I.
Purpose: Enumeration of Total
Plate count in Animal feed, Bakery and Confectionery products, Hard
boiled sugar confectionery, Candies, Gum base, Chewing and Bubble gum, Bread
Honey and Honey products, Food additives, Preservatives and antioxidants, Gums,
Fruits and vegetables products, Canned fruits, Dried fruits and vegetables
powder, Industrial cultures (Bakers yeast), Fruits & vegetable products,
Beverages, Milk, Sweetened milk, Dairy products, cereals and pulses, malt
extract, wheat flour, energy food, flour products, meat and meat products,
snacks, processed foods etc.
II. Reference:
1. IS:
5402:2002/ ISO 4833:1991
2. IS: 10232:2003/ ISO: 6887:1999
3. USFDA-BAM Chapter 3
III. Apparatus and Equipment
Oven (Dry sterilization),
Autoclave (wet sterilization), Water bath (45°C ±
0.5°C),
Incubator (30°C), Incubator (35°C),
Total delivery pipette (1ml), Colony counter, pH meter, petridishes 90mm, test
tubes, flasks or bottles.
IV. Media/Reagents
1. Plate
count agar
2. Phosphate
Buffer
V. Procedure
1. Weigh
25 g or 10 g of sample in 225 ml or 90 ml of sterilized phosphate buffer (IS:
15188) using a sterile spatula.
2. Serially
dilute the sample and plate in replica within 15 min. In case of liquid samples, pipette out
directly 1ml of sample.
3. Pour
10 to 15 ml of PCA Agar (45 °C ± 0.5°C);
mix well by rotating clock wise and anticlock wise. Let the mixture stand till solidified and
then place inverted plates (not more than 4 in one pile) at 30 ± 1°C for
72 hr ± 3hr.
However, as per BAM procedure, incubate the plates at 35°C for 48 ± 2hr
VI (A). Calculation performed (BIS method)
Count the isolated visible
colonies using colony counter, the colonies in each plate containing not more
than 300 colonies.
a) General case-
Petridishes containing between 15 and 300 colonies
Retain dishes containing not
more than 300 colonies at two consecutive dilutions. It is necessary that one
of these dishes contain at least 15 colonies.
Calculate the number N of
microorganisms per milliliter or per gram of product, using the following equation:
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∑ C
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N =
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(n1 + 0.1 n2)
x d
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Where
∑ C = Sum
of colonies counted on all the dishes retained
n1 =
number of dishes retained in the first dilution
n2 =
number of dishes retained in the second dilution
d = dilution
factor corresponding to the first dilution
Round the result calculated to two significant figures.
b) Estimation
of small numbers
If the two dishes, contains
less than 15 colonies calculate the arithmetic mean m of the colonies counted
on both dishes. Report the result as follows:
- Estimated number of
microorganisms per milliliter
NE = m (liquid
products)
-Estimated number of
microorganisms per gram:
-
NE = m x d-1 (other
products), where d is the dilution factor of the initial suspension.
c) No
colonies
If the two dishes corresponding to the test sample
(liquid products) or the initial suspension (other products) contain no
colonies, report the result as follows:
- Not
detected where the detection limit is more than 1 cfu/ml in case of liquid
samples and more than 10 cfu / gram in case of solid samples.
VI. (B) Calculation performed (BAM method)
Select
spreader free plate(s). Count all colony-forming units. Record dilution (s)
used and calculate the aerobic plate count using the formula given above.
All
Plates with more than 250 colonies:
Record
the count as Too Numerous To Count (TNTC). When plates from two dilutions yield
more than 250 cfu each but fewer than 100/cm2, estimate the aerobic
counts from the plates (EAPC) and multiply by dilution factor.
Spreaders
Ø Chain
of colonies, not too distinctly separated that appears to be caused by
disintegration
Ø One
that develops in a film of water between agar and bottom of dish
Ø One
that develops in a film of water at edge or on surface of agar
If the area covered by spreaders including total area of repressed
growth exceeds 50 % of plate area, report plates as spreaders. When it is
necessary to count, count each chain as a single colony and each source as one
colony. Combine the spreader count and compute APC (Aerobic plate count).
Plates with no CFU
When plates from all dilutions have no colonies, report APC as less
than 1 times the corresponding lower dilution.
Laboratory accident
When plate(s) from a sample known to be contaminated or otherwise
satisfactory, record the results as laboratory accident.
All Plates with fewer than 25 CFU
When plates from both dilutions yield fewer than 25 CFU each, record
actual plate count and expressed the count as less than 25 x 1/d when d is the
dilution factor for the dilution from which the first counts were observed.
VII. Remark:
Limits specified as per the specifications.
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