Chitra Srikantan

Department of Biotechnology

IIT Madras

Exploring Research-At School Level

It is to explore the practical aspect of the theoretical knowledge gained

Research

Broad spectrum antibiotic

By-product of bacteria Streptomyces erythreus

Produced inside the cells

Can be screened for preliminary activity

Production of Erythromycin

Two repositories of Microorganisms storage and supply in India

NCIM –Pune

MTCC- Chandigarh

How to procure the Microorganism?

The websites clearly tell on how to buy the strains. The cost will be <Rs. 1000.

The organism will be sent in a glass vial as lyophilised tube.

Separately, the medium for growth and maintenance will be mentioned in the catalogue, which can be bought prior. (Ex: Nutrient broth, Luria Bertani broth)

Can be stored in freezer as glycerol stocks viable upto 4 months. Fresh glycerol stocks to be made.

How to buy? And Cost?

To hold the microorganism in no growing- but still viable stage.

It is stored in microcentrifuge tubes/cryovials under 80% glycerol.

Autoclave 80% glycerol. To vials, add 200µL glycerol and 800µL of culture. Seal and store in freezer.

Reviving from glycerol stocks. Thaw the culture and inoculate it into fresh autoclaved medium. Grow in specified conditions.

Glycerol stocks

Harvest the cells. By using a centrifuge/ by allowing to settle under gravity, inside fridge and filter using Whatman No.1 filter paper.

The cells are then ground with a mortar pestle, and suspended in a extraction solvent, chosen based on literature for erythromycin (Ex: 50% Isoamyl alcohol)

That organic phase is separated and evaporated in a reflux apparaus

The remaining product is dissolved in solvent

Extraction of product

Four other known bacteria like Bacillus subtilis, Pseudomonas, Klebsiella and Streptococcus (All avirulent strains) can be collected from any educational /research institutes in proximity.

They are also to be grown in the required conditions and stored.

They should be later grown in Petriplates containing the solid medium (Agar)

Antibiotic activity

Cut small discs of Whatman No.1 paper using Punching Machine.

Impregnate the discs with the extracted Erythromycin

Vary the amount of extract in each disc

Disc Assay

Now place these discs in the petriplates containing the bacterium.

Incubate again at same conditions.

Zones of inhibition will be observed

Antibiotic Disc

The potent nature of the antibiotic can be understood with the antibiotic disc assay

Result

Most plants possess a store house of varying biochemicals with potent activity

These can be explored and screened .

Phytochemical extraction

Collect the fruits, leaves, plant parts (If possible)

Separate the fruits as seeds, peel, whole fruit.

Dry them at 40-50°Cin Hot Air oven for 1 week

Extraction and screening of Pomegranate

The dried plant material was powdered using mixer grinder, and subjected to soxhlet extraction with 99% ethanol and chloroform for 24hours.

The mixture was evaporated to dryness in a rotary flash evaporator and stored in refrigerator.

The condensed extracts were used for preliminary screening of phytochemicals.

Ethanolic and Chloroform Extract

The peel, whole fruit and seeds powder was boiled in

distilled water for 15-20 minutes, kept in roomtemperature overnight and filtered. The filtrate was evaporated to dryness in hot

air oven and stored in refrigerator. The condensed extracts were used for

preliminary screening of phytochemicals

Aqueous extract

Extracts were tested for the presence of active principles such as Triterpenoids, Steroids, Glycosides, Saponins, Alkaloids, Flavonoids, Tannins, Proteins, Free Amino Acids, Carbohydrate and Vitamin C.

Phytochemical analysis of different Crude extracts

Liebermann Burchard test - Crude extract was mixed with few drops of acetic anhydride, boiled and cooled.

Concentrated sulphuric acid was then added from the sides of the test tube and observed for the formation of a brown ring at the junction of two layers.

Green coloration of the upper layer and the formation of deep red color in the lower layer would indicate a positive test for steroids and triterpenoids respectively.

Test for Steroids and Triterpenoids

Keller Killiani Test – Test solution was treated with few drops of glacial acetic acid and Ferric chloride solution and mixed.

Concentrated sulphuric acid was added, and observed for the formation of two layers. Lower reddish brown layer and upper acetic acid layer which turns bluish green would indicate a positive test for glycosides.

Bromine water test - Test solution was dissolved in bromine water and observed for the formation of yellow precipitate to show a positive result for the presence of glycosides.

Test for Glycosides

Foam Test – Test solution was mixed with water and shaken and observed for the formation of froth, which is stable for 15 minutes for a positive result.

Test for Saponins

Hager's Test – Test solution was treated with few drops of Hager's reagent (saturated picric acid solution).

Formation of yellow precipitate would show a positive result for the presence of alkaloids.

Test for Alkaloids

Ferric chloride test – Test solution when treated with few drops of Ferric chloride solution would result in the formation of blackish red color indicating the presence of flavonoids.

Alkaline reagent Test – Test solution when treated with sodium hydroxide solution, shows increase in the intensity of yellow color which would become colorless on addition of few drops of dilute Hydrochloric acid, indicates the presence of flavonoids.

Test for Flavonoids:

Lead acetate solution Test – Test solution when treated with few drops of lead acetate (10%) solution would result in the formation of yellow precipitate.

Gelatin Test – Test solution when treated with gelatin solution would give white precipitate indicating the presence of tannins.

Test for Tannins

Biuret Test – Test solution was treated with 10% sodium hydroxide solution and two drops of 0.1% copper sulphate solution and observed for the formation of violet/pink color.

Test for Proteins

Benedict's test – Test solution was mixed with few drops of Benedict's reagent (alkaline solution containing cupric citrate complex) and boiled in water bath, observed for the formation of reddish brown precipitate to show a positive result for the presence of carbohydrate.

Test for Carbohydrate

DNPH Test – Test solution was treated with Dinitrophenyl hydrazine dissolved in concentrated sulphuric acid. The formation of yellow precipitate would suggest the presence of vitamin C.

Test for Vitamin C

Combining the above tests, the screening of novel chemicals is possible

The plant extracts can be screened for the presence of the activities. Based on that, one of the extracts maybe chosen for antibiotic disc assay.

Thus, proving some of the traditional medicines used in India.

Screening of phytochemicals for cytotoxic activity

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