PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
1.0 OBJECTIVE:
To lay down a procedure for Microbiological analysis of Raw Water & Purified Water.
2.0 SCOPE:
This SOP is applicable for Microbiological analysis of Raw Water & Purified Water in
Microbiological Laboratory of Quality Control Department.
3.0 RESPONSIBILITY:
Officer / Executive – Microbiology
4.0 ACCOUNTABILITY:
Head – QC
5.0 ABBREVIATIONS:
Hrs. Hours
IP Indian Pharmacopoeia
ml Milliliter
No. Number
NMT Not More Than
QA Quality Assurance
QC Quality Control
SOP Standard Operating Procedure
SCM Soyabean Casein Digest Medium
spp. Species
USP United State Pharmacopoeia
UV Ultra Violet
cfu Colony Forming Unit
TVC Total Viable Count
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
6.0 PROCEDURE:
Prerequisite for microbiological analysis of Raw water & Purified water:
[Link]. Requirements
1 Water Sample for microbiological analysis
2 Calibrated Micropipette 100-1000 Micro litre
3 Sterilized Micropipette Tips 100-1000 Micro litre
4 Sterile SS filtration Assembly
5 Sterile Vacuum filtration flask
6 Sterile forceps
7 Sterile 0.45 µ Membrane Filter
8 Sterile SS Manifold filtration assembly
9 Sterile silicone tube and cork
10 Vacuum pump
12 SS Bucket
13 Sterile water
14 Preincubated R2A Media Plates
15 Preincubated Soyabean casein digest medium tube, Enterobacteria Enrichment broth,
MacConkey broth, Rappaport Vassiliadis Salmonella Enrichment broth, Cetrimide
agar, Mannitol salt agar, MacConkey agar, Xylose lysine deoxycholate agar, Violet
red bile glucose agar plates
16 Sterile Glass test tube
17 Sterile petri plate
6.1 TOTAL VIABLE COUNT:
6.1.1 Sample the Raw water & Purified water as per SOP, Titled “Procedure for sampling of Water
for Microbiological and Chemical Analysis”. Prepare required media as per SOP, Titled
“Receipt, Approval and Preparation of Culture Media”
6.1.2 For Raw Water Analysis: - Take the 1 ml sample in 9 ml sterile water for Total Viable
count and mix properly. After mixing take 1 ml of diluted raw water into two pre-sterilized
Petri plates, and take 1 ml of sterile water into one pre-sterilized Petri plates for negative
control and pour 20-25 ml sterilized R2A media ((cool up to 45°C, checks with IR gun)) and
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
rotate the plate gently in clockwise and anticlockwise direction for proper mixing of sample.
Label the plates/tubes with Negative control/Sampling Point, Date of Testing and Media
Reference Number.
6.1.3 Incubate the R2A media plates at 30 to 350C for 5 (five) days for Total viable count.
6.1.4 For Purified Water Analysis: Pre-wet the filter using approximately 10 ml of sterile water
and Filter 1 ml of Purified water through 0.45µ sterile membrane filter and place the
membrane filter onto pre-incubated R2A plate for Total Viable Count and For negative
control; Pre-wet the filter using approximately 10 ml of sterile water and Filter 1 ml of sterile
water through 0.45µ sterile membrane filter and place the membrane filter onto pre-incubated
R2A plate. Label the plates with Sampling Point / Negative control, Date of Testing and
Media Reference Number.
6.1.5 Incubate the R2A plates at 30 to 350C for 5 (five) days for Total viable count.
6.2 OBSERVATIONS AND RESULTS:
6.2.1 Raw Water and Purified Water: Examine the plates and count the number of colonies with
the help of colony counter. Express the count in term of the number of microorganisms per ml
of raw water and purified water.
6.3 TEST FOR SPECIFIED MICROORGANISMS:
6.3.1 Pretreatment of Sample for Raw Water:
Pre-wet the filter using approximately 10 ml of sterile water and Filter 100 ml of raw water
sample through 0.45 µ sterile membrane filter. Transfer the membrane filter into 100 ml
Soybean Casein Digest Medium.
6.3.2 Incubate the SCM tube at 20-250C for 2-5 hrs. after 2-5 hrs. incubation; perform the test of
Bile-Tolerant Gram-Negative Bacteria After that incubate the SCM at 30-350C for 18-24 hrs
for further analysis.
Test for Bile-Tolerant Gram-Negative Bacteria (Enterobacteria) for Raw Water:
Transfer 1 ml of sample into 100 ml Enterobacteria Enrichment Broth Mossel and Incubate at
30 to 350C for 24 to 48 hrs.
6.3.3 After completion of Incubation period, Streak on the plate of Violet Red Bile Glucose Agar
and incubate at 30 to 350C for 18 to 24 hrs.
