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Quantitative Examination of Water:

• Part A: Enumeration of Bacteria using the Plate Count Technique (PCA)

• Part B: Enumeration of Fecal Coliform using the Membrane Filter Technique (MFT)

• Enumeration of Fecal Coliform using the Most Probable Number Technique (MPN)

by Waterose

Index:

1. Introduction:
2. Methods:
3. Results:
4. Discussion:
5. References:
6. Postscript:

Figures:

1. Map 1. Drainage Basin
2. Map 2. Sample Sites
3. Figure 3. Comparison of Analysis Results for Sample 1 and Sample 2 at Site 2.
4. Figure 4. Comparative Illustration of the results of the analysis of the samples for the Major Project Group 6 sampling sites.

Tables:

Table 1. Total Bacteria Counts from Plate Count Test on PCA plates, incubated at 37° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

Table 2. Presumptive Most Probable Number of Coliform Counts incubated in Lactose Broth at 37° C for 48 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

Table 3. Confirmed Most Probable Number of Total Coliform Counts incubated in Brilliant Green Lactose Broth at 37° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

Table 4. Most Probable Number of Faecal Coliform Counts incubated in EC Broth at 44.5° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

Table 5. Faecal Coliform Counts from Membrane Filter Test on M-FC Agar, incubated at 44.5° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

Table 6. Compilation of Class Data for coliform tests.

Introduction:

The quantitative analysis of water quality is important to monitor the sanitary quality. Water quality may be analysed by the amount and types of bacteria present in water samples. It is not practical to test for all types of organisms, hence, it is a common practice to test for specific indicator organisms such as the Escherichia coli (E.coli) coliform group (Atlas, 1998). The coliform group is defined as "aerobic, Gram-negative, non-sporing rods which ferment lactose with the formation of gas within 48 hours at 37° C" (Royal Roads, 1997). Faecal coliforms are defined as "coliforms that originate in the intestinal tract of humans and animals, and grow and ferment lactose with the formation of gas at 44.5° C within 24-48 hours" (ibid.). The three types of experimental methods included total plate counts, membrane filter tests, and the most probable number tests.

There are different standards and guidelines used to determine the quality of water. The maximum acceptable levels for drinking water are 10 total coliforms per 100 mL and 0 faecal coliforms per 100 mL (Royal Roads, 1997). There are different standards for water used for recreational purposes, which are further differentiated as primary contact and secondary contact activities (MacRae, 1997). Primary contact includes activities, such as swimming or windsurfing, where the user is in direct contact with the water (ibid.). Secondary contact includes activities such as boating or fishing where the user is not in direct contact with the water (ibid.). Under the Guidelines for Canadian Recreational Water Quality, 1992; the maximum acceptable levels are 500 total coliforms per 100 mL and 200 faecal coliforms per 100 mL for primary contact; and, 8 million total coliforms per 100 mL and 400 faecal coliforms per 100 mL for secondary contact (ibid.).

The study area was primarily in the Colwood Creek drainage basin with a few samples from outside this drainage basin. The Colwood Creek drains through residential and recreational lands, the Royal Roads University grounds, and into the Esquimalt Lagoon. The garden ponds, Hatley Creek and Bee Creek also drain into the Esquimalt Lagoon. The lagoon is a wildlife sanctuary and a recreational area. The basin area is identified on Figure 1, Map of City of Colwood, Creek Basin Area (Dillon,1997).

Figure 1 City of Colwood Drainage Basin

(Modified from City of Colwood Community Plan, 1997)

The Esquimalt Lagoon is an area that is very important to the local ecosystem and to anthropogenic uses. Royal Roads University has been monitoring coliform levels in the lagoon. Recent analysis of coliforms suggests that the lagoon would fall within the Health Canada, Guidelines for Canadian Recreational Water Quality, 1992 for secondary contact activities such as boating or fishing, but excluded from primary activities such as swimming or windsurfing (MacRae, 1997). Consequently, Royal Roads University retained a group of students, the Major Project Group 6, to undertake an analysis of the potential sources of inputs of nutrients that may be correlated to the high levels of coliforms in the lagoon.

Major Project Group 6 co-ordinated the ecosystem study of the lagoon with this examination of local water undertaken by the class as a whole, hence there is a focus on analysis of the Colwood Drainage basin and analysis of the duplicate samples. The duplicate set of samples for Site 2 were analysed by the writer and another analyst in the classroom group. The site locations are identified on Figure 2, Map of City of Colwood (Colwood,1997). This report may provide some insight to the ecosystem study and will be further incorporated into the final report of Major Project Group 6 while meeting the objectives of the experiment outlined in the experimental guidelines.

