Water Sample Analysis Report
1.0 Test: Microbiological Analysis
2.0 Objective: To determine the microbiological quality of a water sample.
3.0 Methodology:
4.0 Total Aerobic Microbial Count:
4.1 Por Plate Method
A 1 ml water sample was transferred to a sterile Petri dish. 15 to 20 ml of sterile liquefied and cooled (45°C) R2A agar media for bacterial count and SCA agar media for fungal count were poured into the Petri dishes. The contents were mixed thoroughly by gently rotating the Petri dishes clockwise and counterclockwise on the LAF platform. R2A agar media was incubated at 35°C for 3 to 5 days (for bacteria) and SCA agar media was incubated at 20 to 25°C for 5 days (for fungus). After incubation, the colonies were counted using a colony counter and the results were recorded.
4.2 Tests for Specified Microorganisms:
100 ml of water sample was filtered through a filter membrane with a pore size of 0.45 µm and a diameter of 47 mm. After filtration, the filtered membrane was transferred to 100 ml of SCDM (Flask A). Flask A was incubated at 30 to 35°C for 18 to 24 hours. After incubation, the test flask was checked for turbidity and proceeded to further tests for specific pathogens.
5.0 Tests for E. coli Species:
After incubation, the broth was shaken and 1 ml (from Flask A) was transferred to 100 ml of sterilized MCB and incubated at 42 to 44°C for 24 to 48 hours. After confirmation of turbidity, the broth was subcultured on a plate of MCA and incubated at 35°C for 18 to 72 hours. The MCA plates were examined for selective colonies, and based on the confirmation, the broth was further subcultured on an EMB agar plate. Colonies from the MCA plate were subcultured on EMB agar plates and incubated at 35°C for 18 to 72 hours. After incubation, the plates were examined and the results were recorded.
5.1 Characteristics of E. coli on Selective Agar:
After incubation, the broth was shaken and 0.1 ml (from Flask A) was transferred to 10 ml of RVSB and incubated at 35°C for 24 to 48 hours. After confirmation of turbidity, the broth was subcultured on a plate of XLD and incubated at 35°C for 18 to 72 hours. After incubation, the XLD plates were examined and selective colonies were recorded.
6.1 Characteristic of Salmonella sp. on Selective Agar:
7.0 Test for P. aeruginosa:
After incubation (from Flask A), a portion of the medium was streaked onto the surface of CA and the plates were incubated at 30 to 35°C for 18 to 72 hours. After incubation, the CA plates were examined and selective colonies were recorded. Subsequently, Gram staining and oxidase tests were performed on colonies suspected of P. aeruginosa.
7.1 Oxidase Test:
A portion of the colony was smeared onto an oxidase disc kept in a sterile Petri dish using a sterile inoculation loop. The development of color on the oxidase disc was observed.
7.2 Characteristics of P. aeruginosa on Selective Agar:
8.1 After completion of the incubation period of Flask A, a portion of the medium was streaked onto the surface of MSA (Mannitol Salt Agar), and the plates were incubated at 30 to 35°C for 18 to 72 hours. Plates were examined for the typical growth of S. aureus on MSA, which is characterized by the presence of yellow colonies with yellow zones around them. These colonies are typically composed of gram-positive cocci arranged in clusters.
9.0 Results-
9.1 Total aerobic microbial count
9.2 Total bacterial count (R2A agar medium) - 27±1.0 colonies/ml of water
9.3 Total fungal count (SCA medium) – Nil
9.4 Specific pathogens: SCDM broth- Turbidity
9.5 E. coli - Present
9.6 MCB- Change in color from dark pink to yellow
9.7 MA- Growth of pink, non–mucoid colonies
9.8 EMB- Growth of green metallic sheen
9.9 Gram staining-Gram-ve rod
9.10 Salmonella spp. - Present
9.11 RVSB- Change in color from light blue to creamy green
9.12 XLD agar- Red colonies with or without black centers observed
9.13 Gram staining-Gram-ve rod
9.14 P. aeruginosa - Present
9.15 CA-greenish color colonies observed
9.16 Oxidase disc-Positive
9.17 Gram staining-Gram-ve rod
9.18 S. aureus- Absent
10.0 Summary
11.0 Precautions: Sampling of Water
11.1 The water sample should be collected in pre-sterilized water sampling bottles after draining (for 1 minute) from all the points of use.
11.2 Before collecting water samples, the sampling point should be sanitized with 70% filtered IPA for a contact time of 1 minute.
11.3 A sample of 250 to 300 ml should be collected aseptically from individual points of use into sterile sampling bottles for microbial analysis.
11.4 After drawing the sample, the analysis should be carried out within 2 hours of sampling. If the analysis cannot be carried out within 2 hours, the water sample can be stored at 2 to 8°C for a period of 12 hours.
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