Culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens. There are different types of media for growing different types of cells.
The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth). Liquid media are often mixed with agar and poured into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. Bacteria grew in liquid cultures often form colloidal suspensions. The final pH for both media is 7.4.
There are two major types of culture media: those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or yeast. The most common culture media for microorganisms are nutrient broths and agar plates; specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells. In this experiment the broth contains:
3.0 g/L “Lab-lemco” powder (a beef extract)
2.0 g/L yeast extract
5.0 g/L peptone (a nitrogen source)
5.0 g/L sodium chloride
2.0 g/L agar powder
An autoclave is an instrument used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C or more, typically for 15–20 minutes depending on the size of the load and the contents. It was invented by Charles Chamberland in 1879, although a precursor known as the steam digester was created by Denis Papin in 1679. The name comes from Greek auto-, ultimately meaning self, and Latin clavis meaning key — a self-locking device.
Downward displacement (or gravity type) - As steam enters the chamber, it fills the upper areas as it is less dense than air. This compresses the air to the bottom, forcing it out through a drain. Often a temperature sensing device is placed in the drain. Only when air evacuation is complete should the discharge stop. Flow is usually controlled through the use of a steam trap or a solenoid valve, but bleed holes are sometimes used, often in conjunction with a solenoid valve. As the steam and air mix it is also possible to force out the mixture from locations in the chamber other than the bottom.
Steam pulsing - Air dilution by using a series of steam pulses, in which the chamber is alternately pressurized and then depressurized to near atmospheric pressure.
Vacuum pumps - Vacuum pumps to suck air or air/steam mixtures from the chamber.
Superatmospheric - This type of cycle uses a vacuum pump. It starts with a vacuum followed by a steam pulse and then a vacuum followed by a steam pulse. The number of pulses depends on the particular autoclave and cycle chosen.
Subatmospheric - Similar to superatmospheric cycles, but chamber pressure never exceeds atmospheric until they pressurize up to the sterilizing temperature.
OBJECTIVE:
- To prepare sterile nutrient agar for culturing microorganisms.
DISCUSSION
Culture media must be stored at the specified temperature, under specified conditions such as pH and humidity.
All prepared culture media and their components should be stored away from light and exposure to direct sunlight should be avoided at all times.
Humidity
Sealed glass and plastic containers are unaffected by normal laboratory humidity. Opened containers of dehydrated powders will be affected by high humidity. Hot, steamy media preparation rooms are not suitable environments to store containers of culture media; particularly containers which are frequently opened and closed.
Unopened containers should be stored at room temperature 15-20°C. Opened containers should have the cap or lid carefully and securely replaced. It is important that opened containers are stored in a dry atmosphere at room temperature.
Preparation culture media
Always use freshly prepared distilled or deionised water. Use warm (50°C) water to hasten the solution of the medium. Rinse all glassware with the distilled/deionised water and make sure that the vessels are clean and free from toxic chemicals.
Open the culture medium container away from draughts and moisture. Avoid inhaling the powder and prolonged skin contact.
Weight the powder quickly, accurately and without creating 'clouds of dust'. Reclose the container as soon as possible.
Agar-free media will usually dissolve on gentle agitation. Media containing agar should be heated to dissolve the agar before autoclaving. Bring the medium to the boil without scorching or burning. Most culture media will require final sterilization in an autoclave at 121°C for 20 minutes.
Although sterilization of culture media is best carried out in a steam autoclave at temperatures between 121-134°Cfor 20 minutes to make sure all pathogen is damaged.
Sterilization is a term referring to any process that eliminates (removes) or kills all forms of life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media. Sterilization can be achieved by applying the proper combinations of heat, chemicals, irradiation,high pressure, and filtration.
A widely-used method for heat sterilization is the autoclave, sometimes called a converter. Autoclaves commonly use steam heated to 121–134 °C (250–273 °F). To achieve sterility, a holding time of at least 15 minutes at 121 °C (250 °F) or 3 minutes at 134 °C (273 °F) is required. Additional sterilizing time is usually required for liquids and instruments packed in layers of cloth, as they may take longer to reach the required temperature (unnecessary in machines that grind the contents prior to sterilization). Following sterilization, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is released. Modern converters operate around this problem by gradually depressing the sterilization chamber and allowing liquids to evaporate under a negative pressure, while cooling the contents.
Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions.
For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers must be left ajar. Alternatively steam penetration can be achieved by shredding the waste in some Autoclave models that also render the end product unrecognizable. During the initial heating of the chamber, residual air must be removed. Indicators should be placed in the most difficult places for the steam to reach to ensure that steam actually penetrates there.
For autoclaving, as for all disinfection or sterilization methods, cleaning is critical. Extraneous biological matter or grime may shield organisms from the property intended to kill them, whether it physical or chemical. Cleaning can also remove a large number of organisms. Proper cleaning can be achieved by physical scrubbing. This should be done with detergent and warm water to get the best results. Cleaning instruments or utensils with organic matter, cool water must be used because warm or hot water may cause organic debris to coagulate. Treatment with ultrasound or pulsed air can also be used to remove debris.
CONCLUSION
As a conclusion ,we able to learn correct methods to prepare sterile nutrient agar for culturing microorganisms. Culture media must be stored at the specified temperature, under specified conditions such as pH and humidity.Direct sunlight have to be avoided at all times from exposure of culture mediaand their component.To prevent humidity of laboratory,all plastic containers are saled.there are specific temperature for sterelization of culture media.The culture media need to be sterelized to make sure all pathogen was damaged.Besides that we managed to know the sterelizing method and also know how to use autoclave.
REFERENCES
thank you, it does really help me a lot... i like it.... (Y) <3
ReplyDeleteNot useful, need more details like type of sterilization methods used
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