STERILIZATION

INTRODUCTION

A physical or chemical process that completely destroys or removes all microbial life, including spores present in a specified region, such as a surface, a volume of fluid or in a compound such as biological culture media

 Sterilization can be achieved with one or more of the following:  heat chemicals radiation high pressure and  filtration 2

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METHODS OF STERILIZATION: PHYSICAL Sterilization by Heat: Most common

method Dry Heat

Simplest method is exposing the item to be sterilized to the naked flame e.g. Bunsen burner- for sterilizing bacteriological loops, knives, blades.

Heat sterilization is the most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents.

It employs higher temperatures in the range of 160-180°C and requires exposures time up to 2 hours, depending upon the temperature employed.

The benefit of dry heat includes good penetrability and non-corrosive nature which makes it applicable for sterilizing glass-wares and metal surgical instruments.

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METHODS OF STERILISATION It is also used for sterilizing non-aqueous

thermo-stable liquids and thermostable powders.

Dry heat destroys bacterial endotoxins (or pyrogens) which are difficult to eliminate by other means and this property makes it applicable for sterilizing glass bottles which are to be filled aseptically.

Examples of Dry heat sterilization are: 1. Incineration 2. Red heat 3. Flaming 4. Hot air oven 5

METHODS OF STERILIZATION Hot-air oven :

Dry heat sterilization is usually carried out in a hot air oven, which consists of the following:

(i) An insulated chamber surrounded by an outer case containing electric heaters. (ii) A fan (iii) Shelves (iv) Thermocouples (v) Temperature sensor (vi) Door locking controls.

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HOT AIR OVEN Operation

(i) Articles to be sterilized are first wrapped or enclosed in containers of cardboard, paper or aluminium. (ii) Then, the materials are arranged to ensure uninterrupted air flow. (iii) Oven may be pre-heated for materials with poor heat conductivity. (iv) The temperature is allowed to fall to 40°C, prior to removal of sterilized material.

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HOT AIR OVEN Principle

Sterilizing by dry heat is accomplished by conduction.

The heat is absorbed by the outside surface of the item, then

passes towards the centre of the item, layer by layer.

The entire item will eventually reach the temperature required

for sterilization to take place.

Dry heat does most of the damage by oxidizing molecules.  

The essential cell constituents are destroyed and the organism

dies.

The temperature is maintained for almost an hour to kill the

most difficult of the resistant spores.

FLAMING

This is an emergency method, the forceps-tips, the surfaces of the scalpels and the needles

may be sterilized by holding the items directly in the flame of a Bunsen burner.

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Advantages of dry heat sterilization A dry heat cabinet is easy to install and has relatively

low operating costs; It penetrates materials It is nontoxic and does not harm the environment; And it is noncorrosive for metal and sharp

instruments. Disadvantages for dry heat sterilization

Time consuming method because of slow rate of heat penetration and microbial killing.

High temperatures are not suitable for most materials. 10

METHODS OF STERILIZATIONMETHODS OF STERILIZATIONMoist Heat (or Steam):

Uses hot water. Moist heat kills microorganisms by denaturing proteins. Moist heat may be used in three forms to achieve

microbial inactivation1. Dry saturated steam – Autoclaving 2. Boiling water/ steam at atmospheric pressure3. Hot water below boiling point4. Pasteurisation

Moist heat sterilization involves the use of steam in the range of 121-134°C.

Steam under pressure is used to generate high temperature needed for sterilization.

Saturated steam acts as an effective sterilizing agent. Steam for sterilization can be either wet saturated steam

(containing entrained water droplets) or dry saturated steam (no entrained water droplets).

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Boiling – quite common especially in domestic circumstances. Boiling is done for metallic devices like surgical scissors,

scalpels, needles etc like instruments. Here substances are boiled to sterilize them.

Pasteurisation Pasteurization is the process of heating the milk at a

temperature of 60 degrees or 72 degrees 3 to four times. Here alternative heating and cooling kills all the microbes

and molds without boiling the milk.

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METHODS OF STERILIZATIONMETHODS OF STERILIZATION

Moist heat: Tyndallization

The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling, cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again.

The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step.

