Assessment of the effect of holding time on total coliforms, and E.coli

Transcription

1 Assessment of the effect of holding time on total coliforms, and E.coli, by Quanti-tray method, and heterotrophic bacteria by heterotrophic plate count method. Mohale Dimakatso Pertunia Lepelle Nothern Water Central Laboratory Microbiology

2 1. Rationale 2. Aim: To determine whether holding time of sample at <8 has an effect on the results of E.coli, total coliforms and heterotrophic counts 3. Objectives I. To determine the effects of 8,24 and 48 hours holding time of sample before analysis II. To determine the effect of holding period on plates after incubation 4. Introduction E.coli, total coliform and fecal coliform are used routinely as microbiological indicators of water quality. Microbiological analysis of water samples must be start within 2 hours of collection or as soon as possible after collection to avoid unpredictable changes in the microbial population. And it is important to adhere to sample collection procedure and holding limits Standard method recommend that Microbiological analysis of drinking water samples for compliance purposes must be analysed immediately after collection, if not possible, iced or refrigerated analysed as soon as possible after collection. An allowed at maximum transit time of 6 hours. If the laboratory is far from the sampling point, samples must be transported in a cooler box with ice packs at a temperature of <8⁰C during transportation for less than 6 hours. Sample storage and preservation is governed by sample matrix of interest and desired results, nature of the parameter of interest and analytical method. Storage time should be considered as a whole, both during transport and in the laboratory. Holding time can have significant impact on the density of microbiological indicators at the time of sample analysis. Holding time and temperature according to the standard method 2005:9060 B, E.coli; must be analysed within 8hrs after collection, or kept overnight (24hours) at <8⁰C, sample storage must not exceed 30hours. Total coliforms analyse on day of receipt, if not refrigerate samples overnight at a temperature of <8⁰C and do not exceed 30 hours holding time from collection to analysis. Heterotrophic plate count must be analysed within 8 hours of sample collection, or within 24 hours when stored at <8⁰C. Lepelle northern water laboratory serves large geographic areas and receives samples from distant water supplies. These samples are packed in a cooler box with ice packs at a temperature of <10⁰C after collection and transported to the laboratory. Some sample arrive late in the laboratory due to long travelling distance and cannot be analysed within 8hour period, they are stored in refrigerator at a temperature of <8⁰C for 24 hours. 5. Literature review In a research titled holding effects on coliform enumeration in drinking water samples, conducted by McDaniels et al in1985 revealed that heterotrophic counts using pour plate method when samples are stored at 5 for 30 and 48 hours, heterotrophic counts declined slightly. And half of the samples declined in counts while half increased but the changes were not statistically significant. In a study conducted by Misty et al in 2003, where they studied the effects of holding time and storage conditions on E.coli using methods Colilert 18 (Iddex quanti-tray) and membrane filtration method. E.coli sample were analysed at 0, 8, 24, 30 and 48 hours after sample collection were three of four sides showed no significant difference in bacterial densities throughout the 48hours study by

3 membrane filtration method. While with colilert method, where five of the seven sides showed no significant difference in E.coli densities between time 0 and the 48 hour holding time. Where they concluded samples stored at 4 and 10⁰C for 8 and 24 hours did not show any signicant difference in bacterial densities, compared to 0hrs storage, until samples had been held at 48 hours. Bacterial (Total, fecal coliforms and E.coli) holding time experiments of up to 62 hours were performed in a study conducted by Brent A in Where holding time test for total and fecal coliforms does not have any significant changes with a holding time of up to 27 hours, and possibly up to 72 hours for fecal coliform when using mfc agar. While E.coli results differ with most previous studies because this study indicated significant difference in three to five samples which was 60% of the samples. This study indicated that water samples remain stable up to 18.5 hours when using colilert 18 method. 6. Method Collect samples and divide them into 3, analyse the first batch and store the other two replicate at 8⁰C for 24 and 48hours. Using Colilert 18 iddex quanti-tray method for total, fecal coliform and E.coli, pour plate for heterotrophic counts. Analyse each and every replicate in duplicate, after incubation count the first replicate and store the other replicate in a fridge at <8⁰C for hours. 7. Quality control Samples analysed according to standard method that verified and validated, blank sample analysed with the samples, duplicate analyses, and use reference cultures according to each specific method. 8. Results Parameter Holding time No of analysed samples No of samples with Significant difference % of significant diff HPC 8-24 hrs % 97% hrs % 97% Total coli 8-24 hrs % 75% hrs % 64% E.coli 8-24 hrs % 84% hrs % 80% % of stability The effect of holding time on HPC, indicted that heterotrophs do not change significantly with holding time up to 48 hours, however samples with low bacterial counts will not be detected after 24 hours holding time i;e samples with 5 and less colony forming units will not be detected after 24 hours holding time. Out of 30 samples only one sample showed significant decrease in colony forming units. While the effect of holding time on total coliforms indicated that 6 out of 24 samples had a significant decrease after 24 holding time. E.coli holding time indicated 16% difference and 20% difference after 24 and 48 hours holding times respectively. However 8 %of the samples indicated, that samples that contains 5 or less E.coli have a slight probability of being undetected.

4 9. Discussion This study indicated that 75-97%of the samples remain microbiologically stable for a period of 24 hours at < 10 C. Bacterial density changes significantly after 48 hour storage. This in agreement with a study conducted by Standridge and Lesar 1977 and Brent A.T 2008, where bacterial densities remained at 18, and 27 hours storage respectively. This study indicated that samples with low bacterial i:e heterotrophs and total coliforms and E.coli density, are likely to be not detected after 24 hour storage, and Furgerson indicated that holding time appeared critical when bacterial densities are low. However Selvakumar et al 2004 indicated holding time could be extended beyond the 24 hour for total coliform, fecal coliform and up to 24hrs for E.coli. Changes in bacterial densities are either decrease or increase. 10. Conclusion Water samples for the detection of HPC,TC,FC, and E.coli can be stored up to 24 hours provided they are stored at <10 C immediately after sampling

5 11. References 1. Audrey E. McDaniels., Robert H. Bordner.,Peter S. Gartside., John R.Haines.,Kristen P.Brenner and Clifford C. Rankin (1985). Holding effects of coliform enumeration in drinkin water samples. Applied and Enviromental Microbiology pp vol 50,.No.4 2. Brent T. Aulenbach (2008). Bacterial holding times for faecal coliform by Mfc agar method and total coliform and Escherichia coli by Coliert-18 Quanti-tray method. Environmental monitoring and assessment, Doi 10:1007/s Misty.L.P., Michelle B.,Mary. A.F.,Lois Shadix. Sharon Gonder, Crystal Rodgers, Yildiz Chambers, Jessica Pulz, Ken Miller, Kevin Connell, and Jon Standridge (2003). Assesment of the effects of holding time and temperature on Escherichia coli Densities in surface water samples. Applied and environmental Microbiology.pp Selvakumar, A.,Borst, M., Boner, M., and Mallon, P. (2004). Effects of sample holding time on concerntartions of microorganisms in water samples. Water environmental research, 76(1), Doi: / X Standeridge, A., and Laser, D.J. (1977). Comparison of four-and twenty-four-hour refrigerated storage of non-potable water for faecal coliform analysis. Applied and Environmental Microbiology, 34 (4), Standard method 2005:9060 B