MICROBIOLOGICAL ANALYTICAL
Introduction

Different types of microorganisms can be found in natural waters: viruses, bacteria, cysts, spores, and worm eggs. Some of these organisms can cause disease in animals and or humans, and so it is important to test the quality of drinking water to ensure that it is safe to drink. Different techniques in different places have been applied in our research to test for microbial contamination.

We use relatively simple, yet established, laboratory methods to test for indicator organisms. The presence of indicator organisms, such as E.coli, indicate the presence of waterborne pathogens. Indicator organisms are measured rather than specific pathogens since there are far too many pathogens to test for in a given water sample. The 2 types of microbiological tests which are frequenly used by students in the field are:

  • Presence/Absence (P/A) Test
  • Membrane Filtration (MF) Test
And the indicator organisms that tested for with these methods are:
  • Total coliform
  • Fecal colifom
  • E.coli

P/A tests are easier to implement than membrane filtration tests; however, membrane filtration tests allow the technician to enumerate the number of bacteria colonies per sample volume rather than simply testing for the presence/absence of indicator organisms. Both tests require minimum equipment and are well-suited for use in field conditions.
Presence/Absence Test

The P/A test tests for the presence/absence of indicator organisms in a water sample. This is usually observed in the form of a color change after an incubation period. Two common P/A tests are:

  • H2S-producing bacteria P/A test
  • Total Coliform and E.coli P/A Test

H2S producing bacteria P/A Test

As described in the work of Manja et al.1, the screening for H2S producing bacteria is a good indication of fecal contamination in the sample. The test is relatively simple to carry out and requires only 24-48 hours of incubation at 25°C. The sensitivity of the test is, however, still debatable. Some research shows that positive results will occur with bacteria concentrations as low as 1 CFU/100ml2, while others show 5 CFU/100ml3 or more. MIT students have used the pre-packaged H2S PathoScreen Medium by HACH for P/A testing. The following lists the equipment and procedures for carrying out the P/A test:

Instruments/reagents

  • 20ml glass sampling bottle
  • candle
  • lighter
  • alcohol
  • nail-clipper
  • HACH PathoScreen Medium for 20ml
Procedures

  • Sterilize sampling bottle in air oven at 170°C for 1 hour or autoclave at 15psi and allowed to cool.
  • Pour approx. 20ml sample into sampling bottle and allow sample to stand for 15 minutes.
  • Wipe medium satchet with rubbing alcohol and flame cutting edge (scissors or nail clipper) for sterilization.
  • Cut medium sachet and pour into sample bottle. Cap and mix.
  • Place sample at about 25°C (or room temperature).
  • Take H2S Bacteria P/A reading at 24 and 48 hours (Black = Positive, Yellow = Negative).
  1. Manja, M.; Maurya, S.; Rao, K. M. (1982) Simple field test for the detection of faecal pollution in drinking water. Bulletin WHO 60:797-801.
  2. Pillai, J.; Mathew, R. G.; Ho, G. E. (1999) H2S paper strip method – a bacteriological test for faecal coliforms in drinking water at various temperatures. Wat. Sci. Tech. Vol. 40, No. 2, pp.403-407.
  3. Grant, M. A.; Ziel, C. A. (1996) Evaluation of a simple screening test for fecal pollution in water. Journal Water SRT – Aqua. Vol. 45, No.1, pp. 13-18.

Total Coliform and E.coli P/A Test

The P/A test for total coliform and E.coli is carried out using the HACH Lauryl Typtose with Bromcresol Purple (LT/BCP) Broth with MUG reagent for 100ml sample. Similar to the H2S test, this test can be carried out easily under the same conditions and results can be obtained after 24-48hrs.

Instruments/reagents

  • 100ml glass bottle
  • candle, lighter, alcohol
  • UV lamp
  • HACH Lauryl Typtose with Bromcresol Purple (LT/BCP) Broth with MUG reagent for 100ml sample
Procedures
  • Sterilize sampling bottle in air oven at 170°C for 1 hour or autoclave at 15psi and allowed to cool
  • Pour approx. 100ml sample into 100ml sampling bottle and allow sample to stand for 15 minutes.
  • Wipe broth bottle with rubbing alcohol for sterilization.
  • Break broth bottle and pour into sample bottle. Cap and mix.
  • Place sample at about 25°C (or room temperature).
  • Take Total Coliform P/A reading at 24 and 48 hours.
  • Murky Yellow = Positive, Purple = Negative.
  • Take E.Coli P/A reading with UV lamp at 24 and 48 hours.
  • Fluoresce = Positive, No fluoresce = Negative.
Membrane Filtration Test

The membrane filtration test is a fast, simple way to estimate bacterial population in water. In the initial step, an appropriate sample volume is passed through a membrane filter with a pore size small enough (0.45µm) to retain the bacteria present. The filter is then placed on an absorbent pad (in a petri dish) saturated with a culture medium that is selective for coliform growth. The petri dish is inverted and placed in an incubator for 24hrs at the appropriate temperature. After incubation, the colonies that have grown are identified and counted with a magnifying glass.

Currently, our team uses Millipore products for its membrane filtration tests. The 2 tests that are being used are:

  • Total coliform and E.coli: m-Coliblue24 broth on 0.45µm filter paper @ 35°C for 24hrs.
  • Fecal coliform: m-FC broth on 0.7µm filter paper @ 44.5°C for 24hrs.

Total Coliform and E.coli MF Test

Instruments/Reagents
  • MILLIPORE portable MF setup
  • m-Coliblue24 medium
  • OXFORD pipette
  • candle, lighter, tweezers
  • incubator.


Procedures
  • Pour m-Coliblue24 medium onto absorbent pad in petri dish. Decant extra medium and label.
  • Flush approx. 30ml of distilled water through filter once.
  • Place 0.45µm filter paper with sterile tweezers.
  • Pipette specified volume of sample into funnel. Stir.
  • Run filtration.
  • Rinse funnel with approx. 30ml of distilled water.
  • Remove filter carefully with sterile tweezers and place into petri dish.
  • Invert and place petri dish into incubator set at 35°C for 24hours.
  • Count number of colony forming units (CFU) under magnifying class and expressed as CFU/100ml.
  • Blue = E.coli, Red+blue = Total coliforms (see image at right)
Note: We typically use m-Coliblue24 broth rather than m-Endo broth, which we used to use for the enumeration of total coliform, because the colonies show up clearer thus making counting easier. The m-Endo broth requires the colonies to either show a silver metallic sheen or appear very dark red in color in order to be counted as total coliforms (see image at right). Based on experience, the results are seldom that obvious and require a lot of judgment during interpretation.

Fecal Coliform Membrane Filtration Test

A similar procedure is carried out for the MF test for fecal coliform. The differences are:

  • Use 0.7µm filter paper
  • Use m-FC broth
  • Incubate at 44.5°C
Fecal colonies will appear dark blue (see image at right).