Bacterial Examination of Water

Question 1

What are enteric bacteria defined as?

Answer 1

• Gram-negative bacilli
• non-spore forming
• oxidase negative
• Aerobes or facultative anaerobes
• Ferment glucose to acid end products.

Question 2

What are Coliforms?

Answer 2

• Enteric bacteria that can ferment lactose to acid and gas

Question 3

What is the relevance of the enzyme β-galactosidase?

Answer 3

It gives coliforms the ability to ferment lactose to acid and gas.

Question 4

What are fecal coliforms?

Answer 4

• Coliforms that carry the enzyme β-glucuronidase

Question 5

Why is E. coli presence in water a good indicator species for fecal contamination?

Answer 5

• E. coli is a reliable fecal coliform
  
  • It is normally found in mammalian intestines, and not normally found in fresh water.
• Fecal coliforms like E. coli “indicate” contamination that, along with the usually harmless E. coli, might also carry potentially pathogenic enteric bacteria, protozoans, and viruses that may have been shed along with fecal coliforms.

Question 6

What was the purpose of the filter discs in the water filtration experiment?

Answer 6

• Bacteria larger than 0.45 μm will be retained directly on the surface of the filter.
• The filter is then placed on M-Endo agar, a selective and differential medium.

Question 7

On the M-Endo agar plate, what is the purpose of Sodium lauryl sulfate and sodium desoxycholate?

Answer 7

• Sodium lauryl sulfate and sodium desoxycholate inhibit Gram-positive cocci and endospore-forming bacteria.

Question 8

What is the carbon source on M-Endo agar plates?

Why is this relevant?

Answer 8

• The only carbon source (carbohydrate) in M-Endo agar is lactose.
• This means that only coliforms are able to grow in the medium.

Question 9

What does E. coli and Enterobacter aerogenes look like on M-Endo agar plates? Why?

Answer 9

• E. coli and Enterobacter aerogenes retained on the filter paper that is put on the plate will form colonies with a characteristic greenish- golden metallic sheen within 24 hours of incubation.
• This sheen is from indicator dyes: basic fuchsin and sodium sulfite form a green metallic sheen on colonies able to ferment lactose to acids and acetaldehyde.

Question 10

• After ID’ing a bacteria contamination as a conform via the mENDO plates, how do you differentiate between non-fecal coliforms and fecal coliforms?
  
  • What is the biochemical reason for this?

Answer 10

• The membrane from the mENDO medium is transferred to NA-MUG medium and incubated for 4 hours at 37°C.
• Production of a halo of bright blue fluorescence around the sheen colonies indicates E. coli.
  
  • The fluorescence is caused by the breakdown of a fluorogen,
    4-methylumbelliferyl-β-D-glucuronide (MUG), which is cleaved by an enzyme specific to E. coli: β-glucoronidase.
  • Fluorescent halo colonies are observed with a long-wave (366 nm) ultraviolet lamp.

Question 11

What is the ideal number of coliform colonies filtered during the process?

Answer 11

• An ideal quantity will result in the growth of about 20 to 80 coliform colonies and not more than 200 colonies of all bacterial types.
• Typical coliform colonies are counted and their density is reported in terms of (total) coliforms per 100 mL of sample.

Question 12

What are the limitations of the filtration method?

Answer 12

• Its effectiveness is decreased in water samples characterized by high turbidity, high concentrations of heavy metals or the presence of toxic materials such as phenols.
  
  • Turbidity caused by the presence of algae or other interfering material may not permit examination of a sample volume large enough to yield significant results.
  • Toxic substances, on the other hand, may cause low coliform estimates.
• Finally, the mENDO method is used to test drinking water and groundwater.
  
  • However, it may not be optimal for testing raw surface waters because high levels of non-target background growth may make plates difficult to read.