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Sterilization monitoring by physical, chemical and biological indicators.

How is the sterilization process monitored?

Sterilization procedures should be monitored through a combination of mechanical, chemical, and biological techniques designed to evaluate the sterilizing conditions and the procedure's effectiveness.

· Mechanical techniques for monitoring sterilization include assessing the cycle time, temperature, and pressure of sterilization equipment by observing the gauges or displays on the sterilizer. Some tabletop sterilizers have recording devices that print out these parameters. Correct readings do not ensure sterilization, but incorrect readings could be the first indication that a problem has occurred with the sterilization cycle.

· Chemical indicators, internal and external, use sensitive chemicals to assess physical conditions such as temperature during the sterilization process.

· Chemical Color Change Indicators - are powder substances with a strictly defined melting point: benzonaphthol (110 ° C), antipyrine (113 ° C), resorcinol and sulfur (119 ° C), benzoic acid (120 ° C). These substances are mixed with a small amount of dry aniline paints (magenta, methylene blue) and placed in sealed glass tubes between sterilized objects. Chemical indicators should be positioned near the center of each load, and toward the bottom front of the autoclave.

· If the temperature in the autoclave was sufficient, the substance in the tube melts and stains the color of the dye that dissolves in this substance. Hence, chemical indicators can give a quick visual reference for heat penetration inside the autoclave.

· Tape Indicators

· Tape indicators are adhesive backed paper tape with heat sensitive, chemical indicator markings. Commonly used heat sensitive markings include diagonal stripes (autoclave tape), and/or the word “sterile”. These markings only appear when the tape has been exposed for a few minutes to normal autoclave decontamination temperatures.

An internal tape indicators should be placed in every sterilization package to ensure the sterilization agent has penetrated the packaging material and actually reached the instruments inside. An external indicator should be used when the internal indicator cannot be seen from outside the package.

Caution: Most chemical indicators and tape indicators can only be used to verify that your autoclave has reached normal operating temperatures for decontamination; they have no time factor. Chemical indicators alone are not designed to prove that organisms are actually killed during a decontamination cycle.

 Indicator test results are shown immediately after the sterilization cycle is complete and could provide an early indication of a problem and where the problem occurred in the process. If the internal or external indicator suggests inadequate processing, the item that has been processed should not be used. Because chemical indicators do not prove sterilization has been achieved, a biological indicator (i.e., spore test) is required.

· Biological indicators (BIs) are the most accepted means of monitoring the sterilization process because they directly determine whether the most resistant microorganisms (e.g., Geobacillus or Bacillus species) are present rather than merely determine whether the physical and chemical conditions necessary for sterilization are met. Because spores used in BIs are more resistant and present in greater numbers than are the common microbial contaminants found on patient care equipment, an inactivated BI indicates that other potential pathogens in the load have also been killed.

Biological indicators are designed to demonstrate that an autoclave is capable of killing microorganisms. EH&S recommends the use of commercially available Bacillus stearothermophilus spores to monitor the effectiveness of steam autoclaves. This test must be performed at least every 90 days.

Bacillus stearothermophilus spores die at 121 ° C for 15 minutes when they are contained in 1 ml of a medium of 106 cells. Tubes with strips of gauze, filter paper, with silk thread, infected with spores, are placed between sterilizable objects. After sterilization, a nutrient broth (MPB) is added into the test tube and the growth of microorganisms is observed. The presence of turbidity is a sign of bacterial grows, hence the autoclave is not capable of killing microorganisms and their spores and vice versa, if nutrient medium remains transparent - the autoclave is capable of killing microorganisms.

Use of Chemical agents in microbial control:

The chemical agents are mostly employed in disinfection and antisepsis. The proper use of these agents is essential to laboratory and hospital safety. Factors such as the kinds of microorganisms potentially present the concentration and nature of the disinfectant to be used and the length of treatment should be considered. Many disinfectants are available and each has its own advantages and disadvantages, but ideally the disinfectant must be effective against a wide variety of infectious agents, at high dilutions and in the presence of organic matter and should not be toxic to people or corrosive for common materials. The disinfectant must be stable upon storage, odorless or with a pleasant odor, soluble in water and lipids for penetration into microorganisms, and have a low surface tension so that it can enter cracks in surfaces.


In 1867 Joseph Lister employed it to reduce the risk of infection during operations and phenol was the first widely used antiseptic and disinfectant. Today phenol and phenolics such as cresols, xylenols, are used as disinfectants in laboratories and hospitals. Lysol is made of a mixture of phenolics which is commercially available disinfectant. They act by denaturing proteins and disrupting cell membranes. Phenolics are tuberculocidal and effective in the presence of organic material and remain active on surfaces long after application. However, they do have a disagreeable odour and can cause skin irritation. Hexachlorophene has been one of the most popular antiseptics because once applied it persists on the skin and reduces skin bacteria for long periods.


Alcohols are the most widely used disinfectants and antiseptics. They are bactericidal and fungicidal but not sporicidal. Ethanol and isopropanol are the two most popular alcohol germicides. They act by denaturing proteins and possibly by dissolving membrane lipids. Small instruments like thermometers can be disinfected by soaking them for 10 to 15 min in alcohol solutions. A 70% ethanol is more effective than 95% as water is needed for proteins to coagulate.


Halogens exist as diatomic molecules in the free state and form salt like compounds with sodium and most other metals. Iodine and chlorine are the most important antimicrobial agents. Iodine is used as a skin antiseptic and kills by oxidizing cell constituents and iodinating cell proteins. Spores can be destroyed at higher concentrations. Iodine is often applied as tincture of iodine, 2% or more iodine in a water-ethanol solution of potassium iodide. Skin scars result and sometimes iodine allergies can result. In today's date, brands like Wescodyne for skin and laboratory disinfection and for wounds is being used as iodine is complexed with an organic carrier to form iodophor; and these are mostly used in hospitals for preoperative skin degerming and in hospitals and laboratories for disinfection.

Chlorine is mostly used as a disinfectant for municipal water supplies and swimming pools and also employed in dairy and food industry. It may be applied as chlorine gas, sodium hypochloride or calcium hypochloride, all of which yield hypochlorous acid (HClO) and then atomic oxygen. The result is oxidation of cellular materials and destruction of vegetative bacteria and fungi, although not spores. One potential problem is that chlorine reacts with organic compounds to form carcinogenic trihalomethanes, which must be monitored in drinking water. Ozone sometimes has been used successfully as an alternative to chlorination in Europe and Canada. Small amounts of drinking water can be disinfected with halazone tablets. It slowly releases chloride when added to water and disinfects it in about half an hour.

Heavy metals:

Heavy metals such as mercury, silver, arsenic, zinc and copper were used as germicides and these have been most recently superseded by other less toxic and more effective germicides. A 1% solution of silver nitrate is often added to the eyes of infants to prevent ophthalmic gonorrhea but now erythromycin is used instead of silver nitrate because it is effective against Chlamydia as well as Neisseria . Silver sulfadiazine is used on burns. Copper sulphate is an effective algicide in lakes and swimming pools. The action of these heavy metals is mostly on the proteins, and they combine often with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins.

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