Water Microbiology : Introduction, Bacterial Flora, Sampling, Method of Analysis, Result Interpretation and Treatment of Water

Microbiology of water

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 analysis is therefore to determine whether faecal 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 indicate that fecal pollution has taken place or not . About water borne 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 origin like cholera ,bacillary dysentery, E. coli diarrhoea, leptospirosis, typhoid and paratyphoid and similarly Parasitic origin amoebic dysenter, cryptosporidiosis, giardiasis, balantidiasis and salmonellosis. Due to virus rotavirus diarrhoea, hepatitis A and Poliomyelitis are common.

Bacterial flora in water

Without faecal contamination:

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

After faecal 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 faeces such as

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

Criteria for an ideal indicator organism

  1. It should be a member of intestinal micro flora 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 waste water 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 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 gas burner, lighter or ignited cotton wool soaked in spirit.
  5. Open the tap and allow water to flow at 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 a 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 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. Sample is examined at the earliest preferably within one hour or should be quickly transported to the laboratory keeping in 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 uses 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 faecal 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 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 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 change in colour 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. Presence of both acid and gas indicates positive reaction whereas absence of either or both these features denotes a negative reaction.
  6. Presumptive positives are read and remaining negative bottles are re-incubated for another 24 hours. The probable number of coliforms are read from the probability tables of McCrady.
  7. From the number and distribution of positive and negative reactions, 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.


water microbiology test
water microbiology test
water microbiology MPN table
water microbiology MPN table
water microbiology MPN table 2
water microbiology MPN table 2
Differential features of coliform bacilli
Differential features of coliform bacilli
water microbiology interpretation of result
water microbiology interpretation of result
water microbiology ISO guideline
water microbiology ISO guideline

Membrane filter method

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

Other  tests used in water analysis

Testing chlorine

Measuring the pH of 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 litre, for a minimum contact period of 30 minutes at pH below 8 and a turbidity of 1 nephelometeric turbidity unit or less, protozoa such as Entamoeba histolytica , Giardia species and Balantidium coli may be present in the drinking water. Coliforms are not the reliable indicator of protozoal contamination.

Chlorination of water
Chlorination of water
chlorination of water for making safe
chlorination of water for making safe

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

water microbiology treatment plant
water microbiology treatment plant
water microbiology: water treatment
water microbiology: water treatment
water microbiology: Purification technology
water microbiology: Purification technology


  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

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