6.3.4 Process Negative Control: Pre-wet the filter using approximately 10 ml of sterile water and
Filter 100 ml of sterile water through 0.45 µ sterile membrane filter. Transfer the membrane
filter into 100 ml Soybean Casein Digest Medium.
Incubate the SCM tube at 20-250C for 2-5 hrs.
After 2-5 hrs. incubation; perform the test of Bile-Tolerant Gram-Negative Bacteria Transfer
1 ml SCM of negative control into 100 ml Enterobacteria Enrichment Broth Mossel and
Incubate at 30 to 350C for 24 to 48 hrs.
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
After that incubate the SCM at 30-350C for 18-24 hrs. for further analysis.
After incubation completion of Enterobacteria Enrichment Broth Mossel streak a loop full on
the Preincubated plate of Violet Red Bile Glucose Agar and incubate at 30 to 350C for 18 to
24 hrs.
6.3.5 Negative control should not show any growth.
6.3.6 After completion of Incubation period; examine the plates. upon examination, if none of the
colonies confirm to the description given in Table-1, the sample meets the requirements for
the absence of Bile-Tolerant Gram-Negative Bacteria (Enterobacteria)
6.3.7 If colonies show characteristic growth as per Table-1, carry out the identification by Vitek-2
compact identification system or outside Laboratory.
6.3.8 Pretreatment of Sample for Purified Water:
Pre-wet the filter using approximately 10 ml of sterile water and Filter 100 ml of Purified
water sample through 0.45 µ sterile membrane filter Transfer the membrane filter into 100
ml Soyabean Casein Digest Medium.
6.3.9 Incubate the Soyabean Casein Digest Medium at 30-350C for 18-24 hrs for further analysis.
6.3.10 Test for Escherichia coli for Raw water and Purified water:
[Link] Shake the enriched SCM tube and transfer 1 ml of pretreated sample (SCM) into 100 ml of
Mac Conkey Broth and incubate at 42 to 440C for 24 to 48 hrs.
[Link] After incubation completion; Streak a one loop full portion from MacConkey broth on the
surface of MacConkey Agar Plate and incubate at 30 to 350C for 18 to 72 hrs.
[Link] After incubation completion; examine the plates. if none of the colonies confirm to the
description given in Table-1, the sample meets the requirements for the absence of the E.
coli.
[Link] Process Negative Control: Pre-wet the filter using approximately 10 ml of sterile water and
Filter 100 ml of sterile water sample through 0.45 µ sterile membrane filter.
Transfer the membrane filter into 100 ml Soyabean Casein Digest Medium and Incubate the
medium at 30-350C for 18-24 hrs. for further analysis.
After incubation completion; transfer 1 ml sample to sterile MacConkey broth and incubate at
42-44°C for 24-48 hrs. After incubation completion; subculture a loop full on pre-incubated
plates of MacConkey agar (MCA) and incubate at 30-35°C for 18-72 hours in inverted
position.
[Link] Negative control should not show any growth.
[Link] If colonies show characteristic growth as per Table-1, carry out the identification by Vitek-2
compact identification system or outside Laboratory.
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
6.3.11 Test for Salmonella spp. for raw water and purified water:
[Link] Shake the enriched tube and transfer 0.1 ml of pretreated sample (SCM) to 10 ml of
Rappaport Vassiliadis Salmonella Enrichment Broth and incubate at 30 to 350C for 18 to
24hrs.
[Link] After incubation completion: Streak a one loop full portion from the Rappaport Vassiliadis
Salmonella Enrichment Broth on surface of Xylose Lysine Deoxycholate Agar Plate and
incubate at 30 to 350C for 18 to 48 hrs.
[Link] After incubation completion; examine the plates. if none of the colonies confirm to the
description given in Table-1, the sample meets the requirements for the absence of the
Salmonella spp.
[Link] If colonies show characteristic growth as per Table-1, carry out the identification by Vitek-2
compact identification system or outside Laboratory.
[Link] Process Negative Control: After incubation completion of negative control Soyabean casein
digest transfer 0.1 ml SCM into 10 ml of sterile RVS Broth and incubate at 30- 35°C for 18-
24 hrs. After incubation completion; subculture a loop full on Preincubated
plates of Xylose lysine Deoxycholate Agar (XLD) and incubate at 30-35°C for 18-
48 hours in inverted position.
[Link] Negative control should not show any growth.
6.3.12 Test for Pseudomonas aeruginosa for raw water and purified water:
[Link] Shake the enriched tube and streak one loop full pretreated sample (SCM) on to the plate of
Cetrimide Agar Medium and incubate at 30 to 350C for 18 to 72 hrs.
[Link] After incubation completion; examine the plates. If none of the colonies confirm to the
description given in Table-1, the sample meets the requirements for the absence of the
Pseudomonas aeruginosa.
