Water Analysis

 

The different methods of water analysis for indicators are

1. Coliforms

1. MPN 

2. Colilert defined substrate test 

3. Differential Coliform Test

4. IMViC 

2. Fecal Streptococci  

1. Azide-dextrose/ Glucose azide broth

3. Clostridium perfringens      

1. Litmus Milk Test

4. Pathogenic bacteria 

Specific Methods, if need arises

(1) Coliforms

1. MPN 

2. Colilert defined substrate test 

3. Differential Coliform Test

4. IMViC 

 

Specific Methods for Coliforms 

1)      MPN Test (Most Probable Number) 

Most Probable Number of coliforms or MPN test is commonly used in estimating microbial population in water and other samples. MPN test for determining the presence of coliforms in water is based on the lactose fermenting ability of coliform bacteria. The test is particularly useful with samples that contain particulate material that interferes with plate count enumeration methods.

MPN is a multiple tube fermentation technique where a series of tubes containing lactose broth are inoculated with water samples and incubated at 37°C for 24-48 h. If coliforms are present, lactose is fermented with the production of gas and acid. 

This test is a statistical estimation and is divided into three parts: the presumptive, confirmed, and completed tests. 

 1)      Presumptive Test

The test is conducted in fermentation tubes filled with MacConkey/ lauryl tryptose broth/lactose broth (of known concentration-double and single strength), which contain inverted Durham tubes for the detection of fermentation gas. The lauryl tryptose broth is selective for gram-negative bacteria due to the presence of lauryl sulfate which inhibits gram-positive bacteria. The selective action of lactose occurs because many bacteria cannot ferment this sugar, whereas coliform bacteria and several other bacterial types can ferment it.

 In the presumptive test, dilutions from the water sample-10 ml. 1 ml and 0.1 ml water samples are added to 10 ml, 9 ml and 9.9 ml lactose broth tubes, respectively. There will be three groups of three or five tubes. After 24 to 48 hours of incubation at 37°C, check for gas production in inverted Durham tubes. Coliforms if present,gas is visible as gas bubbles in Durham’s tube . 40-390 million/ml coliforms required to produce visible gas.

The number of Presumptive positive tubes counted and expressed after consulting McCrady’s probability tables.

 A presumptive test is called so, since other than coliforms, some organisms found in water can ferment lactose rapidly with production of gas either singly, or by synergistic (gram positive and gram negative growing together) action. 

Thus fermentation of lactose with the production of gas provides presumptive evidence of the presence of coliforms. It must be confirmed, in the next stages. 

2) Confirmed test 

This test confirms the presence of coliform bacteria when either a positive or doubtful presumptive test is obtained.

 1. A loopful of growth from a positive presumptive tube is transferred into a tube of brilliant green lactose bile (BGLB) broth  containing inverted Durham’s tube and incubated at 37°C for 24-48 hours. Brilliant green inhibits gram positive bacteria and so inhibits any synergistic reactions, if present. Gas formation in the Durham tube is a confirmed test for coliforms.

2. A plate of EMB agar is streaked with a loopful of growth from a positive tube, and incubated at 37°C for 24 hours. Eosin methylene blue agar media contain two dyes Eosin and methylene blue incorporated in a lactose agar base. If coliforms are present in water sample, lactose is fermented and typical colonies appear. Typical coliform bacteria (E. coli and Enterobacter aerogenes) exhibit good growth on this medium and form characteristiuc colonies.

Three types of colonies

1. Typical E. coli colonies – small, nucleated, dark centered E. coli with or without metallic sheen, a sheen is due to the precipitation of eosin in the medium, a result of the very high amount of acid produced from fermentation.

2. Enterobacter colonies – opaque, non-nucleated, large, mucoid, pink Enterobacter colonies.

3. Any other colonies - Salmonella typhi exhibits good growth but the colonies are colorless.

     Sample from positive presumptive tube can also be inoculated onto Endo agar and incubated at 37^oC for 24 h. Endo agar has the dye Basic fuchsin with sodium sulfite incorporated in a lactose agar base. Coliform bacteria (E. coli and Enterobacter aerogenes) exhibit good growth on this medium and form deep red color metallic sheened colonies. 

 

                                                                    E.coli on Endo agar

                                                                    E.coli on EMB agar

3) Completed test

     This test helps to further confirm  positive confirmed test results. A typical coliform colony (especially, E.coli colony, if present)  from an EMB agar plate is inoculated into a tube of brilliant green bile broth and on the surface of a nutrient agar slant. They are then incubated at 37°C for 24 hours. After 24 hours, the broth is checked for the production of gas. A Gram stain is made from organisms on the nutrient agar slant.

