The optimization and application of two direct viable ...

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The optimization and application of two direct viable count methods for bacteria in distributed drinking water JOSEECOALLIER,MICHELEPREVOST,AND ANNIEROMPRE E C O I ~ Polytechrlique

de Montre'al,Department of Civil Eizgirzeering, Environment, C.P. 6079, succ~~rsale Centre-Ville, Montreal, QC H3C 3A7, Canada

AND DANIELDUCHESNE Ville de Laval, 2 , rue Hotte, Laval, QC H7L 2R3, Canada

Received April 7, 1994 Revision received July 15, 1994 Accepted July 19, 1994 M., ROMPRE, A,, and DUCHESNE, D. 1994. The optimization and application of two direct viable count COALLIER, J., PR~VOST, methods for bacteria in distributed drinking water. Can. J. Microbiol. 40: 830-836. The optimal incubation conditions for the direct viable count method with nalidixic acid were determined. They do not differ from those proposed in the literature for a laboratory strain and a mixed bacterial population isolated from drinking water. The direct viable count method with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was performed under in situ conditions. The bacteria were incubated with CTC at a concentration of 1 mM for 4-6 h at the temperature of the water in the pipes and without the addition of an exogenous substrate.The results obtained for a laboratory strain using the two direct count methods were similar. However, for a mixed bacterial population, the counts were always higher with the CTC method than with the nalidixic acid method. Key words: drinking water, CTC, nalidixic acid, direct viable count. COALLIER, J., PREVOST, M., ROMPRE, A., et DUCHESNE, D. 1994. The optimization and application of two direct viable count methods for bacteria in distributed drinking water. Can. J. Microbiol. 40 : 830-836. Les conditions optimales d'incubation pour le dCnombrement des bactCries viables par I'acide nalidixique ont CtC dCterminCes. Celles-ci ne different pas beaucoup des conditions retrouvCes dans la IittCrature tant pour une population bactCrienne mixte provenant d'eau potable que pour une souche de laboratoire acclimatke. Le dCnombrement direct des bactCries viables avec le chlorure de 5-cyano-2,3-ditolyltetrazolium (CTC) peut &trerealist5 dans des conditions in situ. Ainsi, les bactCries sont incubCes en prCsence de CTC h 1 mMpendant 4-6 h sans apport de substrat exogkne et h la tempkrature de I'eau h I'intCrieur des conduites. Des essais ont montrC qu'avec une souche de laboratoire, les dtnombrements par l'acide nalidixique et par le CTC ne different pas beaucoup tandis qu'avec une population bactCrienne mixte, les dknombrements par rkduction de CTC sont toujours plus ClevCs. Mots clex : eau potable, CTC, acide nalidixique, dCnombrement direct de bactCries viables.

Introduction A drinking water distribution system is a natural ecosystem where bacteria and other microorganisms live and reproduce even if conditions are not optimal for their growth. Several factors related to drinking water treatment will stress the bacteria, triggering the development of certain survival mechanisms (LeChevallier and McFeters 1985; McFeters et al. 1986; Coallier et al. 1989). One of these mechanisms is a reduction of the bacteria's metabolic rate that leads to a state of dormancy (Byrd et al. 1991). With traditional culture methods, it is not possible to differentiate between the bacteria's different physiological states (Roszak and Colwell 1987a). Therefore, it becomes very difficult to distinguish between dormant and stressed or injured bacteria (Maul et al. 1989). Nearly 90% of the bacteria present in drinking water are not enumerated by heterotrophic plate counts (McFeters et al. 1986). Injured bacteria are affected by reagents used in the culture medium and are unable to form colonies on an agar medium (LeChevallier and McFeters 1985). When the stress is overcome, the bacteria can mend their cellular lesions, continue normal development, and restore their capability to grow on solid media (Hurst 1977; McFeters et al. 1986). Water suppliers thus face a very important problem concerning the underevaluation of the Printed in Canada / Imprime au Canada

