[Solved] lactobacillus acidophilus isolation and identification

Lactobacillus acidophilus bacteria is a part of the Lactobacillus genus which is a gram positive, facultative anaerobe, rod-shaped, non-spore forming bacteria group.  Lactobacillus is a major part of the lactic acid bacteria group. Lactobacillus acidophilus like most of the Lactobacillus species exists naturally in the body but it is found primarily in the intestines and the vagina.

L. acidophilus can be added to food like cheese and yoghurt where it acts as a probiotic and can also be used for treatment of bacterial vaginosis, lung infection, certain types of diarrhea and eczema. This bacterium grows at optimum temperature of 370C. My sample can be obtained from the intestine or the vagina, but these two body parts also contain other type of bacteria which could make it very difficult to totally isolate the bacteria.

Asides these problems, the intestine which is inside the body would be hard to get to and taking a swab of the vagina is not the healthiest thing to do, hence the best way to obtain my sample would be from a live culture of the bacteria which can be found in active yoghurt. Active yoghurt usually has a combination of lactic acid bacteria which can include; Lactobacillus bulgaricus, Lactobacillus acidophilus, Streptococcus thermophilus, Bifidus and Lactobacillus casei. I would be using a Yoplait yogurt which contains live and active culture of S. thermophilus, L. acidophilus and L. bulgaricus.

To be able to isolate the L. acidophilus, I would be culturing in a media that selects for the Lactobacillus specie. To isolate the bacteria that I have chosen, I will be implementing De Man, Rogosa and Sharpe agar, often abbreviated to MRS agar. The MRS agar is selective for the growth of Lactobacillus genus. This agar contains Sodium acetate which would suppress the growth of other bacteria. MRS agar also contains yeast, meat extract and peptones provide a source of carbon, nitrogen and vitamins for the bacteria growth. The yeast extract also contains vitamins and amino acids specifically required by Lactobacilli.

The MRS agar also contains Polysorbate 80 which acts as a surfactant to aid nutrient uptake by Lactobacilli and magnesium sulfate which provide cations used in metabolism. After obtaining my sample from the yogurt, I will isolate my organism in the following steps: Inoculate MRS agar with a cotton swab containing the sample. Incubate at 37 degrees Celsius till the next lab. Examine the media and any region with white color is the Lactobacilli specie I want.Gram stain: This is to show that the Lactobacilli specie I want is gram positive. I expect to see results where the bacteria take up crystal violet color and hence look purple under the microscope with a rod like shape.

If my Gram stain is positive, I will continue to perform a catalase test. Catalase test are used to differentiate between Staphylococci and Streptococci bacteria. The aim of this test is to see which of the genus can break down hydrogen peroxide. Since my isolated sample should not contain either of this specie, this test should give negative result. Next, I would perform an endospore staining test.

This test is to show if there is any endospore formation due to harsh conditions. This is not usually found in L. acidophilus hence I expect to see a negative result here where we have no green colors showing vegetative state spores. If this step is successful, I would move to the next step. 5. Next, I will perform a carbohydrate fermentation test. From my sample in the MRS agar, I would use my loop to put some sample in each of the carbohydrate solutions which include; mannitol, sucrose, lactose and glucose. The bacteria I want can ferment glucose, sucrose and lactose but would produce no gas in all cases. Mannitol is not fermented.

I expect to see a color change that reflects their fermentation. If any of these tests come out wrong, it means my bacteria was not correctly isolated. 6. Finally, I would perform a starch hydrolysis test on the bacteria from the MRS growth and then, smear the starch plate. If the starch gets hydrolyzed, the α-amylase is produced by the bacteria a clear zone will be produce around the bacteria will appear when gram’s iodine. I expect to see a negative result for this one as my bacteria would not be able to hydrolyze the starch. At the end of these tests, I should have narrowed down my sample and predict that my result would be L. acidophilus.


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