Freshtrop Fruits Ltd (Unit-IV)

SOP: Detection of Heat Resistant Molds

1. Objective:
To check for the presence of heat resistant molds in finished product.
2. Scope:
This Standard Operating Procedure (SOP) is applicable for samples tested in microbiology
laboratory area of quality control department.
3. Responsibility:
Microbiologist, Quality Control (QC) head
4. Requirements:
A. Media and chemicals:
1. Malt Extract Agar
Composition: (Grams/lit)

Malt extract 3 gm
Mycological peptone 5 gm
Agar 15 gm
Final pH (At 25°C) 5.4+ 0.2

2. Phosphate buffered dilution water

Composition: (Grams/lit)
a. Stock solution:

KH2PO4 34 gm
Water 500 ml

Adjust pH to 7.1 with 1N NaOH. Bring volume to 1 liter with water.

OR

3. Butterfield’s Phosphate buffered dilution water.

Use- It is used for preparation of microbiological dilution blanks.
b. Working solution:
Take 1.25 ml of above stock solution and bring volume to 1 liter with distilled water
(Take 100 ml of buffer in a glass bottle / conical flask for each sample and sterilize it.)
B. Glassware and apparatus:
1. Scissor.
2. Sterile glass pipette of 10 ml capacity.
3. Water bath adjusted at 70°C.
4. Sterilized rubber bubbler.
5. Micropipette and sterile tips.
6. Sterile empty Petri plates.
Document No: FFL/QC-MB/SOP-37 Issue No. [Date]: 06 [02/01/2021] Revision No. [Date]: 02 [04/02/2022]
Page: 1 of 3
Prepared By: In-charge Quality Reviewed By: Head Quality Approved By: GM Operation
 Freshtrop Fruits Ltd (Unit-IV)

SOP: Detection of Heat Resistant Molds

5. Procedure:
A. Sample preparation and plating:
1. Prepare required media as per SOP for media preparation. (FFL/QC-MB/SOP-20)
2. Wipe the sample bag with 70% Iso propyl alcohol (IPA) & mix well before analysis.
3. Aseptically cut the sample bag and take 100 gm or (ml) of sample with sterile glass pipette.
4. Add the sample in 100 ml of sterile phosphate buffer and mix properly.
5. Keep the sample bottle in water bath at 70°C for 30 min to activate the spores if present.
6. Place a bottle with distilled water equipped with calibrated thermometer and with volume equal to the
test volume in the water bath.
7. Start to monitor the time only after the temperature of control sample reaches 70°C.
8. Remove the test sample bottle and cool rapidly to room temperature.
9. After incubation, take 1 ml of sample in 4 sterile Petri plates each and add 20 to 25 ml of sterile malt
extract agar cooled at 44 to 47°C into each Petri plate.
10. Carefully mix the sample with the medium by gently swirling the plate clockwise, anticlockwise, to
and fro and taking care that the contents do not touch the lid. Allow the mixture to solidify by
leaving the Petri plates, standing on a cool horizontal surface of laminar air flow (LAF).
11. Allow the media plates to solidify.
12. Invert the plates and incubate at 30°C±1°C for 30 days.
13. After incubation, observe all the plates for growth.

B. Observation:
Observe the plates for growth of molds.
C. Result interpretation:
Interpret and record the result as heat resistant molds “Present or absent”/100 gm or (ml) of sample.

D. Precautions:
1. Follow entry procedure in micro-testing area.
2. Give training to personnel, dealing with microbial material.
3. Wear gloves while performing analysis and sanitize used tips in beaker containing disinfectant.
4. If there is spillage of any sample the immediately clean it with 70% IPA to prevent contamination.
5. Do not work in illuminated Ultra Violet (UV) lamp.
6. Sanitize the lab daily and restrict repetitive entry in micro-testing area.
7. Fire extinguisher should be known by all laboratory personnel.

Document No: FFL/QC-MB/SOP-37 Issue No. [Date]: 06 [02/01/2021] Revision No. [Date]: 02 [04/02/2022]
Page: 2 of 3
Prepared By: In-charge Quality Reviewed By: Head Quality Approved By: GM Operation
 Freshtrop Fruits Ltd (Unit-IV)

SOP: Detection of Heat Resistant Molds

6. Annexure:
Not applicable

7. Reference:
1. Juice producers (IFU). Method no. 4. April 1996. p. 01-15
8. Revision History:

Date Revision No Reason for revision

04.02.2022 02 Added Butterfield’s phosphate Buffered dilution water

for preparation of microbiological dilution blanks.

Document No: FFL/QC-MB/SOP-37 Issue No. [Date]: 06 [02/01/2021] Revision No. [Date]: 02 [04/02/2022]
Page: 3 of 3
Prepared By: In-charge Quality Reviewed By: Head Quality Approved By: GM Operation