CHAPTER I

ISOLATION, CLONING, IN VITRO CULTIVATION

AND GROWTH STUDIES
 21

Morphological, biochemical and immunological studies of protozoans

are done with cultured materials and so isolation and cultivation in
laboratory base medium are considered as primary requisite. The diversity
of kinetoplastid flagellates either free living or parasitic leads to their
difference in nutritional requirements. The nutritional analysis reveals to
(1) find out unique metabolic mechanisms (2) analyse the factors mediating
the intracellularity in vetebrates (3) identify on unidentified growth
factor(s) which is(are) absolute or only stimulatory and inspire synthesis
of chemotherapeutic agents acting as antimetabolites (Hutner et^ al_., 1979).
Nevertheless determination of the nutritional requirements of kinetoplastid
flagellates is providing difficult (Newton 1976).

Leishmania donovani promastigotes and other two flagellates (i.e.,

Bodo indica n.sp. and Herpetomonas indica n.sp.), studied in the disserta­
tion, exist and multiply within the acidic hydrolytic environment of the
gut of the insects. They are invaluable for diagnostic purpose and also for
biochemical and immunological studies of the parasites and the disease. So
it is necessary to device vitro system to cultivate the kinetoplastid
flagellate but not act harmfully on the organisms (Hutner et^ al_., 1979).
The nutritional differences are mirrored in the variety of culture media
employed in their vitro cultivation. The temperature and pH are
considered as important criteria for isolation and cultivation of
kinetoplastid flagellates (Wallace et^al_., 1983).

The present chapter deals with the isolation, cloning, cultivation,

growth in different media and effect of pH and temperature on Bodo indica
n.sp., Leishmania donovani and Herpetomonas indica n.sp.

1.1. MATERIALS AND METHODS

1.1.1. Bodo indica n.sp.

1.1.1.1. Isolation

A large number of giant waterbug Lethocerus indicus (Belostomatidae:

Hemiptera) (Figure 1) was collected from different parts of West Bengal.
Lethocerus indicus are very rapacious, feeding upon small fish, tadpoles,
young frogs and insects. The flies move readily from one source of water
to another and are often attracted to light and met with far away from
a) Dorsal view
b) Ventral view
 a

Figure 2. External feature of Chrysomya megacephala (Fabricius) .

a) Dorsal view .
b) Ventral view ,
 22

water. They are 11 cm (approximate) in length. The flies were anaes­

thetized with ether and kept in Benzalkonium chloride (Sigma) solution
(1:100 w/v) for 5 minutes, then washed three times with sterile distilled
water. The insects were then dissected asceptically on 0.1 M NaCI solution
and examined microscopically. Theflagellates, ifpresent were found
swimming in the lumen of gut. Small pieces of hindgut, contained flagel­
lates, were transferred to tubes containing diphasic modified glycerol beef
extract medium.

The composition of solid phase of the medium was : peptone (Oxoid)

2.0 gm, bacto beef (Oxoid) 1.5 gm, NaCI 0.6 gm, agar (Djfco) 1.8 gm,
glycerol 3.0 ml distilled water 97.0 ml . The pH was adjusted to 6.8 to
7.0 with ,IN Nap or IN HCI .

Penicillin (Gibed) 1000 unit/ml and Streptomycin (Gibco) 1000 ug/ml

of the media were added. The medium wasincubated at 25°C. Liquid
phase contained distilled water (5 ml).

1.1.1.2. Cloning

Cloning was done by two methods

(a) Limiting dilution methods

(b) Isolation of a single organism.

a. Limiting dilution method

Solid phase of modified glycerol beef extract medium was prepared.
Melted solid phase of the above said medium (0.5 ml) was pipetted out
into each well of the plastic culture tray. It was allowed to solidify,
wrapped with plastic wrap and stored at 4°C in a closed plastic container
until needed.

