SXHL288 Microbiology of water

Microbiology of water

The microbiology laboratory that you visit as you study the Water Quality Monitoring topic is presented as a series of pages

accessible using the tabs along the top of the screen. On each tab there is a button to open the ‘Overview’ of the experimental

procedure to help you to carry out each study and interpret the data you obtain. These overview pages from each activity are

reproduced in this guidance document for easy reference.

You should read through the relevant topic materials before starting each of the online experiments.

You should read the overview materials before starting each experiment. For ease of reference, these overviews are

also presented in Appendix 1.

1 Sample Selection page On the Sample Selection tab, you should start by reading the onscreen text and then the ‘Overview of experiment’ text that is

opened using the relevant onscreen button. The overview text provides detailed instructions and additional information

relevant to the assays used in this study.

You can select each sample from this page using the pull-down menu on this first page, as is shown in Figure 1, where Sample 1

has been selected and is described. The ‘Reset Experiment’ button clears any data stored in the laboratory, and should be used

to change between samples in each experiment.

Figure 1: View of the opening tab, from which water samples 1-3 can be selected using the pull-down menu. Details of each sample are noted

on screen.

Each experimental tab requires you to perform certain tasks and to collect the data generated and to do so, you will need to

return to this first tab to change between samples.

2 Aerobic colony count This experiment is in three stages:

Dilution of the collected water sample

Plating the samples and dilution onto growth plates

Counting the bacterial colonies on plates

Follow the instructions presented in the ‘Overview of technique’ page (also in the Appendix) To carry out an aerobic colony

count for each sample, start by selecting the appropriate sample on the sample selection tab. Using your pointing device, select

the pipette and drag it into the relevant tubes (called Falcon tubes) as required to create sequential dilutions of 1/10 and 1/100

of the original sample.

To collect and eject using the pipette, you should ‘click’ on the top of the pipette (the red piston on the top, see Figure 2 (left)).

You will need to change tips using the ‘Change tip’ button. If you make a mistake, you can simply reset the experiment back to

SXHL288 Microbiology of water the start using the ‘Reset technique’ button. Use the ‘Check dilution’ button to check whether you have successfully carried out

the dilution before moving onto plating the samples on growth plates (Figure 2, right).

Figure 2: Views of the dilution page (left) and plated bacterial counts (right) in the Aerobic colony count experiment. Moving the pipette from

the original sample tube to the dilution tubes is achieved by selecting the pipette. You can collect and eject liquid using the plunger and change

the tip between operations using the appropriate button. For plating, you can select to plate any of the samples using the pull down menu

below each plate.

Colonies are counted from enlargements of each page viewed using the ‘Count’ button. As you select colonies on screen the

count is recorded for you. Be careful not to double-score colonies as the counter records ‘clicks’ (Figure 3).

Figure 3: The counting window for one plate during a counting exercise, showing 11 colonies that have been counted highlighted as they are

selected (dark). The count is kept onscreen, top left hand corner, and this window closed using the Done button.

To obtain replicate data: The ‘Reset technique’ clears the current plates and presents you with an empty set to retest the same

sample. You can then choose to replate from any dilution to obtain data from experimental replicates.

All data can be recorded in Table 1.

3 Most probable number Following the instructions in the Overview text and, use the pull down menus to select which sample is analysed in each plate.

When you have chosen the contents of each tray, select the Incubate button.

The experiment can be reset using the Reset Technique button. You should also analyse suitable controls and record your data

under normal (Figure 4, left) and UV (Figure 4, right) light, ensuring you record the number of wells as required in the

instructions.

SXHL288 Microbiology of water Figure 4: Example of wells seen under normal (left) and UV (right) light. The counting tool labels wells with an ‘X’

To establish the MPN for each sample, use the look up table (also in the appendix). All data can be recorded in Table 2. You can

reset the experiment to perform replicates.

4 Detection of enterococci To test for the presence of enterococci, a confirmatory test can be performed in this experiment. For the appropriate water

sample to be tested (selected on the first page), label the plate and select the Incubate button. After incubation, you can count

the numbers of colonies as before (Figure 5). The confirmatory test is performed by selecting the Confirm button.

Figure 5: A confirmatory test is available for the presence of Enterococcus faecalis.

The control samples will help you interpret your data, for example, the positive control shows a positive confirmatory test

(figure 5), as detailed in the relevant explanatory notes.

Figure 5: The positive control for the enterococci confirmatory test gives colonies with a large black-brown halo.

All data can be recorded in Table 3.

SXHL288 Microbiology of water

5 Pathogen detection To confirm the presence of E coli 0157 in a sample, this experiment requires you first to isolate a single colony from a plate of

bacteria. To do this, you use a small inoculation loop that is first sterilised (Figure 6a) to carry bacteria from a single colony onto

a new bacterial growth plate (Figure 6b). [Note that on a PC, your cursor acts as the inoculating loop]

Figure 6: First stages in the experimental process to confirm the presence of E coli 0157 in a sample. (a) The inoculation loop must be

sterilised before and after handling bacteria by placing it in the Bunsen flame before being used to scrape a single colony from the CT-SMAC

plate (b). Bacteria are streaked across the plate to progressively reduce the number of cfus ( (c) (i)-(iv)) sufficiently to generate single well-

isolated colonies.

The Show Me button provides a diagrammatical representation of how the collected bacteria are streaked across the plate

(Figure 6c).

This is done by:

Sterilising the loop (Figure 6a)

Picking a suitable colony from the plate (Figure 6b)

Streaking the loop with bacteria in one portion of the plate (Figure 6c(i))

Sterilising the loop

Streaking across one section of the first streak (dragging some of the bacteria from it)(Figure 6c(ii))

Sterilising the loop

Streaking across one section of the second streak (dragging some of the bacteria from it)(Figure 6c(iii))

Repeating to fill as much of the plate as possible (Figure 6c(iv))

Incubating the plate.

With each streak, the number of bacteria decreases, so that eventually there will be only a small number on the final few

streaks. Your aim is to obtain a single colony after incubation that is isolated away from any other colony. This ensures that you

test a pure specimen of bacteria. This is picked using your cursor/sterilised loop.

A feedback report will indicate successful picking of a suitably isolated single colony, allowing access to the confirmatory test

using the Latex agglutination button.

SXHL288 Microbiology of water

Selecting the Latex agglutination button opens a new window, with your selected pure colony being taken into this assay. Use

the Mix button to initiate the test. Record your data.

Figure 7: An example of a positive test for E coli 0157 with the latex agglutination test.

This final test is used to confirm the presence of pathogenic E coli 0157 bacteria in a sample.

SXHL288 Microbiology of water

Appendix 1 Overview texts from the Microbiology interactive experiment

Sample Selection Page

Aerobic colony count

Step 1: Dilution

Step 2: Plating and counting

SXHL288 Microbiology of water

Most probable number

Look-up table for the MPN assay

SXHL288 Microbiology of water

Enterococcus detection

Pathogen detection