Bacteriological Test of Water

Freshwater swimming pools, whirlpools, and naturally occurring fresh and marine surface waters are all examples of recreational waters. These waters must be tested for the presence of coliform and/or heterotrophic bacteria.

Although the presence of coliform bacteria in water suggests that it is potentially unsafe to drink. Other bacteria isolated from recreational water may pose public health risks via body contact, injection, or inhalation.

Pathogenic and nonpathogenic bacteria suggested as water quality indicators include the coliform group, pseudomonas species, streptococcus, and staphylococcus, among others.

Coxsackie A and B. adenovirus, hepatitis A, and a variety of other gastroenteritis viruses are among the viral diseases associated with untreated recreational water.

Bacteriological Test of Swimming Pool

A swimming pool is a small body of water contained within a holding structure. The water is generally safe to drink and has been disinfected. The water could come from thermal springs or treated water. The modern pool has a recirculating system that filters and disinfects the water. Pools should be classified as either disinfected or untreated.

Swimming Pool Monitoring Requirements

In general, monitor pool water quality for changes in chemical and physical characteristics that may cause irritation to bathers’ skin, eyes, and mucosa barriers or impair disinfection. Water quality is determined by the efficiency of disinfection, the number of bathers in the pool at any given time, and the total number of bathers per day. Microorganisms of concern are typically those from the bather’s body and its orifices and include those causing infections of the eye, ear, upper respiratory tract, skin, and intestinal or genitourinary tracts.

During peak bather load periods, residual disinfectant and turbidity levels must be determined on a regular basis. Alternatively, when the turbidity exceeds 1 NTU and the heterotrophic plate count (HPC) exceeds 500 colony forming units/mi. Normal skin flora that is likely to contain pseudomonas streptococcus and staphylococcus are indicators of health risk. These organisms are responsible for a large proportion of swimming pool-related illnesses.

Heterotrophic plate count (HPC) for the swimming pool should be less than 500 colony-forming units/mi.

The turbidity value of the swimming pool should be ideally less than 1 NTU.

You may like reading: Acidity of Water – How to Measure It

Criteria for Bacteriological Water Testing Sample Collection

Collect samples for bacteriological examination of swimming pool water in sterilized bottles containing 0.1 to 0.4 ml of 10% sodium thiosulfate solution. Collect samples in the area and at the time of maximum bather load, and at a depth of about 1M in a sterilized bottle.

Heterotrophic Plate Count

The heterotrophic plate count (HPC), also known as the standard plate count, is a method of estimating the number of live heterotrophic bacteria in water and measuring charges during treatment, distribution, or in swimming pools. Colonies can form from pairs of cells, clusters, or single cells. For this experiment, three different methods and media are used.

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Microbiological testing methods

The following 3 methods are widely used for the determination of bacteriological analysis of water:

  1. Pour plate
  2. Spread plate and
  3. Membrane filter method

Media Required for the Water bacteriology experiment:

Plate count (Tryptonc glucose yeast agar). This is a high-nutrient-agar, widely used. Other than this simple RA or NWRI agar can also be used. Also, these materials are required:

Tryptone=5.0 g
Yeast extract=2.5 g
Glucose=1.0 g
Agar=15.0 g
Water= 1L
pH should be 7.1± 0.2 before adding agar. Autoclave it at 121°C for 15 min
Water bacteriology test ingredients

Membrane filter required for Water bacteriology test

M-HPC agar. High/nutrient medium only for membrane filter along with:

Peptone = 20.0 g

Gelatin = 20.0 g

Glycerol  = 10.0 g

Agar  = 15.0 g

Reagent-grade water  = 1 L

Mix all ingredients except glycerol. Adjust pH to 7.1 if necessary heat slowly to boiling to dissolve ingredients add glycerol slowly, shake it and then autoclave at 121°C for 15 min.

Worth reading post: How to Measure Alkalinity of Water

In the next paragraphs I will discuss microbiological testing methods for water:

  1. Pour Plate Method

Selection of dilution: Select the dilutions so that the total number of colonies on a plate will be between 30-100 for example if the plate count is 3000 prepare dilution 104.

Sample dilution: Use a sterile pipette for initial sample preparation. Prepare dilution and put it into the plates (previously made). The cold agar (40°C) was overlayed per plate. Shake the Agar clockwise and anticlockwise. Let the Agar solidly and then place the plates in an incubator at 37°C in n inverting position as shown in the figure:

Pour Plate Method
Pour Plate Method

Counting of colonies

Count all colonies on selected plates promptly after incubation. If counting is delayed temporarily. store plates at 5′ to 10°C for no more than 24 hr, but avoid this as routine practice. Compute bacterial count per milliliter by the following equation in CFU (colony forming unit).

Counting of colonies formula
Counting of colonies formula

2. Spread Plate Method

Preparation of plates: Pour 15 ml of the desired medium into a sterile Petri plate, and let agar solidify. Pre-dry plates into inverted position so that water loss takes place.

Sample dilution: (same as Pour plate method)

Select suitable dilutions and put 0.1 or 0.5 ml sample onto the surface of the agar Petri plate. Using a sterile glass rod, distribute inoculum over the surface unless inoculum is observed completely into the medium. Incubate plates at 37°C ± 1° for 24 to 48 hours.

Counting: Colonies were counted and noted down.

3. Membrane Filter Method

Preparation of Plates: Dispense 5 ml of the sterile medium into 50 x 9 mm Petri plates. Let solidify at room temperature. Select a sample size to give a 20 to 200 CFU filters.

Procedure

  1. Filter appropriate volume through a sterile 47 mm, 0.45 pm, gridded membrane filter, under partial vacuum. Rinse funnel with thrice 20-30 ml portion of sterile water. Place the filter on agar in the Petri plate.
  2. Incubate plates at 35° ± 0.5°C for 48 h if using M-HPC agar, or longer if using any other medium.
  3. Report cell as CFU/ml. on the membrane filter.

Conclusion:

Bacteriological Test of Water is one of the most important water quality tests, especially for swimming pools. In this article, We can assess the bacteriological water quality by using 3 Water bacteriology tests, which are discussed in detail above. Moreover, it should be noted that the Turbidity value of the swimming pool should be ideally less than 1 NTU and the Heterotrophic plate count (HPC) for the swimming pool should be less than 500 colony forming units/mi.

Read More tests here: Environmental Engineering

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