Detection and characterization of filterable heterotrophic bacteria from rural groundwater supplies

Abstract

Aims:The chemical/physical environment of groundwater may contribute to the existence of a subpopulation of small-sized bacteria (®lterable bacteria) that fails to be trapped on conventional 0á45 lm-pore-size membrane ®lters during routine bacteriological water quality analyses. Efforts were directed to determining an ef®cient recovery method for detection of such cells. Methods and Results: Individual groundwater supplies in a rural setting were examined by a double membrane ®ltration procedure to determine the presence of heterotrophic plate count (HPC) bacteria capable of escaping detection on conventional pore size (0á45 lm) membrane ®lters but retained on 0á22 lm-pore-size ®lters. Since optimum cultural conditions for recovery of ®lterable bacteria are not well de®ned, initial efforts focused on evaluation of various media (R2A, m-HPC and NWRI) and incubation temperatures (15, 20, 28 and 35°C) for speci®c recovery of ®lterable bacteria. Maximum recovery of small-sized HPC bacteria occurred on low-nutrient concentration R2A agar incubated for 7 d at 28°C. Similarly, identical cultural conditions gave enhanced detection of the general HPC population on 0á45 lm-pore-size ®lters. A 17-month survey of 10 well water supplies conducted with the cultural conditions described above resulted in detection of ®lterable bacteria (ranging in density from 9 to 175 cfu ml )1 ) in six of the groundwater sources. The proportion of ®lterable bacteria in any single sample never exceeded 10% of the total HPC population. A majority of the colonies appearing on the 0á22 lm membrane ®lters was pigmented (50±90%), whereas the proportion of colonies demonstrating pigmentation on the larger porosity ®lters failed to exceed 50% for any of the samples (19±49%). Conclusions: A reliable recovery method was developed for the detection of ®lterable bacteria from groundwater. During a subsequent survey study using this procedure, ®lterable bacteria were detected in a majority of the groundwater supplies examined; however, the density of ®lterable bacteria in any single sample never exceeded 10% of the total HPC population. Identi®cation of randomly selected isolates obtained on the 0á22 lm ®lters indicated that some of these ®lterable bacteria have been implicated as opportunistic pathogens. Signi®cance and Impact of the Study: We have determined the presence of small-sized HPC bacteria in ground water that may go undetected when using standard porosity membrane ®lters for water quality analyses. Further study is needed to assess the signi®cance and possible health risk associated with presence of ®lterable bacteria in drinking water supplies from groundwater sources.

Identification of the culturable and nonculturable bacterial population in ground water of a municipal water supply in Germany

“…The cultured bacteria mainly belonged to the Proteobacteria (a, b and c), Flavobacteria or Actinobacteria. Most of them have been isolated from ground or drinking water by other researchers (Starr et al 1981;Rheinheimer 1991;Bowman et al 1993;Lorbeer and Babel 1996;Defives et al 1999;Richardson 1999;Squier et al 2000;Garrity 2001;Lillis and Bissonnette 2001;Leclerc and Moreau 2002). Kalmbach et al (1997) also found a-, band c-Proteobacteria in drinking water.…”

Effect of different heterotrophic plate count methods on the estimation of the composition of the culturable microbial community

“…Many questions that arose at the time when HPC methods were established (including media suitability, relationship between the bacteria in water samples and the corresponding HPC counts) remain the focus of discussion today ( Bartram et al, 2003 ). Some studies experimentally evaluated the effect of varying the cultivation parameters (i.e., media type and temperature) and found differences in plate counts ( Lillis & Bissonnette, 2001 ; Allen, Edberg & Reasoner, 2004 ; Bartram et al, 2004 ; Reasoner, 2004 ; Inomata, Chiba & Hosaka, 2009 ).…”

Review: Microbial biocenoses in pristine aquifers and an assessment of investigative methods

“…bacteriophages to simulate viruses, and microspheres to simulate bacteria and protozoans (e.g. Auckenthaler et al 2002;Craun et al 2002;DeBorde et al 1998;Edberg et al 1997;Flanigan and Rodgers 2003;Flynn 2003;Golas et al 2002;Herwaldt et al 1992;Lillis and Bissonnette 2001;Lisle and Rose 1995;Mahler et al 2000;Nasser et al 1993;Rossi et al 1998;Schaffter and Parriaux 2002;Szewzyk et al 2000).…”