To perform sterilization of glassware, preparation and sterilization of media

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Aim – To perform sterilization of glassware, preparation and sterilization of media.

Requirements:

  • Instrument: Hot air oven and Autoclave.
  • Glassware’s: Beaker, Conical flask, Glass rod, Petridish.
  • Chemicals: Agar, Peptone, beef extract, glucose.

Theory:

All living forms and other biological agents, such as fungi, bacteria, viruses, spore forms, prions, and unicellular eukaryotic organisms like plasmodium, are eliminated, removed, killed, or rendered inactive through sterilisation. Heat, chemicals, irradiation, high pressure, and filtration are all methods of sterilisation. Sterilization can be defined as the process of purging an item, surface, or medium of all live organisms, including those in the vegetative and spore states.

Microorganisms can develop on culture media with the aid of nutrients and under the control of physical growth conditions. In fact, many bacteria are unable to grow in any known culture medium, and not all microorganisms can grow in a single culture medium. Obligate parasites are some organisms that cannot develop in artificial culture medium. Examples include Chlamydia, Rickettsia, Mycobacterium leprae, and Treponema pallidum.

Procedure:

Sterilization of Glassware’s by Hot Air Oven – Firstly, wash all the glassware before sterilization process. Articles to be sterilized are first wrapped or enclosed in containers of cardboard, paper or aluminium. Then place all glassware’s in hot air oven. Then, the materials are arranged to ensure uninterrupted air flow. Maintain the ideal temperature for killing the microorganisms at 160°C for 45 minutes or 170°C for 18 minutes or 180°C for 7.5 minutes etc. The temperature is allowed to fall to 40°C, prior to removal of sterilized material.

Sterilization of Glassware’s by Autoclave – Firstly, wash all the glassware before sterilization process. Then place all glassware’s (surgical dressings, sheets, surgical and diagnostic equipments, containers, closures, aqueous injections, ophthalmic preparations etc.) in the autoclave. Autoclave uses steam to kill the microorganisms by coagulation mechanism. Maintain the optimum temperature and pressure –

S.NOTEMPERATUREPRESSURE LB/SQ.INCHMINIMUM HOLDING TIMES(IN MINUTES )
1.115-1161030
2.121-1231515
3.126-1292010
4.134-138323

Preparation and Sterilization of Culture Media (Nutrient Agar Media)

  1. Weigh all the ingredients accurately.
  2. Take 1.5gm agar-agar in a conical flask and add 100 ml water in it and heat it till the agar-agar dissolves in water.
  3. Then, add 0.5 gm peptone and 0.3 gm beef extract in it and mix well.
  4. After that add 0.25 gm glucose and maintain the pH of the medium to 7.2pH.
  5. Allow to stand for some time for solidification.
  6. After solidification the conical flask in closed with the help of cotton plug and wrapped with the help of brown paper before autoclaving (Sterilization).
  7. Then place the culture media into the autoclave for sterilization for 15 minutes at 121-123°C.
  8. After sterilization allow to cool the autoclave.
IngredientQuantity Taken
Peptone0.5 gm
Beef Extract0.3 gm
Agar-agar1.5 gm
Glucose0.25 gm
WaterUpto 100ml

Result: The sterilization of glassware, preparation and sterilization of media was performed.

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