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Sterilization is the process of killing all forms of microbial life in or on the given object or preparation. Microbiologically, sterile material is one that contains no living organisms at all and the term sterile is therefore an absolute one. Accordingly, an object is sterile or non sterile but it can never be semisterile or almost sterile. [1]

1. Antiseptic: A substance that arrests sepsis i.e prevents the growth or action of microorganisms by inhibiting their activity without necessarily destroying them. These can be applied on human body.
2. Bactericide: An agent that kills bacteria.
3. Bacteriostat: An agent that arrests or retards the growth of bacteria.
4. Disinfection: A process that removes infection potential by destroying microorganisms but not ordinarily bacterial spores.
5. Disinfectants: These are generally meant for application on inanimate objects.
6. Germicides: A substance that kills disease microorganisms (i.e. pathogens / germs) but not necessarily bacterial spores.
7. Sterility: The absence of viable organisms.
8. Viable: Live and growing bacteria (or microorganisms) + spores
9. Vegetative microorganisms: Growing organisms.

Sterilization Parameters:
Now let us have a simple overview regarding the sterilization parameters which help us in better understanding of the sterilization processes.
The exponential relationship between number of organisms living and the extent of treatment is studied from the inactivation kinetics of pure culture of organisms by exposing them to physical or chemical sterilization procedures. Survivor curves have been used to develop inactivation data for a specific sterilization procedure using suitable biological indicator. This data is helpful in establishing a sterilizing regimen for a specific preparation. [2]

D value: It is the parameter calculated as the time taken for one log (90%) reduction in the number of microorganisms.[2]
Z value: It calculates the temperature or dose of radiation sterilization required to produce a one log (90%) reduction in D value for a particular organism. [2]
F value: This value is used to compare the lethality of different heat sterilization procedures.[2]


Sterilization process can be basically separated as terminal and non terminal process based on the stage at which the preparation is subjected to the process pf sterilization. [2]

Terminal Sterilization: Different types of terminal sterilization techniques employed are Physical sterilization and Chemical sterilization.[2]

Non terminal Sterilization: It includes the filtration procedure.[2]

Based on the principle of mechanism involved in the process they are further classified as follows:

Physical Sterilization: This class includes heat sterilization and radiation sterilization as well.
Heat sterilization procedure is the one in which the destruction of microorganisms mainly occurs due to the high temperatures employed. This process includes
(a). Moist heat sterilization.
(b). Dry heat sterilization. [2]

Radiation Sterilization: This process is accomplished by exposure to ultraviolet (UV) light or high-energy ionizing radiation such as gamma rays and accelerated electrons i.e particulate radiation as well.. [2]

Chemical sterilization: The procedures which involve treatment of the preparations to be sterilized with certain chemicals in either gaseous form or in liquid form are categorized under this class.[2]


Moist heat sterilization is otherwise refered as steam sterilization under pressure.
Mechanism of killing of microorganisms:
Heat in the form of saturated steam under pressure is the most practical and dependable agent for sterilization. Bacterial death by moist heat sterilization is due to denaturation and coagulation of essential protein molecules (enzymes) and cell constituents.[1].
Steam sterilization under pressure is carried out in an autoclave, which is an airtight, jacketed chamber designed to maintain a high pressure of saturated hot steam. Because the autoclave permits the attainment of high moist-heat temperatures and because heat exchange by steam is more rapid than by dry heat, this method of sterilization is more efficient than the dry-heat method. Sterilization of aqueous solutions, glassware, and rubber articles is best done by steam under pressure. This process is not suitable for sterilizing solutions of drugs that are thermolabile in nature.[3]
Conditions to be followed for the moist heat sterilization according to The USP XXI and BP 1988 are given below:
* Pressure: 15 lb / square inch (psi)
* Temperature: 121 C
* Time: 15 minutes

