Using Colilert And DST To Detect Total Coliforms And E.Coli

Colilert is a commercially available enzyme-substrate that simultaneously detects the total coliforms and E.Coli. (Lab Manual, 2015). It is found in presence/Absence format (PA) or most-probable number (MPN).

In general, the defined substrate technology works simultaneously with two enzymes substrates:

• Chromogen
• Fluorogen

Chromogen is an enzyme substrate that reacts with the enzyme that is found in total coliforms. Similarly, Fluorogen is an enzyme substrate that reacts with an enzyme that is commonly found in E.Coli.

Furthermore, placing these two enzymes in a 24-hour incubation at 35, the coliform-positive would turns yellow in color. Meanwhile, an E.Coli-positive causes it to fluoresce from ultra-violet light (Lab Manual, 2015).

Without a doubt, the colilert method is approved for drinking water since it helps to monitor all kinds of water, especially those with high-suspended sedimentation. Lastly, the PA is mostly appropriate for ground water or drinking-water studies (Lab Manual, 2015).

Dilutions: Explanations and Examples of Common Methods.

Guidelines for Canadian Drinking Water Quality - Summary Table." Guidelines for Canadian Drinking Water Quality.

CVG3132 Physical and Chemical Unit Operations of Water and Wastewater Treatment Laboratory Manual.

Overview of Colilert and DST

Indicator microorganisms are biological detectors help to target pathogens. In general, pathogens are hard to isolate and are small in number. In order to identify pathogens that are easier and safety detected (Lab Manual, 2015). There are certain requirements that need to be met in order for the indicator microorganism to be most efficient. Firstly, the indicator must be found in the fecal contamination as it occurs (Lab Manual, 2015).

Moreover, the population of the indicator must be greater than or equal to the population of the pathogens, while having similar survival characteristics like the pathogens (Lab Manual, 2015). In addition, the indicator should not be able to reproduce outside of the host. Once these samples have been collected, these samples should be stored in a cold and dark area (i.e. ice chest or refrigerator between 1- 4  (Lab Manual, 2015). While performing this experiment, it is crucial to constantly sterilize and clean your surroundings, especially when you are working with samples that contain bacteria.

One method that can be used in this experiment that can sterilize equipment is through the use of methanol and allows it to evaporate from the equipment. Make sure to indicate that methanol is highly toxic to bacteria and can interfere with a sample (Lab Manual, 2015).

Firstly, a Membrane Filter Technique offers the advantage of isolating discrete colonies of bacteria. Moreover, it allows testing of large sample volumes. Reduces the amount of preparation time in comparison to a lot of traditional methods used in wastewater.  

In the real world, the MF Technique is useful for monitoring bacteria in pharmaceuticals, cosmetics, electronics, food, and beverage industries (Pall Corporation, 2014).  Moreover, it allows the removal of cidal or bacteriostatic agents that are mainly removed in Pour Plate, Spread Plate or MPN Techniques (Pall Corporation, 2014).  

In general, the MF Technique passes through a filter using a filter funnel and a vacuum system. In addition, the filter and organisms are placed in a petri dish that includes a culture medium (Lab Manual, 2015).  Through the period of incubation, a bacterium reproduces on the surface of the membrane and grows bacterial colonies that are countable.

There must be less than 200 bacterial colonies on the filter to consider this method accurate; one can state that there are too many to count. Similarly, if there are not that many coliforms, one can state that there is less than 1 per 100mL (Lab Manual, 2015).  For Accuracy, make sure to shake the sample prior to filtering, as this allows the separation of all bacteria found.

Indicator Microorganisms and Dilution Methods

Colilert is a commercially available enzyme-substrate that simultaneously detects the total coliforms and E.Coli. (Lab Manual, 2015). It is found in presence/Absence format (PA) or most-probable number (MPN).

In general, the defined substrate technology works simultaneously with two enzymes substrates:

• Chromogen
• Fluorogen

Chromogen is an enzyme substrate that reacts with the enzyme that is found in total coliforms. Similarly, Fluorogen is an enzyme substrate that reacts with an enzyme that is commonly found in E.Coli.

Furthermore, placing these two enzymes in a 24-hour incubation at 35, the coliform-positive would turns yellow in color. Meanwhile, an E.Coli-positive causes it to fluoresce from ultra-violet light (Lab Manual, 2015).

Without a doubt, the colilert method is approved for drinking water since it helps to monitor all kinds of water, especially those with high-suspended sedimentation. Lastly, the PA is mostly appropriate for ground water or drinking-water studies (Lab Manual, 2015).

Meanwhile, the MPN method helps to count from 1 to 2,400 MPN per 100mL of sample. These tests help give a bigger range with the dilution of 1:100 or 1:10 ratios with the assistance of sterile water (Lab Manual, 2015).

This formula of dilution, also known as the dilution factor, can be used where one solute is present, this formula should be solved through stoichiometry if a reaction takes place (Quansys Biosciences, 2013).  The dilution formula can be written as follows:

Where: V1 = Volume of starting solution needed to make new solution (mL)

C1 = Concentration of starting solution

V2 = Final volume of new solution (mL)

C2 = Final concentration of new solution

The objective of this experiment is to determine the membrane filtration technique and defined substrate technology (DST).

Refer to page 17 of the CVG3132 lab manual.