bacterial population present in drinking water. The total direct count, frequently used in microbial ecology for the evaluation of the total number of bacteria (Hobbie et al. 1977; Porter and Feig 1980; Jones et al. 1989),is used to evaluate the bacterial population present in distributed drinking water (Servais et al. 1992; Desjardins et al. 1991; Mathieu et al. 1992). Total direct counts by epifluorescence, however, do not allow for a differentiation between living and nonliving bacteria. Direct viable count methods may provide a more reliable solution. Kogure et al. (1979) have proposed a method in which water samples are incubated with nutrients (yeast extract) in the presence of an antibiotic, nalidixic acid. The bacteria consume the yeast extract while growing but the nalidixic acid prevents cell division by inhibiting DNA synthesis (Goss et al. 1964). The bacteria sensitive to nalidixic acid elongate during incubation because other cellular components (RNA, proteins) are not affected. The elongated cells are then enumerated by epifluorescence microscopy after staining with acridine orange (Hobbie et al. 1977). Other antibiotics such as ciprofloxacin, lomefloxacin (Olson et al., unpublished results), piromidic acid, and pipemidic acid (Kogure et al. 1984) have also been used. As a consequence of the bacterial diversity in distribution systems, a variable response to the antibiotic is observed, making elongation difficult to evaluate (Thorsen et al. 1992).

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Zimmermann et al. (1978) developed a technique for enumerating total and active respiring bacteria. During bacterial respiration,the redox dye INT (2-(p-iodopheny1)-3-(p-nitropheny1)-5-phenyltetrazolium chloride) is reduced inside the cell to an insoluble INT-formazan granule by the elctron transport system. The INTformazan granules can be observed as insoluble red deposits inside the bacteria. It is very difficult to see the intracellular INT-formazan granules in bright-field microscopy because the bacteria present in treated drinking water are very small. Recently, Rodriguezet al. (1992) used anotherredox dye, CTC (5-cyano-2,3ditolyl tetrazolium chloride), to enumerate active respiring bacteria. CTC seems to be superior for use with environmental populations because the reduced form of CTC, CTC-formazan, is a fluorescent red granule easily detected by epifluorescence microscopy (Rodriguez et al. 1992). In this paper, the optimal conditions determined for direct viable count methods of bacteria in drinking water distribution systems will be reported. Subsequently, the different methods (heterotrophic plate counts (HPC), total direct counts, and the direct viable counts with nalidixic acid and CTC) will be compared to determine the method that most closely simulates actual distribution system conditions.

Materials and methods Bacterial strains A strain of Enterobacter cloacae, isolated from a drinking water distribution system, was maintained at 4°C in a minimal medium with acetate (1 mg C/L) (McFeters and Camper 1988). Before each test, the strain was adapted by incubating 1 mL of the cellular suspension in 99 mL of a phosphate buffer (American Public Health Association 1992) containing yeast extract of 0.25% (stock solution 2.5 g/100 mL) for 24 h at 20°C. This culture was then diluted before use. A mixed bacterial population present in samples obtained from the distribution system was also used. The samples were collected at a particular location corresponding to a 10-hresidence time in the distribution system, in sterile bottles containing sodium thiosulfate (0.1 mLof a 10% solution in 120-mL sampling bottles). Bacterial enumeration Total direct counts For acridine orange direct counts (AODC), the method described by Hobbie et al. (1977) was used. Samples of drinking water or from an E. cloacae culture (2-10 mL) were collected and spiked with formaldehyde (final concentration 2% v/v) for preservation and kept at 4OC until used. An aliquot of the fixed samples was filtered through a 0.2-km Nuclepore polycarbonate black filter. The filter was then stained with acridine orange (1 mL of a 0.01% w/v stock solution) for 2 min and rinsed with 4 mL of ultra pure water. The filter was then cut and placed on a slide between two drops of low fluorescent immersion oil (Cargille type A) before being covered with a clear glass cover slip. The number of bacteria were estimated from the counts of 10 microscopic fields using at least three or four replicates of the samples, using the lOOx oil immersion lens. ANikon microscope equipped with a halogen lamp, a 470- to 490-nm excitation filter, and a 520-nm cut off filter was used. The number of bacteria per millilitre of sample was then calculated using the following formula:

whereT is the number of bacteria/mL, N is the number of bacterialfield, Af is the surface of filtration (mm2), a is the surface of microscopic field (mm2), and V is the volume of sample filtered (mL). Direct viable counts by cellular inhibition with nalidisic acid The sample was diluted (1:s v/v) in a phosphate buffer and incubated with 0.025% yeast extract (w/v) and nalidixic acid (Winthrop Pharma)

at final concentrations varying between 2 and 20 mg/L. The nalidixic acid solutions were prepared as follows: solutions of 2,5, and 10 mg/L were made using a 2 g/L stock solution of 0.05 M NaOH and a 20 mg/L solution from a 10 g/L stock solution of 0.05 M NaOH (Al-Hadithi and Goulder 1989). The samples were incubated at 20°C for 6-48 h. After incubation, the bacteria were fixed with formaldehyde (2%, final concentmtion) and the elongated bacteria were enumerated on 10 microscopic fields using at least three or four subsamples by the AODC method. The elongated bacteria were measured with a calibrated microscopic scale placed on one of the eyepieces. Direct viable counts of active respiring bacteria by reduction of CTC The sample was incubated in the dark with or without substrate at 20°C except when the in situ temperature was simulated. The CTC (8 m a , molecular weight 311) was added to the sample at fmal concentrations varying between 0.5 and 5 mM. Incubation times varied between 2 and 24 h. After incubation, the bacteria were fixed with formaldehyde (final concentration 2% v/v) and filtered through a Nuclepore filter. The red fluorescent CTC- formazan granules produced by respiring bacteria were visualized with epifluorescence microscopy using a 420- to 490-nm filter for excitation and a 590-nm cut off filter, or the filters used with acridine orange, on 10 microscopic fields for at least three or four replicates. Heterotrophic plate counts Culturable bacteria were enumerated on an R2A medium. Different dilutions of the drinking water distribution system samples were filtered in triplicate through a 0.45-krn membrane in accordance with procedures of the American Public Health Association (1992). The membranes were then placed on an agar medium and the Petri dishes were incubated for 48 h at 35°C or for 7 days at 20°C. The filtration plant and drinking water distribution system Samples of treated water were obtained from the Chomedey treatment plant (Ville de Laval) and from its distribution system. The Chomedey filtration plant (1 80 000 m3/day) is fed by the des Prairies River. The water undergoes settling, sand and anthracite filtration, and ozonation. The ozonated water is disinfected with chlorine. The water samples were taken at the effluent of the plant site and at different points along the distribution system. The residence times of samples at these points in the distribution system were determined using the RINCAD software package. HPC, total direct counts, direct viable counts with CTC and nalidixic acid, and chlorine residuals (American Public Health Association 1992) were measured at points corresponding to residence times between 0 and 15 h. For nalidixic acid, incubation conditions for the direct viable counts were 10 mg/L nalidixic acid and 0.025% yeast extract and incubation at 20°C for 24 h. For CTC, conditions were as follows: 1 mM CTC and incubation at in situ temperature for 6 h without exogenous nutrients. Data analysis Analysis of variance and Duncan's new multiple range tests were done with the SAS (SAS Institute Inc., Cary, N.C.) and SuperAnova software programs.

Results Influence of nalidixic acid concentration on direct viable counts Enterobacter cloacae The effect of different concentrations of nalidixic acid (between 2 and 20 mg/L) on the elongation of E. cloacae cells was investigated. The results presented in Table 1 give the total counts of the sample before incubation with nalidixic acid and the number of viable and total cells after 6 h of incubation with the antibiotic. The highest number of viable cells was obtained with a 2 mg/L nalidixic acid concentration. However, bacterial growth was observed during incubation at this low nalidixic acid concentration (F = 9.49, P < 0.005). At low nalidixic acid concentrations, the inhibitory effect of the acid on certain bacteria

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MICROBIOL. VOL. 40. 1994

TABLEI . Total number of bacteria (AODC) and total number of viable cells (direct viable counts) of an Etzferohacfercloucue population incubated


for 6 h with different concentrations of nalidixic acid (NA) Concentration of NA ( m a )

Elongated cells ((cells/mL)x I 07) 7.872 1.25 6.262 1.34 6.0821.18 4.23 L0.89

Initial 2 5 10 20

Total cells ((cells/mL)x lo7)

Elongated cells/total cells after incubation

Elongated cells/initial total cells

0.82 0.77 0.76 0.66

7.292 I .I4 9.602 1.38 8.1521.62 8.022 1.22 6.4321.10

1.07 0.86 0.83 0.58

NOTE:Values are given as means 2 SE.