At the time of cloning 0.3 ml of distilled water were added to each

well containing 0.5 ml glycerol beef extract medium. The flagellates were
counted using a Neubauer haemocytometer (Fein-Optic, Blankenburg, East
Germany) by diluting the organisms 10 to 100 folds in 2% formalin in PBS.
The organisms were diluted to a final concentration of 16 cells/ml, 0.3 ml
of the final cell suspension was pipetted out into each culture well, lid
was replaced, wrapped with plastic wrap and was placed in a plastic
 23

chamber at 25°C. After 2 weeks of interval, 1 drop was removed using a

sterile Pasteur pipette to a microscope slide and examined under a phase
contrast standard 18 microscope (Carl Zeiss, West Germany). About 200 ul
of the materials were transferred from the positive well onto another
modified glycerol beef extract slants. The original well was refed and
grown (Lefkovits 1979).

b. Isolation of a single organism

In order to get a pure line culture of a species, a single flagellate
was transferred into a fresh culture tube containing medium. Micro pipettes
were used for transferring the flagellates

1.1.1.3. Different media used for the cultivation, growth studies and
maintenance
The organisms were routinely maintained in diphasic modified
glycerol beef extract medium. For the study of cultivation and growth
characteristics four different diphasic media were used. The media were
modified glycerol beef extract medium, SNB9 medium, McConnell's medium
and modified Tobie's medium.

a. Modified glycerol beef extract medium

The composition and the method of the preparation of this medium
was same as described as above.

b. SNB9 medium
This medium was originally devised by Diamond and Herman (1954).
The solid phase of the medium contained Neopeptone (Difco) 9 gm, NaCI
2.5 gm, agar 9 gm, distilled water 450 ml. The solid ingredients were
dissolved in distilled water with heat, 5 ml were put in each tube,
autoclaved and then kept at an inclined position for the preparation of the
slant.

The liquid phase was a broth of the same composition as the medium
above except agar. This liquid phase was added to the slanted agar tube.
 24

c. McConnell's medium
McConnell's medium was prepared according to the method of
McConnell (1963). At first for the preparation of solid phase bacto beef
(Oxoid) 2.5 gms were dissolved in 100 ml distilled water, boiled and
filtered through filter paper. Then Neopeptone (Difco) 2 gm, NaCI 0.5 gm
and agar (Difco) 2.0 gm were added and autoclaved. Five ml of the above
medium were poured into each sterile culture tube and kept at an inclined
position for the preparation of the slant.

The liquid phase was a broth of the same composition as the solid
medium but without agar.

d. Modified Tobie's medium

Modified Tobies medium was different from that of Tobie et^ a]_.
(1950). The composition of the medium and the method of it's preparation
was according to Ghosh et_ al_. (1987). The compositions of the solid phase
were NaCI 0.5 gm, bacto beef (Oxoid) 5 gm, peptone (Oxoid) 2 gm, Agar
(Difco) 1.5 gm.

The liquid phase was composed of NaCI 0.8 gm, KCI 0.02 gm, CaCI2
0.02 gm, Kf^PO^ 0.03 gm and glucose 0.5 gm. Both solid and liquid phase
were autoclaved separately. It may be worth mentioning here that glucose
of the liquid phase was autoclaved separately. Glucose was added to the
rest part of the liquid phase and overlaid on the solid agar medium.

pH
The pH of all the above media were adjusted to 6.8-7.0 with IN
NaOH or IN HCI.

Antibiotics
Streptomycin (Gibco) 100 Ug/ml and Penicillin (Gibco) 100 units/ml
of media •> were used as antibiotics in all the medium for routine
cultivation and growth study. Sometimes 5-fluorocytosine (Fluka Ag) 200
yg/ml of media was also used as the antifungal agent.

Inoculation of the media

0
All the media were inoculated with 10 organisms per 5 ml which
were subcultured 370 times on modified glycerol beef extract medium over
a 4 years period.
 25

1.1.1.4. Growth experiment

Three different growth experiments were carried out with each

medium. The number of organisms were determined by counting directly
under standard 18 microscope (Carl Zeiss, West Germany) using Neubauer
haemocytometer (Fein-Optic, Blankenburg, East Germany).

