LAB 6 - Detection of Faecal Contamination in Water

Question 1

Why is it often not possible to detect pathogenic microbes directly in environmental samples and what is the solution to this

Answer 1

• Because they may only be present in very low numbers
• Instead detect microbes known to normally occur in the gastrointestinal tract and use this as an indicator of faecal pollution

Question 2

Examples of thermotolerant coliforms

Answer 2

• Citrobacter
• Enterobacter
• Escherichia
• Klebsiella

Question 3

What is the Australian standard for assessing faecal contamination

Answer 3

Based on the detection of E. coli

Question 4

Why is E. coli considered the most sensitive indicator of faecal pollution

Answer 4

• It is present in the gut of human and other warm-blooded animals
• It does not generally inhabit other environments
• It is numerically dominant amongst all coliforms
• Tests to detect it are specific, easy, cheap, well established and in wide use.

Question 5

Australian standard for drinking water

Answer 5

States that no E. coli can be present in any 100mL sample of water

Question 6

Reason for using dilutions in the experiment

Answer 6

Dilutions are included in case there are high numbers of bacteria in the sample, which could lead to confluent growth on agar plates rather than distinct separated colonies

Question 6

First step in detection of faecal contamination of water

Answer 6

• A water sample (and associated dilations) is passed through a filter to trap the bacteria in the water onto the filter surface
• The filter is then transferred to the surface of an agar medium that is selective for coliform bacteria
• Agar incubated at 44 degrees which is selective for thermotolerant coliform bacteria including E. coli.

Question 7

Agar medium used

Answer 7

Membrane Faecal Coliform (m-FC) agar

Question 8

Stains incorporated in medium

Answer 8

• React to acid formation during lactose fermentation by faecal coliforms (FCs), resulting in colonies with various shades of blue.
• This step enables the detection and enumeration of presumptive FCs in the sample

Question 9

Second step in detection of faecal contamination of water

Answer 9

• Selected presumptive colonies from the m-FC plates are inoculated into Peptone water and incubated
• E. coli contains the enzyme tryptophanase, which cleaves tryptophan and produces indole.
• Indole can be detected using Kovac’s reagent

Question 10

Positive indole result

Answer 10

Highly suggestive of E. coli, as not many thermotolerant coliforms are tryptophanase positive.

Question 11

Negative control of experiment

Answer 11

Enterobacter aerogenes

Question 12

Positive control of experiment

Answer 12

E. coli

Question 13

Viable count calculation (CFU/100ml)

Answer 13

• Divide the number of colonies by the sample volume filtered (which in our experiment was 100ml)
• Then multiply the number by 100 so that your final value is expressed as CFU/100ml.
• Next, multiply by the dilution factor to determine the CFU/100ml in the original water sample (e.g. for 1/100 dilution times by 100)

Question 14

Would the results you obtained be adequate for a health authority to approve the water for drinking?

Answer 14

No, contain E. coli

Question 15

What is a major limitation of these testing procedures, even when performed by approved laboratories?

Answer 15

Delay in obtaining results (due to incubation periods, meaning that water contamination can go undetected for several days while tests are being processed)