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METHODS OF STERILIZATIONMETHODS OF STERILIZATIONMoist heat: Autoclaving Standard sterilization method in

hospitals. The Autoclave works under the

same principle as the pressure cooker where water boils at increased atmospheric pressure i.e. because of increased pressure the boiling point of water is >100°C.

The autoclave is a tough double walled chamber in which air is replaced by pure saturated steam under pressure.

Before using the autoclave, check the drain screen at the bottom of the chamber and clean if blocked. If the sieve is blocked with debris, a layer of air may form at the bottom of the autoclave, preventing efficient operation

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AUTOCLAVEAUTOCLAVE The air in the chamber is flushed out and filled with

saturated steam. Water is boiled to produce steam, which is released

through the jacket and into the autoclave's chamber. Hot, saturated steam enters the chamber and the

desired temperature and pressure, usually 121°C. At this temperature saturated steam destroys all

vegetative cells and endospores. Moist heat is thought to kill so effectively by

degrading nucleic acids and by denaturing enzymes and other essential proteins.

It also may disrupt cell membranes. The chamber is closed tightly the steam keeps on filling into it and the pressure gradually increases.

The items to be sterilized get completely surrounded by saturated steam (moist heat) which on contact with the surface of material to be sterilized condenses to release its latent heat of condensation which adds to already raised temperature of steam so that eventually all the microorganisms in what ever form are killed.

The usual temperature achieved is 121 °C at a pressure of 15 pps.ie. at exposure time of only 15-20 mins. By increasing the temperature, the time for sterilizing is further reduced.

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METHODS OF STERILIZATIONMETHODS OF STERILIZATION

Advantages of Autoclave Temperature is > 100°C therefore spores are

killed.

Condensation of steam generates extra heat (latent heat of condensation).

The condensation also allows the steam to penetrate rapidly into porous materials.

Note: that autoclavable items must be steam permeable. Can not be used for items that are lacking water.

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RADIATIONRADIATIONElectromagnetic radiation

Gamma rays UV rays

Particulate radiation Accelerated electrons

The major target for these radiation is microbial DNA. Gamma rays and electrons cause ionization and free radical

production while UV light causes excitation. U.V. light has limited sterilizing power because of poor

penetration into most materials. Generally used in irradiation of air in certain areas eg. Operating Rooms and T.B. laboratories.

Ionizing radiation- e.g. Gamma radiation: Source Cobalt60 has greater energy than U.V. light, therefore more effective. Used mainly in industrial facilities e.g. sterilization of disposable plastic syringes, gloves, specimens containers and Petri Dishes.

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RADIATION Gamma ray Sterilizer: Gamma rays for sterilization are

usually derived from cobalt-60 source, the isotope is held as pellets packed in metal rods, each rod carefully arranged within the source.

This source is housed within a reinforced concrete building with 2 m thick walls.

Articles being sterilized are passed through the irradiation chamber on a conveyor belt and move around the raised source.

Penetrates deep into objects and is an excellent sterilizing agent.

It destroys bacterial endospores and vegetative cells of both prokaryotic and eukaryotic origin but not against viruses.

Gamma radiation from a cobalt 60 source is used in the cold sterilization of antibiotics, hormones, sutures and plastic disposable supplies such as syringes, and Petri dishes.

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RADIATIONRADIATION

Ultraviolet radiation: The optimum wavelength for UV sterilization is 260 nm. A mercury lamp giving peak emission at 254 nm is the suitable source of UV light in this region.

But this does not penetrate glass, dirt films, water and other substances very effectively.

UV radiation is used as a sterilizing agent only in a few specific situations, like UV lamps are placed on the ceilings of rooms or in biological safety cabinets to sterilize air and other exposed surfaces.

Commercial UV units are available for water treatment. Pathogens and microorganisms are destroyed when a thin layer of water is passed under the lamps (water purifiers).

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INFRARED RADIATION

Infrared radiation (IR) is a thermal radiation, i.e. when absorbed by some article its energy is converted to heat and therefore it is often known as radiant energy.

A tunnel having an IR source is used for this purpose. The instruments and glass wares are kept in trays are

passed through this tunnel keeping on the conveyor belt, at a controlled speed exposing them to a temperature of 1800C for 17 minutes, thereby achieving the sterility.

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This is a continuous process and is used in hospitals for regular supply of sterile syringes and other apparatus.

