Water Bacteriology: Introduction, Testing Methods, Test Requirements, Test

Water Bacteriology: Introduction, Testing Methods, Test Requirements, Test Procedure, Result Interpretation and Keynotes

Introduction of Water Bacteriology

Water Bacteriology is necessary to investigate the supplies of drinking water for the possible faecal contamination that may cause diseases like typhoid (Salmonella Typhi), cholera (Vibrio cholerae), bacillary dysentery or shigellosis(Shigella spp.),campylobacteriosis (Campylobacter spp.), amoebiasis (Entamoeba histolytica), cryptosporidiosis (Cryptosporidium), Giardiasis (Giardia), and helminthiasis, etc. It is rather a lengthy procedure to investigate for all. Therefore it is recommended to culture the indicator bacteria which are the intestinal commensals as for example E. coli, Enterococcus faecalis, Clostridium perfringens, etc. Bacteriological water analysis is not only an indicator for the water is safe for human consumption but also that bathing and recreational waters are safe to use or not. The technical staff working in the laboratory must be able to give instruction for the collection of water samples and refer the samples to the centre for bacteriological analysis.

Testing methods of Water Bacteriology

Multiple Tube Method
Filtration Method

Test Requirements for Water Bacteriology

Test requirements depend on the nature of the testing method, but here we discuss in detail of Multiple Tube Method.

Sterle 250ml glass bottles with screw caps
Brown wrapping paper
String
Cotton wool
70% Ethanol and thermometer
3% solution of Sodium thiosulfate in distilled water-Two drops of sodium thiosulfate solution is dropped in the sampling bottle and then the bottle is autoclaved with the paper cover tied with the string which neutralizes chlorine in water samples.
Water Sampling: Source of Water to be sampled should have the following information -The temperature of the water should be checked at the site of collection and noted down on the request form. All the information must be given as to the source of water supply and site, exact locality, whether the water is for drinking purposes or whether the sources are newly dug well etc.

Water sources are divided into 3 basic types for the purpose of sampling.

Water from a tap or hand pump

Wipe off the dirt from outside the tap.
Make sure the tap does not causes splashing.
Turn on the tap to maximum flow and allow the water to flow for a minute or two.
Turn off the tap and sterilize it for a minute with flame. Dip the cotton wool in 70% Ethanol, disinfect the tap by flaming using the burning cotton wool swab.
Fill 3/4th container with water by holding it under the water jet.
Leave small airspace to facilitate shaking at the time of inoculation prior to analysis.
Stopper the cap and label the container.
Put the paper back over the stopper and tie it around the neck of the bottle.

2. Water from a reservoir

Open the container under sterilized conditions.
Fill it by holding it by the lower part, submerging it to a depth of about 20cms, with the mouth facing slightly upwards. If there is a current, the bottle should face the current.
Collect the water to fill the 3/4th of the bottle.
Stopper the bottle and label it and wrap it with paper and tie it with the string.

3. Water from a dug well

Attach a stone of suitable size to the sampling bottle with a piece of string.
Tie a 20 meters length of clean string on the bottle and to a stick.
Open the bottle as described above and lower it into the well.
Immerse the bottle completely in water without touching the side of the well and lower down it down to the bottom of the well.
Pull it out when the bottle is filled.
Discard a little water to provide airspace.
Stopper and label the bottle.

Dispatch of Samples for Water Bacteriology

Dispatch the sample on the same day.
The sample should be packed in the wooden box in an upright position with the paper or foam around so that the bottle does not break.

The following two steps methods are adopted-

Presumptive Coliform count
Confirmative E. coli test.

Presumptive Coliforms count

Two methods are available for this

Multiple tube method.
Membrane filtration method.

Multiple Tube Mehtod (MTM)

Liquid media used are-

MacConkey broth (Single and double strength)
Brilliant green bile broth
Lauryl Tryptose broth

Composition of MacConkey broth

Single strength (SS)

Sodium taurocholate: 5 gm
Peptone: 20 gm
Sodium chloride: 5 gm
Lactose: 10 gm
Bromocresol purple 1% solution in ethanol:5 ml
Neutral red 1% aqueous solution: 4 ml
Distilled/ deionized Water: 1000 ml

Preparation of MacConkey broth (SS)

Dissolve the bile salt, peptone, and Sodium chloride.
Steam for 2 hours, cool, and transfer to the refrigerator overnight.
Add lactose and when dissolved.
Adjust the reaction to pH 7.4
Add indicator.
Distribute 5ml and 1 ml in test tubes with Durham tubes.
Autoclave at 121° C for 15 minutes.

