In What Ways Is The Membrane Filter Method For Coliform

Bacteriological assay of water is one component of drinking water quality analysis. H2o is screened for the presence of fecal contamination by testing for the presence of an indicator microorganism.

Indicator microorganisms are ones that have the post-obit properties:

The microorganism is not establish in water and will exist present in the water simply when a contamination event has occurred; and
The density of the microorganisms present should be proportional to the degree of contamination.

E.coli which is a normal flora nowadays in the intestine of humans and animals meets the above requirements and then detection of East.coli and/or related bacteria termed "coliforms" is done using many different techniques as a role of bacteriological analysis of h2o. I of the techniques is membrane filtration method.

Membrane filtration for determining the coliform count (Paradigm source: https://world wide web.sciencedirect.com )

Membrane filtration method

In this method, water from unlike samples are passed through a membrane filter with a pore size of 0.45 µm and the membrane is incubated on an agar plate. Bacterial cells trapped on the membrane will grow into colonies that can exist counted, and a bacterial density of the water samples can be calculated. Depending on the source of the water dissimilar filtration volumes of water samples are used.

Table 1: Typical sample volumes for membrane filtration analysis

Sample blazon	100 ml	x ml	1 ml	0.1 ml	0.01 ml	0.001 ml
Treated drinking water	X
Partially treated drinking water	X	10
Protected source water		X	X
Recreational water			X	X
Surface water			X	X
Wastewater			10	Ten	X
Discharge from sewage handling plant			X	X	X
Ponds, rivers, storm h2o runoff				X	Ten	10
Raw sewage				10	X	X

Notation:

Pocket-sized volumes should be added to the filtration apparatus together with a minimum of 9 ml of sterile diluent to ensure adequate dispersal across the surface of the filter membrane
1.0, 0.1, 0.01 and 0.001-ml volumes are filtered later first preparing series dilutions of the sample.

Since large volumes of the sample tin can be filtered, the membrane filtration technique tin observe the presence of a very low number of bacteria. However, the turbidity of the h2o may limit the volume of samples that is practical to filter. High numbers of groundwork bacteria or toxic substances if present may interfere with the test and outcome in underestimation of the density of coliforms.

Full coliforms and fecal coliforms:
- Detection of total coliforms and fecal coliforms:
  - Detection of full coliforms using mEndo agar LES
  - Detection of fecal coliforms using mFC agar
- Materials required
  - Apparatus
  - Consumables
- Water sample collection Precautions
- Procedure of membrane filtration technique
  - References and further reading

Total coliforms and fecal coliforms:

They are the members of the family Enterobacteriaceae. Coliforms may include bacteria of the following genera: Escherichia, Enterobacter, Klebsiella, Citrobacter, and Serratia. Fecal coliforms are the subset of total coliforms which are found inside the digestive tract and shed through feces. Fecal coliforms can ferment lactose with the production of acrid and gas at 44.5°C within 24 hours. They are also said to be thermotolerant coliforms equally they tin can grow at a higher temperature. Bacteria belonging to the genera Escherichia and Enterobacter are considered fecal coliforms.

Detection of full coliforms and fecal coliforms:

Total coliforms and fecal coliforms are detected using selective and differential civilization medium. For the detection of total coliforms mEndo agar LES is used and for fecal coliforms mFC agar is used. On mEndo agar, coliforms will form cerise colonies with a metallic sheen and on mFC agar, fecal coliforms will form dark blue colonies.

Detection of total coliforms using mEndo agar LES

Coliform colonies in mEndo agar (note green metallic sheen)

This medium contains lactose and a pH indicator (that changes color when acid is produced due to fermentation of lactose). Membrane filter is incubated at 35°C for 22-24 hours for the detection of full coliforms.

Coliforms typically will produce metal (aureate) sheen, which is due to the extensive production of aldehydes and acid from the fermentation of lactose.

Atypical total coliforms colonies appear as night red, mucoid or have a dark center only without a metallic sheen. E.coli will form colonies with a metal sheen.

Detection of fecal coliforms using mFC agar

Fecal coliform colonies in mFC agar plates

This medium contains bile salts which inhibit other bacteria except enteric. It also contains rosolic acid which inhibits leaner other than fecal coliforms. Aniline blue is used as a pH indicator which gives blue color in acidic pH.

Membrane filter is incubated at 44.5°C for 22-26 hours in mFC agar for the detection of fecal coliforms.

Fecal coliforms grade bluish colonies in this medium (acrid is produced due to the fermentation of lactose).E.coli will form flat night blue colonies.

Materials required

Apparatus

Incubator(s) or water-bathroom(s)
Membrane filtration apparatus, complete with vacuum source (electrically operated pump, paw-pump or aspirator) and suction flask.
Autoclave for sterilising prepared culture media.
Humid-pan or bathroom (if filtration apparatus is to be disinfected in boiling h2o betwixt uses).
Laboratory balance
Racks for bottles of prepared civilisation media and dilution water. These must fit into the autoclave.
Distilling appliance with storage capacity for at least 5 litres of distilled h2o.
Fridge for storage of prepared culture media.
Hot-air steriliser for sterilising pipettes and glass or metallic Petri dishes.
Thermometer for checking scale of incubator or water-bath.
Pipette cans for sterilising pipettes.
Boxes for Petri dishes for use in hot-air steriliser.
Reusable bottles for civilisation media.
Measuring cylinders, chapters 100 ml and 250 ml.
Reusable pipettes, glass, capacity 1 ml and 10 ml.
Bottles to contain nine-ml volumes of buffered dilution water.
Flasks for grooming of culture media.
Wash-canteen.
Blunt-edged forceps.
Pipette bulbs.
Spatula.
Container for used pipettes.
Brushes for cleaning glassware (several sizes).
Fire extinguisher and first-aid kit.
Miscellaneous tools.
Waste bin.