[Link] If colonies show characteristic growth as per Table-1, carry out the identification by Vitek-2
compact identification system or outside Laboratory.
[Link] Process Negative Control: After incubation completion of negative control Soyabean casein
digest medium subculture a loop full on pre-incubated plates of Cetrimide Agar (CTA) and
incubate at 30 to 35°C for 18 to 72 hours in inverted position.
[Link] Negative control should not show any growth.
6.3.13 Test for Staphylococcus aureus for raw water and purified water:
[Link] Shake the enriched tube and streak one loop full pretreated sample (SCM) on to the plate of
Mannitol Salt Agar Medium and incubate at 30 to 350C for 18 to 72 hrs.
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
[Link] After incubation completion; examine the plates. If none of the colonies confirm to the
description given in Table-1, the sample meets the requirements for the absence of the
Staphylococcus aureus.
[Link] If colonies show characteristic growth as per Table-1, carry out the identification by Vitek-2
compact identification system or outside Laboratory.
[Link] Process Negative Control: After incubation completion of negative control Soyabean casein
digest medium i subculture a loop full on pre-incubated plates of Mannitol salt agar plate
(MSA) and incubate at 30 to 35°C for 18 to 72 hours in inverted position.
[Link] Negative control should not show any growth.
TABLE-1
Specified Positive Growth Gram Staining
Media Name
Microorganism Characteristics Characteristics
Medium colour turns to
MacConkey Broth
yellow.
E. coli Gram Negative Rod
Pink/red coloured non-mucoid
MacConkey Agar
colonies.
Rappaport Vassiliadis
Medium colour turns to light
Salmonella Enrichment
green
Salmonella Broth Gram Negative Rod
Xylose lysine Red colonies with or without
Deoxycholate Agar black centers.
Pseudomonas
Cetrimide Agar Greenish yellow colonies Gram Negative Rod
aeruginosa
Yellow colonies surrounded
Staphylococcus aureus Mannitol Salt Agar Gram Positive Cocci
by yellow zones.
Enterobacteria
Enrichment Broth, Medium color turns to yellow.
Bile Tolerant Gram
Mossel Gram Negative
Negative Enterobacteria
Violet Red Bile
Pink/red colonies
glucose Agar
(Note: If there is a holiday on the day of release/transfer of media plates, take the observation/ transfer
of media plates on next working day.
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
7.0 ACCEPTANCE CRITERIA:
Purified Water:
Total viable count Alert Limit 60 cfu/ml
Action Limit 80 cfu/ml
Specified Limit 100 cfu/ml
Specified Microorganisms Escherichia coli Should be absent
Salmonella spp. Should be absent
Pseudomonas aeruginosa Should be absent
Staphylococcus aureus Should be absent
Raw Water:
Total Viable Count Alert Limit 300 cfu/ml
Action Limit 400 cfu/ml
Specified Limit 500 cfu/ml
Specified Microorganisms Bile-Tolerant Gram-Negative Should be absent
Bacteria (Enterobacteria)
Escherichia coli Should be absent
Salmonella spp. Should be absent
Pseudomonas aeruginosa Should be absent
Staphylococcus aureus Should be absent
8.0 ANNEXURES:
ANNEXURE No. TITLE OF ANNEXURE FORMAT No.
Annexure – I Microbiological Analysis Record of Purified Water
Annexure – II Microbiological Analysis Record of Raw Water
ENCLOSURES: SOP Training Record.
9.0 DISTRIBUTION:
Controlled Copy No. 01 Quality Assurance
Controlled Copy No. 02 Microbiology
Master Copy Quality Assurance
10.0 REFERENCES:
United State Pharmacopoeia 39
Indian Pharmacopoeia 2014
British Pharmacopoeia 2012
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
11.0 REVISION HISTORY:
CHANGE HISTORY LOG
Revision Change Control Details of Changes Reason for Effective Updated
No. No. Change Date by
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
ANNEXURE-I
MICROBIOLOGICAL ANALYSIS RECORD OF PURIFIED WATER
Date of Testing: Method: Membrane Filtration Date of report:
Tested By: Incubator ID.:
Observed
A. R. No→ by /date
Test Media Incubation
Name Reference condition Sampling
point→
R2A/ 30-350C
TVC Observation
for 5 days
Enrichment for Test for Specified Microorganisms: Filter 100 ml water sample by 0.45µ membrane and transfer the membrane
into SCM Media
Test performed by/date:
SCM/ 30-350C for Observation
18-24hrs.
PRIMARY TEST FOR SPECIFIED MICROORGANISM:
Test performed by/date:
Salmon RVS/ 30-350C for
Observation
ella 18-24hrs.
