Coliforms and E. coli or thermotolerant bacteria are a group of gram negative, oxidase negative,bacilli shaped non spore forming bacteria mainly from enterobacteriaceae family. They commonly serve as indicator microorganism of faecal contamination of water.Presence of coliform bacteria in water usually indicates the pathogenic contamination of which may cause severe adverse health effects and sometimes chronic community acquired infection.

In RO water, UV filtrate water used for consume & other purpose (household,office premises,factory,hospitals & other public places) we observed most water sample may chemically satisfactory but most of the time it shows bacterial contamination by perform water testing in our lab.

Drinking water can be contaminated with bacteria even after RO-UV filtration,if the membrane filtration unit is not properly cleaned or maintained, failing of UV efficacy or it is not serviced regularly.Lacking of knowledge of servicing or cleaning of Water purifier Agency may also indirectly involved in water bacterial contamination.

At first RO membrane cleaning should start from back washed followed by washing with 10% dilute Hydrochloric acid.Next after washing repeatedly(3-4 times),it is then leached 1% Sodium hexametaphosphate(SHMP) solution for 1 hour.Then after draining out the SHMP solution it is then washed with enough of potable water & now the filter cartridge is ready further use. It should be done in every 1-3 month depends on the usage. Most of the service operators does not follow this process & don’t give proper time(1 hr) to clean the filter which ultimately supports the growth of the microorganisms.Most of the time household,office premises,factory,hospitals & other public places don’t clean or maintain the water filtration unit for which bacteria is found in water.

We detect coliform bacteria from water whether it is present or absent which is very specific and can detect very few number of bacteria from a water sample by membrane filtration technique, followed in our lab- ProResearch Testing & Laboratory.

Instrument used For Detection of Coliform and E. coli by Membrane Filtration Technique from Water:

1. Weight Balance : To weigh the media that supports the growth of bacteria.
2. pH meter: Used during media preparation.Proper pH ensure the expected microbial growth.
3. Autoclave : For sterilization of equipments at 121°C & 15 lbs pressure( it is the minimum & standard temperature which kill microorganism & its spore) and for disinfection of all equipments at same temperature & pressure after completion of testing.
4. Heating Mantle : Use to boil media to dissolve at 70°C-80°C.
5. Hot Air Oven: For drying of equipments.It is generally used as dry sterilization at 150 °C for about 2 hours.
6. BioSafety Cabinet: Use to perform the whole testing in a sterilized environment.The testing should performed inside the biosafety cabinet only otherwise contamination can be occured which ultimately leading to the inaccurate result.
7. Vacuum Pump: In membrane filtration, a vacuum pump generates a pressure differential that compels the liquid to flow through the membrane.
8. Bunsen Burner or Spirit Lamp: Generate heat which is use to sterilize forcep for avoid contamination.
9. Bacteriological Incubator(30°C±2°C or 37°C±2°C): Incubators are equipment used in biological laboratories to grow, maintain or revive bacteria or microorganisms.Incubators have specific temperature range optimum for growth and aerobic or anaerobic conditions. Incubators are also available with shelves & fan with specific distance to ensure uniformity of temperature throughout the chamber.

Chemical,Equipment & Glassware used For Detection of Coliform and E. coli of Water:

• 1. Membrane Filtration Unit: A certain volume of liquid passed through a sterile membrane filter usually with pore size of 45 micrometer, which can easily trap any microorganism larger than this size in it & can be detected if placed on a suitable agar medium & growth condition.
• 2. Sterile Petri Plates : To give microorganisms a clean, nutrient-rich environment in which to develop and expand so that specialists can examine and observe them. Growth media is pour into this plates.
• 3. Sterile Conical flask : To prepare media.Capacity of the flask will depend on the amount of media prepared.Here we use 250ml capacity for 100ml media.
• 4. Measuring Cylinder : To measure distilled water during media preparation.We use 100 ml measuring cylinder for this test.
• 5. Nitrocellulose membrane: This is a special kind of paper like material mainly composed of cellulose nitrate with pore size 45 micrometer that allows water to flow freely through it but trapping microorganism within it.
• 6. Oxidase Test Strips: Used to identify the presence of coliform & E.coli bacteria in water sample by producing color.
• 7. Sterile Forcep : To hold the membrane filter to avoid contamination.
• 8. Distilled Water : To prepare media.Distilled water should be autoclaved & contamination free.
• 9. 70% Isopropyl Alcohol or 70% IPA:Use as sanitizer to perform testing properly.Add 70 ml isopropyl alcohol in 30ml of distilled water.
• 10. Gloves,Cap & Mask : To maintain proper sterility inside the biosafety cabinet.Bare hands can contaminate the equipments which affect the result accuracy. If you don’t wear gloves & masks can affects on analyzer’s body also.

