Page 144 - phytochemistry general program

Modern plant tissue culture is performed under aseptic conditions under filtered
air, using laminar air flow cabinets. The lab should be a clean area and cleaned on
regular basis. Living plant materials from the environment are naturally

contaminated.

Surface sterilization of staring materials (explants) in chemical solutions (usually
alcohol or bleach) is required.

Media, glassware and metallic devices all must be sterilized.

Thermolabile substances sterilized by ultrafiltration.

Sterilization protocols used in P.T.C.

The different methods can be classified as follows:

a- Dry heat

This method is used only for glasswares metal surgical instruments or other
materials that are not charred by high temperatures. A moderate temperature,
approximately 160 oC (320 oF) for a period of 4 hours is sufficient for sterilization.
The objects are carefully wrapped in heavy-duty aluminum foil before being placed

in the oven.

b- Wet heat

This procedure employs an autoclave operated with water vapour under pressure.
A steam pressure of 15 lb/in2 and a temperature of 121oC (250oF) is applied for 20
min. do not start timing the 20 min. period till the autoclave has reached the proper

temperature. This method is used for the sterilization of stable liquid media.

c- Ultrafiltration

This method is used for the sterilization of heat labile (thermolabile) substances
such as proteins, some growth hormones, vitamins and sugars. Solutions of the
thermolabile substances must be sterilized by ultrafiltration at room temperature,

then added to the autoclave medium with a sterile pipette.

There are several different kinds of ultrafilters, the most common are:

i- Sintered glass filters but they are both difficult to clean and expensive.

ii- The nuclepore filter; this is made of polyethylene film punctured with holes of a
given diameter. They are cheaper and disposable. Pore size of these filters are

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