Microbiological Analysis of Tube-Well Water in a Rural Area of Bangladesh

Abstract

Five tube-wells in Matlab, Bangladesh, were selected for analysis of selected biophysicochemical parameters. The results showed that all tube-well water samples contained zooplankton and bacteria. Results for some of the parameters were outside the accepted limits recommended by the World Health Organization for drinking water. It is concluded that water from tube-wells should be treated if used as drinking water.

Tube-well water is used primarily as a source of drinking water by the vast majority (90%) of the rural population in Bangladesh (16). A tube-well is a small-diameter cased well fitted with a cast iron suction hand pump (1). These tube-wells have been installed in Bangladesh at various depths, depending on availability and the level of groundwater. In many cases, immediate environmental conditions are unfavorable; e.g., the distance of tube-wells from latrines or sewage-contaminated ponds or tanks may be insufficient to avoid contamination of the well water with human-pathogenic bacteria. Tube-wells have failed to protect against gastrointestinal diseases in Bangladesh, despite regular use of tube-well water for drinking (14). Recent studies have demonstrated that underground water systems are increasingly vulnerable to both microbiological and heavy metal contamination, especially by arsenic, in Bangladesh. Such problems arise even in developed countries. For example, in 1994, an outbreak of cryptosporidiosis occurred in a rural community in Washington State, where water was supplied by two deep, unchlorinated wells (4).

Besides chemical contaminants, eukaryotic microorganisms (protists) are also a significant component of microbial communities inhabiting groundwater aquifers. This is not unexpected, considering that many protists feed heterotrophically, via either phagotrophy (bacterivory) or osmotrophy (able to grow in the dark on dissolved organic carbon). Protistan numbers in water are usually low (<10² per g [dry weight]) in pristine, uncontaminated aquifers but may increase by several orders of magnitude in polluted aquifers with a high organic content. Small flagellates (typically 2 to 3 μm in size in situ) are by far the dominant zooplankton in aquifers, although amoebae and occasionally ciliates may also be present in relatively lower numbers (17). In Bangladesh, tube-wells have been installed in every village to provide a drinking water source other than rivers and streams, since centralized water treatment systems are not available, at least for the foreseeable future. Therefore, villagers use tube-well water as an assumed safe drinking water source. Since there is a paucity of information concerning protists and other microorganisms in tube-well water, this study was undertaken to address that information gap.

In Matlab, Bangladesh, five tube-wells of various depths (60 to 180 ft) and presently in use in geographically separated villages and assumed safe drinking water sources were selected for study. Plankton analysis was done by filtering 50 liters of tube-well water through a plankton net (20-μm mesh size). The concentrated samples (50 ml) were collected in 4-oz sterile glass bottles. In addition, 500-ml water samples were aseptically collected directly from the tube-wells, using sterile Nalgene plastic bottles. All samples were transported directly to the laboratory at the International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh, in a transport box filled with cool packs (Johnny Plastic Ice, Pelton Sheperd, Stockton, Calif.). The plankton samples were counted within 48 h, using a Sedgewick-Rafter counting chamber, a nanoplankton counting chamber, and a light microscope, according to procedures described by Elliot (5) and Greenberg et al. (6).

Samples of water (100 ml) were filtered through a 0.22-μm-pore-size membrane filter (Millipore Corp.), and the filters were placed on membrane fecal coliform (MFC) agar plates. Plates were incubated at 37 and 44°C for 18 to 24 h to estimate numbers of total and fecal coliforms, respectively.

From each tube-well water sample, 100 μl was inoculated onto nutrient agar using the drop plate technique (8), and inoculated plates were incubated at 37°C for 18 to 24 h. Total viable bacteria were enumerated and recorded as CFU per milliliter.

Acridine orange total count.

Total direct counts of bacteria in the water samples were obtained by the method of Hobbie et al. (7) using Nucleopore filter membranes and fluorescence microscopy.

Direct viable count.

One milliliter from each of the tube-well water samples was enriched with 50 μl of 2% yeast extract and 10 μl of nalidixic acid (1 mg/ml) and incubated at room temperature overnight in the dark, after which the samples were fixed with formalin (2%). Five-microliter portions of the fixed samples were placed on a polytetrafluoroethylene-coated slide and air dried. After fixation with methanol, the samples were stained with 0.01% acridine orange in the dark for 3 min (13). Both nonelongated and elongated cells were counted as total bacteria and only elongated cells were counted as viable bacteria, using a fluorescence microscope and according to the procedures described by Islam et al. (11).

Physicochemical parameters.

Conductivity, salinity, and total dissolved solids (TDS) were measured using a portable meter (HACH C0150 conductivity meter, model 50161). The pH of the water samples was measured using a digital pH/multivolt meter (Orion Research model 611). Hardness was estimated by the titrimetric method of Greenberg et al. (6). The arsenic content of the tube-well water was also measured, using a commercial kit (MerckoQuant; E. Merck, Darmstadt, Germany), following the manufacturer's instructions.

