To lay down the procedure for microbiological analysis of water
2.0 SCOPE
This SOP shall be applicable for microbiological analysis of water in the Microbiology Laboratory.
3.0 RESPONSIBILITY
3.1 Microbiologist and above shall be responsible for the preparation and execution of this SOP
3.2 QC & QA Head shall be responsible for the checking and approval of this SOP.
3.3 shall be responsible for the authorization of this SOP.
4.0 ACCOUNTABILITY
Head QC
5.0 PROCEDURE
5.1 Sampling of Purified water:
5.1.1 The water sample shall be collected in pre-sterilized water sampling bottles after draining (1.0 lit with each point) from all the points of use.
5.1.2 Before collection of water samples sampling point shall be sanitized with 70 % filtered IPA giving the contact time of 1 minute.
5.1.3 Sample of 250 to 300 ml is collected aseptically from individual points of use, into sterile water sampling bottles for microbial analysis.
5.1.4 After drawing the sample, the analysis shall be carried out within 2 hrs of sampling. If the analysis is not carried out within 2 hrs the water sample can be stored at 2 – 8 ° C for a period of 12 hrs.
5.2 Total Aerobic Microbial Count:
BY POUR PLATE METHOD:
5.2.1 Ensure that 1ml of sample shall be taken into a sterile Petri plate.
5.2.2 About 15- 20 mL of molten sterile SCDA/PCA media shall be poured into the Petri plate in duplicate and, mix the sample properly by rotating the plates. Allow to solidifying the media at room temperature.
5.2.3 After solidification, the sterile SCDA/PCA plate shall be incubated in an inverted position at 32.5± 2.5ยบ C for 48-72 hrs for the total aerobic microbial count.
5.2.4 After completion of the incubation period observe the plates for the bacterial count and report the results as colony-forming units/ml (CFU / ml) considering the dilution factor if. Applicable.
5.3 Tests for Specified Microorganisms:
5.3.1 Take 100 ml of water sample and filter it through filter membrane having 0.45 ยต pour size and 47 mm dia. After filtration transfer the filtered membrane into 100 ml SCDM. (Tube A)
5.3.2 Incubate the tube in an incubator at 30-35°C for 18 to 24 hrs. (Tube A)
5.4 Tests for E. coli Species
5.4.1 After completion of the incubation period shake the broth and transfer 1 mL (from Tube A) to 100 mL of sterilized MacConkey broth & incubate it at 42 to 44° C for 24 to 48 hrs.
5.4.2 Subculture on a plate of MacConkey agar and incubate the plates at 30 to 35° C for 18 to 72 hrs.
5.4.3 The growth of pink, nonmucoid colonies indicates the possible presence of Escherichia coli. This shall be confirmed by an identification test.
5.4.4 If there is no growth of such type of colonies, the identification test are negative it indicates an absence of E. coli and the sample passes the test for E.coli.
Characteristic of E. coli on selective agar
|
Name of Media |
Colony characteristic |
Gram Staining |
|
|
Mac
Conkey Broth |
Acid
formation (Color change) &gas formation (bubbles in |
NA |
|
|
Mac
Conkey Agar |
Growth
of pink, non – mucoid colonies observed / doesn’t observe. |
Gram
Negative Rods |
|
|
EMB
Agar |
Growth
of green metallic sheen |
Gram
Negative Rods |
5.5 Test for Salmonella species
5.5.1 After incubation shake the SCDM broth and transfer 0.1 mL (from Tube A) to 10mL of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30 to 35° for 18 to 24 hrs.
5.5.2 Subculture on a plate of Xylose Lysein deoxycholate agar and incubate at 30 to 35° C for 18 to 48 hrs.
5.5.3 Well-developed red colonies with or without black centers indicate the possibility of salmonella spp.
5.5.4 If any colonies as described above are produced. Then proceed for the secondary test.
5.5.5 Secondary Test
Using a sterile inoculation loop, transfer the suspected colonies onto a sterile Triple Sugar Iron agar medium slant by first streaking the surface of the slant and stabbing the loop well beneath into the surface. At the same time inoculate a tube of Urea Broth.
