01 Bacteria Yeast and Mold Count Qcg0001

OASIS LABORATORIES PRIVATE LIMITED, DEHRADUN
SOP No.:
QUALITY CONTROL DEPARTMENT Page 1 of 4
QCG0001
Supersedes : Effective:
STANDARD OPERATING PROCEDURE
00 April 2010
MICROBIL TESTING PROCEDURE FOR
Version No. : Review date:
TOTAL BACTERIAL, YEAST AND MOLD
01 March 2013
COUNT
1.0 OBJECTIVE: To describe the procedure for microbial testing for total
bacterial, yeast and mold counts in raw materials and
finished products.
2.0 SCOPE : Bacteria, Yeast and mold count.
3.0 RESPONSIBILITY : Q.A. / Q.C INCHARGE OFFICER
4.0 PROVEDURE :
Total Bacterial Count
Reagent : Buffer solution or 0.1% peptone solution.
Dissolve 3.56 gm of potassium dihydrogen orthophosphate 4.30 gm sodium chloride,

7.23 gm of sodium orthophosphate and 1.0 gm peptone in 1.0 liter distilled water. Or
1.0 gm peptone in 1.0 liter distilled water. Adjust pH 7.0 of above solution by o.1 N
NaOH or dulute orthophosphoric acid as required.
Distribute 90 ml of buffer solution or 0.1% peptone buffer in 2.50 ml conical flask or

150 ml test tube. Plug conical flask and test tube with nonabsorbent cotton plug.
Autoclave all flask and test tubes at 15 Ib. pressure for 20 minutes.
Soyabaean casein digest agar preparation. Prepare soyabean casein digest agar as
per standard operating procedure.
Cool agar at 40 C in a water bath before use for pour plates.
Method : Transfer aseptically 10 gm/ml sample as per requirement in to a 90 ml

Sterile buffer / 0.1% peptone solution containing flask.
Designation Signature Date
Prepared by Chemist - QC
Reviewed by Manager - QC
Approved by Manager - QA
SOP No.:
QCG0001
00 April 2010
01 March 2013
COUNT
Mix will to dissolved or suspend the sample.
Above dilution is 1:10.
Transfer aseptically 10 ml of above dilution i.e.1:10 to other 90 ml of sterile buffer /0.1

peptone solution containing flask.
Mark with marker pen on petridish for respective dilute transfer aseptically 1.0 ml
solution from 1:10 and 1:100 dilution to the sterile petridishes in duplicate.
Pour 20 ml of sterile agar in above mark petridish when temperature of sterile agar is
40 C aseptically.
Mix and allow the media to solidify at room temperature.
Invert the petridishes and incubator at 30 to 35 C for 48 to 72 hours in incubator.
After incubation period, examine the plates for growth.
Count the number of bacterial colonies in all the plates.
Express the average of two plates in terms of the number of bacteria per gm/ ml of
sample. If no growth is observed for two dilutions i.e.1:10 & 1:100; express the
results not more then 10 bacteria per 10 gm or 10 ml of sample.
Note : If count is higher then 300 colonies per plates dilute the sample further to
1:1000 or more.
Total yeast and mold count
Reagent : Buffer solution or 0.1 % peptone solution.

Dissolve 3.56 gm of potassium dilrydrogen orthophosphate 4.30 of sodium
chloride, 7.23 gm of sodium orthophosphate & 1.0 gm peptone in 1.0 lit. Distilled
water.
Or
1.0 gm peptone in 1.0 liter distilled water. Adjust pH 7.0 of above solution by 0.1 N
NaOH or dilute orthoposphoric acid as required.
SOP No.:
QCG0001
00 April 2010
01 March 2013
COUNT
Distribute 90 ml of buffer solution or 0.1 % peptone buffer in 2.50 ml conical flask

or 1.50 ml test-tube.
Plug conical flask & test-tube with nonabsorbent cotton plug.
Autoclave all flask & test-tube at 15 Ib pressure for 20 minutes.
Sabouraud Dextrose Agar Preparation: Prepare sabouraud dextrose agar as per

standard operating procedure.
Cool agar at 40 - 45 c in a water bath before use for pour plates.
Method: Transfer aseptically 10 gm sample as per requirement in to a 90 ml

sterile buffer /0.1% peptone solution containing flask.
Mix well to dissolve or suspend the sample.
Above dilution is 1:100
Transfer aseptically 10 ml of above dilution i.e. 1:10 to other 90 ml of sterile buffer

0.1 % peptone solution containing flask.
Mix well, dilution is 1:100
Mark with marker pen on petridish for respective dilutions.

Transfer aseptically 1 mi solution from 1:10 & 1:100 dilution to the sterile
petridishes in duplicate.
Pour 20 ml of sterile agar in above mark petridish when temperature of sterile

agar in 40 to 45C aseptically.
Mix & allow the media to solidify at room temperature for 48 to 72 hours in
incubator. Invert the petridishes & incubate at 30 to 35 C for 48 to 72 hours in
incubator. After incubation period examine the plates for molds & yeast growth.

SOP No.:
QCG0001
00 April 2010
01 March 2013
COUNT
Count the number of mokis & yeast colony in all the plates.
Express the average of two plates in terms of the number of molds & yeast per
gm of sample.
If no growth observed for two dilutions i.e. 1:10 &1:100 express the results not
more then 10 molds & yeast per 10 gm of sample
Note : If count is higher then 300 colonies per plates dilute the sample feather to
1:1000 or more.


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OASIS LABORATORIES PRIVATE LIMITED, DEHRADUN

2.0 SCOPE : Bacteria, Yeast and mold count.

3.0 RESPONSIBILITY : Q.A. / Q.C INCHARGE OFFICER

Total Bacterial Count

Reagent : Buffer solution or 0.1% peptone solution.

Dissolve 3.56 gm of potassium dihydrogen orthophosphate 4.30 gm sodium chloride,

Distribute 90 ml of buffer solution or 0.1% peptone buffer in 2.50 ml conical flask or

Cool agar at 40 C in a water bath before use for pour plates.

Method : Transfer aseptically 10 gm/ml sample as per requirement in to a 90 ml

Mix will to dissolved or suspend the sample.

Above dilution is 1:10.

Transfer aseptically 10 ml of above dilution i.e.1:10 to other 90 ml of sterile buffer /0.1

Mix and allow the media to solidify at room temperature.

Invert the petridishes and incubator at 30 to 35 C for 48 to 72 hours in incubator.

After incubation period, examine the plates for growth.

Count the number of bacterial colonies in all the plates.

Total yeast and mold count

Reagent : Buffer solution or 0.1 % peptone solution.

Distribute 90 ml of buffer solution or 0.1 % peptone buffer in 2.50 ml conical flask

Plug conical flask & test-tube with nonabsorbent cotton plug.

Autoclave all flask & test-tube at 15 Ib pressure for 20 minutes.

Sabouraud Dextrose Agar Preparation: Prepare sabouraud dextrose agar as per

Cool agar at 40 - 45 c in a water bath before use for pour plates.

Method: Transfer aseptically 10 gm sample as per requirement in to a 90 ml

Mix well to dissolve or suspend the sample.

Above dilution is 1:100

Transfer aseptically 10 ml of above dilution i.e. 1:10 to other 90 ml of sterile buffer

Mix well, dilution is 1:100

Mark with marker pen on petridish for respective dilutions.

Pour 20 ml of sterile agar in above mark petridish when temperature of sterile

Designation Signature Date

Designation Signature Date

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