GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL COMBINED MOLDS AND YEASTS COUNT
GTP No.
GTP014-00
Effective Date
Reference
USP/EP/BP/JP
Page No.
1.
1 of 3
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.
2.
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.
3.
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.
GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL COMBINED MOLDS AND YEASTS COUNT
GTP No.
GTP014-00
Effective Date
Reference
USP/EP/BP/JP
Page No.
2 of 3
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.
GENERAL TESTING PROCEDURE
TITLE: TEST FOR TOTAL COMBINED MOLDS AND YEASTS COUNT
GTP No.
GTP014-00
Effective Date
Reference
USP/EP/BP/JP
Page No.
3 of 3
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.
4.
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".