--- title: "Multiple tube fermentation (MPN) technique" subject: "Water Supply Engineering" chapter: 4 key: "semester_5/water_supply_engineering:mtf-mpn-procedure" source: https://www.bctengineer.com/q/semester_5/water_supply_engineering/mtf-mpn-procedure site: BCT Engineering --- # Multiple tube fermentation (MPN) technique Water Supply Engineering · Chapter 4: Quality of Water - Asked in: 2081 Bhadra, 4 marks; 2080 Bhadra, 5 marks; 2073 Shrawan, 4 marks; 2075 Asoj, 4 marks; 2076 Asoj, 4 marks (asked 5 times) - Video: [MULTIPLE TUBE FERMENTATION (MTF) - Water Systems Technology](https://www.youtube.com/watch?v=uPZ_YFWUzoE) — AWETDSN mlau - Answer checked by Claude Opus 5.5 (high effort) on 2026-10-14 - Page: https://www.bctengineer.com/q/semester_5/water_supply_engineering/mtf-mpn-procedure - Whole chapter: https://www.bctengineer.com/md/semester_5/water_supply_engineering/chapter-4 ## Question Describe in detail the multiple tube fermentation technique for the determination of E-coli / the MPN of a water sample in the laboratory. ## Short answer The multiple tube fermentation test estimates MPN of coliforms per 100 ml from lactose fermentation. Inoculate lactose broth tubes with sample volumes, incubate at 35 C for 48 hours, confirm positives in BGLB or EC broth, then complete on EMB agar. Read MPN from tables; drinking water must be 0 per 100 ml. ## Answer The **multiple tube fermentation (MTF) technique** estimates the number of coliform bacteria in a water sample as the **Most Probable Number (MPN)** per 100 ml, using a statistical table. It uses the ability of coliforms to ferment lactose with acid and gas at 35 degrees C within 48 hours. It is done in three stages. #### Apparatus and media Test tubes with inverted Durham tubes, pipettes, incubator at 35 +/- 0.5 degrees C, water bath at 44.5 degrees C, lactose broth (or lauryl tryptose broth), brilliant green lactose bile (BGLB) broth, EC broth, EMB agar, sterile dilution water. #### 1. Presumptive test 1. Choose sample volumes by the expected pollution. For example, for drinking water: 5 tubes each of 10 ml, 1 ml and 0.1 ml (a 15-tube series). 2. Add the volumes to tubes of lactose broth (double strength for the 10 ml portions) and mix. 3. Incubate at 35 degrees C for 24 hours; if no gas, continue to 48 hours. 4. Gas in the Durham tube plus acid (colour change) is a **positive presumptive** result. No gas after 48 hours is negative. #### 2. Confirmed test 1. Transfer a loopful from each positive tube to BGLB broth (for coliform), and to EC broth for faecal coliform / E. coli. 2. Incubate BGLB at 35 degrees C for 48 hours; incubate EC broth at 44.5 degrees C for 24 hours. 3. Gas formation confirms coliform (BGLB) or faecal coliform / E. coli (EC). #### 3. Completed test Streak positive confirmed cultures on EMB agar and incubate for 24 hours. Typical E. coli colonies are dark with a green metallic sheen. A Gram stain shows Gram-negative non-spore forming rods, and lactose broth again produces gas. #### Calculation of MPN Count the number of positive tubes in each dilution and read the MPN per 100 ml from the standard MPN table for the combination, for example 5-2-1 (10 ml, 1 ml, 0.1 ml) gives an MPN of about 70 per 100 ml (the 95% confidence limits are also given). For other dilutions, use Thomas' formula: $$ \text{MPN}/100\ \text{ml} = \frac{\text{positive tubes}\times100}{\sqrt{(\text{ml sample in negative tubes})\times(\text{ml sample in all tubes})}} $$ Drinking water should show an MPN of 0 per 100 ml. ## Check yourself The answer's key points as multiple-choice questions, with the correct option and why. 1. What property of coliforms does the MTF technique use? A. Growing only on membrane filters B. Fermenting lactose to acid and gas at 35 degrees C within 48 hours C. Producing indole at 20 degrees C D. Reducing nitrate in 6 hours Answer: B. Gas in the Durham tube plus acid colour change indicates coliform lactose fermentation. (Key point: Principle) 2. What is the correct order of the stages? A. Presumptive, confirmed, completed B. Confirmed, presumptive, completed C. Completed, presumptive, confirmed D. Presumptive, completed, confirmed Answer: A. The test goes from lactose broth, to BGLB/EC broth, then EMB agar streaking. (Key point: Stages) 3. Faecal coliform / E. coli is confirmed by gas in EC broth incubated at: A. 35 degrees C for 48 hours B. 22 degrees C for 72 hours C. 44.5 degrees C for 24 hours D. 44.5 degrees C for 5 minutes Answer: C. The higher temperature selects faecal coliforms. (Key point: EC test) 4. On EMB agar, typical E. coli colonies appear: A. pink and mucoid B. white and dry C. golden yellow without sheen D. dark with a green metallic sheen Answer: D. Dark colonies with a green metallic sheen, Gram-negative non-spore rods, and gas in lactose broth complete the test. (Key point: Completed test) 5. How is the MPN obtained for a 5-tube, 3-dilution series? A. Count positive tubes per dilution and read the standard MPN table B. Divide the total tubes by sample volume C. Measure gas volume in Durham tubes D. Count colonies on the plate Answer: A. The positive combination, e.g. 5-2-1, is looked up in the MPN table (about 70 per 100 ml). 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