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Bacteriology of Water

Water Microbiology is an important test for checking the quality of our drinking water. The above picture is showing a testing procedure of drinking water whether having coliform bacteria or other gram-negative bacilli or not.

Introduction of Water Microbiology

Drinking water is acceptable and fit for drinking when it is clear, colorless, odorless, and without disagreeable taste. It should microscopically be free from pathogenic organisms and thus water microbiology is useful.

Bacterial flora in the aquatic environment

Largely saprophytic organisms, some of which belong to the genera Micrococcus, Pseudomonas, Serratia, Flavobacterium, Chromobacterium, Acinetobacter,  Alcaligenes, etc. Biofilm development in streams, water pipe networks, and industrial processes. Some opportunistic pathogens such as Legionella may be ubiquitous in the water environment.

Examination of Water Microbiology

WHO defines water supply surveillance as ‘ keeping a careful watch at all times, from the public health point of view, over the safety and acceptability of drinking water supplies. It involves two complementary activities (a) sanitary inspection and (b) water quality analysis. Although water can contain unwanted chemicals, the greatest risk to human health is from fecal contamination of water supplies. The most important aspect of the analysis is therefore to determine whether fecal contamination is present or not. Water supplies should regularly test to conform their freedom from contamination. Reliance places on testing the supply for microorganisms which indicates that fecal pollution has taken place or not. About the waterborne route, humans become infected by ingesting pathogenic bacteria, viruses, or parasites in water polluted by human or animal feces or urine. Examples of common water-borne disease bacterial origins like cholera, bacillary dysentery, E. coli diarrhea, leptospirosis, typhoid and paratyphoid, and similarly Parasitic origin amoebic dysentery, cryptosporidiosis, giardiasis, balantidiasis, and salmonellosis. Due to the virus rotavirus diarrhea, hepatitis A and Poliomyelitis are common.

Bacterial flora in water

Without fecal contamination:

Natural water bacteria (mainly derived from the air by rains): Micrococcus, Pseudomonas, Serratia, Flavobacterium, Acinetobacter soil bacteria (washed into water): Bacillus subtilis, Bacillus megaterium, Klebsiella spp.

After fecal contamination:

Intestinal bacteria ( through sewage): E. coli, Klebsiella, S. faecalis, Clostridium perfringens 

Sewage bacteria proper: P. vulgaris, Cl. sporogens, Filamentous bacteria ( e.g. Nocardia spp.)

Bacteriological indicators

It is based on  organisms indicative of water pollution by humans/animals feces such as

1. Coliforms: e.g. E. coli, Klebsiella spp., Enterobacter, Citrobacter
2. Fecal or thermotolerant coliforms
3. Faecal Escherichia coli
4. Fecal streptococci:  They are regularly found in feces though their number is much less than E. coli.
5. Clostridium perfringens

Criteria for an ideal indicator organism

1. It should be a member of the intestinal microflora of warm-blooded.
2. It should be present when pathogens are present and absent in uncontaminated samples.
3. It should be present in greater numbers than the pathogen.
4. It should be at least equally resistant as the pathogen to environmental insults and to disinfection in water and wastewater treatment plants.
5. It should not multiply in the environment.
6. It should be detectable by means of easy, rapid, and inexpensive methods.
7. The indicator organism should be non-pathogenic and present no risk to the analyst.

Sample for water microbiology

Water sources can be divided into three basic types for the purpose of sampling-

a) Water from a tap or fixed hand pump

b) Water from a reservoir (river, lake, tank)

c) Water from a dug well

Sampling from a tap or pump outlet for water microbiology

1. Remove any attachments from the tap that may cause splashing.
2. Wipe off the dirt from outside the tap.
3. Turn on the tap at maximum flow rate and let the water flow for 1-2 minutes.
4. Sterilize it for a minute with flame using a gas burner, lighter, or ignited cotton wool soaked in spirit.
5. Open the tap and allow water to flow at the medium rate for 1-2 minutes.
6. Open the container for collecting the sample and fill the water by holding the bottle under the water jet. Leave small airspace to facilitate shaking at the time of inoculation prior to analysis.
7. Stopper the cap and label the container.

Sampling from reservoir

1. Open the bottle under sterilized conditions.
2. Fill it by holding it by the lower part, submerging it to a depth of about 20 cm, with the mouth facing slightly upwards. If there is a current, the bottle should face the current.
3. Stopper the bottle and label it.