Figure 2 Sample Locations

(Modified from City of Colwood Community Plan, 1997)

Materials and Methods:

All materials and methods used are described in "Experiment 11: The Quantitative Examination of Water" the Environmental Science Lab Manual, 1997 (Royal Roads, 1997).

A brief overview of the methods are:

Part A: Plate Count

• Total Bacteria Plate Count on PCA plates incubated at 37° C for 24 hours using dilutions of 1:1 and 10^-1.

Part B: Membrane Filter Technique

• Faecal Coliform Count on M-FC plates incubated at 44.5° C for 24 hours using dilutions of 1:1 and 10^-1 .

Part C: Most Probable Number Technique

• Presumptive test for total bacterial coliform count in lactose broth incubated at 37° C for 48 hours.
• Confirmed test for total bacterial coliform count in brilliant green lactose bile broth incubated at 37° C for 48 hours.
• Faecal Coliform test in EC broth incubated at 44.5° C for 24 hours.

Site Selection and Field Sampling:

There are three distinct types of site selection and field sampling techniques covered at different levels of discussion in this preliminary study: class group, Major Project Group 6, and Site 2 duplicate sample set.

The class group obtained recreational water samples from sites of their choice, some of which are in the Colwood Creek drainage basin. These included Glen Lake, Colwood Lake, Colwood Creek which is also referred to as Cottonwood Creek, Bee Creek, Hatley Creek, and the Royal Roads garden ponds.

Major Project Group 6 obtained duplicate samples of recreational water from 6 sites that have been selected to analyse potential sources of inputs of coliforms into Esquimalt Lagoon. The sites are identified on Figure 2 Map. Site one is located at the mouth of the lagoon. The sample was collected on the incoming tide to examine if there was a high level of coliforms incoming from the sewage outfall plume which extends 40 metres offshore from the lagoon. Site two is located at the north end of Colwood Creek where it enters the grounds. Site three is located at the south end of Colwood Creek where it enters the lagoon. Site four is located at the south end of the Garden Creek where it inters the lagoon. Site five is located at the south end of Hatley Creek where it enters the lagoon and site six is located at the south end of Bee Creek where it enters the lagoon.

One set of duplicate recreational water samples was obtained from site 2, located at Colwood Creek at the north boundary of the Royal Roads University property and the tests were completed by two separate analysts. The second sample was collected for quality control.

All of the water samples were collected in plastic bottles that had been auto-claved. The samples were refrigerated and analysed within 8 hours of collection.

Results:

The results of the two samples analysed from Site 2:

Table 1. Total Bacteria Counts from Plate Count Test on PCA plates, incubated at 37° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

The results for analysis of total bacteria is summarised are summarised in Table 1. The total coliforms were not within the acceptable limits for recreational water standards for primary and secondary contact. The water was not within acceptable limits for drinking water standards There were three types of colony morphologies observed. The dominant colony type was a large beige cocci, the sub-dominant colony type was a small yellow smooth cocci, and there was one isolated white smooth cocci colony type. The results were consistent between the sample and the duplicate sample.

Table 2. Presumptive Most Probable Number of Coliform Counts incubated in Lactose Broth at 37° C for 48 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

The results for the presumptive most probable number of coliform counts are summarised in Table 2. The results were not consistent between the sample and the duplicate sample. The total coliform counts were within the acceptable limits for recreational water standards for primary and secondary contact. The water was not within acceptable limits for drinking water standards.

The presumptive results were identical to the confirmed results in Table 3.

Table 3. Confirmed Most Probable Number of Total Coliform Counts incubated in Brilliant Green Lactose Broth at 37° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

The results for the confirmed most probable number of total coliforms are summarised in Table 3. The results were not consistent between the sample and the duplicate sample. The total coliform counts for Sample 1 and Sample 2 were within the acceptable limits for recreational water standards for both primary and secondary contact for recreational water standards. The water was not within acceptable limits for drinking water standards.

The confirmed results are identical to the presumptive results in Table 2.

Table 4. Most Probable Number of Faecal Coliform Counts incubated in EC Broth at 44.5° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

The results for the presumptive most probable number of faecal coliform counts are summarised in Table 4. The results were not consistent between the sample and the duplicate sample. The faecal coliform counts for Sample 1 were not within the acceptable limits for recreational water standards for primary contact, but were within the limits for secondary contact. Sample 2 was within the acceptable limits for both primary and secondary contact for recreational water standards. The water was not within acceptable limits for drinking water standards.

