Download In vitro cultivation

Invitro Culture of Parasites
By:Tabassum Urooj
M.Sc. IV Semester
Deptt. Of Zoology
University of Lucknow
Contents
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Introduction
Basic Problem of Cultivation
Technique employed
Material used
Techniques for adult worms
Culture vessels & Conditions
Medium
Result
Attempts to improve cultivation condition
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.
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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.
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.
Technique employed
• General principle
• In cultivation method it is important to distinguish between conditions
& media which allow growth, development & maturation of
trematode parasite.
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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.
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.
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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.
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.
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.
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.
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.
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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.
Attempts to improve cultivation
condition
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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.
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