1.1.1.5. Effect of pH
To study the growth parameter, effect of pH on glycerol beef
extract medium was studied. To find out the optimal pH, the range of pH
selected were 5, 5.5, 6, 6.5, 7, 7.5 and 8.

1.1.1.6. Effect of temperature

For studying the effect of temperature all the four selected media
were used. To determine the range of tolerance following temperatures were
applied: 20°C, 25°C, 28°C, 32°C and 35°C.

1.1.2. Herpetomonas indica n.sp.

1.1.2.1. Isolation

A large number of Chrysomya megacephala (Calliphoridae : Diptera)

(Figure 2) were collected from different parts of West Bengal. The insects
were anaesthetized with ether and kept in Benzalkonium chloride (Sigma)
solution (1 :1000 w/v) for 5 minutes,then washed thrice with sterile
distilled water. The gut was removed asceptically in 0.1M NaCI solution
and examined under microscope for the presence of flagellates. The
flagellates were found attached to the gut wall. The gut, contained
flagellates, was teased and small pieces were transferred to tubes
containing diphasic NNN medium (Novy and MacNeal, 1904; Nicolle, 1908).
Streptomycin (Gibco) 200 yg/ml and Penicillin (Gibco) 200 unit/ml of media
were used during isolation. The pH of the medium was 7.0-7.2. The
medium was incubated at 25°C temperature.

1.1.2.2. Cloning
Cloning was done as described above for Bodo indica n.sp. In case
of Herpetomonas indica n.sp. instead of modified glycerol beef extract
medium NNN medium was used.
 26

1.1.2.3. Different media used for the cultivation, growth studies and
maintenance
The isolated organisms were routinely maintained in modified Ray's
medium {Ray, 1932). Here also four diphasic media such as SNB9 medium,
McConnell's medium, modified glycerol beef extract medium and modified
Tobie's medium were used to study the cultivation and growth. One liquid
{semi defined) medium i.e., Schneider's medium was used to study the
cultivation and growth.

a. Ray's medium
About 2 kg beef heart, purchased from local slaughter house, was
pulverized and suspended in 1 litre of double distilled water and cooked
in a pressure cooker for 30 minutes. The supernatant was filtered and
further autoclaved at 15 lbs for 15 minutes. The supernatant was frozen
until use. Agar agar 2 gm (used for Drosophila culture medium) was cut
into small pieces and soaked in large amount of distilled water at 4°C for
overnight. Next morning the water was removed from the flask and the
semidried agar was mixed with freshly prepared beef heart infusion 50
ml, NaCI 0.6 gm, asparagin (Sigma) 1 gm, lablemco powder (Oxoid) 2 gm
and peptone (Oxoid) 2 gm. The pH of the medium was adjusted to 7.0-7.2
with IN HCl or IN NaOH. The medium was then steam sterilized for 45
minutes in an autoclave continuously 3 times with a gap period of 8-9
hours. Then it was mixed with 3.3 ml defibrinated rabbit blood and
pouring into the tube which were kept in slant condition. Streptomycin
(Cibco) 100 yg/ml and Penicillin (Gibco) 100 unit/ml of media were used.
For checking the sterility slants were kept at 37°C for 48 hours and then
preserved at 4°C until use.

b. Modified glycerol beef extract medium

In addition to ingredients described earlier defibrinated rabbit
blood 10% was added to the solid phase of the medium.

c. SNB9 medium
The composition was same as described for Bodo indica n.sp. In
addition defibrinated rabbit blood 20% was added to the solid phase of
the medium.
 27

d. McConnell's medium
The method and compositions of this medium were same as described
earlier. Here defibrinated rabbit blood 15% was added to the solid phase
of the medium.

e. Modified Tobie's medium

The ingredients required for preparing this medium were also same
as described earlier. In addition defibrinated rabbit blood 10% was added
to solid phase of the medium.

pH

The pH of all the above media were adjusted to 7.0-7.2 with IN NaOH
or IN HC1.

f. Schneider's medium
The medium was prepared according to the method described by
Hendricks et_ aj_. (1979). Heat inactivated FCS (Gibco) 30% was added
ascepticaily to Schneider's tissue culture medium. The whole material was
filter sterilized through the 0.22 pm millipore nitrocellulose filter paper.