Media Used For Detection of Coliform and E. coli of Water::

Growth Media: Growth medium can be solid, liquid or semi-solid, consists of specific nutrients that selectively or non-selectively support bacterial growth under a specific temperature and growth condition.In this test we use a specific medium called Coliform Chromogenic Agar (CCA Agar), cause it selectively supports the growth of coliform bacteria including Escherichia coli. Further we can distinguish other coliform bacteria with Escherichia coli by specific colour.

Sample Storage and Preparation Required For Detection of Coliform and E. coli of Water:

Storage	Samples should be carried in temperature controlled cooled box with ice pack (to inhibit microorganism growth) and shall be analysed immediately after receiving in lab.If not, samples should be stored and refrigerated immediately at 4°C-8°C.This temperature inhibit microbiological activities which states is as close to reality.
Preparation	Bring the sample in room temperature before processing to make the micro-organisms viable and shake well (at least 10-12 times) for even distribution of micro-organisms along the matrix.

Procedure While Doing For Detection of Coliform and E. coli of Water:

Media Preparation	Weigh 3.092 gm Coliform Chromogenic Agar into the conical flask. Measure 100 ml distilled water and pour into it. Adjust the pH 6.8±0.2 at 25°C using HCL or NaOH,cause high ranges pH & temperature may affect the enzyme activity and color development,leading to inaccurate result. Heat to boiling until dissolve the media properly.If it is not dissolved the media won’t be solidified. Allow to cool around 45°C to prevent condensation and preserve sensitive components.
Preparation of sterile environment inside the Biosafety Cabinet	Turn on the UV before starting the testing process about 15-20 minutes to sterile all the equipments & surfaces. Clean the surface with 70% IPA. Analyzer should wear proper gloves,apron,mask & cap.
Pouring of media into petri plates	Aseptically(to prevent cross contamination) pour 15-20 ml media into sterile plates in biosafety cabinet & let it solidify.
Run Water sample through Membrane Filtration Unit	Sanitize hands,cap of sample container with 70% IPA to avoid cross contamination.Heat forcep in burner to make it sterile. Put a sterile nitrocellulose membrane on the MF unit. Place a funnel and lock the MF unit properly.Make sure water does not well out from the funnel. Pass desired quantity of sample (100 ml preferably) through the unit. Unlock the unit and hold the nitrocellulose membrane with a sterile forcep. Place the membrane on CCA Agar plate. Perform the test at least in duplicate set with a media control(Media without sample).
Incubation	Transfer the plates in incubator. Incubate the plates at 37°C±2 & observe the plates after 24-48 hrs.
Observation	Positive sample will show dark blue to purple(for Escherichia coli) on this medium and pink coloured colonies (for coliform bacteria). In media control plate if growth of any microorganisms are not seen, it indicates media is not contaminated & the testing is satisfied.
Confirmation Test	Take identical single colony using a sterile loop & rub on oxidase strips. Colour changes after 30 seconds or no color change will indicate positive for E.coli & coliform,i.e oxidase negative. Development of quick purple colour will indicate negative result(E.coli & coliform absent) ,i.e oxidase positive.

Purpose of testing For Detection of Coliform and E. coli of Water: :

Water Quality	Escherichia coli is an indicator microorganism of fecal contamination. E. coli generally found in human feces. Presence of E. coli in any water means that the water has been contaminated with human fecal waste and never should consume.
Health Issue	Several pathogenic strains of Escherichia coli like E. coli O157 can cause serious health issues like diarrhoea, colitis, haemorrhagic symptoms etc. So drinking water must be check for E. coli to avoid infected.
Disease Outbreak	A regular checking of water quality prevents disease outbreaks. Any indications of presence of E. coli or coliforms in drinking water allows for quick actions like chlorination or bleaching or boiling.
Community ,household,office premises,hospitals or any public places	Presence of E. coli or coliform in water also indicates improper water treatment or leakage in sewage, agricultural washout or animal waste added in water,not maintaining the RO FIltration unit, water pipeline or reservoir is not cleaned etc.