TABLE 2. Total number of bacteria (AODC) and total number of viable cells (direct viable counts) in drinking water samples incubated with different concentrations of nalidixic acid (NA) Concentration of NA Elongated cells (mg/L) ((celIs/m~)xlO~)

Date of sampling

Temp. ("(2)

92-07-14

23

Initial After incubation

92-09-1 5

21


92- 11-24

4


Total cells ((cells/mL)x105)

Elongated cells/ total cells after incubation

Elongated cells/ initial total cells


was lost and bacterial growth increased the direct viable counts. At nalidixic acid concentrations higher than 2 mg/L, the viable population no longer increased. After 6 h of incubation, the number of elongated or viable cells was higher when E. cloacae was incubated with low nalidixic acid concentrations (5 and 10 m g b ) .

A distl-ibution system mixed population Table 2 shows the results of three different experiments on the effect of nalidixic acid concentration on drinking water sampled at the same point in different periods of the year (cold and warm water). The nalidixic acid concentrations varied between 5 and 20 mg/L. The bacteria in the mixed environmental population did not seem to have any resistance to the nalidixic acid concentrations used, since the total count after incubation was never higher than the initial population number. In some cases, total cells decreased in numbers after incubation. There was a low percentage (3-15%) of viable cells in these samples in contrast to the results obtained with E. cloacae where 52-100% of the cells were viable. Reduced bacterial lengths were also observed with heterotrophic populations. At a concentration of 10 mg/L, the E. cloacae cell length increased to a mean value of 11.1 p m compared with a mean value of 6.2 p m for a mixed drinking water population. The elongation was particularly weak in the first experiment and did not allow for the determination of the optimal nalidixic acid concentration. Subsequent experiments performed in September and November showed that the lowest concentrations (5 and

10 m&) favoured bacterial elongation. Since it was very difficult to determine the optimal concentration, the choice was made with regards to bacterial elongation length. Results showed that when bacteria were incubated with a 5 mg/L nalidixic acid solution, the viable bacteria had a mean length of 5.6 p m whereas with a 10 mg/L acid concentration, the mean length was 6.2 pm. Despite this small difference, the 10 mg/L concentration was selected as optimal because with this concentration there was less of a chance to counter possible false higher counts arising from antibiotic-resistant bacteria in the distribution system.

lnfluerzce of incubation time on direct viable colrrzts with nalidixic acid The influence of incubation time was also studied using two types of bacterial populations: a mixed population from a distribution system and an E. cloacae strain. No significant differences were observed (F = 1.13, P > 0.05) between viable cells of E. cloacae after different incubation times (Fig. IA). After 18 h of incubation, the direct total count was significantly higher (P < 0.001) than at the other incubation times. Nevertheless, the ratio of viable to total cells remained constant. Experimental errors, such as incubation or filtration of a larger sample volume, could account for this increase. When a mixed population was incubated with nalidixic acid, an increase in the incubation time generated a gradual increase in the number of viable cells (Fig. 1B). The highest number of viable bacteria occurred after 48 h of incubation, and this increase was linked to a significant rise in the total count. The growth of

COALLIER ET AL


(A) E. cloacae

12 18 Incubation time (hours)

0

6

24

0

6

18 24 Incubation time (hours) 12

48

--

elongated cells

total cells

FIG. 1. Effect of incubation time on the total number of bacteria and the total number of viable cells of an Enterohacter cloacae population ( A ) and in drinking water samples (B) incubated with nalidixic acid (bacteria were incubated with 10 mg/L nalidixic acid; error bars represent standard error of the mean). TABLE3. Effect of incubation time and concentration of CTC on the enumeration of viable cells in drinking water samples (50 mg/L acetate, incubation at 20°C)

