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Dip slide technique for rapid qualitative estimation of fecal coliforms in water and wastewater
PII: S0043-1354(98)00271-1
Wat. Res. Vol. 33, No. 4, pp. 989±994, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/99/$ - see front matter
DIP SLIDE TECHNIQUE FOR RAPID QUALITATIVE ESTIMATION OF FECAL COLIFORMS IN WATER AND WASTEWATER S. SANDHYA*, T. S. UMA and K. SUBBARAO National Environmental Engineering Research Institute, CSIR Complex, Chennai 600113, India (First received January 1995; accepted in revised form June 1998) AbstractÐA simple, rapid, inexpensive method for qualitative estimation of fecal coliforms (FC) in water and wastewaters is presented. The estimation of FC by the dip slide technique (DST) gives results within 8 h. The DST is compared with the conventional membrane ®lter (MF) procedure. The ecacy of the proposed technique is evaluated by statistical analysis. # 1999 Elsevier Science Ltd. All rights reserved Key wordsÐfecal coliforms, dip slide technique, membrane ®lter technique
INTRODUCTION
The fecal coliforms (FC) are the indicator organisms used to assess the microbiological quality of drinking waters and of euents from wastewaters treatment units. The U.S. Environmental Protection Agency (USEPA) has speci®ed the MF and multiple tube dilution (MTD) methods for the enumeration of fecal coliforms from waters and wastewaters (APHA, 1985). The standard methods are time consuming and require appropriate dilution and incubation followed by con®rmation of FC in samples. Keeping in mind, the eorts required in FC estimation by conventional methods, various researchers have proposed a simple, potable, inexpensive procedure for fecal coliform estimation. Dange et al. (1988) have proposed a one hour portable test using radioactive P-32. Recently, USEPA has proposed the use of the presence±absence (P±A) coliform test (APHA, 1985) and the autoanalysis coliert (AC) test (Covert et al., 1989). Development of genetic procedures for environmental detection of coliforms by DNA recovery, ampli®cation by PCR and detection with gene probe is an entry to a new detection methodology (Bej et al., 1990). However, these methods are rather complicated and need highly sophisticated equipment and results obtained are just the estimate and not the actual viable counts. Mara (1972) has reported the use of ``Oxid'' and ``Uricult'' agar dip slides for estimation of fecal coliforms from polluted water. However, this method requires 24 h for completion. This has prompted authors to develop a simple, dip slide technique for rapid estimation of fecal coliforms *Author to whom all correspondence should be addressed. 989
from water and wastewater, which might prove to be of great value for monitoring the samples routinely and during an epidemic situation.
MATERIALS AND METHODS
Preparation of dip slides Glass plates of 87 mm long, 62 mm wide and 1.4 mm thick were used for fabrication of the dip slide on the lines of a sedgwick rafter cell. At one end of the slide a cavity 72 mm long, 59 mm wide and 1.0 mm deep was made by ®rmly pasting narrow glass strips by araldite to accommodate the rapid agar medium as shown in Fig. 1. All the slides were made with minimum statistical errors. The glass-chamber of 117 mm long, 63 mm wide and 102 mm deep with snugly ®tting lid at the top was fabricated to keep the slides under sterile condition in vertical position. Medium The rapid agar medium of Francis et al. (1974) was used, the composition is given in Table 1. Procedure Clean, dried chamber and slides, wrapped separately, were sterilised in a hot air oven at 1658C for one hour. The slides were arranged in rows in horizontal position on two glass rods ®xed on two opposite sides, with the cavity on the top. The cavity of the slide was ®lled with rapid agar medium. The agar was allowed to solidify and set ®rmly. The slides are always prepared on the previous day and checked for their sterility. A ``dip slide'' was dipped in a beaker containing samples and excess of liquid was allowed to drain o and kept in the chamber. The slides in the glass chamber were incubated at 41.5 20.58C for 7±8 h. During this period colonies of fecal coliforms bacteria developed and appeared as yellow or orange against the deep bluishgreen background of the medium. The colonies developed on each slide of respective dilution of the samples were counted and counts were multiplied by the respective dilution factor. Since all the slides have identical surface area with same agar medium, it is but logical to assume
Fig. 1. Dip slide and glass chamber.
