Equipment used for sterilization

Introduction

Sterilization is a term referring to any process that eliminates (removes) or kills all form of life, including transmissible agent (such as fungi, bacteria, viruses, spore form, etc) present in a specific region, such as a surface, a volume of fluid, medication, or in a compound such as a biological culture media. (Gibraltar, 2010)sterilization can be achieved with one or more of the following; heat, chemicals, irradiation, high pressure and filtration. Sterilization is distinct from disinfection, sanitization and pasteurization in that sterilization kills or inactivate all forms of life.

One of the first steps towards sterilization was made by Nicolas Appert who discovered that through application of heat over a suitable period slowed the decay of foods and various liquids, preserving them for safe consumption for a longer time.  In general, surgical instrument and medications that enter an already aseptic part of the body (such as the blood stream, or penetrating the skin) must be sterile. Example of such instrument includes scalpels, hypodermic needles and artificial pacemakers. This is also essential in the manufacture of parental pharmaceuticals.

The aim of sterilization is the reduction of initially present micro organisms or other potential pathogens. The degree of sterilization is commonly expressed by multiples of the decimal reduction time, or D-value, denoting the time needed to reduce the initial number  to one tenth (10^-1) of its original value, (Brown, Amy Christian, 2007). The D-value is a function of sterilization conditions and varies with the type of micro-organism, temperature, water activity, PH etc.

Theoretically, the like hood of survival of an individual micro-organism is never zero. To compensate for this, the overall method is often used. Using the over kill method, sterilization is performed by sterilizing for longer than is required to kill the bio-burden present on or in the item being sterilized. This provides a sterility assurance levels (SAL) equal to the probability of a non sterile unit. For high-risk applications such as medical devices and injections, a sterility assurance levels of at least 10^-6 is required (Bharati, 2009).

A widely used method for the heat sterilization is the autoclave sometimes called a converter or steam sterilizer; autoclave use steam heated or 121⁰C-134⁰C under pressure to achieve sterility the article is heated in a chamber by injected steam until the article reaches a time and temperature set point (Steris Life Science, 2013) proper autoclave treatment will inactivate all resistant bacteria spores in addition to fungi, bacteria and viruses but is not expected to eliminate all prions which vary in their resistance, for prion elimination various recommendation state 121-132^oc for 60 minutes or 134^oC for at least 18 minutes (Brown, 2007).

Dry heat can be used also to sterilize items but as the heat takes much longer to be transfers to the organisms; both the time and the temperature must usually be increased, unless forced ventilation of the hot air is used. The standard setting for a hot air oven is at least two hours at 160^oc,a rapid method heats air to 190^oc for 6 minutes for unwrapped objects and 12 minutes for wrapped objects (Amy Christian, 2002). Dry heat has the advantage that it can be used on powder and other heat stable items that are adversely affected by steam.

Flaming is done to loops one straight wires in microbiology labs. Leaving the loop in the flame of a Bunsen burner or alcohol lamp until it glows red ensure that any infectious agent get inactivated; this is commonly used for small metal or glass object but not for large objects, a variation on flaming is to dip the object in 70% or higher ethanol, then briefly touch the object to a Bunsen burner flame, the ethanol will ignite and burn off rapidly (Molins, Ricardo A. 2001).Incineration will also burn any organisms to ash; it is used to sterilize medical and other bio-hazardous waste before it is discarded with non-hazardous waste.

Methods of sterilization

It is divided into three groups or methods

1. Physical method
2. Chemical method
3. Mechanical method

Physical method: This involves the use of heat; it is divided into dry heat and moist heat.

• Dry heat: It requires much longer exposure time and higher temperature than moist heat. Under dry heat we have hot air oven, flaming, red heat and incineration.
• Moist heat: It destroys all micro organisms and their spore. Under moist heat we have boiling at 100%, steam under pressure, tyndallization.

Chemical method: Many chemical agents are referred to as disinfectant, a term that is applied to substance which destroys micro-organisms or inanimate objects,chemical agent function as sterilizing agent by the following mechanism.

• Interfering with the enzymatic system of the organisms (enzyme poisons).
• Disruption of the cell membrane

• Coagulation of protein

• Damage of RNA and DNA

Chemical agents used for sterilization are

1. Alcohol: ethanol 70%, isopropanol 70%, propanol 60%
2. Aldehydes: formaldehyde
3. Halogens (chlorine, iodine and their derivatives

Mechanical method: This method is used to sterilize heat sensitive fluid. It is particularly useful for solution containing toxics, enzymes, drugs and sugar. Mechanical filter include

• Seit filter
• Sintered glass filter

• Membrane filter

• Pasteur chamber filter etc.

