Penentuan Yeast Dan Molds Enumeration of Yeasts and Molds in Food - Dilution Plating Technique

PENENTUAN YEAST DAN MOLDS
Enumeration of Yeasts and Molds in Food--Dilution Plating

Technique
A. Equipment and materials

1. Basic equipment (and appropriate techniques) for preparation of
sample homogenate, see Chapter 1
2. Equipment for plating samples, see Chapter 3
3. Incubator, 25°C
4. Arnold steam chest
5. pH meter
6. Water bath, 45 ± 1° C

B. Media and Reagents
Media
1. Dichloran rose bengal chloramphenicol (DRBC) agar (M183)
2. Dichloran 18% glycerol (DG18) agar (M184)
3. Plate count agar (PCA), standard methods (M124); add 100 mg
chloramphenicol/liter when this medium is used for yeast and mold
enumeration. This medium is not efficient when "spreader" molds are
present.
4. Malt agar (MA)(M185)
5. Malt extract agar (Yeasts and Molds) (MEAYM) (M182)
6. Potato dextrose agar (PDA), dehydrated; commercially available
(M127)
Antibiotic solutions
Antibiotics are added to mycological media to inhibit bacterial growth.
Chloramphenicol is the antibiotic of choice, because it is stable under
autoclave conditions. Therefore, media preparation is easier and faster due to
the elimination of the filtration step. The recommended concentration of this
antibiotic is 100 mg/liter medium. If bacterial overgrowth is apparent,
prepare media by adding 50 mg/liter chloramphenicol before autoclaving and
50 mg/liter filter-sterilized chlortetracycline when the media have been
tempered, right before pouring plates.
Prepare stock solution by dissolving 0.1 g chloramphenicol in 40 ml distilled

water; add this solution to 960 ml medium mixture before autoclaving. When
both chloramphenicol and chlortetracycline are used, add 20 ml of the above
chloramphenicol stock solution to 970 ml medium before autoclaving. Then,
prepare chlortetracycline stock solution by dissolving 0.5 g antibiotic in 100
ml distilled water and filter sterilize. Use 10 ml of this solution for each 990 ml
of autoclaved and tempered medium. Refrigerate in the dark and re-use
remaining stock solutions for up to a month. Stock solutions should be
brought to room temperature before adding to tempered medium.
C. Procedures:
Sample preparation
Analyze 25-50 g from each subsample; generally, larger sample sizes increase
reproducibility and lower variance compared with small samples. Test
individual subsamples or composite according to respective Compliance
Program for the food under analysis. Add appropriate amount of 0.1%
peptone water to the weighed sample to achieve 10-1 dilution, then homogenize
in a stomacher for 2 min. Alternatively, blending for 30-60 sec can be used but
is less effective. Make appropriate 1:10 (1+9) dilutions in 0.1% peptone water.
Dilutions of 10-6 should suffice.
Plating and incubation of sample
Spread-plate method. Aseptically pipet 0.1 ml of each dilution on pre-

poured, solidified DRBC agar plates and spread inoculum with a sterile, bent
glass rod. DG18 is preferred when the water activity of the analyzed sample is
less than 0.95. Plate each dilution in triplicate.
Pour-plate method. Use sterile cotton-plugged pipet to place 1.0 ml

portions of sample dilution into prelabeled 15 × 100 mm Petri plates (plastic
or glass), and immediately add 20-25 ml tempered DG18 agar. Mix contents
by gently swirling plates clockwise, then counterclockwise, taking care to
avoid spillage on dish lid. After adding sample dilution, add agar within 1-2
min; otherwise, dilution may begin to adhere to dish bottom (especially if
sample is high in starch content and dishes are plastic) and may not mix
uniformly. Plate each dilution in triplicate.
From preparation of first sample dilution to pouring or surface-plating of final

plate, no more than 20 min (preferably 10 min) should elapse. Note: Spread
plating of diluted sample is considered better than the pour plate method.
When the pour plate technique is used, fungal colonies on the surface grow
faster and often obscure those underneath the surface, resulting in less
accurate enumeration. Surface plating gives a more uniform growth and
makes colony isolation easier. DRBC agar should be used for spread plates
only.
Incubate plates in the dark at 25°C. Do not stack plates higher than 3 and do
not invert. Note: Let plates remain undisturbed until counting.
Counting of plates
Count plates after 5 days of incubation. If there is no growth at 5 days, re-

incubate for another 48 h. Do not count colonies before the end of the
incubation period because handling of plates could result in secondary growth
from dislodged spores, making final counts invalid. Count plates containing
10-150 colonies. If mainly yeasts are present, plates with 150 colonies are
usually countable. However, if substantial amounts of mold are present,
depending on the type of mold, the upper countable limit may have to be
lowered at the discretion of the analyst. Report results in colony forming units
(CFU)/g or CFU/ml based on average count of triplicate set. Round off counts
to two significant figures. If third digit is 6 or above, round off to digit above
(e.g., 456 = 460); if 4 or below, round off to digit below (e.g., 454 = 450). If
third digit is 5, round off to digit below if first 2 digits are an even number
(e.g., 445 = 440); round off to digit above if first 2 digits are an odd number
(e.g., 455 = 460). When plates from all dilutions have no colonies, report mold
and yeast counts (MYC) as less than 1 times the lowest dilution used.
Isolate individual colonies on PDA or MA, if further analysis and species

identification is necessary.

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PENENTUAN YEAST DAN MOLDS

Enumeration of Yeasts and Molds in Food--Dilution Plating

A. Equipment and materials

Prepare stock solution by dissolving 0.1 g chloramphenicol in 40 ml distilled

Plating and incubation of sample

Spread-plate method. Aseptically pipet 0.1 ml of each dilution on pre-

Pour-plate method. Use sterile cotton-plugged pipet to place 1.0 ml

From preparation of first sample dilution to pouring or surface-plating of final

Count plates after 5 days of incubation. If there is no growth at 5 days, re-

Isolate individual colonies on PDA or MA, if further analysis and species

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