The results were significantly different from the faecal coliform counts analysed by the membrane filter method summarised in Table 5.

Table 5. Faecal Coliform Counts from Membrane Filter Test on M-FC Agar, incubated at 44.5° C for 24 hours from Cottonwood Creek North at Galloping Goose Trail Boundary of Royal Roads University Property (Site 2).

The results for the faecal coliform counts from the membrane filter test are summarised in Table 5. The results were consistent between the sample and the duplicate sample. The number of faecal coliforms were within acceptable limits for recreational water standards for both primary and secondary contact. The water was not within acceptable limits for drinking water standards.

The results were significantly different from the faecal coliform counts analysed by the presumptive most probable number faecal coliform results summarised in Table 4.

Combination of Duplicate Sample Analysis for Site 2:

The results for all tests completed on sample 1 and duplicate sample 2 are illustrated in Figure 3.

Figure 3. Comparison of Analysis Results for Sample 1 and Sample 2 at Site 2.

The results of the samples analysed from the class group and Major Project Group 6:

Table 6. Compilation of Class Data for coliform tests.

The Comparative results of the samples analysed from Major Project Group 6:

The results of the analysis of the samples for the Major Project Group 6 sampling sites are illustrated in Figure 4.

Figure 4. Comparative Illustration of the results of the analysis of the samples for the Major Project Group 6 sampling sites.

Discussion:

The results are addressed in three contexts: Site 2 : Sample 1 and Sample 2 results, Major Project Group 6 results, and the class results.

Site 2:

The two samples for Site 2 were collected at the same location/time and were analysed by identical procedures; thus, similar results were predicted. The total bacteria count should exceed the total coliform count which should exceed the faecal coliform count.

The total plate count of colony forming units was not identical but was reasonably close. The average is 105,000 cfu/100 mL, thus the two samples are +/- 13,500 cfu/mL of the average. The two samples had identical colony morphologies with the exception of a single colony for the duplicate sample number 2.

The presumptive total coliform count and the confirmed total coliform count for each sample were identical; however, the total coliform counts were significantly less for the duplicate sample. The total coliform counts were less than the total bacteria which was expected. The difference between the results for sample 1 and sample 2 may be due to the number of coliforms in the samples, or may be due to subjective interpretation of identification of a positive test by the two different analysts. The fact that the confirmed test equalled the presumptive test further supports this supposition.

Similarly, the presumptive faecal coliforms for sample 1 were significantly higher than those for sample 2, which were consistent with the total coliforms analysis. The faecal coliforms counts were less than the total coliform counts.

This trend continued with the membrane filter analysis of the faecal coliform count. The total faecal coliform count was significantly less than the coliform count and sample 1 had a higher faecal coliform count than sample 2.

Based on the lower total coliform counts and lower faecal coliform counts in sample 2, it is highly probable that sample 2 did have less coliforms per 100 mL, even though sample 2 had an overall higher bacteria count. It is inconclusive as to whether the most probable number technique or the membrane filter technique is more accurate; however, the membrane filter technique results are very close between different analysts.

This type of quality control protocol appears to be adequate for detecting the presence of bacteria, total coliforms, and faecal coliforms due to the similarity of the results as illustrated in Figure 3. Further analysis with increased numbers of samples may possibly reveal a statistically significant quantitative correlation between total bacteria, total coliform, and faecal coliform.

Major Project Group 6:

The Major Project Group 6 results examined specific sites for levels of coliforms and faecal coliforms in relationship to the Esquimalt Lagoon. There were several key issues of concern: the levels of coliforms in the source lakes and streams, in the lagoon inlet, and in the lagoon.

Duplicates samples were obtained from each of the six sampling sites; however, due to the material constraints of a limited number of tubes of incubating media, only a portion of the samples were analysed. Samples were analysed for sites 1-6 and duplicate samples were analysed for sites 1 and 2. The quality control concerns regarding the duplicate samples for site 2 were previously discussed in the previous section. The quality control concerns regarding the duplicate samples for site 1 will be discussed under separate cover and are outside the scope of this report.

