Question

When performing a Multiple Tube Fermentation Technique, what does a positive Completed test include?

Answer 1

Multiple tube fermentation technique or most probable number method is a method used to determine the quality of ground water or surface water. It is commonly tested to know the water quality, whether it contain any type of pathogenic coliform bacteria or not. The basic idea is to inoculate the water sample in a media. If the sample contains any coliform bacteria, it will form colony and with the amount of coliform bacteria, the concentration of culture also increases.

Pathogenic coliform strains which are aerobic or faculative anaerobic, gram negative who are rod shaped non spore forming bacteria which can ferment lactose at 35℃ after 48 hours of incubation can be determined by this method.

Multiple tube fermentation technique has three main stages- Presumptive stage, confrmed stage and completed stage.

1) Presumptive stage: A series of lauryl tryptose broth which are used as primary fermentation tubes are inoculated with gradually increasing quantity of sample to be tested. The samples are kept at room temparature for 24-48 hours and the gas formation is analysed. Some form gas formation and ones whoch no gas formed are incubated for another 48 hours and examined if any gas if formed. Gas formation at any of these time can be considered as a positive result.

2) Confirmed test: All the tubes which showed positive result in presumptive test will undergo confirmed test. In this method, fermentation tube with brilliant green lactose bile broth is inoculate with the samples showing gas formation in the presumptive test. The tubes are incubated for 48 hours at 35℃. The incubated tubes are examined for gas formation and those with gas are considered as positive result.

3) Completed test: Ite is performed in plates showing a positive result in the completed test. Series of plates having eosin methylene blue are streaked with the samples. Those plates are incubated for 24 hours at 35℃. After incubation, one or more typical colonies which are nucleated are inoculated to a lauryl tryptose broth fermentation tube and nutrient agar slant. These tubes are incubated for 24 hours-48 hours at 35℃.
Coliforms containing tubes produce a greenish metallic sheen and without sheen indicate non- coliform colonies.

After all these, the agar plates corresponding to the tube in which gas is formed , are used to take sample and is observed microscopically. The rod shaped, gram negative, non spore forming coliform bacteria can be seen. From all tgese test, it can be confirmed that the sample contain coliform bacteria and has poor quality.

This method is used for determining the presence of coliform bacteria and its quantification. The serial dolution used in this method can determine the amount of microbe present . As quantity increases, the dilution upto which growth shows will increase.

The lactose present in the tube is used by the micorbe to convert it into gas and acid. Preaence of acid is determined by the colour change and gas is identified by bubble formation.The amount is determined by the number of tubes showing positive result and comparing it with the standard statistical table.