5.5.6 Incubate in an incubator at 35 ± 2ยบ C for 18 to 24 hrs
5.5.7 If an examination shows evidence of having alkaline (red) slants and acid (yellow)butts with (or) without concomitant blackening of the butt from the hydrogen sulfide, together with the absence of a red color in the Urea broth that indicates the presence of Salmonellae spp.
5.5.8 This shall be confirmed by identification test.
5.5.9 If there is no growth of such type of colonies or the identification test is negative it indicates absence of salmonella and the sample passes the test for salmonella spp
Characteristic of Salmonella species on selective agar
5.6 Test for Pseudomonas aeruginosa:
5.6.1 After completion of incubation period OF Tube A Streak a portion of the medium from tube A on the surface of Cetrimide Agar and incubate the plates at 30 to 35° C for 18 to 72 hrs
5.6.2 A greenish color colony indicates the possibility of presence of Pseudomonas aeruginosa.
5.6.3 If any colonies as described above are produced. Gram’s staining and oxidize test must be carried out with colonies suspected of Pseudomonas aeruginosa.
5.6.4 Oxidase test:
Smear a portion of colony with the help of sterile inoculation loop across on oxidase disk kept in sterile Petri dishes.
5.6.5 If there is no development of pink color, changing to purple, the specimen meets the requirement of the test for absence of Pseudomonas
Characteristic of Pseudomonas aeruginosa on selective agar
5.6.6 If none of the plates shows colonies having the characteristics described above, the sample meets the requirements for freedom from Pseudomonas aeruginosa.
5.7 Test for Staphylococcus aureus:
5.7.1 After completion of incubation period of tube A Streak a portion of the medium from tube A on the surface of Mannitol Salt Agar surface and incubate the plates at 30 to 35° C for 18 to 72 hrs
5.7.2 Examine the plates for the growth typical off Staphylococcus colonies on the selective agar medium colonies, which shows the following characteristics.
Characteristic of staphylococcus aureus on selective agar
5.7.3 If none of the plates contains colonies having the characteristic expressed above, the sample meets the requirement for freedom from Staphylococcus.
5.7.4 If any colonies are positive for Staphylococcus perform gram’s staining and coagulase test using single typical colony from selective agar media.
5.7.5 Secondary test:
coagulase test :
With a sterile inoculation loop, transfer suspect colonies from selective agar media to a tube containing 0.5 ml of rabbit or horse plasma.
5.7.6 Keep negative control without addition of culture to 0.5 ml of rabbit horse plasma.
5.7.7 Keep positive control by transferring Staphylococcus culture with the help of a sterile inoculation loop in 0.5 ml of rabbit or horse plasma.
5.7.8 Incubate in an incubator at 35 to 37ยบ C for 24 hrs and observe for coagulation at 3 hrs intervals.
5.7.9 If no coagulation is observed in the test sample meets the requirements for absence of S.aureus.
6.0 REFERENCES
N.A
7.0 ANNEXURE
N.A
8.0 ABBREVIATIONS :
SOP : Standard Operating Procedure
QCD : Quality Control Department
Dept. : Department
QC : Quality Control
QA : Quality Assurance
QCD : Quality Control Department
No. : Number
N.A : Not Applicable
I.P.A : Iso Propyl Alcohol
5.5.1 After incubation shake the SCDM broth and transfer 0.1 mL (from Tube A) to 10mL of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30 to 35° for 18 to 24 hrs.
5.5.2 Subculture on a plate of Xylose Lysein deoxycholate agar and incubate at 30 to 35° C for 18 to 48 hrs.
5.5.3 Well-developed red colonies with or without black centers indicate the possibility of salmonella spp.
5.5.4 If any colonies as described above are produced. Then proceed for the secondary test.
5.5.5 Secondary Test
Using a sterile inoculation loop, transfer the suspected colonies onto a sterile Triple Sugar Iron agar medium slant by first streaking the surface of the slant and stabbing the loop well beneath into the surface. At the same time inoculate a tube of Urea Broth.
