---
title: "Indicator organisms and membrane tube fermentation"
subject: "Water Supply Engineering"
chapter: 4
key: "semester_5/water_supply_engineering:indicator-organisms-mtf"
source: https://www.bctengineer.com/q/semester_5/water_supply_engineering/indicator-organisms-mtf
site: BCT Engineering
---

# Indicator organisms and membrane tube fermentation

Water Supply Engineering · Chapter 4: Quality of Water

- Asked in: 2072 Kartik, 8 marks; 2069 Asar, 8 marks; 2068 Chaitra, 8 marks (asked 3 times)
- Video: [Indicator organism (fecal coliform, total coliform)](https://www.youtube.com/watch?v=DUXIWKGCKyU) — Shomu's Biology
- Answer checked by Claude Opus 5.5 (high effort) on 2026-10-14
- Page: https://www.bctengineer.com/q/semester_5/water_supply_engineering/indicator-organisms-mtf
- Whole chapter: https://www.bctengineer.com/md/semester_5/water_supply_engineering/chapter-4

## Question

What are indicator organisms? Describe in detail the (membrane tube) multiple tube fermentation technique for the determination of E-coli / coliform in the laboratory by the MPN method.

## Short answer

Indicator organisms are easily detected microbes whose presence shows faecal pollution, such as coliforms and E. coli. The MTF technique uses presumptive, confirmed, and completed tests: lactose broth with gas, BGLB/EC broth confirmation, and EMB agar with metallic sheen. MPN is read from tables; drinking water must show 0 per 100 ml.

## Answer

#### Indicator organisms
Indicator organisms are micro-organisms whose presence in water shows faecal pollution and so the possible presence of pathogens. Testing for individual pathogens is difficult, so an easily detected indicator is used. The best-known are **coliform group bacteria** and **E. coli** (faecal coliform); others are faecal streptococci and Clostridium perfringens.

An ideal indicator is always present when pathogens are present and absent in safe water, is more abundant and lives longer than pathogens, does not multiply in water, and is easy to detect.

#### Multiple tube fermentation (MPN) technique
The MTF technique estimates the **Most Probable Number** of coliform per 100 ml based on gas production from lactose.

**Stage 1: Presumptive test**
1. Prepare a series of tubes of lactose broth with inverted Durham tubes. Inoculate 5 tubes each with 10 ml, 1 ml and 0.1 ml of sample (use higher dilutions for polluted water).
2. Incubate at 35 degrees C for 24 hours; if there is no gas, incubate for another 24 hours.
3. Gas and acid in the tubes show a positive presumptive result.

**Stage 2: Confirmed test**
1. Transfer a loopful from each positive tube to brilliant green lactose bile broth, and incubate at 35 degrees C for 48 hours. Gas shows coliform.
2. For faecal coliform / E. coli, transfer to EC broth and incubate at 44.5 degrees C for 24 hours; gas shows E. coli.

**Stage 3: Completed test**
Streak on EMB agar and incubate at 35 degrees C for 24 hours. E. coli forms dark colonies with a metallic green sheen. Gram stain should show Gram-negative, non-spore forming rods, and growth in lactose broth should again produce gas.

**Calculation of MPN**
The number of tubes giving a positive result in each dilution is noted (for example 5-1-0), and the MPN per 100 ml is read from the standard MPN table (5-1-0 gives about 33 per 100 ml). For other combinations Thomas' formula is used:

$$
\text{MPN}/100\ \text{ml} = \frac{\text{positive tubes}\times100}{\sqrt{(\text{ml in negative tubes})\times(\text{ml in all tubes})}}
$$

**Interpretation:** drinking water must have 0 MPN of E. coli per 100 ml.

## Check yourself

The answer's key points as multiple-choice questions, with the correct option and why.

1. What is an indicator organism?
   A. An organism whose presence shows faecal pollution and possible pathogens
   B. A pathogen that causes cholera
   C. An organism that clears turbid water
   D. A chemical that changes colour with pH
   Answer: A. Indicators are used because testing individual pathogens is hard. (Key point: Indicator definition)

2. Which property does an ideal indicator have?
   A. Multiplies rapidly in clean water
   B. Does not multiply in water and lives longer than pathogens
   C. Dies faster than pathogens
   D. Is hard to detect
   Answer: B. It must be abundant, persistent, non-multiplying and easy to detect. (Key point: Ideal indicator)

3. A positive presumptive test is shown by:
   A. clear broth with no change
   B. green metallic sheen on membranes
   C. gas in the Durham tube plus acid
   D. blue colour with starch
   Answer: C. Lactose fermentation to acid and gas in lactose broth within 48 hours. (Key point: Presumptive)

4. Gas in BGLB broth incubated at 35 degrees C for 48 hours confirms:
   A. only viruses
   B. hardness
   C. only E. coli
   D. coliform
   Answer: D. EC broth at 44.5 degrees C is used for faecal coliform / E. coli. (Key point: BGLB)

5. In Thomas' formula, the numerator is:
   A. positive tubes x 100
   B. negative tubes x 100
   C. total ml x 100
   D. positive tubes / 100
   Answer: A. MPN/100 ml = positive tubes x 100 / sqrt(ml in negative tubes x ml in all tubes). (Key point: Thomas formula)

6. Safe drinking water by this test must show:
   A. 33 MPN per 100 ml
   B. 0 MPN of E. coli per 100 ml
   C. Up to 70 MPN per 100 ml
   D. Any value if chlorinated
   Answer: B. The standard is zero E. coli per 100 ml. (Key point: Interpretation)

## Related in Water Supply Engineering

- [Multiple tube fermentation (MPN) technique](https://www.bctengineer.com/question.md?key=semester_5%2Fwater_supply_engineering%3Amtf-mpn-procedure) — Water Supply Engineering, chapter 4, asked 5×
- [Indicator organism; membrane filter technique](https://www.bctengineer.com/question.md?key=semester_5%2Fwater_supply_engineering%3Aindicator-organisms-membrane-filter) — Water Supply Engineering, chapter 4, asked 2×
- [MPN per 100 ml numerical](https://www.bctengineer.com/question.md?key=semester_5%2Fwater_supply_engineering%3Ampn-numerical-k78) — Water Supply Engineering, chapter 4