Antibiotics :
Streptomycin (Gibco) and Penicillin (Gibco) at same concentration of
previous study, were used. Sometimes antifungal agent, 5 Flurocytosine,
(Fluka AG) was used at a concentration of 20 Ug/ml of media.

Inoculation of the media

6
All the media were inoculated with 1x10 organisms per ml which
were subcultured 190 times on modified Tobie's medium for a 2 year
period.

1.1.2.4. Growth experiment

Three different growth experiments were carried out with each
medium. Using Neubauer haemocytometer (Fein Optic, Blankenburg, GDR) the
number of organisms were determined by counting under standard 18
microscope(Carl Zeiss, West Germany).
 28

1.1.2.5. Effect of pH
To determine the optimal pH, the range of pH used were 5, 6, 7
and 8.

1.1.2.6. Effect of temperature

All the four selected media were used to study the effect of
temperature. The range of temperature used were 20°C, 25°C 28°C, 32°C
and 35°C.

1.1.3. Leishmania donovani

1.1.3.1. Isolation
Leishmania donovani was isolated from kala-azar patient by bone
marrow aspiration.

Sites
Second piece of the body of the sternum.

Iliac crest at the region of posterior superior iliac spine.

Other sites like the tibial tuberosity or the spinal process of the
vertebrae were not routinely used. The iliac crest was a preferred site,
especially in children.

Process
The patient was advised to lie on the bed upside down. Good
exposure of the site was made. The posterior superior iliac spine was
located. The skin over the spot was cleaned with antiseptics as follows :
spirit, iodine, spirit. Lignocaine (2-3 drops) was drawn into a sterile 5
cc syringe and a sterile 22 gauze needle was fixed to it. With this the
skin was anaesthetised and then the needle was pushed further till it
touches the periosteum. While withdrawing the needle, Lignocaine is
pushed into the periosteum and the soft tissue, so that the area was well
anaesthetised within 2-3 minutes. The syringe was removed keeping the
needle in place as a marker for the sites of insertion of the bone marrow
aspiration. The bone marrow aspiration needle was slowly inserted from
the site of puncture of the skin with a boring movement of the wrist. A
loss of resistance denoted that the needle had entered the marrow cavity
 29

(the guard of the needle was adjusted prior to insertion to avoid the much
penetration). The stiliete was taken out and its tip was rubbed on a piece
of cotton so that the marrow materials were visualized. A 5 cc glass
syringe was attached to the opening of the needle tightly and suction was
applied. With entry of the marrow insight the needle and syringe, the
patient experienced pain. Just at this point the needle and syringe both
were removed. The marrow material was inoculated into the tubes
containing modified Tobie's medium as it showed higher rate of isolation
(Ghosh et^ al_., 1987) and kept at 25°C. The site of puncture was sealed
with Benzene. The patient was advised to take rest on bed and given
analgesics.

1.1.3.2. Cloning
For cloning NNN medium was used. The method was similar as
described earlier.

1.1.3.3. Different media used for cultivation, growth studies and

maintenance
The media used for studying the cultivation and growth of L.
donovani were same as also used for H. indica n.sp.

1.2. RESULTS

In modern parasitological research for characterization

(morphological, biochemical and immunological) it is always encouraged to
isolate and cultivate the organisms/strains. Success of isolation process of
the strains/organisms depends primarily on the choice of media, pH of the
media and incubating temperature which help to adapt the strains from one
environment (jn^ vivo i.e., insect gut) to another (|£ vitro).

