Advance preparation
Day 1
1. Set up and autoclave the NCBE
Bioreactor as described in the User Guide, using 450
ml of
starch yeast
broth. After autoclaving, allow the
vessel to cool, and attach sterile syringes
etc. as instructed in ther Bioreactor
manual.
2. Prepare an inoculum of Saccharomyces
diastaticus in a McCartney bottle containing 20
ml of
starch yeast broth. Incubate at 28-30
oC.
Day 2
3. Place the Bioreactor in a water
bath at 28oC. Allow the
broth inside to warm to this
temperature, then inoculate the
Bioreactor using the culture prepared in (2) above.
Running the Bioreactor
4. Bubble air through the culture medium to ensure
that the conditions within remain aerobic.
Add antifoam
as required to prevent undue frothing. Take care to ensure
that the air
filters do not become blocked
with liquid.
Monitoring starch breakdown
5. Take small samples of broth from the Bioreactor
at regular intervals, using the method
described in the Bioreactor manual.
6. Put the sample into a centrifuge
tube, cover with Cling film to prevent
the formation and
escape of aerosols. Spin
down the yeast cells in a properly-balanced centrifuge. Take some
of the cell-free
supernatant to test for glucoamylase activity.
7. Add an equal volume of 1%
starch solution to the cell-free
broth. Incubate at 30oC
for 6
hours
or overnight if more convenient.
8. Use a semi-quantitative glucose
test strip to measure the concentration of glucose in the
reacted mixture.
9. Dispose of used test samples and glucose test strips
into disinfectant.
Safety
Standard microbiological safety procedures, including aseptic techniques, must be observed by teachers, technicians and students when carrying out this work.
Particular care should also be taken when using the centrifuge, and steps such as those described above should be taken to prevent the formation of aerosols.