Document No:

F. A.S.T.
TEST METHODS TM-
Effectivity Date:
MANUAL
SECTION: Revision No.:
Microbiological Analysis of Dairy Products 0
LABORATORIES SUBJECT: Pages:
Coloring, Flavoring or Other Added Material 1 of 2

I. Reference: Standard Methods for the Examination of Dairy Products

16th ed. Chapter 9. Microbiological Methods for Dairy Products

II. Responsibility: Microbiologist

III. Scope : This is applicable to coloring, flavoring, extracts or other added materials such as nuts,
cake, pastry, confection, or fruits for the determination of standard plate count, and
coliform count.

IV. Principle: Known dilutions of sample are allowed to grow on specific media at suitable temperature
and period of time.

V. Apparatus: 1. Incubator
2. Water bath
3. Autoclave
4. Toploading balance
5. Colony counter

VI. Reagents : 1. Phosphated dilution water

2. Standard methods agar
3. Violet red bile agar

VII. Methodology:

A. PREPARATION OF SAMPLE

1. Heat sterile 99 ml phosphate dilution water to 40 - 45C.

2. Weigh 1 g of well-mixed dry colors into a sterile 99 ml dilution water and mix thoroughly. Owing
to the inhibitory properties of some components of extracts and flavors, more colonies may appear
on plates of higher rather than lower dilutions.
3. Weigh representative 11-g portions of nuts, cake, pastry, confection, or fruits directly into a wide-
mouthed container before adding 99 ml of dilution water.
4. Allow the mixture to soak for 5 minutes and shake vigorously, then plate.
5. Prepare serial dilutions as needed.

B. STANDARD PLATE COUNT

1. Pipet 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes.
2. Pour the inoculated plates with 12 – 15 ml of tempered Standard Methods Agar and mix
thoroughly all samples or dilutions by making about 25 complete up-and-down or back-and-forth
movements.
3. Allow agar to solidify.
4. Invert and incubate plates at 32  1C for 48  3 hours.
5. Count plates with 25 – 250 colonies and multiply by corresponding dilution.
6. Record the computed count and report as colony-forming units ( CFU ) per gram or per ml
 Document No:
F. A.S.T.
TEST METHODS TM-
Effectivity Date:
MANUAL
SECTION: Revision No.:
Microbiological Analysis of Dairy Products 0
LABORATORIES SUBJECT: Pages:
Coloring, Flavoring or Other Added Material 2 of 2

sample.

C. COLIFORM COUNT

1. Pipet 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes.
1. Pour the inoculated plates with 12 – 15 ml Violet Red Bile Agar and allow to solidify.
2. Overlay an additional 3 – 5 ml of VRBA to inhibit surface growth and spreading of colonies.
3. Allow to set. Invert and incubate at 32º  1C for 18 – 24 hours.
4. Count purple-red colonies that are 0.5 mm larger in diameter and surrounded by zone of
precipitated bile acids.
5. Multiply the colonies counted by the corresponding dilution.
6. Record the computed count and report as colony-forming units ( CFU ) per gram or per ml
sample.

VIII. Sterility Controls :

Check sterility of agar, dilution water, pipets and air in plating area by pouring control plates with
specific media.