Microbiology @ IHS Laboratory (BioLab).
The BioLab is equipped to analyse and test for microbial contamination of water. Various plate count tests are available to estimate bacterial colony forming units (cfu), which is a measure of the total bacterial population in a water sample. Multiple tube dilution technique is used to estimate most probable number (MPN) of total coliforms, some of which would indicate environmental and faecal contamination of water. Confirmatory tests are available to identify indicator organisms associated with environmental and sewage contamination. The bacterial endotoxin assay is available to test the level pyrogens (endotoxins) in water for dialysis.
The plate count methods rely on growth of bacterial colonies on a variety of nutrient mediums incubated at specific temperature for various durations, so that colony becomes visible to the naked eye. But too many colonies, some of which may coalesce with each other would be difficult to count. Hence multiple Petridishes with chosen nutrient medium are inoculated with different dilutions of test sample and the colony count is multiplied by the dilution factor.
In the most probable number (MPN) tests, rows of tubes containing lactose broth are inoculated with water samples measuring 10 ml, 1 ml, and 0.1 ml. During incubation, coliform organisms, if present, would produce gas. A small inverted tube (Durham tube) is placed in each test tube to capture as gas is produced by coliform bacteria. Durham tubes float when filled with gas. The number of test tubes in each row, showing definite signs of gas production is identified and a statistical table is used to arrive at the point estimate and range of coliform bacteria in the sample.
The bacterial endotoxin test (BET), based on the fact that the blood of the horseshoe crab (Limulus or Tachypleus) gels or clots when it comes in contact with endotoxin. The Limulus amebocyte lysate (LAL) reagents are used to test for endotoxin using gel-clot method.
The IHS Laboratory is accredited by the National Board of Accreditation for Testing and Calibration Laboratories (NABL) India.
Lab Id: T-4179; Certificate Number: TC-7658; Issue Date: 02-01-2025; Valid Until: 01-01-2029.
The IHS Laboratory is gradually developing itself as an easily accessible Public Health Laboratory for laboratory tests that are relevant to safeguarding and improvement of people’s health. Keeping with this objective, new test services are introduced to meet felt needs. The IHSL follows sound quality assurance mechanisms and adopts standard test methods for all tests including newly introduced tests. However, immediate accreditation by NABL may not be feasible, for various reasons. Thus, at any point of time, the laboratory would have a set of NABL accredited parameters, and other parameters for which NABL accreditation is not yet available. To avoid any misrepresentation, the IHSL we adopt a strict policy of using NABL symbol in test reports only if all test results included in the report are within the scope the accreditation. For more details see;
The IHSL Policy for Use of NABL Symbol and Claim of NABL Accreditation on Test Reports.
BioLab - Scope of Service
Test Parameters Accredited by NABL, vide certificate number TC-7658, date 02-01-2025, valid until 01-01-2029.
| Sl | SvCd | Parameter | Test Material | Test Method |
|---|---|---|---|---|
| Plate Counts: | ||||
| SPC | Standard Plate Count | Water for Food Processing, Groundwater, Drinking Water, Surface Water, Irrigation Water, Swimming Pool Water. | IS 1622 - 1981, RA 2019, cl 3.2 Pour plating & incubation at 37°C, 24h:
