- First, create an ANSI C++ algorithm (Balagurusamy 2004) in the Turbo C++ v.3.0 software (Borland International Inc., USA) to calculate the correlation between real time hypergravity values and corresponding values of centrifugal speed (in RPM or revolutions per minute) to be used for exposing the Limnaea eggs to increased gravity conditions (relative centrifugal force or RCF) inside an ultracentrifuge. Following algorithm must be entered into the Turbo C++ v.3.0 software to run this, “Program for Calculation of Centrifugal RPM with respect to Hypergravity”:
float RCF, rpm, r, b, e;
cout<<”Program for calculation of REVOLUTIONS PER MIN. with respect to HYPERGRAVITY”<<”\n\n”;
cout<<”\t”<<”Enter the value of Avg. radius (r) of centrifugal tube:”;
cout<<”\t”<<”Enter the value of HYPERGRAVITY (RCF) e.g. 5 for 5g:”;
cout<<”\n\t”<<”The value of Revolutions per Minute (rpm) is:”<<sqrt(rpm)<<endl<<”\n”;
- Once the above algorithm has been entered into the software one may run the program after which the program will ask for two values i.e. (1) Value of Average radius (r) of centrifugation rotors and (2) The value of Hypergravity required.
The average radius of the centrifugation rotor is provided by the manufacturer with the fixed-angle rotors. On the other hand, value of hypergravity depends upon the type of investigation. Since hypergravity is the level of gravitation force experienced by astronauts in space-crafts, there are specific minimum or maximum values of hypergravity which may be entered into the program. Following is the array of such values (multiples of G=9.8m/s2) (Chatterjee 2002):
Hypergravitation experienced at first booster stage of aircraft: +9G
Hypergravitation experienced at second booster stage of aircraft: +8G
Hypergravitation causing fall in blood pressure to 40mm Hg: +4G
Hypergravitation that may cause fracture in vertebrae: +20G
- Any of above values or similar values may be entered into the C++ program as per the nature of investigation. Once a particular hypergravity value is entered, the program will give corresponding RPM value.
Conventionally, if the ultracentrifuge is run at this calculated RPM, the Limnaea eggs and embryos would experience that particular value of hypergravity in the centrifuge tube as would be experienced by the astronaut in a spacecraft.
In order to obtain the eggs of Limnaea, visit freshwater ponds with floating lotus or water lily (Nymphaea) leaves. Turn the leaves upside-down and observe for presence of transparent egg masses attached on the leaves under surface, to ensure the presence of snails in the pond.
Identify and confirm the taxonomic status of Limnaea snails.
Now, bring these Limnaea snails and the leaves along with the pond water to laboratory.
Filter the pond water and fill it into the troughs. Lay down the lotus leaves on the water’s surface.
Allow the snails to lay eggs under the leaves, to permit collection of freshly laid eggs.
After fresh eggs have been laid, detach these egg masses carefully from the leaf’s under-surface by scrapping with a scalpel or forceps and transfer into a Petri dish containing filtered pond water.
Now observe various eggs under the light microscope and segregate each of them into specific groups depending upon the developmental or cleavage stage in which the embryo exists.
Initially, note the time intervals between various cleavages. At room temperature, time interval between each cleavage is 90 minutes.
As soon as the cleavage stage is identified, put the eggs into the centrifuge tube and centrifuge in an ultracentrifuge at corresponding centrifugal speed (in RPM), calculated via the above ANSI C++ algorithm.
Precisely note the time for which the eggs were exposed to centrifugation and remove them accordingly when a particular cleavage stage should exist as per the inter-cleavages timings noted previously.
Immediately observe the eggs under light microscope to find as to whether the expected cleavage stage has taken place or not. Note the timings accordingly.
- Analyze the corresponding values for each cleavage stage in normal condition as well as in hypergravity conditions.