Sampling from a dug well for water microbiology

1.  Attach a stone of suitable size to the sampling bottle with a piece of string.
2. Tie a 20-meter length of clean string on the bottle and to a stick.
3.  Open the bottle as described above and lower it into the well.
4. Immerse the bottle completely in water without touching the sides of the well and lower it down to the bottom of the well.
5. Pull it out when the bottle is filled.
6. Discard a little water to provide airspace.
7. Stopper and label the bottle.
8. 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.
9. It Should be positively examined within 6 hours of collection.
10. Note: While sampling chlorinated water 0.5 ml of sodium thiosulphate solution (18 gm/L) should be added to sampling bottles to neutralize the residual chlorine present in water.

Principle of Water Microbiology

The following tests use for bacteriological analysis of water-

Plate count

Detection of coliform bacteria and E. coli –

a. Presumptive coliform count: Multiple tube technique

Differential coliform test- Called Eijkman test usually employed to find out whether the coliform bacilli detected in the presumptive test are E. coli or not

Membrane filtration method

Detection of fecal streptococci :

Examination of Cl. perfringens , and

Test for pathogenic bacteria

Methods of analysis for water microbiology

Presumptive coliform test: By using 2 methods

multiple tube method and

membrane filter method

Multiple-tube method

In this method, different amounts of water to be tested are added to tubes containing a suitable culture medium. Bacteria present in the water reproduce and produce acid with or without gas. From the number of tubes inoculated and the number with a positive reaction, the most probable number (MPN) of bacteria present in the original water sample can determine statistically. It is applicable to all kinds of water: It can use with clear, colored, or turbid water.

MacConkey broth

Lauryl tryptose broth

Minerals modified glutamate medium containing lactose and bromocresol purple

Methods of analysis

Set up the following volumes of different strengths of MacConkey broth in tubes/bottles each having an inverted Durham tube to detect the presence of gas and add a specified volume of water as mentioned:

1. One 50 ml of water to 50 ml of double strength medium. 5, 10 ml quantities each to 10 ml double strength medium.
2. 5, 1 ml quantities each to 5 ml single strength medium.
3. Incubate the tubes/bottles at 37.0°C for 18-24 hours.
4. Observe the change in color and appearance of gas in Durham tubes in bottles.
5. Media receiving one or more of the indicator bacteria show growth and a color change which is absent in those receiving an inoculum of water without indicator bacteria. The presence of both acid and gas indicates a positive reaction whereas the absence of either or both these features denotes a negative reaction.
6. Presumptive positives are read and the remaining negative bottles are re-incubated for another 24 hours. The probable number of coliforms is read from the probability tables of McCrady.
7. From the number and distribution of positive and negative reactions, the count of the most probable number (MPN) of indicator organisms in the sample may be estimated by reference to statistical tables.
8. The test gives presumptive coliform count as the reaction observed may occasionally be due to the presence of some organisms other than coliforms.

Membrane filter method

A measured volume of water is filtered through a membrane specially made of the cellulose ester. Bacteria returned to the surface of the membrane. Inoculated (face upwards) membrane in a suitable medium and incubate for 15-24 hrs. Colonies to be counted on the membrane will be numbers between 20 and 200.

Other  tests used in water analysis

Testing chlorine

Measuring the pH of the water

Testing for turbidity

Note-Viruses in water are destroyed by chlorination. When the concentration of free residual chlorine is at least 0.5 mg per liter, for a minimum contact period of 30 minutes at pH below 8 and a turbidity of 1 nephelometric turbidity unit or less, protozoa such as Entamoeba histolytica , Giardia species, and Balantidium coli may be present in the drinking water. Coliforms are not a reliable indicator of protozoal contamination.

Water treatment

Useful methods for water treatment are as follows.

water treatment plant surface water supply

Purification technology applying reverse osmosis, ultra-filtration and UV radiation

References

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996186/
2. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/water-microbiology
3. https://science.jrank.org/pages/7311/Water-Microbiology.html
4. https://www.frontiersin.org/articles/10.3389/fmicb.2016.00045/full
5. https://www.hindawi.com/journals/jeph/2018/2139867/
6. https://www.researchgate.net/publication/27469016_Water_Microbiology
7. https://www.rapidmicrobiology.com/test-method/theory-and-practice-of-microbiological-water-testing
8. https://www.iso.org/ics/07.100.20/x/
9. https://cfpub.epa.gov/si/si_public_record_Report.cfm?Lab=NRMRL&dirEntryID=100233
10. https://www.mdpi.com/1660-4601/7/10/3657