Lab Exercise: Examining Water Quality: Most Probable Number & Colilert Test Kit Lab OBJECTIVES 1. Understand the use of MPN to determine likely fecal water contamination. 2. Understand the use of MUG, ONPG and β-galactosidase in the Colilert test kit. 3. Understand the use of positive and negative controls in this experiment. 4. Determine the cleanliness of the experimental water sample. BACKGROUND Most Probable Number (MPN) is a procedure used to estimate bacterial populations in the environment (including bacterial contamination of water). This procedure is based on the application of the Theory of Probability with the following given assumptions. 1. The organisms are randomly and evenly distributed throughout the sample; 2. The organisms exist as single entities, not as chains, pairs or clusters and they do not repel one another; 3. The proper medium, temperature and incubation conditions have been selected to allow even a single viable cell in an inoculum to produce detectable ; 4. The population does not contain viable, sub-lethally injured organisms that are incapable of in the culture medium used. Samples are diluted prior to use in order to reduce the number of positive tubes to a manageable number. The number of tubes prepared is generally based on the expected population contained within the sample. Reliable results occur when all tubes at the lower dilution are positive and all tubes at the higher dilution are negative for, as the dilution scheme has accurately bracketed the population, much as the case in dilution plating. Typically, this results in serial dilutions for MPN analysis in sets of 3, 5 or 10 MPN tube series, with more tubes increasing accuracy. MPN values are an indirect measurement of the number of viable organism in a given sample. However, MPN counts are useful for their technical ease and are particularly useful when low concentrations of organisms (<100/ml) are encountered in such materials as milk, food, soil and water where a low concentration of organisms and/or particulate matter of the matrix may interfere with accurate colony counts. Once dilutions have been performed, samples that are positive for microbial are indicated by gas production and/or visible turbidity. In this lab, you will be inoculating a series of 3 MPN tubes with the sample serial diluted (i.e., undiluted; a 10-1 dilution; and a 10-2 dilution). You will be looking for the presence of aerobic or facultatively anaerobic, Gram negative, non-endospore forming rods that ferment lactose and produce gas within 24 hours at 35 C. Organisms having these characteristics are typically considered indicator organisms for fecal contamination of water. However, there are many organisms that fit this description but are not indicative of fecal contamination. For this reason, positive MPN results are a putative result for fecal contamination. Confirmation of this must be done by further biochemical and physiological analysis of the cultured microorganisms. More sophisticated testing can be directly on water samples through the use of test kits which examine the bacteria s ability to metabolize lactose (and indicator substrates) into specific chemical compounds. Remember from earlier laboratory discussions that when bacterial cells are exposed to lactose, the lac operon is induced, in order that the cells may begin the process of metabolizing this sugar. One of the enzymes coded for in the lac operon is β-galactosidase, which converts the disaccharide lactose to the monosaccharides glucose and galactose. Thus, the catabolism of lactose into glucose and galactose in a given water sample is typically indicative of the presence of fecal contamination. Cells will also produce β- galactosidase when exposed to the nutrient-indicator ONPG (ortho-nitrophenyl-β-d-galactopyranoside), which mimics the lactose molecule and is broken down by β-galactosidase into ONP (ortho-nitrophenyl-β-dpyranoside), which is yellow in color ( Figure 1). In this way, the conversion of ONPG to ONP, which is easily detectable with the naked eye (Figure 2), is an indicator reaction for the presence of coliform contamination. (For reference, the ONPG
