1. Invitro Culture of Parasites By:- Tabassum Urooj M.Sc. IV Semester Deptt. Of Zoology University of Lucknow

2. Contents Introduction Basic Problem of Cultivation Technique employed Material used Techniques for adult worms Culture vessels & Conditions Medium Result Attempts to improve cultivation condition

4. In vitro cultivation Many aspects of Physiology & Biochemistry of parasitic organisms can be studied only when they are grown in invitro culture free from contamination of other organisms. No. of terms have been developed to designate particular aspect of culture techniques. A culture with other species of organisms is termed as Monogenic. While with other species are termed as polygenic. The term invitro has been used to describe a culture involving a liquid or solid medium in a tube or silver container.

5. Basic Problem of cultivation Cultivation of Trematode in vitro presents a no. of problems due to their complex life cycle. Biological habitat of Trematode Many species such as intestinal fluke live in non sterile habitat so that antibiotic treatment is necessary before forming or establishing culture. An alternative solution is to the medium in which the culture using those larval stages for ex. Metacercariae which occurs only in sterile environment. In the natural habitat the metabolic waste product of a worm can easily be removed from the site their production and result of circulation of body fluid. A successful in vitro method must provide condition for rapid removal of toxic waste product. Life cycle of trematode involves several hosts that means that at each stage of development require different physiochemical condition & have different nutritional requirement.

7. Technique employed General principle In cultivation method it is important to distinguish between conditions & media which allow growth, development & maturation of trematode parasite. The maintenance of organism's in vitro condition which permits the metabolism to operate at a level sufficient to keep cells & tissue alive, but not sufficient to allow growth & maturation in case of metacercariae or continued sperm and egg production in case of adults. To know the pattern of development in the normal host and this should be normally the first step in attempt at in vitro culture.

8. Material used In vitro work has been carried out by using either adult worm or metacercariae first disadvantage is that if they are intestinal forms they require treatment with antibiotics. on the other hand since the upper bile duct in a sterile site, For Example :- Fasciola hepatica may easily be obtained in a sterile condition from an infected liver by opening the host & dissecting the liver. Schistosomes provide useful material for invitro work since they occur in the blood vessels & liver from which they can be easily removed. For Ex. :- Schistosoma mansoni. Metacercariae also provide valuable material for experiment and many species occur in sterile condition or situation inside the intermediate host and have the added advantage that they often occur in large number.

10. Techniques for Adult worms Once sterility is achieved many adult trematode will survive for considerable period in balanced saline solution provided suitable conditions like PH, Oxygen tension, osmotic pressure and temperature are maintained at satisfactory method for waste material developed. For ex.:- Fasciola hepatica - The specimens can be made hidden in freez saline for 18-34 days at 390C in a special culture tube. Under such condition cellular differentiation become abnormal which in few hours as adjusted by histological and cytological condition of the test. This organ is particularly sensitive to abnormal environment condition and its cytological examination often provides valuable area as to the efficiency of culture method adopted.

11. Culture vessels and conditions Good results can be obtained using a standard 20 ml M carton type bottle with a flat internal base and a rubber stopper. It consist of a tube whole lower open end is covered by a small sac of cellulose membrane and so has the advantage of permitting waste product to diffuse easily from the vicinity of worms.

12. Medium It consists of essentially inactive rabbit serum Earle's saline, glucose and red cells. The addition of other nutrients such as chick embryo extract or liver extract which have proved useful in tissue culture or culture of other parasites particularly nematode has a detrimental effect on development and these are therefore not suitable to such medium.

14. Result Under the best condition remarkable degree of growth can be obtained and schistosomidae can consistently develop to at least stage IV characterized by the appearance of sperm in male and occasionally to stage V characterized by the appearance of egg cell protein in the vitelline cell of female. `Pairing will occur regularly in the medium fully formed eggs are produced cultured schistosomidae reach stage V but worms do not grow as large or as rapidly as those grown in the mouse.

15. Attempts to improve cultivation condition The failure of culture technique describe above to produce fully mature worms would be due to a number of cases. The major or important reason for failure of culture technique is lack of nutritional factor or factors of unsuitable physical conditions such as local accumulation of excreted metabolites as mentioned in addition of various growth stimulants such as chick embryo extract or liver extract has generally proved inhibitory and not stimulatory to further development.

16. Acknowledgement I greatly pleasure to submit the project report on invitro culture of parasites in the Department of Zoology University of Lucknow, Lucknow. I am greatly thankful to professor Nirupama Agarwal and other senior professors who have guided me to report on that topic. Special thanks to our parasitologist Dr. A. M. Saxena who is specialist of parasites guided and suggested me to make a proper way to that project. I am also thankful to Dr. Amita Kanaujia who helped me to report the project on time. Thanks! Tabassum Urooj