After letting the four sample incubate for 24 hours at 35°C, no colonies were clearly observed on the filter. On the 10% sample some dark dot could be observe but it was not enough to count as a colony. The distribution of the colonies is shown in the following table.   

Table 1: Results of the Membrane Filtration Technique

Sample	Colonies
Control (Distilled water)	0
0.1% sample	0
1% sample	0
10% sample	Less than one

For this part, the sample was incubated in the Colilert Quanti-tray for 24 hours at 35?. The sample was then compared to the color comparator for color change. The presence of total coliform bacteria is shown when the sample turn to yellow. Only one large well of the colibert Quanti-tray turned to yellow.

The second part of the experiment was to identify if E. coli was present in the sample. The Colibert Quanti-tray was expose to the 365nm UV light and no fluorescent wells was detected.

Membrane Filtration Technique

The number of wells that contain coliform bacteria and the result for the presence of E. coli are shown in the table 2.

Table 2: Well counts of the Colilert Quanty-tray.

Positive Result	Large Well Count	Small Well Count
Total coliforms (yellow)	1	0
E. coli (fluorescence)	0	0

The calculation for the control sample, the 0.1% sample and the 1% sample are not possible because the filter has no trace of bacteria on it. At the opposite, the 10% sample showed some trace that can be defined as ''less than one'' colony. The result per 100ml of original sample is calculated as follow.

Colonies per 100ml of original sample =Defined Substrate Technology 

In this part, the most probable number (MPN) is use to identify the number of bacteria contained in the sample. To determiner this number, the use of the IDEXX Quanti-Tray/2000 MPN Table to determine the MPN for total coliform bacteria and E. coli bacteria. The table is shown in the figure below

Using the data of the Table 2 and the IDEXX Quanti-Tray®/2000 MPN Table, the amount of total coliform bacteria is 1.0 MPN/100ml and the density of the E. coli bacteria is less than 1.0 MPN/100ml.The sample was not diluted for this analysis. Therefore, the density of bacteria per 100ml of sample is given directly with the chart.  The results are shown in the table 3.

Table 3: The MPN for total coliform and E. coli bacteria

Positive Result	Large Well Count	Small Well Count	MPN/100 mL
Total coliforms (yellow)	1	0	1.0
E. coli (fluorescence)	0	0	Less than 1

 Discussion

The first part of experiment of the membrane filtration method ha four major samples which were unable to show the presence of coliform. The samples included a control sample, dilution sample of 10%, dilution sample of 1% and dilution sample of 0.1%. Some bacterial colony were found to exist on the 10% dilution sample. Nevertheless, none of the were coliform able to be differentiated by the red color and mettalic sheen. The use of non-dilited sample is assumed to could have helped to find coliform on the sample. The experiment is able to show that there was less than 10 coliform available per 100 ml.

Second part of the experiment was the DST method. At this part, no E-Coliform were present. This meant that the most probable number of E-Coliform which can be found in a 100 ml is less than 1. In this part of the experiment, only one large positive to the total coliform was found in our sample. According to the table, we can conclude that one MPN of the total coliform per 100 ml is able to exist.

Results of the Membrane Filtration Technique

The major observation made while comparing the two tests is that they have the same range and this proves that they are done in the right manner. The Canadian guidelines for drinking water quality is that the samples aren't good for consummation. The guide means that the concentration of the total coliform and E-Coli are not mostly detected in a sample of 100 ml.

Contamination is one of the source of error in this lab experiment. Nevertheless, this source of error was not a major problem in this experiment due to the low results in the experiment. In addition, another are which can be source of error in the experiment is the shaking step which happens before the diluting and filtration. Lack of proper shaking means that the bacteria will agglomerate together and form a big colony instead of multiple colonies as required.

The need for high number of equipment and more disinfection step makes the membrane filtration method more involving although its more precise. DST method is not much precise and its more easier to perform. DST method is usually preffered for routine water quality testing since it is not likely to be manipulated and thus have less chances of contamination. Moreover, the method is easier to perform and provides results of both total coliform and E. Coli from the same test.

Conclusion

In conclusion, from the experiment, the sample was found to have low quantities of coliform. The end results evident from the two-techniques, which included the membrane filtration and defined substrate technology were the same. The experiment was successful. More accurate result would have been achieved eda with use of undiluted samples especially for membrane filtration method. The sample from this experiment did not meet the Canada standard in relation to water quality. This meant that the DST method was found to be more appropriate for routine water quality testing.

References

"Dilutions: Explanations and Examples of Common Methods." Quansys Biosciences. Quansys Bioscience, 8 Nov. 2013. Web. 3 Feb. 2015. <https://www.quansysbio.com/dilutions>.

"Guidelines for Canadian Drinking Water Quality - Summary Table." Guidelines for Canadian Drinking Water Quality. Health Canada, 26 Nov. 2014. Web. 3 Feb. 2015. <https://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/sum_guide-res_recom/index-eng.php>.

"Membrane Filter Technique." Membrane Filter Technique. Pall Corporation, 6 July 2014. Web. 3 Feb. 2015. <https://www.pall.com/main/laboratory/literature-library-details.page?id=7290#26538>.

Mackenzie L. D., "Water and Wastewater Engineering", McGraw-Hill, New York c2011.

Narbaitz, Roberto. CVG3132 Physical and Chemical Unit Operations of Water and Wastewater Treatment Laboratory Manual. Lab handbook. Department of Civil Engineering, University of Ottawa. Ottawa, ON. Winter 2015. Print.