Estimation of fecal coliforms in water Table 1. The composition of speci®c rapid agar Components
g/l
Proteose peptone Yeast extract Lactose Sodium chloride Sodium lauryl sulfate Bromothymol blue Agar
5.0 3.0 10.0 7.5 0.05 0.3 15.0
pH 7.3.
that the quantity of liquid sticking to the agar surface of one of the slides will be the same as that of the other slides. The estimation of fecal coliforms by consecutive dipping of slides has already been studied and discussed by Sandhya et al. (1990). RESULTS
The following parameters were ascertained which include eects of immersion time, eect of FC population on DST, temperature and veri®cation of colonies for FC. The application of DST in FC estimation in a wastewater treatment plant and in drinking water sources was investigated. Eect of time immersion of the dip slide in a suspension of bacteria upon numbers retained on the dip slide during agitation
991
The results of the experiment conducted in triplicate are presented in Fig. 2. No increase in numbers retained by dip slides was observed by agitating the samples. The eect of changes in suspension population density upon the number of bacteria retained by the dip slides was shown to be a linear relationship (Fig. 2). This evidence suggests that the retention of bacteria in liquid ®lms on agar surfaces after immersion in a bacterial population is a simple dilution eect and the number retained is dependent upon suspension population density. The relationship is unaected by the time of immersion of the dip slide and the disturbance. At the same time extrapolation of these results to surfaces other than rapid medium is not valid. The existence of a similar relationship between bacterial population densities and retention of bacteria in tooth enamel, has also been noticed. Correlation between the bacterial population and colonies on the dip slide Five dierent volumes of sewage sample (120 103 CFU/ml) were added to 100 ml of sterile distilled water, then the dip slides in triplicate were dipped and incubated. Similarly ®ve dierent
A dip slide loaded with rapid medium was immersed for various times (up to 20 s) in diluted sewage (30 colony forming units (CFU)/ml in MF count) with agitation on a magnetic stirrer. The slides were then incubated for 8 h.
Fig. 2. Eect of time of immersion in suspension of bacteria on numbers retained by dip slides.
Fig. 3. Eect of population on number of colonies on dip slides.
992
R. S. Sandhya et al.
Table 2. Eect of incubation temperature on the development of fecal coliforms on dip slides Temperature (8C)
Time of incubation (h)
Number of organisms on dip slides (103)
16 8 8
42 170 190
37 20.5 41.5 20.5 44.2 20.5
samples of sewage (60 103 CFU/ml) were analyzed to ascertain the correlation between the bacterial population and the number of colonies on the dip slide. The correlation at two dierent bacterial population densities is depicted in Fig. 3. The extent of colonization on the surfaces is in¯uenced not only by the innate capacity of bacterial species to sorb, but also by the number of cells available. At higher dilution, the fecal coliforms estimation by DST, indicates a correlation (r = 0.9961) between the population and the size of inoculum. The experiments showed that the higher the population the more sensitive is the DST technique. It has been suggested that for absorption and actual colonization of bacterial cells on surfaces, signi®cantly higher numbers of cells are essential. Eect of incubation temperature on fecal coliform colonies development on dip slides The slides loaded with rapid medium were immersed in an appropriate dilution of sewage sample and incubated at 37, 41.5 2 0.5 and 44.2 2 0.58C temperatures. The time required for development of visible colonies was considered as the optimum time and counting of the colonies was carried out (Table 2). There was no signi®cant increase in numbers at a higher temperature (44.2 + 0.58C) during incubation. Coupling this optimum incubation temperature with the rapid, speci®c medium has permitted quantization of fecal
Fig. 4. Comparison of fecal coliforms numbers estimated by conventional MF and DST in wastewater treatment units.
coliforms in 8 h. Van Donsel et al. (1969) used a gradient temperature incubation and determined that 41.58C was the optimum for quantization of fecal coliforms in 8 h. Veri®cation of colonies on dip slides for fecal coliforms The orange/yellow colonies developed within 8 h on the dip slide were picked and subjected to a con®rmation test for FC by the standard procedure (APHA, 1985). All 575 colonies were tested and results are given in Table 3. Almost all the colonies developed on the dip slide within 8 h of incubation at the selective temperature of 41.5 2 0.58C were invariably con®rmed as fecal coliforms (99.75%) and 93.75% as E. coli type I, thus omitting the tedious procedure of con®rmation. The often faced drawback of false positive test of FC in conventional methods can be avoided in the direct dipping method.
