Sterilization - Agribusiness Education and Research International

Sterilization:

The process of destroying all forms of microbial life from the environment is called sterilization.

Objectives of Sterilization:

To maintain the sterile condition
To avoid contamination

Source of infection:

Infection or contamination can occur in three different ways:

By air: The air contains a large number of suspended micro-organisms in the form of fungal and bacterial spores.
By explants: Explants itself is an important source of contamination. The outer surface of the plant tissue may bear pathogen spore.
By worker: The human body (skin, hand, etc) carries several microorganisms.

Causes of infection:

Talking while working.
Contact between explants and fingers.
Poor sterilization of media, instruments, and explants.
Obstruction of clean air by any culture vessel inside the hood.
The neck of culture tubes, flask, jar, etc. if not systematically sterilized.
Use of explants which has fallen on the working table.

Sterilization Methods:

Via the Heat
A) Dry heat: It is normally done for 2-3 hours in an oven at 165⁰c to170⁰ Different glass-ware is usually sterilized by dry heat, such as; petri dish, pipettes, etc.

(b) Moist heat

Boiling: Most microbes, except bacterial spores and fungal propagules, typically need 10-15 minutes to be destroyed. Some instruments, such as forceps, needles, scissors, etc., are sterilized for certain times by boiling water.
Free steam: Free stem is usually applied without pressure in a coarse container containing stem. Large-scale soil can be sterilized by free steam passing through.

iii. Compressed steam: This is normally achieved in an autoclave for 15 minutes under 15 PSI (Per Square Inch) at a temperature of 121⁰c. This process is usually used to sterilize cultural media, water, and glassware.

Intermittent sterilization (Tyndalization):

In this method, materials are heated on 3 to 4 consecutive occasions for a few minutes in the stream (100⁰c) giving 2-4 hours of the interval at a temperature favorable for germination. These methods are used for those organisms that are not destroyed by the sterilization method of dry heat and moist heat.
By Chemical:

Usually, 70 % of ethyl alcohol is used to sterilize the hand, glassware, table surface, inoculation chamber, or laminar airflow surface. Formaldehyde is used to sterilize the room, the large apparatus, and the chamber of inoculation.

By filtration:

Without altering their chemical and physical properties, such solutions can not be sterilized by heat or by chemicals. The most useful phenomenon in such situations is sterilization by filtration. Millipore filter, Asbestos pad filter, and chamber land filter are widely used filters.
By radiation: UV (Ultra Violet) light is a good sterilization agent for glassware, media, equipment, etc. All radiation from 150-3900 A includes the ultraviolet portion of the spectrum. Wavelengths around 2650 A has the highest bactericidal efficacy. It is absorbed by the nucleic acid and also sterilizes glassware by inhibiting the replication of DNA, X-ray, β-ray and γ-ray.

Types of Sterilization with objectives and equipment used

Sl. No.	Types of Sterilization	Objectives	Equipments Used
01	Wet/Steam Sterilization	To sterilize different media	Autoclave, pressure cooker
02	Dry Sterilization	To sterilize glassware, forceps, needles etc.	Drying oven
03	Filter Sterilization	To sterilize vitamin, hormone	Different filter and vacuum pump
04	Alcohol Sterilization	To sterilize worker hand and laminar airflow	Chemicals used e.g. alcohol, rectified sprit
05	Flame Sterilization	To sterilize forceps, knife, planting rod etc.	Alcohol and striking rod
06	Chemical Sterilization	To sterilize explants.	Different sterilants e.g. Na OCl

Autoclave:

It is a machine for sterilization that is used for both dry and steam sterilization. By the use of an autoclave or pressure cooker, most of the nutrient media are sterilized. The Autoclaving Media Standard Conditions are 121⁰c with a pressure of 15 PSI for 20 min. For test tubes or any other containers with a capacity of 20-50 ml of nutrient media, these conditions are followed. An autoclave has a temperature range of 115⁰c -135⁰c.

Autoclave ‘S Operation Principle:

The autoclave is filled with water up to phase at first.
The materials to be sterilized are loaded into the autoclave.
The lid is fastened and locked.
Then the autoclave is connected to the electrical line.
The operation value is opened to allow the air out of the chamber.
The thermometers are tracked until the temperature reaches 100⁰c or 2120F and then the opening value closes. At this time, all of the air had been discharged from the chamber.
The thermometer is tracked until the temperature exceeds 121⁰c at a pressure of 15 lbs per square inch. This state is maintained for 15 minutes.
Then the electric light is to be off.
After the pressure scale shows that there is no more vapor pressure, the lid is opened and the sterilized substance can be removed.

Precautions at the time of autoclaving:

In an autoclave/pressure cooker, check the level of water at the bottom.
Ensure that the autoclave/pressure cooker lid is closed properly.
Ensure the air exhaust is running normally.
Do not accelerate pressure reduction after the time needed for autoclaving. If the temperature is not lowered steadily, it is important for the media to boil again. And because of the quick and forced release of pressure, the material in the containers could burst out.
Bottles should not be firmly screwed and they should be loose on their tops. These bottles are held in the laminar airflow after autoclaving and the tops of these bottles are tightened upon cooling.

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