1.2.1. Bodo indica n.sp.

1.2.1.1. Isolation
Twenty specimens of Lethocerus indicus out of 110 dissected between
April 1985 and November 1986 were found infected with flagellates. The
rate of infection seemed to be high during rainy season. Out of 8 isolates
inoculated into culture one was successfully subcultured 370 times and
maintained over a period of four years.
 30

1.2.1.2. Cloning
The isolate was cloned 5 times, twice by serial dilution and thrice
by isolation of a single organisms with a capillary pipette. Each clone
was made from a stock which had previously been cloned.

1.2.1.3. 2E vitro cultivation in different media

The results obtained for the growth of B. indica n.sp. in various
media are plotted in Figure 3. All the media exhibit highly efficient
growth within 4 days of culture. Using modified glycerol beef extract
medium B. indica n.sp. was subcultured continuously at 4-6 days'
interval. Concentration of cells were 5.5xl06/ml on 4th day. In SNB9
medium, McConnell's medium and modified Tobie's medium maximum growth
was found on 4th day but the number of cells was higher in SNB9 medium
than McConnell's medium and modified Tobie's medium.

1.2.1.4. Effect of pH
The pH 6.8-7.0 was found most suitable for the growth of B. indica
n.sp. However, the organisms can grow both in acidic pH (5.0) and
alkaline pH (8.0), but the number of organisms were poor.

1.2.1.5. Effect of temperature

Effect of temperature experiments were initiated with organisms after
its subculture 370th on modified glycerol beef extract medium.

a. Cultivation at 25°C
It was found that E3. indica n.sp. grew abundantly at 25°C. Three
different shapes in following proportions were found : carrot shape 30-
32%, sausage shape 65-70%, oval or round shape 2-3%. Initial latent period
of growth was found between 24-48 hours, followed by an exponential
phase of growth and a stationary and then declining phase which was
commenced on day 5. The doubling times during log phase were between 12
and 24 hours, depending on the medium.

b. Cultivation at 20°C
Theproportional distribution of the three shapes in each of 10
serial passages in modified Tobie's medium, McConnell's medium and SNB9
medium varied little from those observed in modified glycerol beef extract
Figure 3. Growth curve of Bodo indica n.sp. on modified glycerol beef
extract, McConnell's, modified Tobie's and SNB9 media at
25°C. Each point is the average of four culture tubes. The
bars represent standard deviation ---- • : Modified glycerol
beef extract medium,a --- a: McConnell's medium, o - — -o !
Tobie's medium,®----- s:SNB9 medium.
 31

medium, the vast majority being sausage shape. The organisms reached log
phase within 24 hours. The declining phase commenced0*^ hours.

c. Cultivation at 28°C
In each of 10 serial passages in four media, three different shapes
were found. The percentages of different forms were as follows : carrot
shape 40-42%, sausage shape 55-57%, round or oval shape 2-3%. The
organisms reached the log phase within 60 hours. The declining phase
commenced after 84 hours.

d. Cultivation at 32°C
In each of 10 serial passages in four different media, three
different shapes were observed. But the percentage of round or oval (33-
35%) and carrot shape (50-55%) were more prevalent than sausage shape
(10-15%). The organisms reached at log phase on 36 hours and started to
decline on 48 hours.

e. Cultivation at 35°C
At 35°C only one form i.e., round form was found. Within three
hours of incubation all the organisms became round. The organisms
survived for 24 hours at 35°C.

1.2.2. Herpetomonas indica n.sp.

1.2.2.1. Isolation
Out of 800 dissected Chrysomya megacephala between June 1984 and
December 1986 only 10 C. megacephala were found infected with flagellates
(H. indica n.sp). The infection seemed unrelated to climatic conditions, as
the infected flies were collected during winter and rainy season. Out of
seven isolates inoculated into culture, three were successfully subcultured
182 times and maintained over a period of 4 years.

