SAFE OR SOILED?
A Bacterial Analysis of the Kelvin and Luggie Rivers
by Timothy Girgis (2614615G)
BIOL 2D Microbiology & Immunology II
Dr. Nicola Veitch
University of Glasgow Glasgow, Scotland
March 4, 2022
1. Introduction
Two microbial profiles can generally be found in water supplies - the expected microbiome, consisting of organisms that grow optimally at temperatures of about 22°C on average, and a contaminated microbiome, consisting of microbes which grow optimally at temperatures of 37°C on average (standard human body temperature) These organisms are generally of faecal origin, and often include Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginos, Salmonella spp., and Clostridium perfringens, among other potentially pathogenic microbes and “faecal coliforms” (Bartram et al., 2003, p. 140).
Differences between the natural microbiome and microbes of faecal origin indicate the degree of water source contamination, particularly from sewage runoff. These differences are represented as ratios and referred to as 22°C:37°C microorganism counts. A 22°C:37°C microorganism count of <10:1 indicates contamination, whereas >10:1 indicates a normal (uncontaminated) microbiome.
To obtain a microorganism count, samples must first be serially diluted and then plated using specific culture media. Chromogenic coliform agar (CCA) is generally considered the industry standard for the determination of potential coliform water source contamination. The tergitol -7 contained in CCA inhibits Gram-positive non-enteric organism growth, thus selecting for coliforms in particular. Moreover, sugars contained in the medium also allow for the differentiation between E. coli and other coliforms. In contrast, yeast extract peptone (YEP) agar is more or less non-selective, supporting broad spectrum growth of both Gram-negative and Gram-positive organisms, as well as fungi. Thus, YEP is the preferred medium for primary isolates, absent suspicion of contamination by fastidious organisms, which are less likely to grow in YEP. These two methods were relied on in this experiment to determine whether or not the River Kelvin and the Luggie are not only potable, but safe for those who may be more susceptible to waterborne infections, such as young children or immunocompromised adults.
2. Methods
2.1
Serial Dilutions
Water samples were obtained by the University of Glasgow MVLS laboratory team from the River Kelvin (a tributary of the River Clyde) and the Luggie (a tributary of the River Kelvin). Serial dilutions were performed by dispensing 0.5 mL of the unfiltered water from the River Kelvin sample into a 1.5 mL microfuge tube, under aseptic conditions. 0.9 mL of distilled water was then added to two additional tubes, and 100 μL of Kelvin water from first tube was transferred into one of these tubes and mixed via vortexing for a dilution of 10-1 (1:10). Finally, 100 μL of water from this 10-1 tube was transferred into the second tube containing distilled water, and mixed via vortexing, for dilution of 10-2 (1:100). The same procedure was repeated for the Luggie water sample. The process is also illustrated in Figure 1 below.
2.2 Sample Plating
100 µL of each Luggie water sample dilution was then spread onto two plates containing Yeast Extract Peptone (YEP) agar, for a total of 6 plates. For each dilution, one of the two plates was incubated at 22°C while the other was incubated at 37°C. A seventh plate
was then prepared by spreading 250 µL of original (undiluted) Luggie water on Coliform Chromogenic Agar (CCA). The process was repeated for the River Kelvin samples. A simple breakdown of the process is illustrated in Figure 2 below.
Figure 2 The spread plate method in three simple steps. Created with Biorender.com.
3. Results
3.1 Analysis of 22°C versus 37°C Microorganism Counts for Each River
For the 30 samples obtained from the River Kelvin, there was an overall larger 22°C viable cell count (M = 57010.00, SD = 68156.85) as compared to the 37°C count (M = 18969.33, SD = 25671.36) which was statistically significant, t(29) = 3.6105, p = 0.001139. The overall larger 22°C counts were plainly visible in nearly every one of the 30 samples provided, as shown in Figure 3 below.
Figure 3 River Kelvin 22°C and 37°C 22°C and 37°C Counts. 22°C count is higher in nearly all samples. The Luggie Water samples also had higher 22°C counts (M = 60920.00, SD = 67918.83) than 37°C counts (M =4920.67, SD = 5077.725). This was again statistically significant t(29) = 4.8686, p = 3.654e-05 although it is noteworthy that the standard deviation of the 37°C count exceeds the mean, which suggests a few outliers may be contributing to a notable portion of the gap between 22°C and 37°C counts. Regardless, the overall trend again applied to nearly all of the 30 Luggie Water samples, as shown in Figure 4 below.
