EXPERIMENT – 2:MAINTENANCE OF ASEPTIC

CONDITIONS

AIM: Study of sterilization technique and aseptic manipulation.

Following precaution are to be taken to avoid microbial contamination:

• Avoid atmospheric air currents which carry spores of contaminated organisms
in sterile area.
• Remove shoes before entering the culture room.
• Wash your hands before entering the culture room.
• Avoid spoilage of medium.
• Clean lab tables to avoid growth of bacteria and fungi.
• Keep laboratory neat and clean.

Washing of glassware:
• Destroy all autoclaved contaminated cultures and remove contamination if
any present.
• Transfer liquified material in a container and transfer the glassware to a crate
& them wash thoroughly.
• Transfer the glassware in another crate containing hot water & rinse them
properly.
• Rinse 3 times under tap water.
• Rinse in distilled water.
• Place glassware in oven for drying.
• Store the glassware properly in a suitable place.
• Dispose the discarded material carefully.

Sterilization of media and instruments:

• Dry heat sterilization (oven sterilization) – glassware, metal instruments &
other materials that are not cleaned by high temperature can be sterilized by
dry heat.
 TEMPERATURE TIME
160oC 45mins or 2-4hrs
170oC 18mins
180oC 1-5 mins
190oC 1-5 mins

For moderate sterilization, oven is set at 160oC for 2-4 hrs.

Wet sterilization (steam sterilization) –

1) Autoclaving :- Steam under pressure is used for sterilization in autoclave while
paper products, glassware, instruments and liquid are sterilized at steam
pressure of 15lb/sq. at a temperature of 121oC (150oF) if applied for 15mins.
The minimum sterilization time increases with an increase in liquid volume. Do
not record timing after sterilization period until the autoclave or pressure
cooker has reached the proper temperature & all residual air has been
displaced by steam. At the end of sterilization period, the pressure must be
permitted to return slowly to atmospheric level. Rapid decompression forces
the liquid to boil out the vessels and containers.

VOLUME OF MEDIA PER VESSEL (mL) MINIMUM AUTOCLAVE TIME (mins)

25 20
50 25
100 28
500 35
1000 40
2000 48
4000 63

2) Boiling water bath :- It may also be used for heat sterilization equipment
where material is placed in boiling water containing sodium bicarbonate (2%
w/v) for 20mins.

3) Ultracentrifugation :- Some media components are unstable at high

temperature & must be sterilized by ultracentrifugation at room temperature.
Usually a small weight is sterilized by passage through membrane filtration
unit attached to a graduate syringe. The appropriate volume of the sterile
liquid is added directly to autoclaved medium. If an agar medium is employed
this is done while agar is warm & in the solid state. For volume a large filter
 holder equipped with vacuum pressure is used. Millipore membrane filters
with 0.22micrometre pore size should be used for the removal of complete
organisms. Following chemical which are thermolabile need to be filter
sterilized: IAA (Indole Acetic Acid), calcium pantothenate, GA3 (Gibberellic
Acid), zeatin, IBA (Indole Butyric Acid), kinetin libase, colchicum, urea, ascorbic
acid, riboflavin, tryptophan, cellulose, pectinase, macro zyme, zip.

4) Working with LAF cabinet :- Put on the UV for at least 15mins to destroy
organisms in the cabinet. Spray the cleaner with 70% ethyl alcohol and surface
with cotton plug moistened with 70% alcohol. Put on the airflow.

Surface sterilization of plant material can be done by rinsing the plant material in
70%-80% ethanol for 1min followed by the sterilization of material with the following
chemicals.
CHEMICAL CONCENTRATION DURATION (in mins)
Calcium hypochloride 9% - 10% 5 to 30
Sodium hypochloride 2% 5 to 30
Hydrogen peroxide 10% - 20% 5 to 15
Bromide water 1% 2 to 10
Silver nitrate 0.1% - 1% 5 to 30
Mercury chloride 0.1% - 10% (mL/L) 2 to 10
Antibiotics 7% - 15% 30 to 60

Take due care to avoid health hazards:

• Never look at the UV tube with naked eyes. A glass barrier between UV and
eye provide complete protection.
• UV radiation can produce irritation to unprotected skin so avoid placing hands
in the transfer chamber when UV is on.
• Mercuric chloride is an extra dangerous poison. A solution of HgCl2 is slightly
volatile at room temperature which may be able to poison the worker.
• Hypochloride solution should be used with care. Inhalation can produce
bronchial irritation & skin contact can be harmful. Never pipette hypochloride
with your mouth. Never use hypochloride in presence of UV because the
resulting chlorine gas is a serious health hazard.
• Hamming instrument in alcohol may cause fire danger. Ethanol is highly
inflammable gas & thus be careful about spilling it.