1.2.2.2. Cloning
The isolates were cloned 5 times, thrice by serial dilution and
twice by isolation of a single organisms with a capillary pipette. Each
clone was made from a stock which had previously been cloned.
 32

1.2.2.3. Jn vitro cultivation in different media

6
The number of organisms was maximum in Tobie's
medium (17x10 )
6
followed by modified glycerol beef extract medium (15.5x10 ), McConnell's
medium (14.5x10®) and then SNB9 medium (14.25x10®) (Figure 4). The

organisms reached at log phase within 6th day.

1.2.2.4. Effect of pH

The optimal pH for the growth of H. indica n.sp. was found to be

7.2. Herpetomonas indica n.sp. could also grow at pH 6.9 (slightly acidic)
and at pH 7.4 (slightly alkaline). But the number of organisms was less.

1.2.2.5. Effect of temperature

The experiments of temperature effect were initiated with the orga­

nisms after its 370th subcultured on modified Ray's medium.

a. Cultivation at 25°C

Herpetomonas indica n.sp. grew abundantly at 25°C. At 25°C four

different forms were observed in each of 10 serial passages. They were
promastigote, paramastigote, opisthomastigote and round form. These forms
were found in different proportion, such as, promastigote 45-50%,
paramastigote 40-45%, opisthomastigote 8-15%, round 1-2%. It was noted
that the number of opisthomastigote increased double at the end of the
exponential growth phase. In Schneider's medium the number of organisms
were very high but after 3 serial passages the number of organisms
gradually reduced. Initial latent period of growth was found between 48-60
hours, followed by an exponential phase of growth and a stationary and
then declining phase which commenced after day 7. The doubling times
during log phase were between 18 and 36 hours depending on medium.

b. Cultivation at 20°C
The proportional distribution of four different forms in each of 10
serial passages in modified Tobie's medium, McConnell's medium, SNB9
medium and Schneider's medium varied little from those observed in
glycerol beef extract medium. The vast majority being promastigote form.
The organisms reached at log phase within 96 hours. The declining phase
commenced after 120 hours.
Figure 4. Growth curve of H- indica n.sp. on modified glycerol beef
extract, McConnell's, modified Tobie's, SNB9 and Schneider's
media at 25°C. Each point is the average of four culture
tubes. The bars represent standard deviation O - -o :
Modified glycerol beef extract medium, O • O : McConnell's
medium,®--------- # : Modified Tobie's medium,A------- A : SNB9
medium, o-------------- ----- '• Schneider's medium.
 33

c. Cultivation at 28°C

In each of 10 serial passages in four diphasic media, four different

forms were found. In Schneider's medium there were round forms 25%
whereas all the other diphasic media showed round form 5-6%, paramasti-
gote form 33-36%, promastigote form 48-50% and opisthomastigote form 8-
12%. The organisms reached at log phase within 96 hours and the declining
phase started after 120 hours.

d. Cultivation at 32°C

At 32°C four different forms were also found in each 10 serial

passages. The percentages of round forms increased (30-32%) than
paramastigote forms 28-30%, promastigote 28-30%, opisthomastigote 1-2%.
Maximum number of cells were observed within 72 hours and the declining
phase started after 96 hours.

e. Cultivation at 35°C

Organisms cultured at 35°C yielded similar growth patterns.

Remarkably high proportions (90%) of round forms appeared in culture.

1.2.3. Leishmania donovani

1.2.3.1. Isolation

Out of the 15 clinically suspected patients, the microscopic

examination of 8 (53.33%) patients revealed the presence of the amastigotes
in smear imprints. These 8 positive samples were tried for isolation
process. Out of 8 isolates inoculated into culture, one (1) was successfully
subcultured 182 times and maintained for a period of over 4 years.

1.2.3.2. Cloning

The isolate was cloned 5 times, twice by serial dilution and thrice
by isolation of a single organisms with a capillary pipette. Each clone
was made from a stock which had previously been cloned.