TABLE^. Effect of different conditions of substrate, incubation time, and temperature on the enumeration of viable cells in drinking water samples by reduction of CTC

Incubation time (h)

Concentration of CTC (mM)

Viable cells ((cells/mL)x104)

Date

2

0.5 1O . 2.0 5.0 0.5 1.O 2.0 5.0 0.5 1.O 2.0 5.0

7.3052.18 7.665 1.20 9.25 52.35 4.875 1.17 9.53 52.02 9.645 1.98 7.48 52.05 8.1252.64 9.865 1.95 10.2051.99 8.4952.52 8.1252.64

4

6

93-02-02

93-02-23

Conditions of incubation Substrate, 20°C, 4 h Substrate,4"C, 4 h No substrate, 4"C, 4 h No substrate, 4"C, 6 h No substrate, 4"C, 8 h No substrate, 4"C, 12 h No substrate, 4"C, 18 h Substrate, 20°C, 4 h No substrate, 20°C, 4 h Substrate, 4OC, 4 h No substrate, 4"C, 6 h

Viable cells ((cells/mL)x104) 1.9050.48 1.71"0.49 2.1750.59 2.7450.64 2.01 20.43 1.7150.44 1.2650.30 2.7050.56 3.2850.7 1 3.85 20.75 3.4750.50


NOTE:Values are given as means % SE.

antibiotic-resistant bacteria after 48 h of incubation was probably responsible for this higher total count. It is therefore not recommended to incubate an environmental sample for longer than 24 h (Al-Hadithi and Goulder 1989; Roszak and Colwell 19876). The number of viable bacteria was highest ( F = 52.84, P < 0.001) after 24 h of incubation whereas the number of total bacteria remained constant.

Optimization of incubation time and CTC concentration for a mixed population of a distribution system CTC concentrations of 0.5, 1.O, 2.0, and 5.0 mM were added to drinking water samples and incubated for 2 , 4 , and 6 h at 20°C. Results (Table 3) show that after 2 h of incubation and disregarding the CTC concentration, the fluorescent granules were smaller than after4 or 6 hand the count was lower ( F = 12.92, P < 0.001). At a 5 mM CTC concentration, the counts were lower ( F = 10.68, P < 0.001) than for the other concentrations. The highest viable counts were obtained with 0.5 and 1.0 mM CTC after 4 or 6 h of incubation.

Influence of in situ conditions on direct viable counts by reduction of CTC The tests were performed on samples taken during the winter at water temperatures varying between 1 and 4°C. The incubation was carried out without substrate at the temperature of the distribution system as opposed to an incubation at 20°C with substrate. The number of viable bacteria observed was significantly higher after 6 h of incubation under in situ conditions thanunder the experimental conditions indicated in Table 4 ( F = 14.62, P < 0.001). An incubation period longer than 6 h lowered the number of viable cells. A second test under in situ conditions gave similar results (Table 4). The incubation of bacteria at environmental conditions favoured an increase in the number of viable cells detected. It would thus seem that the in situ incubation had a greater impact on viable bacteria counts than the addition of nutrients. Indeed, the lower counts were obtained when a drinking water sample was incubated with CTC and substrate at 20°C ( F = 8.22, P < 0.001). Comparison of direct viable count methods Figure 2A shows the results of nalidixic acid and CTC viable counts for an E. cloacae strain. There was no difference between the number of viable cells evaluated using these two methods.


CAN. J. MICROBIOL. VOL. 40, 1994

Direct viable count - N.A.

Direct viable count - CTC

Total direct count

FIG.2. Comparison of the enumeration of viable cells with CTC and nalidixic acid (N.A.) in an Enterobacter cloacae population ( A ) and in drinking water samples (B) (error bars represent standard error of the mean).

-

0

5

10

15

Residence time (hours)

....*...