5.5.6 Incubate in an incubator at 35 ± 2ยบ C for 18 to 24 hrs
5.5.7 If an examination shows evidence of having alkaline (red) slants and acid (yellow)butts with (or) without concomitant blackening of the butt from the hydrogen sulfide, together with the absence of a red color in the Urea broth that indicates the presence of Salmonellae spp.
5.5.8 This shall be confirmed by identification test.
5.5.9 If there is no growth of such type of colonies or the identification test is negative it indicates absence of salmonella and the sample passes the test for salmonella spp
Characteristic of Salmonella species on selective agar
|
Name of Media |
Colony
characteristic |
Gram Staining |
|
|
RVSB |
Growth
in terms of turbidity observed |
NA |
|
|
Brilliant
Green Agar |
Small,transparent
and colourless or opaque,pimkish or white surrounded by pink |
Gram
Negative Rods |
|
|
Xylose
lysine deoxycholate agar |
Red
colonies with or without black centers observed |
Gram
Negative Rods |
5.6 Test for Pseudomonas aeruginosa:
5.6.1 After completion of incubation period OF Tube A Streak a portion of the medium from tube A on the surface of Cetrimide Agar and incubate the plates at 30 to 35° C for 18 to 72 hrs
5.6.2 A greenish color colony indicates the possibility of presence of Pseudomonas aeruginosa.
5.6.3 If any colonies as described above are produced. Gram’s staining and oxidize test must be carried out with colonies suspected of Pseudomonas aeruginosa.
5.6.4 Oxidase test:
Smear a portion of colony with the help of sterile inoculation loop across on oxidase disk kept in sterile Petri dishes.
5.6.5 If there is no development of pink color, changing to purple, the specimen meets the requirement of the test for absence of Pseudomonas
Characteristic of Pseudomonas aeruginosa on selective agar
|
Selective Media |
Morphology |
Oxidize test |
Gram’s stain |
|
Cetrimide agar |
Generally greenish Colonies observed |
Positive |
Negative rods |
5.6.6 If none of the plates shows colonies having the characteristics described above, the sample meets the requirements for freedom from Pseudomonas aeruginosa.
5.7 Test for Staphylococcus aureus:
5.7.1 After completion of incubation period of tube A Streak a portion of the medium from tube A on the surface of Mannitol Salt Agar surface and incubate the plates at 30 to 35° C for 18 to 72 hrs
5.7.2 Examine the plates for the growth typical off Staphylococcus colonies on the selective agar medium colonies, which shows the following characteristics.
Characteristic of staphylococcus aureus on selective agar
|
Name of Media |
Colony characteristics |
Gram Staining |
|
Mannitol Salt Agar |
Yellow colony with yellow zones |
Positive cocci (in clusters) |
5.7.3 If none of the plates contains colonies having the characteristic expressed above, the sample meets the requirement for freedom from Staphylococcus.
5.7.4 If any colonies are positive for Staphylococcus perform gram’s staining and coagulase test using single typical colony from selective agar media.
5.7.5 Secondary test:
coagulase test :
With a sterile inoculation loop, transfer suspect colonies from selective agar media to a tube containing 0.5 ml of rabbit or horse plasma.
5.7.6 Keep negative control without addition of culture to 0.5 ml of rabbit horse plasma.
5.7.7 Keep positive control by transferring Staphylococcus culture with the help of a sterile inoculation loop in 0.5 ml of rabbit or horse plasma.
5.7.8 Incubate in an incubator at 35 to 37ยบ C for 24 hrs and observe for coagulation at 3 hrs intervals.
5.7.9 If no coagulation is observed in the test sample meets the requirements for absence of S.aureus.
6.0 REFERENCES
N.A
7.0 ANNEXURE
N.A
8.0 ABBREVIATIONS :
SOP : Standard Operating Procedure
QCD : Quality Control Department
Dept. : Department
QC : Quality Control
QA : Quality Assurance
QCD : Quality Control Department
No. : Number
N.A : Not Applicable
I.P.A : Iso Propyl Alcohol
Hi. I’m Writer of Researchsop.com. ’ ’ Please share these SOPs to all concern pharma people for their development. I like to fullfill the need of curious people. These things inspire me to make things looks better.
0 comments:
Post a Comment