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Enumeration SOP-Subtilis

This document outlines the standard test procedure for determining the total viable count of Bacillus subtilis (P3) in a sample, detailing the necessary equipment, preparation steps, and analysis methods. It specifies the responsibilities of the Quality Officer and Head of Operations, and includes guidelines for calculating colony forming units (CFU). The procedure ensures accurate measurement and reporting of bacterial counts in samples.

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irshad
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2 views

Enumeration SOP-Subtilis

This document outlines the standard test procedure for determining the total viable count of Bacillus subtilis (P3) in a sample, detailing the necessary equipment, preparation steps, and analysis methods. It specifies the responsibilities of the Quality Officer and Head of Operations, and includes guidelines for calculating colony forming units (CFU). The procedure ensures accurate measurement and reporting of bacterial counts in samples.

Uploaded by

irshad
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Document Number

STANDARD TEST PROCEDURE PELL/STP/


011_2.0
DETERMINATION OF TOTAL Document Level
COLONY FORMING UNITS P3 03

1 Objective
To determine the total viable count for Bacillus subtilis (particularly
P3) in a given sample.
2 Scope
This document describes the procedure of carrying out total viable
count for Bacillus spp. reported in billion CFU/gm.
3 Responsibility
Quality Officer
4 Accountability
Head of Operations
5 Abbreviation and Definition
CFU: Colony Forming Unit
6 Procedure
6.1 Requirements
6.1.1 Autoclave
6.1.2 Incubator at 30°C
6.1.3 Weighing balance
6.1.4 Laminar air flow cabinet (PSI)
6.1.5 Calibrated micropipettes and tips (Abdos)
6.1.6 Vortex mixer
6.1.7 Sterile petriplates (90 mm diameter) (Tarsons)
6.1.8 Peptone Bacteriological (Finar)
6.1.9 Peptone Diluent for initial suspension and serial dilution (Annexure
9.1)
6.1.10 Plate count agar (Himedia) (Annexure 9.2)
6.2 Preparation of Peptone diluent
6.2.1 Prepare the diluent as mentioned in Annexure 9.1.
6.2.2 Transfer 400 ml of the Peptone diluent to media bottles and cap bottle
loosely.
6.2.3 Autoclave for 20 min at 122 °C.
6.3 Preparation of Plate count agar medium
6.3.1 Prepare the diluent as mentioned in Annexure 9.2.
6.3.2 Autoclave for 20 min at 122°C.
6.4 Sample Preparation
6.3.3 Transfer 1 gm of sample into 99 ml peptone diluent and mix using
homogeniser at 10,000 rpm for 1 minute. The dilution of the sample
will be 10-2.

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Document Number
STANDARD TEST PROCEDURE PELL/STP/
011_2.0
DETERMINATION OF TOTAL Document Level
COLONY FORMING UNITS P3 03

6.3.4 Withdraw a sample aseptically and observe under the microscope for
clump formation. If clumping is observed sonicate in bath type
sonicator for two to three minutes.
6.3.5 Transfer 0.9 ml peptone diluent into sterile Eppendorf for serial
dilution.
6.3.6 Aseptically add 0.1 ml of the homogenous suspension from the above-
mentioned flask and mix with 0.9 ml of diluent using vortex mixer.
Note that the sample should be completely dissolved or in the
suspended state while drawing, this dilution will be 10 -3.
6.3.7 Repeat step 6.3.3 until the final dilution is expected to contain about
30 colony forming units (CFU)/ml.
[ NOTE—Take care to plate the sample preparation dilution within 10 to
20 min of preparation.]
6.4 Analysis
6.4.1 Aseptically transfer 1.0 ml of the sample preparation separately into
appropriately labelled sterile petri plate, then pour 15-20 ml of the
molten Plate count agar medium, then gently swirl the plates to mix
the sample preparation and the Plate count agar medium.
6.4.2 Prepare one blank plate that contains only Plate count agar medium
6.4.3 Allow the plates to sit at room temperature until the Plate count agar
medium solidifies, then invert the plates and incubate them at 30 ± 2
°C for 24± 4 h.
6.5 Calculation of CFU
6.5.1 After 24 h of incubation, count the colonies on the prepared plates.
6.5.2 Plates containing between 30 and 300 colonies are considered ideal
for counting.
6.5.3 Count only colonies that appear as follows. Surface colonies should be
1 mm to 5 mm in diameter, white to cream in color, convex, with
entire margins and smooth surfaces. Colonies inside the Plate count
agar medium should be 0.5 mm to 1 mm in diameter and should
appear as cream-colored pinpoints in the Plate count agar medium.
6.5.4 Calculate the average number of colonies per plate, then multiply the
average number of colonies counted by the reciprocal of the dilution
factor to obtain the CFU/g of the sample. The presence of any colonies
not conforming to this description should be noted. Both blank plates
should be entirely free of any type of colonies.
[NOTE—In the case of blank plates that contain colonies, the entire
procedure must be repeated, potentially including the preparation of the
Diluent and the Plate count agar medium, depending on which plate(s)
contain colonies.]
7 Appendices
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Document Number
STANDARD TEST PROCEDURE PELL/STP/
011_2.0
DETERMINATION OF TOTAL Document Level
COLONY FORMING UNITS P3 03

7.1 Diluent
Peptone: 1.0 g
Distilled water: 1000 ml
Final pH: 7 (Adjust with lactic acid)
7.2 Plate Count Agar
Tryptone: 5.000
Yeast extract: 2.500
Dextrose (Glucose): 1.000
Agar: 9.000
Distilled water: 1000 ml
Final pH (at 25°C): 7.0±0.2
Dispense in an appropriate capacity flask and autoclave at 15 lbs pressure
(121°C) for 15 minutes.
8 Reference
NA
9 Review History
Date Version/Revision No. Reason of Review
01-12-2019 PELL/STP/011_1.0 Initial Release
27-08-2021 PELL/STP/011_2.0 Structure and format change

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