- - -A-- -

0

5

10

15

C4

Residence time (hours)

Viable .....direct count - CTC Total direct count (AODC) -......-O.-. Viable direct count - N.A. HPC 2 d, 35°C

----ft---- HPC 7 d. 20°C Free residual chlorine

FIG.3. Evolution of total, viable (CTC and nalidixic acid (N.A.)), and culturable (HPC) cells in a full-scale drinking water distribution system in cold (A) and warm (B) water. Results, however, were different when the two methods were compared for drinking water samples. For a mixed drinking water population, the viable counts using CTC were always higher than those using nalidixic acid (Fig. 2B, F = 47.83, P < 0.001).

Bacterial enumeration in a jilll-scale distribution system using HPC, total direct counts, and direct viable counts with CTC and nalidixic acid The bacterial counts at two sampling periods, one in cold water and one in warm water, as well as the residual free chlorine values, are shown in Figure 3. Viable counts using CTC and nalidixic acid gave higher results at 20°C. The same observation was made with the HPC for samples incubated for 48 h at 35°C. It should be noted that the counts for the AODC and HPC for samples incubated for 7 days remained constant in both cold and warm water. The HPC for samples incubated at 35°C for 48 h were lower than those for a 7-day incubation period at 20°C. Evaluations of bacterial populations were lower by the HPC method than by epifluorescence microscopy counts. In addition, it was noted that the HPC were the most sensitive to residual oxidants. The total

direct counts (AODC) gave the highest values. The viable bacterial counts were therefore in between the total direct counts and the HPC. The CTC method gave higher results than the nalidixic acid method. In warm water, the two viable bacterial count methods produced similar results for the water samples obtained at the plant effluent (residence time 0). In cold water, results were different. Generally, bacterial numbers increased with residence time in the distribution system. This increase appeared to be much more pronounced with HPC than with epifluorescence microscopy wherein the counts increased slightly all along the distribution system.

Discussion The optimal concentration of nalidixic acid to enumerate the viable bacterial cells for an environmental population found in the present study did not differ significantly from values proposed in the literature (Kogure et al. 1979; Maki and Remsen 1981; Peele and Colwell 1981; Roszak and Colwell 19876; Byrd et al. 1991) nor from those used for a laboratory strain adapted to a particular substrate and incubation temperature. The incubation time with nalidixic acid for a drinking water population must be


COALLIER ET AL.

much longer than for a laboratory strain to compensate for the effect of adaptation, a slow generation time, and a much longer latency phase (Singh et al. 1990). Various objections have been raised concerning direct viable counts by cellular inhibition. The differences in sensitivity using mixed populations and the variable effect of the antibiotics on these bacteria are often mentioned as important limitations of the method (Roszak and Colwell 1987a; Thorsen et al. 1992). Counts by cellular inhibition underestimate the proportion of viable bacteria, since Gram-positive and some Gram-negative bacteria are resistant to the antibiotic. A proportion o f the bacterial population is therefore not detected as viable (Roszak and Colwell 1 9 8 7 ~ ) However, . it should be noted that in a drinking water environment, Gram-negative bacteria make up the major portion of the bacterial population (Hall et al. 1990). It is also thought that the resistant bacteria represent only a small fraction of the population, since generally no bacterial growth is observed during incubation. When compared with results for the other two methods (Fig. 2), the nalidixic acid measurements were found to be less constant over the 4-month sampling period. This led to the following question: are we observing a variation in the number of viable cells in the population or rather a variation in the population composition and sensitivity to nalidixic acid? It is known that each bacterial species making up a population has a certain sensitivity threshold to antibiotics. In addition, this threshold may vary depending on the physiological state of the bacteria and on environmental factors. The bacteria were removed from their natural environment and incubated under entirely different conditions than those existing in a drinking water distribution system. Bacteria living in cold water ( 6 ° C ) and in an oligotrophic environment were incubated at 20°C in the presence of a rich substrate (yeast extract). The addition of a highly concentrated substrate may have produced a nutritional stress on the bacteria used to develop an oligotrophic environment. Incubation at 20°C promotes the multiplication of bacteria that are able to grow at this temperature. These bacteria are not necessarily those that are active in the distribution system when the water temperature is less than 5°C (during 5 months of the year). It can be difficult to distinguish between a normal cell and an elongated cell. An elongated bacterium may be smaller than the population average and be missed during a count. In addition, a bacterium with an elongated shape may be counted even if the antibiotic does not affect it (Al-Hadithi and Goulder 1989; Singh et al. 1990). The direct viable count method based on the reduction of a formazan compound counts a wider spectrum of bacteria. All bacteria having an electron transport system in the respiratory chain will reduce the CTC and form formazan granules, thus being easily enumerated by epifluorescence microscopy. The detection of CTC reduction by bacteria is sensitive to changes in experimental conditions, especially the incubation time and the CTC concentration. The lower number of bacteria obtained when the CTC concentration reached 5 mM is perhaps related to a toxic effect of CTC at higher concentrations or to the presence of impurities or deposits that made the counts difficult. A similar observation has been reported by Rodriguez et al. (1992). They found thatthe incorporation of CTC by a Pseudomorzas putina strain decreased rapidly when the CTC concentration exceeded 6 mM. It is also important not to exceed the optimal incubation time. When the desired incubation time is surpassed,

