Control of Microorganisms 1 The process of sterilization

Control of Microorganisms 1

The process of sterilization is important in microbiology to prevent contamination from extraneous organisms Sterilization is essential : in hospitals for maintaining asepsis, in laboratories for ensuring safety from contaminating organisms. in food and drug manufacture to prevent microbial growth and also 2

Sterilization is also defined as a process by which an article, surface or medicine is made free of all microorganisms either in the vegetative or the sporing forms. The sterile object is totally free of viable microorganisms, spores and other infectious agents. 3

Definitions of commonly used Terms: Disinfection: It is killing, inhibiting or removal of microorganisms that may cause disease. Disinfectants : These are agents, usually chemical, used to carry out disinfection. Sanitation: It is the reduction in the microbial population to the level that is considered to be safe by public health standards. 4

Antisepsis: It is the prevention of infection or sepsis by killing or inhibiting the pathogen growth. Antiseptics: These are chemical agents applied to tissue. These are not as toxic as disinfectants. Germicide: A chemical agent which kills germs; most of pathogens but not endospores. Bactericide, Fungicide, Viricide and Algicide: Chemicals which kills bacteria, fungi, viruses and algae respectively. Bacteriostatic and Fungistatic: Chemicals which do not kill but prevent the growth of bacteria and fungi respectively. 5

Conditions That Affect the Efficiency of Antimicrobial agents Destruction and inhibition of microorganisms depend on the factors: 1. Population Size. 2. Population Composition. 3. Concentration and intensity of an antimicrobial agent 4. Duration of exposure 5. Temperature 6. Local environment 6

Methods of Sterilization 7

The different physical agents are: 1. Sunlight 2. Drying 3. Heat 4. Filtration 5. Radiation 6. Vibration 8

i ) Dry Heat : a) Flaming: Different materials used in microbiological laboratories such as forceps, scalpels are passed over the Bunsen burner for bringing about sterilization before use. b) Red heat: Inoculation loop or wires are usually made hot before it can be used for inoculation. c) Incineration: It is an effective method for rapidly destroying materials such as surgical dressings, animal carcasses and any other material used in hospital. It is usually done in a special device called incinerator. 9

d) Hot- Air Sterilization: This is used where it is either undesirable or unlikely that steam under pressure will make direct contact with materials to be sterilized. Only dry articles such as glass wares, bandages, instruments , mineral oils, talcum powder may be sterilizes by this method. The apparatus used for this type of sterilization may be a special electric or gas oven. Normally for laboratory glass wares, a 2 hour exposure to a temperature of 160°C is employed. 10

ii) Moist Heat: a)Temperature below 100°C : This is mainly used in pasteurization and temperature applied is either 63°C for 30 minutes ( holder’s method) or 72°C for 15 seconds ( flash Method). By this process all non sporing pathogenic organisms present in milk are destroyed. b) Temperature at 100°C: Boiling: The vegetative forms are killed at 90 -100°C but spore forming bacteria require considerable periods of boiling. Sterilization may be promoted by the addition of 2% sodium carbonate to the water. In cases where boiling is considered to be adequate , the material should be immersed in water and boiled 11 for a period of 10 -30 minutes.

c) Steam at atmospheric Pressure ( 100°C ) : An atmosphere of free steam is used to sterilize culture media which may decomposed if subjected to high temperatures. Special devices such as Koch’s steam sterilizer or Arnold steam sterilizer can be used for sterilization. One exposure of 9 minutes usually ensures complete sterilization but for media containing sugars and gelatin, an exposure of 100°C for 20 minutes on three successive days is used. This is known as Fraction Sterilization or Tyndallization. The principle is that the first exposure kills all vegetative bacteria and spores present will germinate and be killed on subsequent exposures. 12

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Arnold steam sterilizer 14

d) Temperature above 100°C: This is commonly obtained by holding the steam under pressure in an instrument called Autoclave. Steam under pressure provides temperatures above those obtainable by boiling Temperatures of steam under Pressure Steam Pressure, Lb/in 2 0 5 10 15 20 Temperature in °C 100 109 115 121. 5 126. 5 15

In routine use of autoclave, the temperature of 121. 5°C at 15 lb / in 2 for 15 minutes is employed. This period is referred to as holding Period during which effective sterilization takes place. Many media, solutions, discarded cultures, and contaminated materials are regularly sterilizes with autoclave. 16

Sterility checks are done by using certain indicators. 1). Chemical indicator-this includes Bowie _Dick tape or Browne’s tubes and 2) Biological indicators- this contains spores strip of Bacillus stearothermophilus. 17