Step-1, Media Preparation: Estimate volume media required @ 150ml/sample to allow for marginal losses during processing and inoculation. Suspend required amount of ready-to-use nutrient agar powder to distilled water and heat the mixture until the agar is completely dissolved. Autoclave the mixture at 121°C for 15 minutes, let it cool and maintain molten media at 43±2°C for plating. Step-2, Inoculation & Plating: Label required number of sterile Petri plates. Plan & prepare required serial dilutions of the sample, which would give 30 to 300 colonies on a plate. Mix sample/dilutions well, before inoculation. Ready two plates for each dilution. Pipette 1.0 ml of sample into sterile Petri dish. Pour 15 ml melted nutrient agar over the sample, tilt & rotate gently to mix sample with molten media. Let the agar-sample mixture solidify at room temperature. Step-3, Incubation: Ensure that the incubator is set at 37°C. Place the plates upside down (inverted), preferably on a separate shelf and incubate for 24h. Step-4, Counting & Reporting: Count colonies formed on surface and within agar, using counting aid (colony counter). Disregard plates with colonies outside the counting range (30 - 300). Report result as the average of all plates falling within counting limit. If number of colonies in undiluted sample < 30 report TFTC. If colonies in all plates are confluent (>300), report TNTC. |
|
| AMC | Aerobic Microbial Count | Purfied Water, Water from Purifier. | IS 14543 rw IS 5402: Plate count agar & 24h incubation at 37℃. | |
| CPC | Cold Plate Count | Purified Water. | IS 23500 part 3 Tryptic soy agar & 48h incubation at 35-37℃. | |
| TVC | Total Viable Count | Water for Medicinal Purposes, Purified Water. | IS 23500 part 3 Tryptic soy agar & 48h incubation at 35-37℃. | |
| HPC | Heterotrophic Plate Count | Water for Food Processing, Drinking Water, Groundwater, Surface Water, Irrigation Water, Swimming Pool Water. | IS 5402 Plate count agar & 72h incubation at 30℃. | |
| Enzyme Substrate Tests: | ||||
| TCDES | Total Coliform Detection by Enzyme Substrate | Drinking Water, Groundwater, Surface Water. | APHA Standard Methods 24th Ed. 9223 B. Enzyme Substrate Test: Use 100 ml transparent, nonfluoroscent, borosilicate glass bottle with screw cap, as incubation bottle. Sterilise the incubation bottle. Pour contents of premeasured Colilert-18 packet into the incubation bottle. Fill 100 ml sample, allowing headspace above 100 ml makr, and close cap tightly. Vigorously shake the bottle back and forth at least 25 times to properly mix the media with the sample. Place bottle in the incubator within 30 minutes of mixing the medium & sample. Prewarm the bottle in water bath at 35±0.5°C for 7 to 10 minutes to bring it to incubation temperature. Then, Incubate at 35±0.5°C for ≥18h. | |
| ECDES | E. coli Detection by Enzyme Substrate | Drinking Water, Groundwater, Surface Water. | APHA Standard Methods 24th Ed. 9223 B. Enzyme Substrate Test. | |
| Multipe Tube Fermentation Tests: | ||||
| MPNTC | MPN (Total coliforms) by multi-tube dilution | Water for Food Processing, Groundwater, Surface Water, Irrigation Water, Swimming Pool Water, Wastewater. | IS 1622 - 1981, RA2019, cl 3.3.1 Multi-tube dilution test (MDT):
Step-1, Media Preparation: Prepare double & single strength MacConkey broth using certified ready-to-use media. Distribute 10 ml of double strength media into 150 x 18 mm tubes; and 10 ml of single strength media into 150 x 15 mm test tubes. Place an inverted Durham's tube into each media tube, plug the test tubes with non-absorbent cotton and autoclave at 115°C for 10 minutes. Step-2, Innoculation: Arrange 5 presterilised double strength MacConkey broth tubes in 1st row; single strength tubes in rows 2 & 3. Innoculate each of the 1st row tubes with 10 ml; 2nd row tubes with 1.0 ml; and 3rd row tubes with 0.1 ml of water sample. Step-3, Incubation: Incubate all tubes at 37°C for 24±2h. Step-4, Observation: Observe & media colour change, if any; position of Durham's tube (bottom/floating), and gas in Durham's tube, if any. Record inference about number of presumptive positive tubes in each row. Step-5, Computation of presumptive total coliforms: Refer Appendix B Table-3 MPN per 100 ml sample and confidence limits using 5 tubes of 10 ml, 5 tubes of 1 ml and 5 tubes of 0.1ml. |