Table 3. Veri®cation of colonies on dip slides for fecal coliforms Item
Number of colonies
Percentage
515 512 480
ÿ 99.75 93.75
Total number of colonies examined Number of colonies con®rmed as FC Number of colonies con®rmed as E. coli type I
Table 4. Fecal coliform count in wastewater treatment plant enumerated by DST Source
Sewage treatment plant Koyambedu Nesapakkam Wastewater treatment plant Fragnance manufacturing company Biscuits manufacturing company
Number of samples tested
FC count (meana)
in¯uent
euent
in¯uent (105)
euent (103)
33 33
33 33
5.8 6.3
5.9 14.0
28 28
28 28
2.8 8.8
2.0 1.1
Estimation of fecal coliforms in water
993
Table 5. Enumeration of fecal coliforms by DST in drinking water sources Source
Number of samples
Number of samples for fecal coliforms positive
Tube well Drinking water supply Open well Lake water
13 14 1 2
Application of DST in evaluation of wastewater treatment units The in¯uent and euent samples from wastewater treatment plants at Chennai were collected and analyzed for fecal coliforms content, Table 4. The dierent wastewater samples (112) were analyzed for their FC content by DST. The data were subjected to regressional analysis with FC removed enumerated by DST being X variable and by MF being the Y variable as depicted in Fig. 4. The two methods agreed with each other to yield an r value of 0.99. Application of DST in FC estimation from drinking water sources A total of thirty samples from dierent drinking water sources as speci®ed in Table 5 have been ana-
negative
DST
MF
5 10 1 1
7 9 1 0
6 3 ÿ 1
lyzed for enumeration of FC by DST and the standard MF technique. The rapid test is as sensitive as the conventional MF test for detection of fecal pollution in dierent drinking water sources (Fig. 5). A comparison of qualitative examination for FC by dip slide and MF techniques showed that in 30 drinking water samples, 82% samples are positive for FC by DST, whereas 93% by MF. The four samples, which were tested negative for FC by DST, whereas by MF technique they were positive. In general for ``qualitative estimation'' of FC from drinking water sources DST can be considered as a simple and rapid technique. For routine monitoring of euent quality and drinking water pollution, dip slides come to the nearest order of magnitude and is suciently accurate.
Fig. 5. Comparison of fecal coliforms numbers estimated by conventional MF and DST in drinking water sources.
994
R. S. Sandhya et al. CONCLUSIONS
Some of the major strengths of the DST are . results in 8 h . 99.97% FC are E. coli with no con®rmation tests needed . sensitivity to FC and E. coli concentration as low as 10 CFU/100 ml . can be used by unskilled, layman without having microbiology background . can be used as disposable slides because of low cost of manufacturing AcknowledgementsÐAuthors are grateful to Professor P. Khanna, Director, National Environmental Engineering Research Institute, Nagpur, India for his valuable suggestions, guidance and allowing to publish the work. The authors are thankful to CPHEEO, New Delhi, India for funding the research. REFERENCES
American Public Health Association (1985) Standard Methods for the Examination of Water and Wastewater,
16th ed. American Public Health Association, Washington DC. Bej A. K., Stean R. J., Dicesare J., Ha L. and Atlas R. M. (1990) Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Appl. Environ. Microbiol. 56, 307±314. Covert T. C., Shadix L. C., Rice E. W., Haines J. R. and Freybery R. W. (1989) Evaluation of the autoanalysis coliert test for detection and enumeration of total coliforms. Appl. Environ. Microbiol. 55, 2443±2447. Dange V., Jothikumar N. and Khanna P. (1988) One hour portable test for drinking water. Wat. Res. 22, 133±135. Francis D. W., Peeter J. T. and Twedt R. M. (1974) Rapid method for detection and enumeration of fecal coliforms in fresh chicken. Appl. Microbiol. 27, 1127± 1130. Mara D. D. (1972) The use of agar dip slides for estimation of bacterial numbers in polluted waters. Wat. Res. 6, 1605±1607. Sandhya S., Phirke P. M. and Deshpande V. A. (1990) Dip slide technique: a rapid methodology for performance evaluation of wastewater treatment system. J. Environ. Sci. Health A (U.S.A.) 25, 325±341. Van Donsel D.J., Twelt R.M. and Gelrich F.E. (1969) Optimum temperature for quantization of fecal coliforms in seven hours on the membrane ®lter. Bacteriol. Proc. Abstr. G46.