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Browne’s tubes 19

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Filtration This is a method adopted to remove organisms from eat labile liquids. This is used for antibiotic solutions, serum preparations and carbohydrate solutions used in culture media. By this technique, one can obtain bacteria free filtrates. This method is also useful where separation of microorganisms is required which less in specimens. 21

Types of Filters a) Earthenware candle filter. Ex: Berkefeld and Chamberland filter Candle Type Filters: Berkefeld are made up of diatomatious earth or Kieselgarh. These filters are available in different pore sizes. The filters are in the form of hollow candle open at one end. b) Asbestos disc filter. Ex: Seitz filter: Thse are made of asbestos and chemically composed of magnesium silicate. These filters are usually in the forms of discs and are available in different pore sizes. 22

Berkefeld Sinter glass filter Seitz filter 23

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c) Sinter glass filter: These are made of finely ground glass which are then made to fuse. They are also available in different pore sizes. d) Collodion or Membrane filter: These are usually made of nitrocellulose and have precise pore sizes from 25μm 0. 025 μm. Advantages of these filters are, they relatively inexpensive , do not clog easily and can filter large volumes of fluid rapidly. They can be autoclaved. Membrane filters are used to sterilize materials likely to be damaged by heat sterilization. Materials like special nutrients that are added to culture media, sera, drugs, and vitamins are regularly filtered through 25 membrane filters.

Membrane filters The Isopore Filter 26

e) High-efficiency particulate air filters ( HEPA ): These are used in the ventilation systems of areas where microbial control is especially important , such as operating rooms, burn units, and laminar flow transfer units in laboratories. These filters remove all organisms larger than 0. 3 μm in diameter. 27

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High-efficiency particulate air filters ( HEPA ) 29

Radiation Four types of radiation-ultraviolet light, ionizing radiation, microwave radiation and in some case strong visible light can be used to control microorganisms. i) Ultraviolet ( UV ) light: UV light consists of wavelength between 40 and 390 nm, but wave length in the 200 nm range are most effective in killing microorganisms by damaging DNA and proteins. UV light does not penetrate glass, cloth, paper or most other materials and it does not reach corners and crevices of laboratory surfaces and materials thus limiting its use. 30

Electromagnetic Spectrum 31

ii) Ionizing Radiation: X rays which have wave length of 0. 1 to 40 nm and gamma rays which have even shorter wavelengths are forms of ionizing radiation. These cause ionization of molecules when they hit them resulting in death of microorganisms and viruses. 32

X-RAY DAMAGE OF DNA 33

UV RADIATION DAMAGE OF DNA 34

iii) Microwave Radiation: This radiation fall at the longer wavelength end of electromagnetic spectrum with the wave length of approximately 1 mm to 1 m. Microwave oven frequencies are turned to match energy level in water molecules. In the liquid state, water molecules quickly absorb the microwave energy and then release it to surrounding materials as heat. Specialized microwave oven is available now that can be used to sterilize the medium in just 10 minutes. 35

iv) Strong visible light: It has bactericidal property because of UV rays in sunlight. Strong visible light which has wave length from 400 to 700 nm can oxidize light sensitive molecules such as riboflavin and porphyrins. The fluorescent dyes eosin and methylene blue can be denature proteins on the presence strong light. Therefore the combination of a dye and strong light can be used to rid materials of both bacteria and viruses. 36

Vibrations: Sonic or sound waves in the audible range can destroy bacteria if waves are of sufficient intensity. Ultrasonic waves or waves with frequencies above 15, 000 cycles per second can cause bacteria to cavitate ( formation of partial vacuum in a liquid). Bacteria so treated disintegrate and their proteins are denatured. The disruption of cells by sound waves is called sonication. Neither sonic nor ultrasonic waves are a practical means of sterilization. 37

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Refrigeration, Freezing and Freeze –Drying: Cold temperatures retard the growth of microorganisms by slowing the rate of enzyme –controlled reactions but do not kill microbes. Refrigeration of foods at 5°C prevents spoilage from microorganisms. However, the storage should be limited to a few days as some bacteria and fungi continue to grow at this temperature. 39

Osmotic pressure: High concentration of salt, sugar or other substances creates a hyper osmotic medium which draws water from microorganism by osmosis. Plasmolysis or loss of water severely interfere with cell function and eventually leads to cell death. The use of sugar in jellies , jams and syrups or salt solution in curing meat and making pickles plasmolyzes most organisms present and prevents growth of new organisms. A few halophilic organisms grow even in such conditions and spoil the food stuff. 40

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