The set of results for site 1, located at the mouth of the lagoon inlet, indicated high levels of total coliforms and faecal coliforms. There was a wide variance between the two samples and between the MPN results and the membrane filter results. This site was selected to evaluate if there were coliforms entering the lagoon on the incoming tide that may possibly be discharged from the outfall which extends 40 meters from the lagoon into the ocean. The results indicated that there were coliforms incoming, but, they may also be accounted for by the presence of local waterfowl. The oddity of the finding is that the outflow from the lagoon carrying coliforms out into the Pacific Ocean would be significantly diluted. In addition, the saline conditions and cool water temperatures would not be conducive to the longevity of the coliforms. This anomaly should be targeted for further study and analysis regarding the source input of the coliforms detected in the incoming tide.

These of results for site 2, located at Cottonwood Creek north, were previously discussed regarding the duplicate sample analysis. The faecal coliform counts were quite high in the Cottonwood/Colwood stream which may be attributed to upstream discharge from urban septic tanks, storm water drain overflow, or the geese that frequent the Colwood Golf Course.

The set of results for site 3, located at Cottonwood Creek south, were higher than the coliform results for Cottonwood Creek north. The higher total coliforms and faecal coliforms downstream may be attributed to slower velocity flow as the stream joins the estuary, or to higher bird populations in the estuary.

The set of results for site 4, located at the Garden Creek south, were relatively low in total coliforms and faecal coliforms. The lower counts may be attributed to reduced velocity of the water flow through the pond system. The coliforms require high temperatures to survive, and any significant communities may be vulnerable to predation in the three ponds of the garden pond system.

The set of results for site 5, located at Hatley Creek south, were low in total coliforms but unusually high in faecal coliforms for the membrane filter count which was outside the expected trend.

Moving westward, the total coliform results for site 6, located at Bee Creek south, were significantly higher than those for Hatley Creek. Bee Creek is adjacent to the City of Colwood and runs through a field that used to be pasture land. It is possible that there is seepage from the urban area, or runoff from the field that would account for the unusually high levels of coliforms and faecal coliforms.

In addition to the project sampling sites, the results from the class results are of interest, particularly those sites in the Colwood drainage basin and on Royal Roads University grounds.

Class Results:

Overall, the trend for the class results was consistent with the expected trend previously identified in that the total bacteria should exceed the total coliforms which should exceed the faecal coliforms.

There were wide ranges of results for groups of like source locations such as the results for Glen Lake, the ponds or for streams. This may be attributed to analytical interpretation of the experimental results by the different analysts. In addition, there was a wide variability between sampling site selection and the indicator organisms that may have been present in the water samples.

There were significant differences between the faecal coliform counts using the most probable number technique and the membrane filter technique. There were a number of factors that may contribute to the differences. The most probable number technique was vulnerable to subjective interpretation of what constitutes a positive test. The membrane filter technique was vulnerable to subjective counting of colonies. In addition, the size of the pore on the membrane filter may affect the results due to the size of the bacteria filtered, and the sedimentation in the water sample.

Conclusion:

The qualitative and quantitative analysis of water for the presence of pathogenic organisms is important to monitor the sanitary quality of water that is used by humans and members in an ecosystem.

One individual sample is insufficient to conclusively determine water quality. Duplicate samples and duplicate sample analysis is necessary to confirm a preliminary analysis. Preliminary analysis by means of plate count technique or most probable number technique may identify areas of concern that may be further analysed by more definitive techniques. Even so, there exists a wide variability of organisms within the water samples or water column which are vulnerable to slight changes within their environmental niche.

The Colwood basin does have coliforms and faecal coliforms present in every input source into the Esquimalt Lagoon, including the incoming tide at the lagoon mouth. Many of the source inputs exceed the recommended levels for recreational waters categorised as areas of primary contact; however, very few exceed the recommended levels for recreational waters categorised as areas of secondary contact.

There are fairly high concentrations in the garden pond system, albeit there is also a wide range in the pond system. There is a trend of increasing concentration of coliforms moving westward on the Royal Roads property towards the City of Colwood which suggests a trend of increased outputs. This area could be targeted for further study.

References:

Atlas, R.M., Bartha, R. Microbial Ecology: Fundamentals and Applications. Fourth Edition. Benjamin Cummings. Menlo Park, Ca. 694 pp. 1998.

City of Colwood. Official Community Plan. 1997. Schedule "A" to Bylaw No. 373. City of Colwood, Planning Department. Colwood, B.C. 155 pp. 1997.

MacRae & Associates Inc. Environmental Health Services. Letter. Water Quality, Royal Roads University Lagoon. August 11, 1997.

Royal Roads Environmental Science Lab Manual. Experiment 11: Quantitative Examination of Water. (pp.11-1 to 11-14.) Royal Roads University. Victoria, B.C. 1997.

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