works in the same manner as IPTG in Lab Exercise 15: pbluescript Transformation: Blue/White Colony Selection.) Figure 1: Chemical representation of the catabolism of either lactose (upper diagram) or ONPG (lower diagram) by the Bacterial enzyme β-galactosidase into their respective catabolites. Figure 2: Left to right: Colilert test results for E. coli (under fluorescent light); a sample containing noncoliform Bacteria; and a sample containing coliform Bacteria (shown under visible light). Additionally, the indicator organism Escherichia coli will use β-galactosidase to convert a second indicatornutrient MUG (4-methylumbelliferyl-β-D-glucuronide) into its fluorescent counterpart (Figure 2). Although this test will confirm the presence of the fecal contaminant, E. coli, the ability to convert MUG to a fluorescent compound is lacking in most pathogenic E. coli strains, and so additional tests are usually required to single them out. A commercially available test kit, Colilert, which contains both ONPG and MUG can be used to easily determine the presence of coliforms as well as E. coli. The sensitivity of this test kit is such that it can detect E. coli and coliforms at concentrations of 1 CFU/ml within 24 hours, with as many as 2x10 6 other bacterial cells/ml present. It is important to note that both of these tests are sensitive only to bacterial water contamination. Fecal contamination can also result in the transmission of viral and eukaryotic pathogens. INTRODUCTION During this laboratory activity, we will be looking for typical biological markers of fecal water contamination, including the enteric bacterium, Escherichia coli. The class will work as a team to investigate the cleanliness of an environmental water sample compared to both positive (water inoculated with E. coli) and negative (sterile water) control samples. We will determine the cleanliness of the experimental water sample using both MPN and Colilert data. Water samples will be inoculated into Durham lactose tubes with Colilert reagents and will be incubated for 48 hours at 35ºC. Data will be collected and analyzed in order to determine the likelihood of fecal contamination of the water. Remember that in lactose broth is only a putative diagnosis, as this is not a selective media- and will allow most bacteria to grow. Further analysis can be done using the more
specific tests where ONPG and MUG are broken down selectively by enteric bacteria generally and E. coli specifically. In order to analyze the data, you will be looking for visible changes in the water samples for: visual color changes, as ONPG is broken down to ONP by fecal/enteric bacterial contaminants; turbidity and gas production, as lactose is consumed and gas is produced by microorganisms; and fluorescence, as MUG is broken down by E. coli. These results can be compared to the expected results in Table 2 below to calculate the most probable number (MPN) of organisms per milliliter of water. Additionally, Table 3 can be used to determine the specific presence of fecal contamination and E. coli as a measure of color change and fluorescence due to the addition of Colilert reagents. Class assignments are as follows: table 1 will be analyzing a positive control, table 2 will be analyzing the experimental water sample and table 3 will be analyzing a negative control. PROTOCOL Team Supplies 1 Colilert pack 100ml water sample (sterile water for 2 teams, environmental water samples for 4 teams) in Milk Dilution Bottles Escherichia coli 3 lactose Durham tubes 1. Experimental teams: add the contents of 1 Colilert pack to the 100ml Milk Dilution Bottle water sample. Label this water experimental. Cap the sample and shake vigorously. 2. Positive and negative control teams: add Colilert pack to 100ml Milk Dilution Bottle sterile water. Label this water control. Cap the sample and shake vigorously. The control water can be shared for the positive and negative control inoculations. Please plan accordingly to share reagents with your neighbors. 3. Collect and label your Lactose Durham tubes. Positive control team: Preparation of Positive Control Samples (Figure 3) 1. Add 10ml control water to 3 Lactose broth tubes. Label these tubes positive control. 2. Add 1 loopful of E. coli to each of the positive control tubes. 3. Incubate the sample at 37 C for 48 hours. 4. Incubate the Milk Dilution bottle at 37 C for 48 hours Negative control team: Preparation of Negative Control Samples (Figure 3) 1. Add 10ml control water to 3 Lactose broth tubes. Label these tubes negative control. 2. Incubate the sample at 37 C for 48 hours. 3. Incubate the Milk Dilution bottle at 37 C for 48 hours