Double strength MacConkey broth

Prepare the double strength (DS) MacConkey broth as shown above. The composition is the same but half amount of water is used.
Distribute 50/50 ml in 5 bottles using 3×3/8 inch test tubes with Durham tubes, and in 10ml tubes using 2× 0.25 inch Durham tubes.

Brilliant green bile broth is also the commonly used media for MTM. It is simple to do. It consists of growing the bacteria in the suitable culture medium in multiple tubes, the gas and acid formation helps to detect the presence of the Coliforms. It also helps to enumerate the bacterial count.

The technique of the Multiple Tube Method for Water Bacteriology

In this method. measured volume (105ml) water is added to the sets of tubes of media as recommended in the standard regimen. The growth medium contains the indicator. From the number of the tubes showing the positive results, the presumptive count can be derived following MacCrady’s table.

Test Procedure of Multiple Tube Method

Approximately 200ml of water from the source is collected in a sterile container.
50ml water is added to one bottle containing a 50ml double-strength medium.
10ml water added to 5 bottles containing 10 ml single strength medium.
1ml water added to 5 bottles containing 5ml single strength medium.
The total volume tested is now 105 ml of water.
The total number of tubes used for water culture is 11. After inoculation of the sample, they are incubated at 37° C for 24 hours.
The next day the bottles are removed and checked for the colour, gas formation and turbidity and the numbers giving position reactions are counted.

Result Interpretation of Multiple Tube Method

By referring to the table the MPN can be derived and the following results are noted down.

Excellent: If MPN is zero (no growth in all the tubes).

Satisfactory: If MPN is 1-5 ( number of tubes showing growth).

Indeterminate: If MPN is 6-10 (number of tubes showing growth) and needs more treatment to purity the sources.

Unsatisfactory: If MPN is above 10, this needs vigorous treatment to clear the contamination.

Membrane filtration technique

The drinking water can be checked for its purity using the membrane filtration technique. For this, a Millipore system has provided a full set required for filtration of the water.

It consists of-

Filter pad of 47mm diameter with 0.4μm pore size. 100ml water is filtered through this.
A funnel and clamp to hold the filter pad
Conical flask with a sidearm.

The procedure of filtration in Water Bacteriology

Water is filtered through the pad using negative pressure by a suction machine.
The filter pad is removed and then placed over the surface of the culture media and incubated. The media used are MacConkey, Lauryl sulphate broth containing lactose and phenol red.
The next day colonies growing on it were identified and enumerated.
Count, the members of the colonies on the pad.
This gives presumptive Coliforms count per 100ml of water samples.

2. Confirmative E. coli test in Water Bacteriology

This test is also called the Eijkman test. This helps to identify faecal E.coli. This consists of culturing a loopful of the growth presumptive positive tubes onto a fresh set of Lactose broth and incubated at 44° C for 24 hours. The results are read by change of colour to pink and gas production showing fermentation of lactase, which indicates the presence of faecal Coliforms.

Quality control: Use Escherichia coli positve control and Pseudomonas aeruginosa for negative control.

Keynotes in Water Bacteriology

Bacteriological water analysis is a method of examining water to estimate the numbers of bacteria present and, if needed, to find out what type of bacteria they are.
Testing of water samples from the different sources also falls into the priorities of the public health laboratories.
Nearly all, tertiary care hospitals have their water bacteriology laboratory set up for assaying the quality of waster using in different sites of the hospital.
It is necessary to supply sterile containers for water sample collection.
Sodium thiosulfate is used for neutralizing chlorine in water samples.
The sample is examined at the earliest preferably within one hour or should be quickly transported to the laboratory keeping in a cool container away from sunlight. It should be processed for culture within 6 hours of collection.
Water culture should be done every week at least for 6 weeks
MPN (Most probable number) may vary from source to source.
ATP testing: An ATP (adenosine triphosphate) test is the process of rapidly measuring active microorganisms in water through ATP detection.
Various techniques for bacteriological water analysis, are the Multiple tube method, ATP testing, Plate count, Membrane filtration and Pour plate method.
MTM (Multiple tube method) is the most commonly used technique for bacteriological water analysis.

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