Mechanism of killing of microorganisms:
The vital constituents of cells such as proteins (enzymes) and nucleic acids are denatured by oxidation. The killing of microorganisms by heat is a function of the time-temperature combination. [1]
Dry heat is the simplest and most economical method of sterilization. This method requires higher temperatures and longer exposure times to achieve the same microbial-killing efficiency as compared with that of steam sterilization. A major problem associated with dry heat
sterilization is non uniform distribution of temperature. A fan is usually installed in the oven to overcome this problem, but unless the fan is properly baffled it may blow around powders that are to be sterilized. Furthermore, dry heat cannot be used with materials that are heat sensitive. It is mainly used for sterilization of glass and metal objects. It can also be used to sterilize thermostable powders and fatty substances.[3]
Conditions to achieve complete sterilization by dry heat sterilization are as follows:
Cycles recommended as per BP 1988 are:
* A minimum of 180 C for not less than 30 minutes.
* A minimum of 170 C for not less than 1 hour.
* A minimum of 160 C for not less than 2 hours.
Mechanism of killing of microorganisms:
The radiation employed for the sterilization may be ionizing or non ionizing radiation. Gamma and X-rays, having energies more than about 10eV, are called ionizing radiations because they have enough energy to knock electrons away from molecules and ionize them. These radiations create free hydrogen radicals, hydroxyl radicals and some peroxides which cause different kinds of intracellular damage. The less energetic ultraviolet radiation cannot ionize and it is absorbed and excites the electrons and rises them to higher energy states, thus finally destroying the cellular structure of the microorganism. When properly operated, the UV radiation technique can reduce the level of airborne bacterial contamination in a room by 90% within 30 min. [3]
Pharmaceutical products are more resistant to degradation by ionizing radiation if they are in a powdered form than in a liquid form. Thermolabile drugs such as penicillin, streptomycin, thiamine, and riboflavin have been effectively sterilized by ionizing radiation. In hospitals, UV radiation is used to control the spread of infection during or after surgical procedures. [3]

This process involves exposure of materials to sterilize gasses such as ethylene oxide, formaldehyde, glutaraldehyde , propylene oxide.
Gaseous sterilization is accomplished by exposure to a gas that kills microorganisms. The most commonly used gas for sterilization is ethylene oxide. Ethylene oxide acts by alkylation. Ethylene oxide is a cyclic ether that has flammable and explosive properties, especially when confined and mixed with oxygen. It is therefore often used in combination with inert gases such as carbon dioxide to avoid hazard. The effectiveness of ethylene oxide in the sterilization process depends on relative humidity, gas concentration, temperature, exposure time, and the extent of contamination. The normal working ranges for the relative humidity and ethylene oxide concentration are 30 to 60% and 500 to 1000 mg/l, respectively. At these ranges, the times required for complete sterilization are 2 to 5 h at 55 C. In practice, an exposure time of 6 h or more is normally used to provide a safety margin. Ethylene oxide sterilization is effective in sterilizing a wide variety of materials including surgical instruments and gloves, plastic syringes, disposable needles, tubing sets, and dialysis units. In addition, it is often used to sterilize thermolabile powdered drugs such as penicillins. However, some drugs, for example, thiamine, riboflavin, and streptomycin, lose potency when treated with ethylene oxide. [3]
Phenol and phenolic compounds produces a variety of effects on the microorganisms. Based on their concentration they exert various effects like disruption of cells, precipitation of cell protein, inactivation of enzymes and lekage of aminoacids from the cells.[1]
Other chemical agents include alcohols, halogens, heavy metals and their compounds, dyes, detergents, quaternary ammonium compounds and aldehydes etc.[1]

This method is used for sterilizing thermolabile solutions, which will otherwise be degraded by other conventional heating methods. The drug solutions are passed through the sterile bacteria proof filter unit and subsequently transferring the product aseptically into the sterile containers which are then sealed. Depth and surface filtration are suitable for prefiltration of pharmaceutical products as they can retain large amounts of particles. To strerilize a product it is often necessary to combine several types of filtration to achieve removal of microorganuisms.[2]

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2005 by CRC Press LLC.
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Sirisha Pingali's picture

Hello praveen You have given a very elaborated note on sterilization. I have few queries What is the best method to kill pyrogens?? Is there any specific pore size for the filter used in filteration sterilization??

Sirisha Pingali

Viswanadha Institute of Pharmaceutical Sciences.