835

bacterial growth can be observed and CTC degradation by the bacteria is possible. CTC reduction does not seem to be influenced by the presence of a substrate. This method can easily be adapted for in situ conditions. The viable bacteria counts obtained under conditions different from the ones prevailing in the original environment probably underestimated the number of viable bacteria. The CTC counts were the highest counts obtained under in situ conditions and this is not surprising as the conditions reflected their natural environment. Conclusions CTC reduction is an effective method for direct viable counts in drinking water. This technique can evaluate viable populations under experimental conditions that are representative of those in a distribution system. The method is fast, requires shorter incubation times than those for cellular inhibition counts, and does not require staining. The direct viable counts by cellular inhibition and total counts can be replaced by the CTC method. At the same time and using the same filter, counts of total and of viable cells are possible by staining the bacteria with DAPI (4',6-diamidino2-phenylindole) after their incubation with C T C (Rodriguez et al. 1992). The problems of variability in the sensitivity of the bacteria to antibiotics and the necessity of strict experimental conditions will always limit the validity of viable counts by cellular inhibition. These limitations will never be overcome even if this method is coupled with sophisticatedcounting methods such as flux cy tometry (Thorsen et al. 1992). It is impossible to incubate bacteria under in situ conditions for the inhibition method: at a low temperature and without exogenic substrate, the generation time for the bacteria is very long (as long as several days), which would increase the incubation time substantially before a significant elongation might be observed. Viable counts by nalidixic acid give similar results to the CTC viable counts with a homogeneous population in a controlled environment. When working with a heterogeneous population with a variable composition, nalidixic acid counts are probably a poor estimate of the number of viable cells. Since several species with variable physiological states exist in a natural environment, it is impossible to apply optimal experimental conditions for each sample. The CTC viable count is not affected by this phenomenon because all bacteria with their electron transport system intact will reduce CTC and produce formazan granules. The results have shown that the direct viable count method is the best method for the evaluation of the bacterial population in drinking water. The HPC method, the most commonly used in the drinking water industry, is capable of detecting only a small percentage of the viable bacteria. Total direct counts with acridine orange are easier to perform than direct viable counts with CTC but it is not possible to differentiate between living and nonliving bacteria. As water suppliers are constantly searching for a method of bacterial enumeration to evaluate treatment plant disinfection efficiencies and the bacteriological quality of distributed water, the direct viable count technique with CTC alone or coupled with the total count stained with DAPI seems to be the most promising solution.

Acknowledgements We thank Sttphane Perron, Denis Allard, Sylviane Desautels, Htlkne Baribeau, and Pierre Galameau for the collection of the samples. We also thank Catherine Poirier and Jacinthe Mailly for their technical assistance, Bernard ClCment for his advice on statistical analysis, and Pierre Servais for his suggestions and

CAN. 1. MICROBIOL. VOL. 40, 1994


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