0
[Link] MCB/ 42-44 C for
Observation
24-48hrs.
0
[Link]/ 30-35 C for
Observation
nosa 18-72hrs.
0
S. MSA/ 30-35 C for
Observation
aureus 18-72hrs.
SECONDARY TEST FOR SPECIFIED MICROORGANISM:
For [Link] Tested by/date:
[Link] MCA/ 30-350C for
Observation
18-72 hrs.
Confirmatory identification test: Observation
For Salmonella Tested by/date:
Salmon XLD/ 30-350C for
Observation
ella 18-48hrs.
Confirmatory identification test: Observation
For Pseudomonas aeruginosa Tested by/date:
Confirmatory identification test: Observation
For Staphylococcus aureus Tested by/date:
Confirmatory identification test: Observation
P→ Characteristic growth observed, N→ No Characteristic growth observed,
CONCLUSIONS
Test Organism E. coli NA
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
Salmonella spp.
P. aeruginosa
S. aureus
A→ Absent, P→ Present
Acceptance criteria of Total Viable count and Specified Microorganisms :
Total Viable count Alert Limit 60 cfu/ml
Action Limit 80 cfu/ml
Specified Limit 100 cfu/ml
Specified Microorganisms Escherichia coli Should be absent
Salmonella spp., Should be absent
Pseudomonas aeruginosa Should be absent
Staphylococcus aureus Should be absent
Remarks: The above samples comply/do not comply as per IH specification.
Microbiologist: Reviewed By:
Date: Date:
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
ANNEXURE – II
MICROBIOLOGICAL ANALYSIS RECORD OF RAW WATER
Date of Testing: Method: Membrane Filtration &Pour plate Date of report:
Tested By: Incubator ID.:
Observed
Media Incubatio A. R. No→ by /date
Test
Referenc n
Name Sampling
e condition
point→
R2A/
Observat P1
30-350C ion P2
TVC for 5
days Average
Count /ml
Enrichment for Test for Specified Microorganisms: Filter 100 ml water sample by 0.45µ membrane and transfer the membrane
into SCM Media
Test performed by/date:
SCM/ 30-350C for
Observation
18-24hrs
PRIMARY TEST FOR SPECIFIED MICROORGANISM:
After 2-5 hrs. at 20-250C of enrich sample for Bile Tolerant Gram Negative Bacteria
Test performed by/date:
EEM/ 30-350C for
Observation
24-48hrs
0
After 18-24 hrs. at 30-35 C of enrich sample for Salmonella, E. coli, P. aeruginosa, S. aureus
Test performed by/date:
Salmon RVS/ 30-350C for
Observation
ella 18-24hrs
0
E. coli MCB/ 42-44 C for
Observation
24-48hrs
0
[Link]/ 30-35 C for
Observation
nosa 18-72hrs
0
S. MSA/ 30-35 C for
Observation
aureus 18-72hrs
SECONDARY TEST FOR SPECIFIED MICROORGANISM:
For Bile Tolerant Gram Negative Bacteria
Test performed by/date:
VBA/ 30-350C for
Observation
18-24hrs
For [Link] Tested by/date:
[Link] MCA/ 30-350C for
Observation
18-72 hrs.
Confirmatory identification test: Observation
For Salmonella Tested by/date:
Salmon XLD/ 30-350C for
Observation
ella 18-48hrs
PHARMA DEVILS
MICROBIOLOGY DEPARTMENT
STANDARD OPERATING PROCEDURE
Department: Microbiology SOP No.:
Title: Microbiological Analysis of Raw Water & Purified Water Effective Date:
Supersedes: Nil Review Date:
Issue Date: Page No.:
Confirmatory identification test: Observation
For Pseudomonas aeruginosa Tested by/date:
Confirmatory identification test: Observation
For Staphylococcus aureus Tested by/date:
Confirmatory identification test: Observation
P→ Characteristic growth observed, N→ No Characteristic growth observed
CONCLUSION
Sampling point →
Bile Tolerant Gram
Negative Bacteria
Test E. coli
Organism NA
Salmonella spp.
P. aeruginosa
S. aureus
A→ Absent, P→ Present, P1→Plate-1, P2→Plate-2,
Acceptance criteria of Total Viable count and Specified Microorganisms :
Total Viable count Alert Limit 300 cfu/ml
Action Limit 400 cfu/ml
Specified Limit 500 cfu/ml
Specified Bile-Tolerant Gram-Negative Bacteria (Enterobacteria) Should be absent
Microorganisms Escherichia coli Should be absent
Salmonella spp. Should be absent
Pseudomonas aeruginosa Should be absent
Staphylococcus aureus Should be absent
Remarks: The above samples comply/do not comply as per IH specification.
Microbiologist: Reviewed By:
Date: Date:
