frequently used for skin antisepsis prior to needle puncture.  They are good organic solvents.  Their benefit is derived primarily in their cleansing action.  The alcohols must have a prolonged contact with the organisms to have an antibacterial effect.  This contact is prevented due to its rapid evaporation. Alcohol is sometimes used as a rinse following a surgical scrub.   Its effectiveness lies in the solvent action and not in its antibacterial properties.  Ethanol (Ethyl alcohol) is employed in the concentration of 70 percent as a skin antiseptic.  It has poor activity against bacterial spores, fungi, and viruses. It is used in the concentration of 60 to 70 percent v/v, for disinfection of skin.   The alcohols do not have reliable sporicidal, virucidal, or fungicidal action; hence, they are not useful for sterilizing surgical instruments.

alcohols as disinfectants


Phenol itself toxic to skin and bone marrow.  The phenolic compounds were developed to reduce their side effects but are still toxic to living tissues.  These compounds, in high concentration, are protoplasmic poison, and act by precipitating the proteins and destroy the cell wall.

Lawrence and Block (1968) reported that their spectrum of activity  includes lipophilic viruses, fungi and bacteria but not spores.   Hence these are approved by ADA for use as surface or immersion disinfectant.These compounds are used for disinfection of in animate objects such as walls, floors and furniture.  They may cause damage to some plastics, and they do not corrode certain metals, such as brass, aluminium and carbon steel.


Benzalkonium chloride (Zephiran) is the most commonly used antiseptic.  Its spectrum of activity is primarily Gram-positive bacteria.  It is well tolerated by living tissues.  It is not widely used because of its narrow spectrum of activity.



Many studies have shown, that, iodophor compounds are the most effective antiseptics,.  Iodine is complexed with organic surface-active agents, such as, polyvinylpyrrolidone (Betadine, Isodine).  Their activity is dependent on the release of iodine from the complex.  The surface agent is film forming; this prevents the solution form staining clothes or skin.

These compounds are effective against most bacteria, spores, viruses, and fungi.  These are the most commonly used surface disinfectants along with hypochlorite.  Concentrated solutions have less free ioidine.  Iodine is released as the solution is diluted.  An appropriate dilution is 1 : 2 : 3 parts of iodophor and distilled water, respectively.


Advantages are 

  1. Low toxicity.
  2. Prolonged residual effect
  3. Inexpensive and
  4. Odorless.

Geraci (1963) reported that these compounds build up on the skin after successive scrubs, and that this provides long la


Literature reported that the storage of instruments is also a problem.  The pattern of storage varies from place to place.  They are either stored in drawers, or in containers, in packs or sterilized trays.  The maintenance of sterility during transportation and storage is of utmost importance.

Packs should be stored with the following considerations

  1. Instruments are kept wrapped until ready for use.
  2. To reduce the risk of contamination, sterile packs must be handled as little as possible.
  3. Sterilized packs should be allowed to cool before storage; otherwise condensation will occur inside the packs.
  4. To prevent contamination from rodents, ants, and cockroaches, the store must be subjected to adequate pest control.
  5. Materials should be stored at least 8” off the floor and 18” from the ceiling.
  6. Sterile packs must be stored and issued in correct date order.  The packs, preferably, are stored in drums which can be locked.  Preset trays and cassettes, are useful as, the instruments can be organized as per the procedure



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