Handling mistakes should avoid during the test of Coliform and E. coli in Water:

• All the works should be done in a sterile environment and using sterile equipment to minimise contamination risk.Maintain the temperature of workplace(where testing is performed) at 25°C±2 to prevent contamination.
• Also,during this test care should be taken during placing of sterile nitrocellulose membrane on MF unit and on the CCA agar plates to avoid any tear of the membrane.
• Sample should be mix thoroughly before analysis to allow even distribution of bacteria along the matrix.
• Avoid any deviation from the suggested sample quantity (100 ml or 250 ml). Do not pass any less or any more water than the suggested amount.
• Improper cleaning & sterilisation of glasswares leads to false results.Washing procedure described.
• Media control must be done to check the viability, productivity and specificity of the media prior testing.

Conclusion of Detection of Coliform and E. coli of Water:

The detection of coliform and E. coli are an important test to determine the quality of water and its eligibility to consume.This presence of any of these organisms indicates a contamination with sewage and consumption of which may cause severe adverse health effects.Absence of bacteria indicated that the water is safe to consume and requires no immediate corrective actions.The test is vital for protecting public health and maintaining water quality standards.By using the method mentioned above, you can easily perform the E. coli & Coliform detection test in any lab or factory or in other places that has the right equipments and chemicals.This testing process gives you accurate results, which are really important for quality control in many areas. Plus, if you find the procedure confusing, you can look at the real-time photo included with this text, which shows you each step and makes it easier to understand.Also, if you want professional help or need more advanced testing, you can contact Pro Research & Testing Laboratory for your testing needs.

FAQ For Detection of Coliform and E. coli of Water:

Q .What is E. coli?

A: E. coli or Escherichia coli are pathogenic gram negative aerobic bacteria, a member of coliform group that normally present in human faeces. But when consume and reach gastrointestinal tract may cause severe adverse health effects like diarrhoea.

Q. Why is E. coli detection important?

A: Presence of E. coli indicates fecal and probably sewage contamination in water and other pathogenic microorganism can also be present in water and require immediate actions.

Q. How does E. coli get into water?

A: Sewage contamination, Agricultural washout, Animal waste, Contaminated distribution system.

Q. What is the acceptable limit of E. coli in drinking water?

A: E.coli & Coliform should be absent/100 ml.

Q. What should I do if I found E. coli in water?

A: Boil the water, clean your filter and disinfect the water system (tank, pipeline, reservoir etc). Membrane filtration unit in RO should be cleaned properly or maintain the efficacy of UV.To confirm that your water is free from bacteria contact the laboratory & test your water.

Q. How does E. coli contaminated water looks like?

A: Absolutely transparent and clean. Just like filtered water or commercially available drinking water which can’t detect in normal condition without tested your water.

Q. Which water shall test for E. coli and coliform?

A: Any water purposed for drinking or to be used in cooking or other consume purpose or for bathing shall test for E. coli and coliform. According to us every household water, water from deep tube well, borewell, tap water, reservoir water, RO water,Package water (20ltr jar),water from other sources for drinking & other purpose shall tested for E. coli and coliform.

Q.Why sterilization is important during the test?

A: To prevent cross contamination & get accuracy of the result.

Q. Why should I choose Membrane Filtration Technique?

A: There are multiple alternative methods of membrane filtration like Multiple Tube Dilution Method, but we believe, the Membrane Filtration Technique is more specific and can detect even one bacteria present in a sample.

Q. Is it important to do confirmation test?

A: Yes, to get proper result it is important to perform the confirmation test.

How We Verified This Testing/Research Procedure :

This testing procedure is done under qualified analyst .Continually monitored by expertise.Repeatedly testing is always done to get accurate result.

Written by
Anwesha Das (M.Sc Microbiology,BU)
Designation – Microbiologist

Reviewed by
Sramana Ghosh (M.Sc. Microbiology, MAKAUT)
Designation – Quality Manager

Verified By
Tathagata Talukdar (M.Sc,Microbiology) University of Calcutta
Designation – Senior/Chief Microbiologist
Experience – 12 Years + of experience including medical microbiology (NABL 15189) and general microbiology (NABL 17025)