Heating at or above 2000C by IR in vacuum is employed as a means of sterilizing surgical instruments.

Cooling is hastened, (after the heating cycle) during the cooling period, by admitting filtered N2 to the chamber. 21

FILTRATIONFILTRATION In order to sterilize solutions which is heat

sensitive, filtration is an excellent way to reduce the microbial population.

The filters simply remove the microbes instead of killing them.

Depth filters Consists of fibrous or granular materials that have

been bonded into a thick layer filled with twisting channels of small diameter.

The solution is passed through the filter which is sucked through this layer under vacuum and microbial cells are removed. The material used mostly is unglazed porcelain, asbestos or other similar materials

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FILTRATIONFILTRATION

Membrane filters Are also used and have replaced depth filters in

recent times. These filters are made up of cellulose acetate,

cellulose nitrate, polycarbonate, polyvinylidene fluoride, and other synthetic materials.

These filters vary in size with pore sizes mostly of 0.2 to 0.5 µm in diameter and used to remove most vegetative cells, but not viruses, from solutions ranging in volume from 1ml to many litres.

These filters are mostly used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics and other heat sensitive solutions. 23

FILTRATION

The other way this method is used is in the laminar flow biological safety cabinets where the air is sterilized by filtration.

These cabinets contain high-efficiency particulate air (HEPA) filters, which remove 99.97% of 0.3µm particles.

The safety cabinets are most useful as the culturing of any organisms requires contamination free air to reduce the growth of other undesired organisms or for the preparation of media, examining tissue cultures etc 24

Phenol and Phenolics Halogens Alcohols Heavy Metals Quaternary Ammonium Compounds Aldehydes Gaseous Peroxygens

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STERILIZATION : CHEMICAL METHODS Useful for heat sensitive materials e.g. plastics and lensed

instruments endoscopes). The sterilising agent 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.

Peracetic Acid liquid sterilization: Peracetic acid was found to be sporicidal at low concentrations. It was also found to be water soluble, and left no residue after

rinsing. It was also shown to have no harmful health or environmental effects.

It disrupts bonds in proteins and enzymes and may also interfere with cell membrane transportation through the rupture of cell walls and may oxidize essential enzymes and impair vital biochemical pathways. 26

CHEMICAL STERILISATION Hydrogen Peroxide Sterilization:

This method disperses a hydrogen peroxide solution in a vacuum chamber, creating a plasma cloud.

This agent sterilizes by oxidizing key cellular components, which inactivates the microorganisms.

The plasma cloud exists only while the energy source is turned on. When the energy source is turned off, water vapor and oxygen are formed, resulting in no toxic residues and harmful emissions.

The temperature of this sterilization method is maintained in the 40-50°C range,

well-suited for use with heat-sensitive and moisture-sensitive medical devices.

The instruments are wrapped prior to sterilization, and can either be stored or used immediately. 27

GASEOUS STERILIZATION Ethylene Oxide Sterilizer:

Ethylene oxide gas readily penetrates packing materials, even plastic wraps and is both microbicidal and sporicidal and kills by combining with cell proteins.

Ethylene oxide alkylates DNA molecules and thereby inactivates microorganisms.

Ethylene oxide may cause explosion if used pure so it is mixed with an inert gas e.g. Neon, Freon at a ratio of 10:90

It requires high humidity and is used at relative humidity 50-60% ; Temperature : 55-60°C and exposure period 4-6 hours.

An ethylene oxide sterilizer consists of a chamber of 100-300-Litre capacity and surrounded by a water jacket.

Air is removed from sterilizer by evacuation, humidification and preheated vaporized ethylene oxide is passed.

After treatment, the gases are evacuated either directly to the outside atmosphere or through a special exhaust system.

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GASEOUS STERILIZATION

Activated alkaline Glutaraldehyde 2%: Immerse item in solution for about 20 minutes if

organism is TB. In case of spores, the immersion period is extended to 2-3 hours.

Batapropiolacetone (BPL) is occasionally used as a sterilizing gas in the

liquid form to sterilize vaccines and sera. Recently vapour-phase hydrogen

peroxide has been used to decontaminate biological safety cabinets.

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ENUMERATION OF BACTERIA- MOST PROBABLE NUMBER (MPN ) TECHNIQUE AND MEMBRANE

FILTER TECHNIQUE

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Most Probable Number & Membrane Filter methods

These are methods used to enumerate the numbers of bacteria in water samples.

