This video will demonstrate how to perform mold and yeast determination
using the Hydrophobic Grid-Membrane Filter method.
Here is the equipment you will need.
Disinfect the lab bench using 70% ethanol.
The agar plate preparation is not included here but it is worth noting that
the sterile antibiotic is prepared as follows:
Install the syringe filter onto the syringe.
Remove the plunger and transfer the antibiotic,
in this case chloramphenicol, into the syringe using Pasteur pipette.
Replace the plunger and slowly push the solution through the filter
and collect into a sterile microfuge tube.
Using a sterile pipette tip, pipette the appropriate amount
of sterile antibiotic to the warm potato dextrose agar media (~55°C).
Mix well before pouring the plates in the laminar flow hood, and leave dry.
Label the agar plates.
Don’t forget to label one of the agar plates as your agar control.
Weigh 10.00 g of apple puree and transfer into a sterile stomacher bag.
Add 90 ml of sterile peptone.
For apple leather, weigh 5.00 g of apple leather
and transfer to a sterile stomacher bag.
Add 40 ml of peptone and 5 ml of sterile pectinase stock solution.
Homogenize using the stomacher for 30 seconds.
Place the apple leather sample into a 42 degrees Celsius incubator for 30 minutes.
For the apple puree sample, aseptically transfer 1.00 ml of 10^(-1) apple puree dilution to
9 ml of peptone to give the 10^(-2) dilution.
Vortex the test tube briefly.
Prepare the sterile filtration apparatus as follows.
For the Y-connector, link a short tubing to
the vacuum flask, a long tubing to the vacuum pump,
and another short tubing with a Y-tubing valve.
Install the filtration unit in place and turn on the vacuum pump.
To begin, ensure that the Y-tubing valve on the short tubing is loosened.
You do not want a strong vacuum during the setup.
Open the filtration unit, use a sterile tweezer
to obtain an ISO-GRID membrane and place it on the unit.
Close the filtration unit and secure it by sliding the ISO-GRID clamp into place and
raising the clamp’s arm to lock in position.
Pipette 10.00 ml of sterile water then 1.00 ml of 10^(-1) apple puree dilution to the
pre-filter and allow to mix.
Tighten the Y-tubing valve slowly to draw the mixture onto the ISO-GRID membrane.
Completely close the Y-tubing valve so the vacuum will
draw the mixture through the membrane filter fully.
When complete, open the filtration unit with the vacuum on,
and aseptically transfer the ISO-GRID membrane
to the potato dextrose agar, with grid-side up.
Repeat filtration for the 10^(-2) apple puree dilution and 10^(-1) apple leather dilution.
Use a different sterile filtration apparatus for the apple leather sample.
When you are done, invert the plates, stack and tape together with proper labels.
Place the plates inverted in an incubator
at the appropriate temperature for the proper duration.
In this case, we incubate at 28 degree Celsius for 24–48 hours.
Properly dispose the samples into a large beaker containing bleach by the sink labelled
as “culture sample” and disinfect the lab bench.
Count the number of positive squares on each ISO-GRID membrane filter and record your results.