Wat. Res. Vol. 33, No. 4, pp. 989±994, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/99/$ - see front matter
DIP SLIDE TECHNIQUE FOR RAPID QUALITATIVE ESTIMATION OF FECAL COLIFORMS IN WATER AND WASTEWATER S. SANDHYA*, T. S. UMA and K. SUBBARAO National Environmental Engineering Research Institute, CSIR Complex, Chennai 600113, India (First received January 1995; accepted in revised form June 1998) AbstractÐA simple, rapid, inexpensive method for qualitative estimation of fecal coliforms (FC) in water and wastewaters is presented. The estimation of FC by the dip slide technique (DST) gives results within 8 h. The DST is compared with the conventional membrane ®lter (MF) procedure. The ecacy of the proposed technique is evaluated by statistical analysis. # 1999 Elsevier Science Ltd. All rights reserved Key wordsÐfecal coliforms, dip slide technique, membrane ®lter technique
INTRODUCTION
The fecal coliforms (FC) are the indicator organisms used to assess the microbiological quality of drinking waters and of euents from wastewaters treatment units. The U.S. Environmental Protection Agency (USEPA) has speci®ed the MF and multiple tube dilution (MTD) methods for the enumeration of fecal coliforms from waters and wastewaters (APHA, 1985). The standard methods are time consuming and require appropriate dilution and incubation followed by con®rmation of FC in samples. Keeping in mind, the eorts required in FC estimation by conventional methods, various researchers have proposed a simple, potable, inexpensive procedure for fecal coliform estimation. Dange et al. (1988) have proposed a one hour portable test using radioactive P-32. Recently, USEPA has proposed the use of the presence±absence (P±A) coliform test (APHA, 1985) and the autoanalysis coliert (AC) test (Covert et al., 1989). Development of genetic procedures for environmental detection of coliforms by DNA recovery, ampli®cation by PCR and detection with gene probe is an entry to a new detection methodology (Bej et al., 1990). However, these methods are rather complicated and need highly sophisticated equipment and results obtained are just the estimate and not the actual viable counts. Mara (1972) has reported the use of ``Oxid'' and ``Uricult'' agar dip slides for estimation of fecal coliforms from polluted water. However, this method requires 24 h for completion. This has prompted authors to develop a simple, dip slide technique for rapid estimation of fecal coliforms *Author to whom all correspondence should be addressed. 989
from water and wastewater, which might prove to be of great value for monitoring the samples routinely and during an epidemic situation.
MATERIALS AND METHODS
Preparation of dip slides Glass plates of 87 mm long, 62 mm wide and 1.4 mm thick were used for fabrication of the dip slide on the lines of a sedgwick rafter cell. At one end of the slide a cavity 72 mm long, 59 mm wide and 1.0 mm deep was made by ®rmly pasting narrow glass strips by araldite to accommodate the rapid agar medium as shown in Fig. 1. All the slides were made with minimum statistical errors. The glass-chamber of 117 mm long, 63 mm wide and 102 mm deep with snugly ®tting lid at the top was fabricated to keep the slides under sterile condition in vertical position. Medium The rapid agar medium of Francis et al. (1974) was used, the composition is given in Table 1. Procedure Clean, dried chamber and slides, wrapped separately, were sterilised in a hot air oven at 1658C for one hour. The slides were arranged in rows in horizontal position on two glass rods ®xed on two opposite sides, with the cavity on the top. The cavity of the slide was ®lled with rapid agar medium. The agar was allowed to solidify and set ®rmly. The slides are always prepared on the previous day and checked for their sterility. A ``dip slide'' was dipped in a beaker containing samples and excess of liquid was allowed to drain o and kept in the chamber. The slides in the glass chamber were incubated at 41.5 20.58C for 7±8 h. During this period colonies of fecal coliforms bacteria developed and appeared as yellow or orange against the deep bluishgreen background of the medium. The colonies developed on each slide of respective dilution of the samples were counted and counts were multiplied by the respective dilution factor. Since all the slides have identical surface area with same agar medium, it is but logical to assume
Fig. 1. Dip slide and glass chamber.