 Equipment used for sterilization

1 Autoclave:

This is a device that sterilizes items by heating them under pressurized steam;in autoclave the items are heated under a pressure of 151b/sq to a temperature of 121⁰c for 15-20 minutes,this condition has been proven sufficient to kill contaminants and infectious agents such as fungal, viruses and bacterial spore.

 Components of the Autoclave

1. Boiler: Large deep cylinder in which apparatus to be sterilized is kept.
2. Basket: Big wire basket that holds material.

• Basket Support: Hold the basket above the water level.

1. Drainage Tap: A tap fitted at base of boiler that drains excess water.
2. Lid: The lid covers and seals the boiler.
3. Lid clamps: Clamps together with rubber washer seal the lid and prevent steam from escaping.
4. Air outlet Valve: There is a valve at the top of the boiler or on the lid that lets air out when water is first heated.
5. Safety value: It let steam escape if the pressure becomes too high to prevent an explosion.
6. Temperature Gauge/pressure Gauge: there is a dial at the top of the lid that shows the pressure, temperature or both.
7. Gauge Graduation: Gauge indicate temperature in ⁰c, while some set indicates pressure at Ibs.

 Sterilization procedures

1. Fill the auto-clave with water to cover the heater.
2. Put bucket containing the material to be sterilized.
3. Close the lid putting the washer in place; screw the lid evenly and firmly not too tightly.
4. Open the air outlet valve.
5. Begin heating
6. Watch the air outlet until steam appears uniformly.
7. Then close the outlet valve, tighten the clamps and reduce the heat slightly.
8. Watch gauge until desired temperature and pressure is attained.
9. Start timing for 15 minutes.
10. Turn off the heat and allow the autoclave to cool.
11. Open the discharge tap immediately but slowly.
12. Open the lids and remove the content.

 Materials that can be sterilized in Autoclave

1. Infected culture for discards.
2. Bacteriology culture media (agar, peptone water)
3. Surgical instrument.
4. Glass wares.
5. Non-thermolabite liquids.

 Hot Air Oven

Hot air oven is an electric or gas instrument used for sterilization of contaminated materials; the hot air oven consist of heating element attached to an insulated cabinet and a fan, the fan circulates the hot air for rapid, uniform and controlled heating of materials,it is also equipped with a temperature regulator and thermostat temperature of 100⁰c for 1^1/2 hours.

 Component of Hot Air Oven

1. Lid: To cover the machine.
2. Glass cover: To prevent the heat.
3. Grate: To hold the materials to be sterilized.
4. Heating element: To heat the system.
5. Base: to hold the machine.Power Indicator: To show if the equipment is on/off.
6. Set Switch: To set the temperature reading.
7. Socket: To connect to source of light.
8. Fans: To re-circulate the heat (air)

 Sterilization procedures

1. Unlock the door and load the substance into the chamber carefully.
2. Do not tilt the flask or vessel in chamber.
3. Lock the door completely and inspect if there is any leakage from door.
4. Adjust and close the vent on top of the equipment for fast heating, adjust the vent when the temperature is stabilized to achieve uniform temperature.
5. Plug cord into properly grounded outlet.
6. Switch on the power button, the corresponding lamp of power will be illuminated at the same time.

 Materials that can be sterilized in Hot Air oven

1. Glass wares e.g. test tubes, Flask, Petri-dish.
2. Fat, oil and grease e.g. petroleum jelly.
3. Powders
4. Forceps, scalpels and scissors.

 Pressure Cookers

Pressure cookers are saucer pan designed to cook food very quickly using steam under pressure, they are used in small laboratories for sterilizing specimen collection equipments and Agar preparation. There are two types

1. Model with revolving valve
2. Model with fixed valve

 Using the model with revolving valve

1. Fill the bottom of cooker with water.
2. Place the materials to be sterilized in the basket on a support
3. Placed the wrapped article upright.
4. Fix on the lid; screw it down with its knob.
5. Place revolving valve on its shaft in the lid.
6. Start heating.
7. Time for 20 minutes.
8. Time off heat to cool.
9. Put off the revolving valve so that air enters.
10. Remove lid and remove materials.

 The model with fixed valve

1. Put material to be sterilized in the basket.
2. Placed the wrapped article upright
3. Open the valve on the lid start heating.
4. As soon as jet of steam escapes, close valve
5. Wait until whistle sound then reduce heat and leave for 20 minutes.
6. Turn of heat
7. Open valve to let air.
8. Remove lid and material.