|
| FCDMT | Fecal (Thermotolerant) coliforms Detection by multi-tube dilution. | Water for Food Processing, Groundwater, Surface Water, Irrigation Water, Swimming Pool Water, Wastewater. | IS 1622 - 1981, RA2019, cl 3.3.1.2b(i) & 3.3.3: Continue after observation of results for MPN (Total coliforms). Subculture, by transferring one or two loopful from lowest dilution presumptive positive tubes to a tube of BGLB broth and incubate in water bath at 44.5°C for 24±2h. Any amount of gas formation in the Durham's tube indicates fecal coliforms. If negative, reincubate and examine again at 48±2h. | |
| ECDMT | E. coli Detection by multi-tube dilution. | Drinking Water, Water for Food Processing, Water for Dialysis, Purified Water, Groundwater, Irrigation Water, Surface Water, Swimming Pool Water, Wastewater. | IS 1622 - 1981,RA2019, cl 3.3.4: Continue after observation of results for Fecal coliforms FCDMT. Subculture, from Fecal coliform positive BGLB tube, in tryptone water at 44.5°C for 24h. Add a few drops of Kovac's reagent. Pink colour indicates E. coli positive & yellow means negative. | |
| MPNFC | MPN (Fecal Coliforms) by multi-tube dilution. | Water for Food Processing, Groundwater, Surface Water, Irrigation Water, Swimming Pool Water, Wastewater. | IS 1622 - 1981,RA2019, cl 3.3.3.2: Continue after observation of results for MPN (Total coliforms). Subculture, from all presumptive positive tubes of coliform test into as many BGLB tubes at 44.5℃ for 24±2h in water bath. Observe for gas formation (FC +ve) or lack of it (FC -ve). Refer Appendix B Table-3 to estimate MPN(Fecal coliforms) per 100 ml sample. | |
| MPNEC | MPN (E. coli) by multi-tube dilution. | Water for Food Processing, Groundwater, Surface Water, Irrigation Water, Swimming Pool Water, Wastewater. | IS 1622 - 1981, RA 2019, cl 3.3.4: Continue after observation of results for MPN (Fecal coliforms). Subculture, from all positive tubes of BGLB broth, in as many tubes of tryptone water at 44.5℃ for 24±2h in water bath. Add few drops of Kovac's reagent into each tube to test for indole production (pink) or lack of it (yellow). Refer Appendix B Table-3 to estimate MPN (E. coli) per 100 ml sample. | |
| Membrane Filtration: | ||||
| ECDMF | E. coli Detection by membrane filtration. | Purified Water. | IS 15185, 2016 RA 2021 / ISO 9308-1, 2014: | |
| YMDMF | Yeast & Mould detection by membrane filtration. | Purified Water. | IS 5403, 1999 RA 2018 / ISO 7954: | |
| SLDMF | Salmonella detection by membrane filtration. | Purified Water. | IS 15187, 2016 RA 2021 / ISO 19250, 2010: | |
| PADMF | P. Aeruginosa detection by membrane filtration. | Purified Water. | IS 13428, 2024: | |
| SADMF | S. Aureus detection by membrane filtration. | Purified Water. | IS 5887 (Part 2), 1976 RA 2022: | |
| Notes: | ||||
Test Parameters Outside the Scope of NABL, Accreditation.
| Sl | SvCd | Parameter | Test Material | Test Method |
|---|---|---|---|---|
| BET | Bacterial Endotoxin Assay (LAL) | Water for Medicinal Purposes, Purified Water. | International Pharmacopoea | |
| Notes: | ||||
Water Sample Collection Bottles for Bacteriological Analysis:
| SBCd | Bottle Name | Details | Volume |
|---|---|---|---|
| CSB | Clean Sterile Bottle | Clean sterile rigid polypropylene bottle without any additive. | 250ml |
| SBT | Sterile Bottle with Thiosulfate | Sterile rigid polypropylene bottle with sodium thiosulfate to neutralise chlorine, for bacteriological analysis (coliform test) of water. | 250ml |
| SBTE | Sterile Bottle with Extra Thiosulfate | Sterile polypropylene bottle with extra sodium thiosulfate to neutralise excess chlorine, for bacteriological analysis of pool water. | 250ml |
| NPT | Non Pyrogenic Tube | Non-pyrogenic glass tube to collect water sample for bacterial endotoxin test (BET) i.e. LAL assay. | 14ml |
| Notes: | |||