Water is unsafe to consume if gas producing lactose fermenting microorganisms present in water. A gas formation may be due to non-coliform bacteria such as Clostridium perfringens, which are gram-positive, and also it may due to gram-negative coliform. The presence of Coliform bacteria is an indication of the unsuitability of water. E.colithat is one of coliform are usually resided in human intestine and known to be fecal in origin. To confirm the presence of gram-negative lactose fermenters in water, the confirmed test is carried out.
In confirm test used medium is the Eosin methylene blue agar medium. Eosin methylene blue agar medium contains methylene blue which inhibits gram-positive bacteria. Hence it will inhibit the growth of Clostridium perfringens. Gram-negative lactose fermenting organisms which grow on this medium will produce nucleated colonies. (Dark centers) All coliforms do not reside in intestines and also all of them are not fecal coliforms. Only E.coli is the fecal coliform. Other 4 organisms which are mentioned in the theory part are normally present in the soil. Hence only E.coli is identified in the confirmed test. Colonies of Escherichia coli and Enterobacter aerogenescan be differentiated on the basis of the size and the appearance of the colonies. Differentiation in this manner is not completely reliable.
Procedure:
1. Pour plate method
First 1ml of the fermented water samples were transferred into sterile Petri dishes, using a sterile pipette. 15ml of Eosin methylene blue agar was poured into the Petri dish and rotate the Petri dish while on the table to mix the water sample with the agar. Next, this was allowed to solidify. Finally, this incubated at 370C.
2. Spread plate method
Sufficient amount of agar was poured into a sterile petri dish. Next, this was allowed to solidify. 0.1ml of the fermented water samples were transferred on to the agar surface. A spreader was dipped in 70% alcohol and was flamed. Finally, the sample was spread over the surface of the agar then lids were closed.
3. Streak plate method
First sufficient amount of agar was poured into a sterile petri dish. Next, this was allowed to solidify. Then a sterilized inoculating needle was dipped in the Durham tube containing test tube. Next, the agar plate was streaked to separate the two types of coliforms. Finally, this was incubated at 370C.
Observation
1. Pour plate method
2. Spread plate method
3. Streak plate method
Discussion:
Coliforms are aerobic or facultatively anaerobic, gram-negative, non-spore forming, rod-shaped organisms that ferment lactose with gas formation within 48 hours at 350C. Coliform bacteria usually present in the intestine of human or warm-blooded animals. These organisms are a heterogeneous group of bacteria composed of Escherichia, Enterobacter, Klebsiella, Citrobacter, and Aeromonas. Expect E.coli other organisms are non-fecal in origin. They usually found in soil. Since coliforms are gram negatives they can easily grow in Eosin methylene blue agar (EMB). Gram-positive bacteria cannot survive in EMB. EMB suppresses the growth of gram positives. Since coliforms are aerobic or facultative anaerobic they can be seen on the top of the agar plate and sometimes inside but close to the top layer. The dye also helps to eliminate other unwanted organisms.
Pour plate method involves platting of diluted samples mixed with melted agar medium. The main principle of diluting the inoculum in tubes containing liquefied agar medium is to have a thorough distribution of bacteria cells within the medium. This medium is maintained in a liquid medium at a temperature of 42-450C. In pour plate method colonies may develop in the medium known as subsurface colonies and on the medium known as surface colonies. The disadvantages of the pour plate method are;
- The picking up of subsurface colonies needs, digging them out of the agar medium thus interfering with other colonies.
- The microbes being isolated must be able to withstand temporary exposure to the 42-450C temperature of the liquid agar medium.
Pour plates are useful in determining the number of viable bacterial cells present in a culture. Also in pour plate method, there is no requirement of previously prepared plates and it is often used to assay bacterial contaminations.
The number of bacteria in solution can be readily quantified by using the spread plate technique. According to this method, the sample is appropriately diluted and a small aliquot is transferred into an agar plate. The difference in between the pour plate and spread plate is only surface colonies develop in the spread plate.
There are many streaking patterns. Those are radiant streak, continuous streak, “T” streak and quadrant streak. Radiant and continuous streak methods are used to culture microorganisms. “T” and quadrant streaking are used to isolate single colonies to study microorganisms. In “T” and quadrant streaking usually, we sterilize the inoculating loop at the end of each line. Good streaking is observed in “T” and quadrant streaking because it isolates a single colony.
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| “T” Streak |
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