Project for biotechnology/biochemistry graduates/post graduates

ISOLATION OF NITROGEN-FIXING CYANOBACTERIA

Aim:
To isolate nitrogen-fixing cyanobacteria from rice fields at Sinhagad foothills (Pune
district, Maharashtra).
Methodology:
Bacterial sample (floating green matter) was collected from the stagnant water in the
rice fields at the foothills of the Sinhagad hills in Pune district, Maharashtra. The
sample was kept in water for three days in the presence of sunlight.
Ashbey’s nitrogen-free agar was selected as the medium for isolation of nitrogen-
fixing cyanobacteria which has the following composition per litre:

Agar:15.0 g
Mannitol:15.0 g
CaCl2.2H2O:0.2 g
K2HPO4:0.2 g
MgSO4·7H2O:0.2 g

100 ml of medium is prepared by adding appropriate amount of components to tap
water in a 250 ml conical flask. pH of the mixture was adjusted to 7.2 at 25°C. The
medium was autoclaved for 15 minutes and was poured in five sterile petriplates.
Bacterial sample was streaked on two plates and the plates were kept at room
temperature in the presence of sunlight. After one week of growth, yellowish green
colonies were picked up and streaked on two more petriplates containing Ashbey’s
nitrogen-free agar. Gram staining of the bacteria was performed after 10 days of
growth on these newly transferred petriplates and the cells were observed at 100X.
Wet mount was prepared to observe the bacterial cells at 100X using oil-immersion
microscopy.
Results:
Direct streaking of bacterial sample on Ashbey’s nitrogen-free agar petriplates
showed growth of many transparent small sized colonies after two days. After one
week of growth in the presence of sunlight, five yellowish green colonies appeared on
the petriplates. The bacterial colony characteristics observed after 10 days of growth
on the two newly transferred petriplates were as follows:
Shape: Round
Size (Diameter): < 1 mm
Margin: Entire
Elevation:Flat
Transparency: Opaque
Gram Staining: Negative
Motility: Non-motile
Consistency: Hard

Medium-sized thin pink rods with tapering ends were observed at 100X after gram
staining of the bacteria was performed. On observing the wet mount of these bacterial
cells at 100X, pale green highly motile bacterial cells were observed. Most of the cells
were rod-shaped and a few appeared to be circular (the circular ones were
continuously changing orientation and appeared as rods within a few seconds).
Discussion:
The isolated organism has most of the characteristics in common with nitrogen-fixing
cyanobacteria. Since the bacterial cells could grow in the medium devoid of any form
of combined nitrogen, it is perceptible that they possess the ability of nitrogen
fixation. The organism is supposed to be capable of performing photosynthesis in the
presence of sunlight in view of the fact that there was no source of carbon for its
growth in the medium. The slow rate of growth of the organism in solid media and the
yellowish green colour of the colonies further support the fact that the bacteria is a
form of blue-green algae. Moreover, as the individual cells were non-transparent and
had a pale green colour as observed at 100X without any staining, it is evident that the
cells possess certain pigments which are vital for photosynthesis. However, some
small sized transparent colonies did appear on the petriplates containing Ashbey’s
nitrogen-free agar. These organisms may be some species of oligotrophic bacteria
which are capable of growing in highly nutrient deficient media.

Growing plants......the Biotech way!!!

Producing pure ayurvedic compounds in a flask ........
Shatvari is a plant that is considered highly medicinal in ayurvedic texts. Its roots produce compounds called saponins which have the medicinal properties. But these are produced only after flowering. And the plant flowers only after 4years. So, nobody wants to grow this plant ....who is going to wait for 4years? So we took a different approach .We established the root callus of this plant in our tissue culture laboratory (we are the first one in the world to have done so, other attempts failed for years). We then made the callus friable and established individual cells in suspension cultures. These cells now grow in suspension and produce pure saponins. No need to grow the plants, no need to wait for 4years for flowering, no need to have a field, no fertilizers, no pesticides. All you need is a small room, a few cheap equipments and you can manufacture the medicinal compunds. Bonus?? You get pure, high value saponins, rather than crude root powder. Neat , isn't it?

What is BIOTECHNOLOGY?

Consciously manipulating and using processes of life for the benefit of mankind is what biotechnology is all about. For example, we can introduce the human insulin into bacteria thereby producing huge quantities of human insulin at a fraction of the cost. This is of great help to diabetics world over who, till some years back, had to purchase cattle insulin which was both costly and less potent. Similarly, we have introduced the Bt gene into cotton making cotton crops less susceptible to pests.
Biotechnology touches virtually all aspects of human life. Through biotechnology we get new and improved medicines at a fraction of the cost, higher quantity of food, improved varieties of food, insect resistant plants, plants making their own fertilizers, fruits that have longer shelf life…. and so on. Gene therapy, which is supposed to be the answer to incurable diseases, is also a variant of biotechnology.
So then, biotechnology is a field that has a say in virtually every field of human endeavor.


When you make an omelet and throw away the eggshell ……

Worldwide millions of eggs are consumed daily and it is a big problem to dispose off the shells. Our aim was only to design a simple method to obtain pure calcium carbonate from the shells. But we woke up to the possibility that the membrane that adheres to the shell can also be exploited for value added products. We have made possible the development of a technique that gives you pure calcium carbonate, and such high value products as collagen, hyaluronic acid and chondroitin sulfate. The last three come in a mixture. Currently we are developing methods to separate the three.