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

 Experiment

Aim: 

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

Requirements: 

Erlenmeyer flask (conical flask),test tube, beaker, petridish, glassrod, cotton etc

•Peptone: 5.0 gm

•Beef extract: 3.0 gm

• Sodium chloride: 5.0 gm

•Agar: 15.0 gm

•Sterile water: 1000 ml

Instruments: Hot Air Oven, Autoclave, weighing balance, Water bath.

Theory:

Sterilizing methods for glassware in a laboratory:

HOT AIR OVEN :

Sterilizing glassware such as bottles, Petri dishes and test tubes, dry heat is required and this is carried out in a hot air oven. The ideal temperature of the oven needs to reach at least 160℃ and the glassware need to regulated at time period of 45 to 50 min . The glassware must not be removed from oven immediately as a slow cooling period is necessary when the temperature has reduced down 50℃.

AUTOCLAVING :

Autoclave are widely used to sterilize instruments, glassware and plastic ware, solutions and media and to decontaminate biological wastes. Because of the physical hazards associated with autoclaving, extra care must be taken to ensure their safe use. The following safety practices are followed when autoclaving laboratory glassware:

◆Never autoclave items containing corrosives or radio active materials.

◆Use only borosilicates, glassware’s, which can be better withstand the stresses of high autoclave temperatures and pressures.

◆Load the auto clave properly as per the manufacturers recommendations.

◆ Individual glassware vessels should be placed within a heat resistant plastic or metal tray on a shelf or rack and never placed directly on the autoclave bottom or floor. 

◆Add ¼ to ½ inch of water to the tray so the glassware will heat more evenly.

◆ Check any plastic caps, tubing or other items to ensure they can be safely autoclaved with the glassware.

◆Fill glassware only half full with liquids to be sterilized. Take into account the volume of liquid to be autoclaved.

PREPARATION AND STERILIZATION OF CULTURE MEDIA

1. The constituents or ingredients of that particular medium are weighed accurately

(i.e.Peptone: 5.0 gm, Beef extract: 3.0 gm, Sodium chloride: 5.0 gm, Agar: 15.0 gm)

2. The required amount of water is measured (i.e. 1000ml) and poured into stainless steel pan or heat resistant beaker and the weighed ingredients are putted one by one into it.

3. The pan is kept on heat source and each ingredients is gradually dissolve by constant stirring with glass rod and heat is reduced to prevent boiling over or burning of the medium.

4. Small amount of preparation is taken out, cooled and pH is checked and if necessary pH is adjusted.

5. The medium is now poured into test tubes usually in 10 ml aliquots in 500 ml erlemeyer flasks (conical flask) or other containers. While in liquefied state, solid media can be added in test tubes, which are allowed to cool and harden in a slanted position producing agar slants.

6. The container are now closed with cotton plugs, screw caps or cap all covers.

7. The medium is now sterilized in an auto clave usually at 121°C for 15 min and allowed to cool to be used for the purpose.

Result:

 The sterilization of glassware and the culture media was done successfully.