Siriki Praveen Kumar's picture

Hi, General pore size of a microbial retentive filter was designated as 0.45uM. But in case of microorganisms which cannot be retained on this pore size, filters with still reduced pore sizes are employed.[1] For example: B.dimuntia can pass through the above mentioned pore size and hence the standard pore size was reduced to around 0.2/0.22 uM-rating.[1] So, 0.2/0.22 uM-rating was considered to be the specified pore size of a sterilizing filter.[1] References: [1]HTTP://AMERICANPHARMACEUTICALREVIEW.COM/VIEWARTICLE.ASPX?CONTENTID=223(Accessed on 06 march 2011). Thank You.


S. Praveen Kumar.

Pharma warriors

Dr. T.E.G.K. Murthy's picture

How to validate sterilization?

Bapatla College of Pharmacy, Bapatla

Siriki Praveen Kumar's picture

hi sir, The validation of a sterilization process mainly depends on two types of data namely, commissioning data and performance qualification data.[1] Commissioning data mainly deals with several factors in relation to the equipment used for sterilization like: (a) Assurance that the equipment has been installed in accordance with specifications. (b) Is the equipment safe to use and whether the equipment functions within predetermined limits. Performance qualification data includes:[1] (a) Assurance that the equipment will produce a product with an acceptable assurance of sterility. (b) Physical performance qualification- it gives the evidence that the specified conditions are maintained through out the sterilization cycle -the required data should be generated from the worst spot in the sterilizer. -the data should prove that the applied procedure do not show any detrimental effects on the product and its packaging. (c) Biological performance qualification-[1] it gives the evidence that the specified sterilization conditions provides sufficient microbiological lethality to the product. It is generally evaluated by the use of biological indicators.Particularly, biological performance qualification is not essential if the process is well defined. REFERENCES: [1]AULTON's PHARMACEUTICS THE DESIGN AND MANUFACTURE OF MEDICINES; Edited by: MICHAEL E. AULTON; THIRD EDITION; CHURCHILL LIVINGSTON ELSEVIER. Page no: 260


S. Praveen Kumar.

Pharma warriors

SS Md Shafi's picture

hi..... what is the mechanism involved in gas sterilization...??

shafi ..

Siriki Praveen Kumar's picture

hi, The most commonly used gas for gas sterilization is "ethylene oxide". Ethylene oxide generally acts by the mechanism alkylation of several organic compounds like enzymes, proteins etc finally making them inactive.It consists of replacement of an active hydrogen atom in an organic compound rendering its action.[1] For example: Several enzymes containing "sulfhydryl" groups are inactivated by ring opening and alkylating reactions of ethylene oxide.[1] References: 1. MICROBIOLOGY BY MICHAEL J.PELCZAR, JR. ; E.C.S.CHAN; NOEL R.KRIEG. FIFTH EDITION, TATA MCGRAW-HILL EDITION 1993 Page no: 502 - 504


S. Praveen Kumar.

Pharma warriors

Sravani kompella's picture

How can we sterilize emulsions and suspensions?
Gangadhar Hari's picture

Is it Practically possible to achieve 100% sterility?
Siriki Praveen Kumar's picture

HI, Taking into account several practical considerations, it is not possible to attain 100% sterility. thankyou.


S. Praveen Kumar.

Pharma warriors

Amol Dhiaman's picture

I want to know that if there is any relation between D,Z,F values. With regards Amol Dhiman Team GLORY

Amol Dhiaman

Siriki Praveen Kumar's picture

Hi, As i already mentioned D value is the parameter calculated as the time taken for one log (90%) reduction in the number of microorganisms. Z value calculates the temperature or dose of radiation sterilization required to produce a one log (90%) reduction in D value for a particular organism. F value is used to compare the lethality of different heat sterilization procedures. There exists a relation between F and Z value, as the F value of a particular process of sterilization is calculated at a particular Z value. For example: The reference value (Fo) of Geobacillus stearothermophilus spores is at 121C is mostly usedf with a Z value of 10C. REFERENCES: AULTON's PHARMACEUTICS THE DESIGN AND MANUFACTURE OF MEDICINES; Edited by: MICHAEL E. AULTON; THIRD EDITION; CHURCHILL LIVINGSTON ELSEVIER. Page no: 243-244 Thank you.


S. Praveen Kumar.

Pharma warriors

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