Consumables

Methanol for disinfecting filtration apparatus using formaldehyde gas (unnecessary in the laboratory, but essential if analyses are done in the field). It is essential to utilize methanol. Ethanol or methylated spirits cannot be substituted.
Membrane filters, 0.45 µm pore size and of diameter appropriate for the filtration apparatus being used and complete with absorptive pads.
Disinfectant for cleaning laboratory surfaces and a container for discarded pipettes.
Civilisation media (mEndo Agar LES and mFC Agar).
Phosphate-buffered dilution water.
Petri dishes, drinking glass or aluminium (reusable) or plastic (dispensable).
Polyethylene bags for wrapping Petri dishes if dry out incubator is used.
Magnifying lens (every bit an assistance to counting colonies after filters are incubated).
Wax pencils for labelling Petri dishes.
Autoclave tape.
Detergent for cleaning glassware and equipment.
Positive control sample (100 ml of h2o with a v ml of a 1:100 dilution of an overnight culture of East.coli)
Negative command sample (sterile water)
Water samples to be tested: Volume of water needed for testing.

Water sample collection Precautions

H2o sample must be collected in a sterile canteen
H2o sample must exist representative of the supply from which it is taken.
Contamination of the sample must be avoided during and later on sampling.
The sample should be tested every bit promptly equally possible after collection.
If at that place is a delay in the examination of the sample, it should be stored at a temperature betwixt 0 and 10°C.

Procedure of membrane filtration technique

Sterilise the tips of the blunt-ended forceps in a flame and allow them to absurd.
Carefully remove a sterile membrane filter from its package, belongings it only by its edge.
Place the membrane filter in the filter appliance, and clamp it in place. (If the apparatus has been disinfected by boiling, ensure that it has cooled down before inserting the membrane filter.)
Mix the sample by inverting its container several times. Pour or pipette the desired volume of sample into the filter funnel. This volume should normally exist called in the light of previous feel, but suggested volumes are given in Table 1. If the volume to exist filtered is less than x ml, information technology should be made upwardly to at least 10 ml with sterile diluent so that the sample will be distributed evenly across the filter during filtration.
Apply a vacuum to the suction flask and draw the sample through the filter; disconnect the vacuum.
Dismantle the filtration apparatus and remove the membrane filter using the sterile forceps, taking care to touch on but the edge of the filter.
Remove the lid of a previously prepared mENDO agar LES plate and identify the membrane, grid side uppermost, onto the agar. Lower the membrane, starting at i edge in order to avert trapping air bubbling between membrane and agar. Mark the petri dish with the sample number or other identification. The sample book should as well be recorded. Apply a wax pencil or waterproof pen when writing on Petri dishes.
Repeat procedure (1 to 8) with the aforementioned volume, but place membranes on mFC plates.
Incubate mEndo agar LES plates at 35 ± 0.v°C for 22 – 24 hours and mFC plates at 44.5 ± 0.ii°C for 22 to 26 hours, all chapeau side down. In gild to maintain the temperature within such a narrow range, a water bath is typically used for incubation of the mFC agar plates. These plates are placed in watertight plastic numberless and then submerged in the h2o bath.

24-hour interval 2 (24 hours after):

Later on 22 – 24 hours, remove the mEndo agar LES plates from the 35°C incubator and count the colonies that are nighttime red, mucoid, have a dark eye or (more typically) produce a metal sheen. These are considered to be full coliform colonies.
From the mEndo agar LES plates, choose 2 full coliform colonies that are isolated on the membrane and confirm that they are Gram-negative rods and not-spore formers.
After 22 to 26 hours, remove the mFC agar plates from the 44.five°C incubator and count the colonies that accept any blue color. These are considered to be fecal coliform colonies.
From the mFC agar plates, choose 2 fecal coliform colonies that are isolated on the membrane. Ostend that they are Gram-negative rods and non-spore formers.
Calculate the total and fecal coliform CFU per 100 ml for each sample every bit described below:
The original density is estimated from the book of sample filtered (or the volume of dilution and the dilution gene), and the number of colonies counted on the membrane.
As counts are reported per 100 ml of sample (not per ml), the per ml values must exist multiplied past a factor of 100.

No. of CFU per 100 ml = [(No. of colonies on the membrane)/(volume filtered)] × 100

If the sample is diluted and the volume of the dilution was filtered, the denominator will be (volume (ml) filtered x dilution)

References and further reading

Water Quality Monitoring – A Practical Guide to the Design and Implementation of Freshwater Quality Studies and Monitoring Programmes (WHO)
Brian Forster, Catalina Arango Pinedo. 2015. Bacteriological examination of waters: membrane filtration protocol. American Society for Microbiology (ASM)
Manual for Bacteriological Analysis of Natural H2o Supply Sources in Disaster Situations. Pan American Health System (PAHO)
Pelczar MJ, Chan ECS, Krieg NR (2007). Microbiology. 5th edn. Tata McGraw-Loma.

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Section of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.