introduction:
total combined molds and yeasts count is one type of fungal purity testing, which is used to count the number of colony forming unit present in an article complying with monograph standards.
preparation of culture media:
sodium hydroxide 1n: dissolve 4.0 g of sodium hydroxide in water to make 100 ml.
hydrochloric acid 1n: dissolve 8.5 ml of hydrochloric acid in water to make 100 ml.
soyabean-casein digest broth medium (scdm)/ sabourauds dextrose agar or sabourauds chloramphenicol agar (sda): reconstitute dehydrated media as directed by the manufacturer and sterilize in an autoclave at 121°c for 20 minutes.
procedure:
sample preparation:
water soluble products: aseptically add 10g of specimen if it’s a solid or 10ml accurately measured, if the specimen is a liquid to make 100ml of sterile soybean casein digest broth medium unless otherwise specified in the individual standard testing procedure, shake and mix the sample preparation to achieve a homogeneous solution/suspension or dilute the specimen in a suitable quantity of diluter unless otherwise specified in the individual standard testing procedure.
water insoluble products: aseptically add 10g of specimen in 100ml of sterile soybean casein digest broth medium with 0.1ml of polysorbate 80, unless otherwise specified in the individual standard testing procedure. shake and mix the sample preparation to achieve a homogeneous solution/suspension or dilute the specimen in a suitable quantity of diluter unless otherwise specified in the individual standard testing procedure.
capsules: aseptically add 10g of capsules in 100ml of sterile soybean casein digest broth medium unless otherwise specified in the individual standard testing procedure, subject the dilution to a water bath at temperature nmt 45 ºc for 15 min. shake every 5 min to achieve a homogeneous solution/suspension or dilute the specimen in a suitable quantity of diluter unless otherwise specified in the individual standard testing procedure.
(note: alternatively sample prepared for the test total aerobic microbial count can be used)
pour plate method:
note: for specimen that is sufficiently soluble or translucent perform pour plate method, otherwise use the membrane filtration method.
test preparation:
pipette 1 ml of the final diluted sample into each of two sterile petri dishes.
transfer to each dish 15-20 ml of sabouraud dextrose agar or sabouraud chloramphenicol agar medium that previously has been melted and cooled to approximately 45°c.
cover the petridishes, mix the sample with agar by rotating the dishes and allow the contents to solidify at room temperature.
negative control:
transfer 15 to 20 ml of sabouraud dextrose or chloramphenicol agar medium without any diluents or sample.
cover the petri dishes and allow the contents to solidify at room temperature.
invert the petri dishes, and incubate at 20°- 25°c for 5-7 days.
after incubation, examine the plates for growth and record the results.
positive control:
pipette 1ml of <100 cfu/ ml of aspergillus niger or candida albicans culture suspension into a sterile petri dish.
transfer 15 to 20 ml of sabouraud dextrose agar or sabouraud chloramphenicol agar medium that previously has been melted and cooled to approximately 45°c.
cover the petridishes, mix the suspension with agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature.
incubate all the petridishes at 20-25°c for 5-7 days at inverted position.
after incubation, examine the plates for growth.
membrane filtration methods:
membrane filtration method only applicable to the sample where more than 1 ml of sample has to be used for total combined molds and yeast count.
arrange sterile filtration apparatus and place membrane with 0.45µm nominal pore size in to the filtration cup.
filter the sample in to membrane filter and rinse the membrane filter with appropriate volume diluent (100 to 300ml)
transfer the membrane filter to the surface of the sabouraud dextrose or chloramphenicol agar medium for total combined molds and yeast count (use one membrane filter).
incubate the plate at 20°- 25°c for 5-7 days.
after incubation, examine the plates for growth and record the results.
interpretation:
negative control should not show any growth.
growth should be present positive control.
select the plate corresponding to a given dilution and showing highest number of colonies less than 50 cfu/ plate, count the number of colonies and express the average for the two plates in terms of colony forming unit per gram or per ml of specimen.
if the count is carried out by the membrane filtration method record the results per sample quantity taken.
if the colonies of bacterial count are detected on the same medium, they are counted as part of total combined yeast and molds count.
if no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as "less than 10 micro-organisms per g or per ml of specimen".