If the organism is a Gram-negative, rod and produces gas in the lactose tube, then it is positive that (fecal) coliforms are present in the water sample.

 

Advantages of MPN

 Easy to interpret- observation of gas bubbles only is needed

 Sample toxins are diluted – since it is a dilution method

 Effective method of analyzing highly turbid samples such as sediments, sludge, mud, etc.

 Less costly and do not require sophisticated labor

 Limitations

 Statistical- high chance of errors

 Laborious  in terms of materials, glassware and incubator space.

 Requires 3-5 days for complete results

  

2. Colilert defined substrate test 

            A newer and more convenient method of detecting coliforms, specifically the fecal coliform E. coli. Here water sample is inoculated into a media containing the two substrates (ortho-Nitrophenyl-ß-galactoside) (ONPG) and 4-methylumbelliferyl-glucuronide (MUG). Coliforms produce the enzyme β galactosidase, which breaks down ONPG to ortho nitrophenol (ONP) and forms a yellow color. Yellow colour  indicates the presence of coliforms  in the sample. 

    E. coli, fecal coliform has the enzyme β-glucuronidase, which braks down MUG to form umbelliferone, which give a blue fluorescent appearance under UV light. 

    So, presence of fecal coliforms is indicated by a yellow color and fluorescent appearance under UV light.

  3. Differential Coliform Test/Eijkman test 

A test used for the identification of coliform bacteria from warm-blooded animals ie., fecal coliforms. Water sample is inoculated into MacConkey medium with inverted Durham’s tube pre-warmed to 37^oC. Incubated at 44^oC for 24 h, in thermostatically controlled water bath, which do not deviate more than +/- 0.5^oC. Gas production detected in Durham’s tube kept inside the broth. E. coli confirmation can be further done by IMViC.

4. IMViC 

A series of tests with the short code IMViC used to differentiate fecal (E. coli) and non-fecal (Enterobacter) coliforms. "I" is for indole; "M" is for methyl red; "V" is for Voges-Proskauer, and "iC" is for citrate. 

Fecal and nonfecal coliforms are differentiated as

                       I  M V iC

     E. coli       +   +    -   -

Enterobacter    -   -   +   +

 I- Indole production  

M-Methyl Red Test 

V-Voges-Proskauer test 

Ci-Citrate Utilization Test 

Indole production The ability to break down tryptophan to indole using tryptophanase enzyme is tetsed.  Addition of Kovac's reagent produces a pink/red layer forming on top of the liquid which is the positive result. So water sample is inoculated to glucose peptone broth and tseted for indoloe production by adding Kovac's reagent 

Methyl Red Test Water sample inoculated into MRVP broth after 24 h incubation at 37^oC. When Methyl Red indicator is added, a red color appears if pH is lower than 4.2 which indicates positive test. 

Voges-Proskauer test Water sample inoculated into MRVP broth; incubated at 37^oC. On addition of α-naphthol and KOH, a pink-burgundy color indicates the presence of acetyl methyl carbinol as a result of lactose fermentation. E. coli does not produce acetyl methyl carbinol, but Enterobacter  does

Citrate Test This test determine the ability of a microorganism to use citrate as its sole carbon source. Simmon's citrate agar is inoculated with the water sample. Green citrate agar turns blue if positive. E. coli is unable to use citrate as its sole carbon source and hence is negative for this test. In short

                       I    M V iC

     E. coli       +   +    -   -

Enterobacter    -   -   +   +

 (II) Specific test for Fecal Streptococci

Water sample inoculated to Azide-dextrose/ Glucose azide broth.  Incubation at 45⁰C and checked for production of acid within 18 h. Confirmation by plating onto MacConkey medium.

(III) Specific test for Clostridium perfringens

Inoculate freshly heated tubes of Litmus Milk with water after preheating the water at 80°C for 10-15 minutes to destroy non-spore formers. Incubated at 35°C for up to 5 days under strict anaerobic conditions; check for stormy fermentation.

Litmus milk - a complex medium with lactose (milk sugar), casein (milk protein) and  litmus (a pH indicator -purple to blue at neutral to alkaline pH and pink at acidic pH). If lactose is fermented, the solution turns pink. If gas is produced during fermentation, bubble or cracks in the milky medium. Intense gas production can lead to stormy appearance in the medium.  

(IV) Pathogenic organisms

Bacteria like Typhoid bacilli, Cholera Vibrio, Viruses such as- Enterovirus & other cytopathogenic viruses.  Protozoa such as E. histolytica, Giardia, Balantidium etc can be suspected. Specific media and tests should be performed as and when required.