Estimation of fecal coliforms in water Table 1. The composition of speci®c rapid agar Components
g/l
Proteose peptone Yeast extract Lactose Sodium chloride Sodium lauryl sulfate Bromothymol blue Agar
5.0 3.0 10.0 7.5 0.05 0.3 15.0
pH 7.3.
that the quantity of liquid sticking to the agar surface of one of the slides will be the same as that of the other slides. The estimation of fecal coliforms by consecutive dipping of slides has already been studied and discussed by Sandhya et al. (1990). RESULTS
The following parameters were ascertained which include eects of immersion time, eect of FC population on DST, temperature and veri®cation of colonies for FC. The application of DST in FC estimation in a wastewater treatment plant and in drinking water sources was investigated. Eect of time immersion of the dip slide in a suspension of bacteria upon numbers retained on the dip slide during agitation
991
The results of the experiment conducted in triplicate are presented in Fig. 2. No increase in numbers retained by dip slides was observed by agitating the samples. The eect of changes in suspension population density upon the number of bacteria retained by the dip slides was shown to be a linear relationship (Fig. 2). This evidence suggests that the retention of bacteria in liquid ®lms on agar surfaces after immersion in a bacterial population is a simple dilution eect and the number retained is dependent upon suspension population density. The relationship is unaected by the time of immersion of the dip slide and the disturbance. At the same time extrapolation of these results to surfaces other than rapid medium is not valid. The existence of a similar relationship between bacterial population densities and retention of bacteria in tooth enamel, has also been noticed. Correlation between the bacterial population and colonies on the dip slide Five dierent volumes of sewage sample (120 103 CFU/ml) were added to 100 ml of sterile distilled water, then the dip slides in triplicate were dipped and incubated. Similarly ®ve dierent
A dip slide loaded with rapid medium was immersed for various times (up to 20 s) in diluted sewage (30 colony forming units (CFU)/ml in MF count) with agitation on a magnetic stirrer. The slides were then incubated for 8 h.
Fig. 2. Eect of time of immersion in suspension of bacteria on numbers retained by dip slides.
Fig. 3. Eect of population on number of colonies on dip slides.
992
R. S. Sandhya et al.
Table 2. Eect of incubation temperature on the development of fecal coliforms on dip slides Temperature (8C)
Time of incubation (h)
Number of organisms on dip slides (103)
16 8 8
42 170 190
37 20.5 41.5 20.5 44.2 20.5
samples of sewage (60 103 CFU/ml) were analyzed to ascertain the correlation between the bacterial population and the number of colonies on the dip slide. The correlation at two dierent bacterial population densities is depicted in Fig. 3. The extent of colonization on the surfaces is in¯uenced not only by the innate capacity of bacterial species to sorb, but also by the number of cells available. At higher dilution, the fecal coliforms estimation by DST, indicates a correlation (r = 0.9961) between the population and the size of inoculum. The experiments showed that the higher the population the more sensitive is the DST technique. It has been suggested that for absorption and actual colonization of bacterial cells on surfaces, signi®cantly higher numbers of cells are essential. Eect of incubation temperature on fecal coliform colonies development on dip slides The slides loaded with rapid medium were immersed in an appropriate dilution of sewage sample and incubated at 37, 41.5 2 0.5 and 44.2 2 0.58C temperatures. The time required for development of visible colonies was considered as the optimum time and counting of the colonies was carried out (Table 2). There was no signi®cant increase in numbers at a higher temperature (44.2 + 0.58C) during incubation. Coupling this optimum incubation temperature with the rapid, speci®c medium has permitted quantization of fecal
Fig. 4. Comparison of fecal coliforms numbers estimated by conventional MF and DST in wastewater treatment units.
coliforms in 8 h. Van Donsel et al. (1969) used a gradient temperature incubation and determined that 41.58C was the optimum for quantization of fecal coliforms in 8 h. Veri®cation of colonies on dip slides for fecal coliforms The orange/yellow colonies developed within 8 h on the dip slide were picked and subjected to a con®rmation test for FC by the standard procedure (APHA, 1985). All 575 colonies were tested and results are given in Table 3. Almost all the colonies developed on the dip slide within 8 h of incubation at the selective temperature of 41.5 2 0.58C were invariably con®rmed as fecal coliforms (99.75%) and 93.75% as E. coli type I, thus omitting the tedious procedure of con®rmation. The often faced drawback of false positive test of FC in conventional methods can be avoided in the direct dipping method.
