Sterilization.ppt

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

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.

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

METHODS OF STERILIZATION

 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.

HOT AIR OVEN

(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

 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

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.

 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.

METHODS OF STERILIZATION

METHODS OF STERILIZATION

Moist Heat (or Steam):

 Uses hot water.

 Moist heat kills microorganisms by denaturing proteins.

 Moist heat may be used in three forms to achieve microbial

inactivation

1. Dry saturated steam – Autoclaving

2. Boiling water/ steam at atmospheric pressure 3. Hot water below boiling point

4. 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).

 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.

METHODS OF STERILIZATION

METHODS 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

METHODS OF STERILIZATION

METHODS OF STERILIZATION

Moist 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

AUTOCLAVE

AUTOCLAVE

 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

METHODS OF STERILIZATION

METHODS 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

RADIATION

RADIATION

Electromagnetic 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

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

RADIATION

RADIATION

 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

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

 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

FILTRATION

FILTRATION

 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

FILTRATION

FILTRATION

 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

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

 Phenol and Phenolics

 Halogens

 Alcohols

 Heavy Metals

 Quaternary Ammonium Compounds

 Aldehydes

 Gaseous

 Peroxygens

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

CHEMICAL STERILISATION

 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

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.

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.

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

FILTER TECHNIQUE

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

 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.

 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;

 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

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

MEMBRANE FILTER TECHNIQUE

MEMBRANE FILTER TECHNIQUE

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

PROCEDURE

1. 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

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.

 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)

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