Water Purification and Sanitary Analysis of Water Sample


  • WHO and UNICEF states that 1 billion population worldwide do not have access to safe, potable water, and 40% of the population lack sanitation procedures.
  • Water purification is needed to remove the pathogens present in water, which can cause water-borne illness.
  • Few bacterial and protozoan pathogens can thrive in water and cause infection in humans.
  • For e.g., Vibrio vulnificus, V. cholera, V. parahaemolyticus, and Legionella are common pathogens usually found in water bodies.
  • Transmission of pathogens is carried out by when water containing pathogen is directly consumed or by consumption of raw seafood.

Steps for water purification:

  • The steps involved in the purification of water depend on the type of impurities present in the water source.
  • Generally, three to four steps are carried out for municipal water supplies:
    • For Suspended Particle removal
      • Raw untreated water contains lots of impurities, a large particle such as sand is removed by sedimentation procedure, and it is carried out in a sedimentation basin.
    • Partially treated water is further treated with chemicals like alum and lime and moved to settling basin.
    • Impurities such as toxic contaminants, organic matter, and microorganisms are removed by coagulation or flocculation. It is basically the chemical precipitation of the impurities.
    • Further, water is passed through rapid sand filters, which trap flocs and fine particles, removes 99% of bacteria present in the water.
    • Disinfection of treated water is carried out, by the process called chlorination, recently replaced by the ozonation process because carcinogens compounds such as trihalomethanes are produced when chlorine reacts with organic matters.
      • This process of purification removes pathogenic bacteria and coliforms.

Detailed steps of water purification

Detailed steps of water purification


  • The use of disinfection, rapid filtration is not capable to remove the cysts of the protists, such as Giardia intestinalis, Cryptosporidium oocysts, Cyclospora, and viruses.
  • Giardia is a common water-borne pathogen that causes diarrhea in humans, it’s removal can be achieved by slow sand filters,
    • This technique includes a bed of sand, through which water is slowly passed, and the microbial layer is covered by the surface of each sand grain.
  • Viruses can be inactivated or eliminated, by 90% further removal is carried out by chemical oxidants, photooxidation, and high pH.

Sanitary Analysis of Waters Samples

  • The disease-causing microorganisms and indicator microorganisms can be detected and monitored by sanitary analysis of samples.
  • The pathogens can be detected directly from the environment or can be obtained from human feces that can be used as indicator organisms,
    • Serves as an index of water contamination by pathogens.
  • The following criteria are needed to be an ideal indicator organism are:
    • Indicator bacteria should be suitable for analysis of all kinds of water samples such as tap, river, groundwater, etc.
    • They should be available where enteric bacteria are present.
    • They should live longer than enteric pathogenic microorganisms.
    • They should not multiply in the contaminated water which can cause inflated value.
    • The assay procedure should be specific and sensitive and capable of detecting low levels of indicator organisms
    • Non- pathogenic in nature
    • The level of indicator bacteria in contaminated water should be directly related to fecal pollution.
  • Coliforms, such as E.Coli belong to the family Enterobacteriaceae accounts for 10% of microorganisms present in the intestines and are commonly used as indicator microorganisms.
  • When enteric indicator bacteria are not detected in a water sample of volume 100ml, the sample is considered potable or suitable for drinking purposes.

Different Test methods are used for the analysis of water samples which are as  follows:

  1. Most Probable Number (MPN) Test

  • This test method is used to detect a group of coliforms such as E. Coli, Enterobacter aerogenes, and Klebsiella pneumonia.
  • Coliforms are facultative anaerobes, gram-negative, non-sporing, rod-shaped bacteria, which can ferment lactose with the formation of gas in 48 hrs at 35°C.
  • The MPN Test includes three consecutive tests, presumptive, confirmed and completed, all process takes 4 days for incubations and transfers.
Presumptive Test
MPN Test- Presumptive Test
  • In Presumptive Test, different volumes of water are inoculated in lactose broth tubes and checked for positive gas production.
MPN Test- Confirmed and Completed
MPN Test- Confirmed and Completed Tests

2. Colilert Defined substrate Test

  • To test coliforms and E.Coli effectively, a defined substrate test is used.
  • In a specialized medium containing nutrients such as o-nitrophenyl-D-galactopyranoside (ONPG) and 4-methylumbelliferyl-D-glucuronide (MUG).
  • For positive results of coliforms, the yellow color is observed within 24 hrs at 35°C, due to hydrolysis of the substrate.
  • A modified MUG i.e., fluorescent substrate, can be easily detected by its fluorescence under UV light.
  • If the test is negative, a water sample can be used for drinking purposes.

Defined substrate test

Colilert Defined substrate Test a.] Uninoculated Sample B.] Positive result for coliforms – as the substrate is turned into yellow color C.] Fluorescence reaction indicating the presence of fecal coliforms3. Presence -Absent Test [P-A Test]

  • Simplified test for detection of coliforms
  • It’s a modification of the MNP Test, in which 100 ml is inoculated in three different types of broths- lactose broth, lauryl tryptose broth, and bromocresol purple indicator.
  • A positive result shows the production of acid which turns the broth into yellow color.

4. Membrane Filtration technique

  • Most common a preferred technique for evaluating the presence of the microorganism in water samples.
  • A membrane filter is used to trap bacteria present in the water sample when it is passed through it.
  • The filter, with trapped bacteria, is transferred on the solid surface medium or in a liquid medium with an absorptive pad.
  • The rapid detection of coliforms present in the sample can be detected by using the proper medium, different coliforms can be differentiated by characteristic colonies observed on the medium.
  • Resuscitation is needed with chlorinated samples, where microorganisms are stressed, therefore the sample is placed for 2 hrs incubation on a pad soaked with lauryl sulfate broth, to provide them a less stressful environment, prior to their growth under selective conditions.

Advantages of Membrane Filtration Technique:

  • Better reproducibility.
  • Easy and Time-saving test.
  • Cheaper at cost than MPN test.
  • Filters can be transferred between different media.
  • Large volumes can be processed.

Disadvantages of Membrane Filtration Technique:

  • Volume samples are limited with the high turbid water sample.
  • Overgrowth cause by background bacteria which are present in the high number
  • Metals and Phenols can adsorb to the membrane filters which causes growth inhibition.

Reference and Sources

  • https://www.scribd.com/presentation/328380188/Microbial-Analysis-of-Water
  • https://www.fda.gov/food/laboratory-methods-food/bam-chapter-4-enumeration-escherichia-coli-andcoliform-bacteria
  • https://ivypanda.com/essays/water-purification-process/
  • https://www.nap.edu/read/11010/chapter/6
  • https://www.slideshare.net/waswawalter/microbiology-and-parasitology-in-nutrition
  • http://fire.biol.wwu.edu/brodham/biol346_S07/labman_week9b.pdf

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