BHAVANS RESEARCH CENTER (MICROBIOLOGY) WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample APHA, 20th Edition

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1. IDENTIFICATION NUMBER: WH-001 2. SCOPE: To enumerate the number of microorganisms present in water sample by pour plate method. 3. TYPE OF SAMPLES TO BE TESTED: 1. Drinking water 2. Packaged drinking water 3. Packaged natural drinking water 4. Packaged mineral water 5. Water for processed food industry 6. Bottled water 4. PRINCIPLE: The general principal of this technique consists of inoculating a known volume of water sample in to a culture medium. Plates are prepared using a specified culture medium and a specified quantity of the test sample; other pairs of poured plates are prepared, under the same conditions, using decimal dilutions of the test sample. The plates are aerobically incubated at 37C for 48h. The number of microorganisms per ml of the sample is calculated from the number of colonies obtained on selected plates. 5. CULTURE MEDIA & DILUENT: 5.1 Diluent: Buffered peptone water (BRC Manual No LMB1) 5.2 Media: R2A agar (BRC Manual No SMR1) 6. APPARATUS AND GLASSWARE: 1. Autoclave 2. Weighing balance 3. Incubator (30C + 2C) 4. Vortex Mixer 5. pH meter 6. Sterile Petri dishes 90mm 7. St. Flasks 150 ml, 250 ml 8. St. Test tubes 9. Water bath at 55C + 1C 10. Colony Counter 11. Variable Microtiter Pipette (1000l & 100l) 12. St. Microtiter Tips (1000l & 200l capacity) 13. St. Scissors, St Spatula 7. REFERENCE STANDARDS AND REFERNCE MATERIAL:
Name of Laboratory Bhavans Research Center (Microbiology) Document Name and No. SOP WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample Issue no-- 02 Issue Dt- 1st Oct 10 Copy no 01 of 01 Approved by Amend no 00 Amend Dt- NA Prepared by Technical manager/ Technical Coordinator/ Scientist Technical Assistant

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BHAVANS RESEARCH CENTER (MICROBIOLOGY) WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample APHA, 20th Edition

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7.1 Reference Standard: Standard Methods for the Examination of Water and Waste Water, APHA, 20th Edition, 1998. 7.2. IS 1622: 1981 7.3 Reference Material: Escherichia coli ATCC 51755 8 DESCRIPTION OF PROCEDURE 8.1 Sample Preparation: 1. Samples are handled appropriately using aseptic techniques in sterile bags, clean instruments like scissors, spatula etc. either sterile or flamed with alcohol are used. 2. If the sample cannot be dealt with immediately maintain sample at temperature below 4C but do not freeze. Maximum elapsed time between collection and analysis must not exceed 24 h. 8.2 Test procedure 1. Carry out serial ten fold dilution of the test sample up to 10-5 2. Pipette in duplicates one ml of each dilution into sterile plates (labeled previously) and add approximately 10 - 12 ml of molten R2A agar held at 45C + 1C. Swirl the plates five to ten times. Allow the plates to solidify. 3. The time elapsing between the end of preparation of the initial suspension and the moment when the medium is poured into the dishes should not exceed 15 minutes. 4. Invert the prepared dishes and incubate them in the incubator at 37OC for 48h. 5. Count the colonies in each dish. Retain dishes containing fewer than 300 colonies at successive dilutions & calculate the number of microorganism per milliliter of the product. 6. Prepare a reference culture of Escherichia coli ATCC 51755 by introducing in a control sterile flask 10 to 100 cells (10-2 dilution of : 10l(0.1O.D.620nm) added to 100ml sterile saline ) per flask. Proceed with the control flask as the test culture to demonstrate that microbial culture is recovered. Prepare a control plate from the same batch of media, with 15ml of uninoculated medium to check its sterility. 9. EXPRESSION OF RESULTS: 9.1. General case Dishes containing colonies between 15 & 300. Calculate the number N of microorganism using the following equation: N= a V (n1 + 0.1n2) d Where, a: is the sum of the colonies on all the dishes from the two successive dilutions which contain at least 15 colonies, V: is the volume of inoculum applied to each dish, in milliliters. n1: is the number of dishes retained at the first dilution.
Name of Laboratory Bhavans Research Center (Microbiology) Document Name and No. SOP WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample Issue no-- 02 Issue Dt- 1st Oct 10 Copy no 01 of 01 Approved by Amend no 00 Amend Dt- NA Prepared by Technical manager/ Technical Coordinator/ Scientist Technical Assistant

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BHAVANS RESEARCH CENTER (MICROBIOLOGY) WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample APHA, 20th Edition

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n2: is the number of dishes retained at the second dilution. d: is the dilution corresponding to the first dilution retained.

9.1 Expression of small numbers of viable organism: If the two dishes, at the level of the test sample
(liquid product) contains less than 15 colonies, determine the arithmetical mean y of the colonies counted on two dishes, express the results as follows: For liquid products: estimated number of microorganisms per milliliter NE = y.

9.2 Expression of no detectable colonies: If the two dishes, at the level of the test sample (liquid
product) contains on visible colonies express the results as follows: Less than 1 microorganism per milliliter (liquid product) 10. DATA ENTRY FORMAT:

Template WH- 001 BRC TFA, Heterotrophic Plate Count of Water Sample
11. TEST REPORT: The test report shall specify the following:
REPORT Bipro no.: Received from: Name of the organization: Title of the test: Sample category: Sample description: Number of samples: Sample code: Date of receipt of samples: Date of analysis: Reference methods: Incubation conditions: Date of Report Making: Results of Analysis

Analyzed by

Verified by

Authorized by

Name of Laboratory Bhavans Research Center (Microbiology) Document Name and No. SOP WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample Issue no-- 02 Issue Dt- 1st Oct 10 Copy no 01 of 01 Approved by Amend no 00 Amend Dt- NA Prepared by Technical manager/ Technical Coordinator/ Scientist Technical Assistant

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BHAVANS RESEARCH CENTER (MICROBIOLOGY) WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample APHA, 20th Edition

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STATEMENT OF COMPLETION: Test results relate only to the sample(s) tested. This analysis has been performed to the best of our ability & our responsibility is limited to proven negligence. Our analytical findings reflect the quality of the same at the time of testing. No responsibility can be expected for the possible consequences of further development of micro organisms which may depend upon storage, handling & weather conditions which may influence the results at a later date/time. STATEMENT OF SAMPLE RETENTION: Samples will be retained for a period of 30 days only, unless specific instructions to the contrary are received. A statement specifying that the test report shall not be reproduced except in full, without the written approval of the laboratory.

Annexure:

Template WH- 001 BRC TFA, Heterotrophic Plate Count of Water Sample

Name of Laboratory Bhavans Research Center (Microbiology) Document Name and No. SOP WH001 BRC-SAF, Heterotrophic Plate Count of Water Sample Issue no-- 02 Issue Dt- 1st Oct 10 Copy no 01 of 01 Approved by Amend no 00 Amend Dt- NA Prepared by Technical manager/ Technical Coordinator/ Scientist Technical Assistant

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