Microbiological Technique Assignment

(Practical)

Topic: 1. Identification of Lactobacillus Acidophilus

2. Total Viable Count

Submitted to: Madam Fareeha

Submitted by: Pharmacology Group

(M.phil & Ph.D)

3rd Semester

The University of Veterinary & Animal Sciences

Total Viable count

Total Viable Count (TVC) gives a quantitative idea about the presence of microorganisms such as bacteria, yeast and mold in a sample. To be specific, the count actually represents the number of colony forming units (cfu) per g ( or per ml) of the sample.

This involves counting the colonies produced by viable cells under favourable growth conditions. In pour-plate method, an aliquot of suitably diluted sample is mixed with nutrient agar at a temperature where it is liquid. Then the mixture is poured into petridishes and allowed to set.

Alternatively an aliquot of the sample is spread over the agar surface of a Petridis using a sterile spreader. Membrane filters can also be used to determine the bacterial numbers. In this method cells are filtered onto membrane filter which is then placed over nutrient agar surface.

Results

Identification of Lactobacillus Acidophilus

Study Background:

Lactobacillus acidophilus and other lactic acid bacteria are important in the fermentation of many foods from dairy products to fruits and vegetables. Fermentation occurs when bacteria break down sugars and carbohydrates to produce alcohol, carbon dioxide and lactic acid. These by-products are responsible for the unique taste of fermented foods and help preserve and increase palatability.

L. acidophilus can also be used as a probiotic or living organism, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition. Lactic acid bacteria use lactose as their main source of carbon to produce energy. Lactic acid bacteria use energy to transfer lactose (main sugar of milk) through their cell membrane. The lactose is metabolized to lactic acid and in some species also acetic acid, ethanol and carbon dioxide. Lactic acid bacteria that only produce lactic acid as an end product are called homofermentative; those that also produce acetic acid, ethanol and carbon dioxide are termed heterofermentive. The by-product of this reaction is energy that the bacteria use for growth. The end products of fermentation ultimately change taste and texture of food.

L. acidophilus is a member of one of the eight main genera of lactic acid bacteria. The genera, Lactobacillus, Streptococcus, Lactococcus, Leuconostot, Bifidobacterium, Carnobacterium, Enterococcus and Sporolactobacillus can then be divided into species, subspecies, variants and strains.

Lactobacillus are able to live in highly acidic environments of pH 4-5 or lower. This pH is well below the pH other lactic acid bacteria can live in and because of this, Lactobacillus is responsible for the final stages of fermentation in products.L. acidophilus is probably the best well-known species of Lactobacillus . L. acidophilus is naturally found in the human and animal GI tract, mouth and vagina. It can also be found in certain dairy products and freeze dried in capsule and tablet form in grocery and health food stores.

Morphology: The overall shape of L. acidophilus is one that is rod shaped. This bacterium occurs in small chains and is usually 0.5 to 0.8 micrometre (1 mm=10-6 metre) across by 2 to 9 mm in length. It is also important to note that L. acidophilus is non-spore-forming.

This is the assignment of our class work as a group for the identification of Lactobacillus acidophilus in laboratory.

Steps:

1. Lactobacillus acidophilus was taken present in powder form in probiotic Sachet named Acotec containing Bifidobacterium, Lactobacillus acidophilus, streptococcus thermophile.

2. Powder was dissolve in sterilized distilled water.

3. Solution was poured on agar media plate under aseptic conditions.

4. Plates were incubated at in incubator for 24 hours.

5. Next day plates were removed from incubator and mixed growth appeared.

6. Culture was purified by streak method on another media plate under aseptic conditions and allowed to grow for 24 hours in incubator.

7. Pure growth was appeared on media plate, Lactobacillus acidophilus was identified according to its morphology and further scheme was followed to identify.

Scheme to identify Lactobacillus Acidophilus

Bifidobacterium, Lactobacillus acidophilus, streptococcus thermophile

Acid FastStainingCarbolfuchsinRed color

Lactobasillus Spp.

Catalase TestBubble formations,

negative

Lactobacillus Acidophile confirmed