Chemical agent used for sterilization

1. Soap and detergent: These are surface agent; when soap and detergent is used on surface, fatty acids absorbs it to the cell membrane and the surrounding water. This cause the cell membrane to undergo strain and become destroyed; however, soap and detergent have considerable bacteriostatic properties and their recurrent soap and detergent should contain Phisohex and chlorine-Phenol combined so that their antiseptic properties can be felt.
2. Alcohol: This produced denaturation of bacteria protein and frequently used as skin disinfectant. The most effective disinfectant occurs at a concentration of 50%, 70% and 95%.
3. Phenol and cresols: In current concentrations; these agents have high bacterial properties, they denature bacteria protein. Lysol is the most widely used of the cresol and employed at the concentration of 2-3%. They are not easily inactivated by the presence of organic matter but have little or no effect on spores, they are at their best when used diluted.
4. Gases: Ethyl oxides is a colourless odourless poisonous inflammable and explosive which if diluted with inert substance such carbon-dioxide becomes an efficient sterilizer of surgical instruments such as plaster material which can not be heat sterilized. It is also used for heart, lung machine and respirator. It kills both vegetative organisms and spores in 8 hours. Formaldehyde is an irritant water soluble gas, lethal to all kinds of vegetative microbes and spores.
5. Glycerol: This chemical agent at 50% solution can be used to kill bacteria but not virus; they are also applied in the preservation of vaccines and agglutination of sera.

 Factors that Influence the degree of killing of organisms

1. Types of organisms: Organisms vary in their ability to withstand chemical and physical treatment e.g spores have coat rich in protein, lipids and carbohydrate. Mycobacteria cell walls are richin lipids.
2. Number of organisms: The total number of organisms which determine the exposure time of killing agent is composed with varying degrees of susceptibility to kill agents, higher numbers of organisms require longer exposure.
3. Concentration of disinfecting agent: A proper concentration of disinfecting agent ensure the activation of target organisms e.g providone-iodine should be diluted with water before use because there is no enough free iodine to kill micro-organisms.
4. Temperature: Disinfectant are generally used at room temperature (20-22⁰c), their activity is increased by an increased temperature and decreased by a drop in temperature.
5. PH: The ph of the material to be disinfected or sterilized can have an effect on the activity of disinfecting or sterilized agent.

 Sterilization control

To ensure that potentially infectious agents are destroyed by adequate sterilization regimes in three levels;

1. Physical: Measuring device control (temperature, time, pressure).
2. Chemical: Substance that undergo a colour change or have melting points with the sterilizing range e.g Browne’s tubes, Brown Dick tape give an immediate indication of a successful or non successful sterilization.
3. Biological: Bacillus stearothermophilus spores (10⁴-10⁶ organisms) survive steam heat at 121⁰c for 15 minutes and is killed at 121⁰c in 13 minutes.
4. Bacillus Substance Spores
5. Reheat and determine the adequate of ethylene oxide or dry heat sterilization.

 Chemical indicator

1. Browne’s tube: are glass tube that contain heat sensitive dyes; these change colour after sufficient time at the desired temperature, before heat exposure, the content of the tube appear red as heating progress the colour changes to green, only when the tube is green sterilization condition can be considered adequate.
2. Bowie dick tape: Is apply to articles being autoclave before heat exposure; the tape is uniformly buff in colour, after adequate heating the tape develops dark brown stripes, the pack on the left has been properly sterilized, that on the right has not.

 Conclusion

Sterilizing medical equipment is of utmost importance in medical field; in laboratories, containers and other laboratory ware must be sterile for research to be accurate. Sterilization prevents the growths of disease, medical sterilization prevents the spread of diseases, if proper medical sterilization is not practiced it can lead to number of medical problems, therefore sterilization in medical laboratory should be stressed more to minimize the contamination by organisms and transmission of disease from one individual to another.

References

Amy, C. (2002):Understanding Food Sterilization and Preparation (3^rd ed) Cengage Learning P.546

Bharati, K (2009): Chemical Disinfection of Medical Laboratory Material In Block SS, ed. Disinfection, Sterilization and Preservation. Philadelphia: Lippincott, Williams and Wilkins, pp 881-917.

Brown (2009):“Steam Sterilization Principles” Steris Life Sciences: Media Sterilization”. In Stephanopoulos G. Biotechnology ZE, Vol. 3, Bio-processing, Weinheim: Wiley. pp. 157-184.

Gibraitar (2010): Infection Control; the role of disinfection and sterilization, J. Hosp. Infect 43:543-55.

Molins, R.A.(2001):Incineration Principles and Applications. Wiley IEE. pp 23.