Table 3. Veri®cation of colonies on dip slides for fecal coliforms Item
Number of colonies
Percentage
515 512 480
ÿ 99.75 93.75
Total number of colonies examined Number of colonies con®rmed as FC Number of colonies con®rmed as E. coli type I
Table 4. Fecal coliform count in wastewater treatment plant enumerated by DST Source
Sewage treatment plant Koyambedu Nesapakkam Wastewater treatment plant Fragnance manufacturing company Biscuits manufacturing company
Number of samples tested
FC count (meana)
in¯uent
euent
in¯uent (105)
euent (103)
33 33
33 33
5.8 6.3
5.9 14.0
28 28
28 28
2.8 8.8
2.0 1.1
Estimation of fecal coliforms in water
993
Table 5. Enumeration of fecal coliforms by DST in drinking water sources Source
Number of samples
Number of samples for fecal coliforms positive
Tube well Drinking water supply Open well Lake water
13 14 1 2
Application of DST in evaluation of wastewater treatment units The in¯uent and euent samples from wastewater treatment plants at Chennai were collected and analyzed for fecal coliforms content, Table 4. The dierent wastewater samples (112) were analyzed for their FC content by DST. The data were subjected to regressional analysis with FC removed enumerated by DST being X variable and by MF being the Y variable as depicted in Fig. 4. The two methods agreed with each other to yield an r value of 0.99. Application of DST in FC estimation from drinking water sources A total of thirty samples from dierent drinking water sources as speci®ed in Table 5 have been ana-
negative
DST
MF
5 10 1 1
7 9 1 0
6 3 ÿ 1
lyzed for enumeration of FC by DST and the standard MF technique. The rapid test is as sensitive as the conventional MF test for detection of fecal pollution in dierent drinking water sources (Fig. 5). A comparison of qualitative examination for FC by dip slide and MF techniques showed that in 30 drinking water samples, 82% samples are positive for FC by DST, whereas 93% by MF. The four samples, which were tested negative for FC by DST, whereas by MF technique they were positive. In general for ``qualitative estimation'' of FC from drinking water sources DST can be considered as a simple and rapid technique. For routine monitoring of euent quality and drinking water pollution, dip slides come to the nearest order of magnitude and is suciently accurate.
Fig. 5. Comparison of fecal coliforms numbers estimated by conventional MF and DST in drinking water sources.
994
R. S. Sandhya et al. CONCLUSIONS
Some of the major strengths of the DST are . results in 8 h . 99.97% FC are E. coli with no con®rmation tests needed . sensitivity to FC and E. coli concentration as low as 10 CFU/100 ml . can be used by unskilled, layman without having microbiology background . can be used as disposable slides because of low cost of manufacturing AcknowledgementsÐAuthors are grateful to Professor P. Khanna, Director, National Environmental Engineering Research Institute, Nagpur, India for his valuable suggestions, guidance and allowing to publish the work. The authors are thankful to CPHEEO, New Delhi, India for funding the research. REFERENCES
American Public Health Association (1985) Standard Methods for the Examination of Water and Wastewater,
16th ed. American Public Health Association, Washington DC. Bej A. K., Stean R. J., Dicesare J., Ha L. and Atlas R. M. (1990) Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Appl. Environ. Microbiol. 56, 307±314. Covert T. C., Shadix L. C., Rice E. W., Haines J. R. and Freybery R. W. (1989) Evaluation of the autoanalysis coliert test for detection and enumeration of total coliforms. Appl. Environ. Microbiol. 55, 2443±2447. Dange V., Jothikumar N. and Khanna P. (1988) One hour portable test for drinking water. Wat. Res. 22, 133±135. Francis D. W., Peeter J. T. and Twedt R. M. (1974) Rapid method for detection and enumeration of fecal coliforms in fresh chicken. Appl. Microbiol. 27, 1127± 1130. Mara D. D. (1972) The use of agar dip slides for estimation of bacterial numbers in polluted waters. Wat. Res. 6, 1605±1607. Sandhya S., Phirke P. M. and Deshpande V. A. (1990) Dip slide technique: a rapid methodology for performance evaluation of wastewater treatment system. J. Environ. Sci. Health A (U.S.A.) 25, 325±341. Van Donsel D.J., Twelt R.M. and Gelrich F.E. (1969) Optimum temperature for quantization of fecal coliforms in seven hours on the membrane ®lter. Bacteriol. Proc. Abstr. G46.