Figure 4 Luggie Water 22°C and 37°C Counts 22°C count is higher in nearly all samples.
3.2 Comparison of 22°C:37°C Microorganism Counts Between Rivers
The difference in 22°C:37°C microorganism counts between the River Kelvin (M = 4.277, SD = 3.498281) and the Luggie Water (M = 12.193, SD = 2.869306) was also statistically significant t(29) = -9.6677, p = 1.413e-10 as illustrated in Figure 5 below.
Figure 5 Comparison of 22°C:37°C Microorganism Counts. Kelvin (right) appears contaminated while Luggie (right) is not, though the data in both cases appears to be slightly skewed by outliers in opposite directions.
3.3 Analysis of CCA Data
The CCA plate data of 24 undiluted 250 µL samples incubated only at 37°C, measured in CFU/100mL, yields an opposite picture to the one painted in section 3.2 – in this case, the River Kelvin samples had a significantly higher CFU/100mL (M = 1233.083, SD = 133.2581) than the Luggie Water (M = 586.6667, SD = 55.69941), and this difference is statistically significant t(14.73) = 15.504, p = 1.586e-10 as can be inferred from Figure 6 below.
Figure 6 Analysis of CCA Plate Sample Data. Water obtained from the River Kelvin has a significantly higher mean CFU/100mL than the mean of samples obtained from the Luggie Water. These are only 37°C counts.
The CCA plates contained 250 µL undiluted samples, and thus the means need first to be converted into CFU/mL for a proper comparison. These converted values, along with the means of the 37°C counts from the YEP plates obtained in 3.2, are given in Table 1 below.
(M ± SD)
Table 1. Sample Means and Standard Deviations of Kelvin and Luggie 37°C microbial counts.
4. Discussion
Based on the converted values in Table 1, the differences in the means of River Kelvin 37°C count from the YEP plate data and the CCA plate data are not statistically significant, t(40) = 1.8800, p = 0.0674. Similarly, the differences in the means of Luggie Water 37°C count from the YEP plate data and the CCA plate data are not statistically significant t(40) = 1.7424, p = 0.0891 It is thus the case that, while both river water samples cultured using the YEP agar
had higher 22°C counts than 37°C counts, the overall quantity of 37°C organisms in the river Kelvin samples is enough to confirm that it is, in fact, soiled The data provided by the CCA plate analysis further supports this, by showing significantly higher levels of 37°C organisms in the River Kelvin than in the Luggie Water. This is even more significant considering that the River Kelvin is a larger river than the Luggie (which is in fact closer to a stream), with a significantly greater volume of water that can be expected to have a more dilutive effect when exposed to contaminants than the Luggie It is therefore clear that, given the significant concentration difference in the levels of 37°C organisms between the Kelvin and the Luggie, the exposure of the Kelvin to sewage runoff is significant. Based on the, the
Under EU Directive 2006/7/EC river water quality (2006), the minimum ("sufficient") threshold for inland water is 330 CFU/100mL for intestinal enterococci spp., and 900 CFU/100mL for E. coli (https://eur-lex.europa.eu/eli/dir/2006/7/oj). In either case, the Kelvin River does not meet the threshold, and is therefore not fit for consumption Immunocompromised individuals, and children with weaker immune systems in particular, should further take measures not merely avoid consumption, but to avoid the River Kelvin altogether.
References
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Biorender.com (n.d.). Serial Dilution Procedure. Available at: https://app.biorender.com/biorender-templates/t-5f4fb4ecc97d3234de8b516c-serialdilution-procedure.
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https://app.biorender.com/biorender-templates/t-601ad56f65dc9f009e2bee3c-spreadplate-method.
McGillivray, I. (n.d.). River Kelvin. [Photograph] Available at: https://res.cloudinary.com/fleetnation/image/private/c_fit,w_1120/g_south,l_text:style _gothic2:%C2%A9%20Iain%20McGillivray,o_20,y_10/g_center,l_watermark4,o_25, y_50/v1467765540/rqk4ngjmtjkcihwuoziq.jpg.
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