All of the tube-well water samples contained a variety of microorganisms (Table 1). Total zooplankton (ciliates, flagellates, and pseudopodans) ranged in number from <1 to 2,830 cells/liter. The highest count of zooplankton was obtained in water samples collected from tube-well 2, and the lowest was for water samples collected from tube-well 5. Pseudopodans were observed in water samples collected from tube-wells 1, 3, and 4. Tube-wells 1, 3, and 5 contained both total coliforms and fecal coliforms. The total aerobic bacterial plate counts in all of the tube-well water samples ranged from 3.0 × 10¹ to 8.5 × 10² CFU/ml. However, the acridine orange total and direct viable counts ranged from 4.3 × 10⁵ to 1.6 × 10⁷/ml and from 9.5 × 10⁴ to 1.3 × 10⁷/ml, respectively. The nonculturable but viable bacterial counts for the water samples from all of the tube-wells were 3 to 5 log units higher than the total aerobic culturable bacterial counts. The pH, conductivity, TDS, salinity, and hardness were in the ranges of 6.27 to 7.07, 1,533 to 1,997 μS/cm, 755 to 994 mg/liter, 0.8 to 1.0 ppt, and 383 to 678 mg/liter, respectively. The arsenic content of the water samples collected from tube-well 2 was >0.1 mg/liter.

TABLE 1.

Biophysicochemical parameters of water samples collected from five tube-wells in Matlab, Bangladesh

Parameters	Value in tube-well:					World Health Organization guideline value
	1	2	3	4	5
Depth (ft)	180	60	180	180	120	NGVa
Total zooplankton/liter	1.4 × 103	2.8 × 103	1.3 × 103	2.1 × 103	<1	NGV
Total viable (culturable) bacteria/ml	3.0 × 101	3.2 × 102	1.3 × 102	6.0 × 102	8.5 × 102	<103
Total coliforms/100 ml	1.7 × 101	<1	1.0 × 101	<1	1	0
Fecal coliforms/100 ml	1.0 × 101	<1	7	<1	1	0
AODTCb/ml	4.27 × 105	1.58 × 107	1.22 × 106	1.41 × 106	2.35 × 106	NGV
AODVCc/ml	9.49 × 104	1.30 × 107	6.76 × 105	6.17 × 105	1.05 × 106	NGV
pH	6.39	7.07	6.27	6.29	6.56	6.5–8.5
Conductivity (μS/cm)	1,676	1,997	1,705	1,654	1,533	NGV
TDS (mg/liter)	780	994	855	819	755	<1000
Salinity (‰)	0.8	1.0	0.9	0.8	0.8	NGV
Hardness (mg/liter)	406	678	412	383	390	<500
Arsenic (mg/liter)	<0.1	>0.1	<0.1	<0.1	<0.1	<0.01

^aNGV, no guideline value. 

^bAODTC, acridine orange direct total counts. 

^cAODVC, acridine orange direct viable counts. 

Zooplankton (including ciliates) were present in all tube-well water samples examined in this study. Biofilms containing aquatic fungi were observed in water samples collected from all of the tube-wells.

Clearly, tube-well water in Bangladesh is not free of microorganisms (Table 1). Moe et al. (15) reported similar conclusions concerning groundwater contamination in a study carried out in the Philippines. In this study, zooplankton were present at thousands of individuals per liter in most of the tube-well water samples examined. In several instances, physicochemical parameters of the tube-wells were outside ranges recommended by the World Health Organization (18). The pHs of water samples collected from tube-wells 1, 3, and 4 were acidic and were lower than recommended by the World Health Organization (18). The hardness of water samples collected from tube-well 2 was above the recommended value, as was the arsenic content. In fact, the arsenic concentration in tube-well 2 water was 10-fold higher than the accepted limit recommended by the World Health Organization (18).

It has been generally believed in Bangladesh that groundwater is relatively free of microorganisms and, therefore, fit for human consumption without treatment. However, the results of this study show clearly that all samples of tube-well water in rural Bangladesh that were examined contained high counts of bacteria and zooplankton, as well as fungi.

The findings of this study are also in agreement with those of Kinner et al. (12) concerning plankton populations in groundwater. The conclusion is that tube-well water in rural Bangladesh cannot be considered safe for drinking unless properly treated.

It has been proposed that simple filtration can be employed to reduce the incidence of gastrointestinal disease caused by enteric pathogens attached to particulates, particularly Vibrio cholerae, which is commensal to zooplankton, when centralized systems for drinking water employing filtration and chlorination are not available, as is the case for the rural villages of Bangladesh (2, 3, 9, 10). Since gastrointestinal diseases transmitted by unsafe drinking water are a global threat, low-technology solutions to the problem of providing safe drinking water provide a short-term, stop-gap solution. The salient point, however, is that tube-wells by themselves do not provide a source of microbiologically or chemically safe drinking water.