The Most Probable Number method is used to check potability (if water is safe enough to be drinking water) of water.

The MPN method looks for the presence of potential pathogenic bacteria that may be in the water due to fecal contamination of the water supply.

Water supplies are generally derived from ground sources and have to be checked for safety levels of bacterial contamination

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MPN method enumerates the enteric bacteria called coliforms, specifically fecal coliforms (E. coli)

Coliforms are Gram negative bacilli that have the ability to ferment lactose with the production of acid and gas.

Fecal coliforms are those coliforms that are normally found in the feces of warm blooded animals (including humans)

MPN method thus enumerates the fecal coliforms in water samples.

E. coli is thus used as an indicator organism.

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MPN test includes 3 levels of testing: Presumptive, Confirmed, Completed.

The presumptive test looks for presence of fecal coliforms in the water sample by inoculating lactose broths with the water sample.

Those tubes that show presence of acid and gas are scored + and those with no acid/gas as -.

Three sets of lactose broths are inoculated with varying dilutions of the sample: First set of 3 or 5 tubes inoculated with 10ml of

sample; Second set of tubes inoculated with 1ml of sample; Third set of tubes inoculated with 0.1ml of sample.

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The combination of positives in the 3 sets is used to figure out the MPN /100ml of water using the table provided.

The tubes that show positive in the presumptive test should be confirmed to contain E.coli.

This done in the confirmed test using the selective/differential medium EMB (that uniquely highlights E.coli growth on it).

The completed test is done only where legal issues are involved wherein the bacterial culture is then identified by a full complement of test including gram stain to show that it is indeed E.coli 35

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MPN

Advantages Disadvantages Relatively simple and sensitive Can count a specific type in the presence of others Can use large sample volumes

Time consuming and labor intensive Requires large volumes of glassware Doesn’t give the “real” value Doesn’t give isolated colonies

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MEMBRANE FILTER TECHNIQUE

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MEMBRANE FILTER TECHNIQUE Samples are filtered through a membrane designed to

retain bacteria Filter is antiseptically transferred from the filtration

apparatus and placed on either a pad saturated with media or agar plate.

Plates are inverted and incubated at 35°C for 22-24 hours.

The media is heated to near boiling All bacteria that produce a red colony with a metallic

(golden) sheen, are considered to be members of the coliform group. However, some non-coliform bacteria (ie. Proteus mirabilis) can produce sheen colonies.

The MF test requires confirmation with LTB and BGLBB. Only colonies that ferment lactose (found in BGLBB) can be confirmed as coliforms. 40

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PROCEDURE1. Collect the sample and make

any necessary dilutions.2. Select the appropriate

nutrient or culture medium. Dispense the broth into a sterile Petri dish, evenly saturating the absorbent pad.

3. Flame the forceps, and remove the membrane from the sterile package.

4. Place the membrane filter into the funnel assembly.

5. Flame the pouring lip of the sample container and pour the sample into the funnel.

6. Turn on the vacuum and allow the sample to draw completely through the filter.

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7. Rinse funnel with sterile buffered water. Turn on vacuum and allow the liquid to draw completely through the filter.

8. Flame the forceps and remove the membrane filter from the funnel.

9. Place the membrane filter into the prepared Petri dish.

10. Incubate at the proper temperature and for the appropriate time period.

11. Count the colonies under 10 - 15 X magnification.

12. Confirm the colonies and report the results. 43

Counting range is 20-80 colonies per membrane

Dehydrated media must be tightly closed and stored properly

Prepared media must be kept in the refrigerator unless the method allows for a different storage temperature

Standard Units = CFU/100 mL (Colony Forming Units)

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ADVANTAGES Permits testing of large sample volumes. Reduces preparation time as compared to many

traditional methods. Allows isolation and enumeration of discrete colonies

of bacteria. Provides presence or absence information within 24

hours. Effective and acceptable technique. Used to monitor

drinking water in government laboratories. Useful for bacterial monitoring in the

pharmaceutical, cosmetics, electronics, and food and beverage industries.

Allows for removal of bacteriostatic or cidal agents that would not be removed in Pour Plate, Spread Plate, or MPN techniques. 45