1.2.3.3. Jn vitro cultivation in different media

The results of the vitro characteristics of L. donovani in

different media (both monophasic and di~phasic) indicated that diphasic
Tobie's medium was the best for the in vitro cultivation of L. donovani
 34

(Figure 5). Leishmania donovani multiplied more than 7 folds after 6th
days of inoculation in diphasic Tobie's medium. Next to modified Tobies,
McConnell's medium was recommended for the In vitro propagation of L.
donovani. All other media including SNB9 medium, modified glycerol beef
extract medium and Schneider's medium were not sufficient for the in
vitro propagation of the parasites in laboratory condition.

1.3. DISCUSSION

The selection of a reliable medium for the primary isolation and

consequently for routine cultivation of parasites is an essential requirement
for any type of research work on laboratory stock culture. In the present
study an attempt has been made to cultivate B. indica n.sp., H. indica
n.sp. and L. donovani. The purpose of the present study is to enumerate
the sensitivity and efficiency of the different media which are already
present in literature with their original constituents as well as with some
modifications.

Small changes in the composition or preparation of media can have

drastic effect on the growth of the parasites (Shaw and Lainson, 1981;
Marin et^ aL, 1982). The results of our study also support this view.

All the diphasic media used in the present study are suitable for
the maintenance of B. indica n.sp., H. indica n.sp. and L. donovani.

The comparative mass cultivation studies show that the highest cell
densities of B. indica n.sp. are obtained in modified glycerol beef extract
medium whereas H. indica n.sp. and L. donovani show maximum growth in
modified Tobie's medium. As the composition of glycerol beef extract
medium and McConnell's medium is almost similar except glycerol it can be
conjectured that glycerol plays an important role for the growth of B.
indica n.sp. The highest growth for hi vitro cultivation of B. indica
n.sp. in SNB9 medium, than McConnell's medium or modified Tobie's medium
indicate that peptone is more important than bacto beef. As the growth in
modified Tobie's medium is not so satisfactory it can be presumed that
glucose or other components in overlay are not necessary for the growth
of B. indica n.sp. So it can be concluded that peptone and glycerol are
most important factors for growth of B. indica n.sp.
Figure 5. Growth curve of Leishmania donovani on modified glycerol beef
extract, McConnell's, modified Tobie's, SNB9 and Schneider's
media at 25°C. Each point is the average of four culture
tubes. The bars represent standard deviation,# — •* :
* Modified glycerol beef extract medium,o----------O : McConnell's
medium,#---------- • : Modified Tobie's medium,#-• — - -*:SNB9 medium
*---------- * ; Schneider's medium.
 35

Results of vitro cultivation of H. indica n.sp. and L. donovani

show that increasing amount of defibrinated rabbit blood (in McConnell's
medium and SNB9 medium) did not support the growth of parasites after
long period of incubation. Original Tobie's medium with slight modification
supports the growth of H. indica n.sp. and L. donovani. So it can be
presumed that glucose or other components in overlayare necessary
specially for the growth of L. donovani and also for H. indica n.sp;

Insect tissue culture media were used to culture Leishmania

promastigotes (Childs et^ al_., 1978; Hendricks and Wright, 1979). All of
the species including membersof L. donovani complex and L. b.
braziliensis could be sufficiently subcultured in this tissue culture
medium. Schneider's medium with 20% FCS appeared to give the best
results (Childs et^ aL, 1978). Maximum yields of promastigotes form
almost all species exceeded 10/mi. But the present study with L.
donovani did not support the above observation. These different results
may be due to the difference of strain. However, H. indica n.sp. showed
enormous growth in Schneider's medium.

Both L. donovani and H. indica n.sp. are now maintained in modified

Ray's medium. This medium is used as it requires minimum amount of
blood (3.3%). It yields maximum number of cells and it is economical.
Freshly prepared beef heart infusion (50% v/v) has some important factors
which ultimately increase the growth with the lowering of the generation
time. The continued development and application of improved agar culture
techniques for the quantitation of Leishmania and Herpetomonas will lead to
more meaningful data on the host-parasite relationships.