Figure 3: Diagram of dilution protocol for preparation of Control Samples. Experimental sample teams: Preparation of Experimental Samples (Figure 4) Undiluted Samples 1. Aseptically pipette 10ml of the experimental water into 3 Lactose Broth tubes. 10-1 Samples 1. Add 1ml experimental water to each of the tubes. 10-2 Samples 1. Add 100 l (0.1ml) experimental water to each of the tubes. 2. Incubate all 9 of the sample at 37 C for 48 hours. 3. Incubate the Milk Dilution bottle at 37 C for 48 hours
Figure 4: Diagram of dilution protocol for preparation of Experimental Samples. DATA AND OBSERVATIONS 1. Collect florescence data (MUG) by reading the absorbance of the samples at 260nm using the Spec20. 2. Collect visual color change data (ONP) by reading the absorbance of the samples at 570nm using the Spec20. 3. Collect turbidity data by reading the absorbance of the samples at 600nm using the Spec20. 4. Input your data into the appropriate table below, and then get class data for the other 5 samples (total of 6: 4 experimental, 1 positive, 1 negative) Experimental sample 1: Sample site Table 1: Data collection for each of the sample test tubes for the experiment. = average value. Experimental sample 2: Sample site Experimental sample 3: Sample site
Experimental sample 4: Sample site Positive control Negative control 5. Count the number of tubes for the experimental water sample that have turbidity and/or gas production. Each turbid tube is considered a positive result. Record these data in a table similar to that shown in Table 2. Experimental sample 1: Table 2: Data collection for each of the sample test tubes for the experiment for turbidity. Mark each tube with with a + and each tube with no with a -. 10 1 are the undiluted samples. Experimental sample 2: Experimental sample 3: Experimental sample 4:
Positive control: Negative control: 6. Calculate the average absorbance for both visual and ultraviolet wavelengths of light for each of the three sample dilutions (i.e. undiluted; 10-1 ; 10-2 ). Input these data into Table 2 Table 1 above. 7. Using Table 4 below, determine the MPN of the samples. 8. Analyze your colorimetric data according to Table 3. appearance less yellow than the positive control more yellow the positive control more yellow & florescent the positive control result negative for total coliforms and E. coli positive for total coliforms positive for E. coli Table 3: Colorimetric changes of water samples due to the addition of the Colilert reagents ONPG and MUG and their subsequent catabolism by bacteria. DISCUSSION 1. Determine the MPN for the water sample using the data collected in Table 2 and analyzing it according to the MPN table (Table 4). Experimental sample 1: Experimental sample 2: Experimental sample 3: Experimental sample 4: Positive control: Negative control: 2. Was the water sample you collected contaminated? 3. What do both yellow color change and fluorescence indicate about a water sample?
positive tubes MPN/ml lower 95% CI a 0-0-0 <3 9.5 0-0-1 3 0.15 9.6 0-1-0 3 0.15 11 0-1-1 6.1 1.2 18 0-2-0 6.2 1.2 18 0-3-0 9.4 3.6 38 1-0-0 3.6 0.17 18 1-0-1 7.2 1.3 18 1-0-2 11 3.6 38 1-1-0 7.4 1.3 20 1-1-1 11 3.6 38 1-2-0 11 3.6 42 1-2-1 15 4.5 42 1-3-0 16 4.5 42 2-0-0 9.2 1.4 38 2-0-1 14 3.6 42 2-0-2 2 4.5 42 2-1-0 15 3.7 42 2-1-1 20 4.5 42 2-1-2 27 8.7 94 upper 95% CI positive tubes MPN/ml lower 95% CI a 2-2-1 28 8.7 94 2-2-2 35 8.7 94 2-3-0 29 8.7 94 2-3-1 36 8.7 94 3-0-0 23 4.6 94 3-0-1 38 8.7 110 3-0-2 64 17 180 3-1-0 43 9 180 3-1-1 75 17 200 upper 95% CI 3-1-2 120 37 420 3-1-3 160 40 420 3-2-0 93 18 420 3-2-1 150 37 420 3-2-2 210 40 430 3-2-3 290 90 1000 3-3-0 240 42 1000 3-3-1 460 90 2000 3-3-2 1100 180 4100 3-3-3 1100 420 a CI is an abbreviation for Confidence Interval, the range of values within which you have a statistically accurate chance of predicting the actual population number. Table 4: MPN Table. Data are collected for tubes displaying for each dilution series. As an example, 3 positive undiluted tubes, 2 positive 10-1 tubes and 0 positive 10-2 tubes would yield an MPN of 93 with lower & upper confidence limits of 18 and 420 respectively.