1. Dip a 5 mm diameter cork
borer into alcohol. Set fire to the
alcohol and let it burn away.
Take care to hold the borer horizontally
while doing this so that flames do not travel up the
centre of the borer and burn
your hand.
2. Hold the lid of a CMC agar plate
slightly to one side and use the borer to cut a well
in the
agar.
Remove the agar plug from the borer if necessary using a mounted needle.
3. Repeat steps 1 and 2 so that you have two wells in the agar.
4. Label each well on the base of the Petri
dish. A suitable code would be: C (Cellulomonas);
W (sterile water,
a 'control').
5. Into the appropriate well place 0.2
ml of either microbial culture or sterile
water, using a
separate sterile
syringe for each. Place the syringes, as they are used, in a
beaker of
disinfectant.
6. Incubate the plates for up to a
week at 25-30oC. Cellulomonas
will produce clear zones up
to 16 mm
in diameter after 48 hours at 30oC.
After incubation
1. Flood the plates with Congo red
solution for 15 minutes, then de-stain
with the salt solution for 10-15
minutes. Unstained areas indicate where the CMC
has been broken down to b-1-4-glucans
that contain seven or fewer glucose residues. The diameter of the
clear zone can be measured to provide a
quantitative comparison of cellulolytic activity.
Safety
Standard microbiological safety
procedures, including aseptic techniques,
must be observed by teachers, technicians and students when carrying
out this work.