This document describes culture methods for cultivating various protozoan parasites. It discusses the purposes of culturing parasites, including for diagnostic, research, and teaching purposes. It provides examples of parasite species that can be cultured, such as Entamoeba histolytica, Giardia lamblia, and Plasmodium spp. The document outlines different types of culture media, including xenic, polyxenic, monoxenic, and axenic cultures. It also describes specific culture media and methods used for cultivating intestinal protozoa like amoebae, as well as haematozoan parasites including Leishmania and trypanosomes.

1. CULTURE METHOD FOR PROTOZAL PARASITE
Pradip Hamal

2. CULTURE METHODS
• In most of the parasitic infections, culture is not a routine identification
technique.
• Very few clinical laboratories offer specific culture techniques for parasites..
• Parasite cultivation is a tricky task, which requires expertise and knowledge of
all kinds of microbiological cultures.
• It is sometimes employed for accurate identification of the parasite species.
• It is more often employed for obtaining large yields of the parasite as a source
of antigen, animal inoculation, drug sensitivity testing, experimental or
physiological studies, and teaching purposes.

3. Purpose or Uses of Culture for Parasites
• For the diagnostic of parasites in sample.
• Serological study
• For the research purpose
• Maintaining the strain of parasites
• Preparation of antigen
• Epidemiological study
• For the teaching purpose

4. Parasites which can cultures in laboratory
• Entamoeba histolytica
• Naegleria fowleri
• Acanthamoeba spp.
• Giardia lamblia
• Trichomonas vaginalis
• Toxoplasma gondii
• Leishmania spp.
• Trypanosoma spp.
• Balantidium coli
• Plasmodium spp.

5. Types of Culture media
• Xenic culture - It refers to culture of parasites grown in association with other
unknown microbial associates, for example stool specimens cultured for E.
histolytica in National Institute of Health medium (NIH). Microbial associates are
known to provide various nutrients required for the growth of parasites. All microbial
associates, however, are not beneficial. It is used for primary growth of parasites.
• Polyxenic culture - Cultivation of parasites in associates with many known
microorganisms.
• Monoxenic culture - If the parasites are grown with a single known bacterium, the
culture is referred to as monoxenic, for example corneal biopsy specimens cultured
with Escherichia coli as a means of recovering species of Acanthamoeba. It can be
used for primary growth as well as a transitional phase in isolation.
• Axenic culture - It is a pure culture without any bacterial associate or any other
metabolizing cells. It is mainly used as isolation medium for the parasites, but can be
used for primary growth also, for example TYI-S-33 medium in case of T. vaginalis. It
is very difficult to achieve and maintain in the laboratories.
• Axenic cultures was first developed by Diamond in 1961. Axenic cultivation has
enabled precise antigenic and biochemical studies on amoebae.

6. Cultivation of Amoebae
1. Balamuth’s Aqueous Egg Yolk Infusion Medium:
• This medium is used to detect the presence of amoebae.
• The specific solutions required are phosphate buffer and whole liver concentrate solutions
• Balamuth’s monophasic liquid medium is also used commonly for cultivation of amoebae and
other intestinal protozoa.
• This is an egg yolk-liver extract infusion medium.
Composition of balamuth’s medium
• Liver concentrate powder: 1 part
• Egg yolk medium: 9 parts
Phosphate buffer
A. Tribasic potassium phosphate B. Monobasic potassium
(K3PO4): 212.27 gm Phosphate (KH2PO4): 136.092 gm
Distilled water: 1000 ml Distilled water: 1000ml

7. Cont……
• Mix the solutions in the ratio of 3 parts tribasic (A) to 2 parts monobasic
(B) to make 1 M phosphate buffer stock.
• Dilute the stock buffer to 0.067 M before use (add 492 ml distilled water to
1 lit mPO4 buffer).
Whole liver concentrate solution.
• Liver concentration powder (Wilson or Lilly): 5 gm
• Distilled water: 100 ml
Suspended the power in cold water and autoclave. Filter through a Buchner
funnel to remove, dispense in 10 ml quantities and reautoclave.

8. Procedure for culture of amoebae
• Tubes should contain 6 to 8 ml of fluid and should be incubated for 4
days at 37ᴼC as a sterility check.
• Before inoculation, a loopful of sterile rice power or starch is added to
each tube.
• To each tube, add stool material (about the size of a small pea), break
it up thoroughly in the medium and incubate at 37ᴼC.
• The culture should be checked at 2, 3 and 4 days by examing 0.1 ml of
sediment under the microscope (low intensity light) for characteristic
motility. Although the initial culture may appear to be negative,
subcultures may reveal organisms.

9. 2. Boeck and Drbohlav’s Locke –Egg Serum (LES) medium
• This is another culture medium used to diagnosis of amoebae.
• The medium as used now, is basically an egg slant, with an overlay of
sterile serum or liver extract in buffered saline.
• A loopful of sterile rice powder is added to the medium just before
inoculation with fresh feces or its saline centrifugal sediment.
• Cultures can be obtained from feces containing cysts or trophozoites.
• The cultures are incubated at 37° C and subculture at 48-hour intervals.
• Amoebae can be demonstrated in the liquid phase in unstained mounts or
stained smears.

10. Locke’s Solution (Autoclave before storage)
• Nacl: 9 gm
• CaCl2: 0.2 gm
• KCL: 0.4 gm
• NaHCO3: 9.0 gm
• Glucose: 2.5 gm
• Distilled water: 1000 ml

11. Preparation of complete medium
• Wash 4 eggs, wipe the shells with 70% alcohol, and break the eggs
into a sterile flask containing glass beads.
• Add 50 ml of Locke’s solution and shake until homogenous.
• Dispense the medium so that a slant of 1 to 1 inches is produced in the
bottom of the tube.
• Plug the tubes and place them in a slant position in an inspissator at
70C until the slant solidifies.
• The tubes can then be autoclaved at 15 lb pressure for 20 minutes.
Discard any damaged slants.

12. Cont..
• Prepare a mixture of 8 parts sterile Locke’s solution to 1 part inactivated
human serum.
• Sterilize the mixture by filtration and incubated at 37ᴼC for 24 to 48 hrs as a
sterility check before use. Cover the slants to a depth of 1 cm with the sterile
solution and inoculate in the same manner as for Balamuth’s medium. LES
medium should have a loopful of sterile rice powder added before
inoculation.
• Antibiotics are also added to the medium to inhibit the over growth of
various organisms. These include; penicillin (1000units/ml) streptomycins 2
mg/ml and acriflavine (0.1 ml of 0.02%).

13. Axenic culture for Intestinal Protozoa
• TY-S-33 medium (T: Trypticase, Y: Yeast extract) for E. histolytica
Composition:
• Trypticase
• Yeast extract
• Dextrose
• NaCl
• L cystine HCL
• Ascorbic acid
• K2HPO4
• Ferric ammonium citrate
• Distilled water

14. Culture of Pathogenic Free Living Amoebae
Acanthamoeba Medium
• For the isolation of Naegleria or Acanthmoeba from tissues or soil samples, the following
procedure is recommended.
Prepare page’s Saline
• NaCl: 60 mg
• MgSO4. 7H2O: 2 mg
• CaCl2.2H2O
• Na2HPO4
• KH2PO4
• D/w: 500 ml
• Autoclave 15 psi for 15 min and store in glass bottle at 4 C.

15. • Non nutrient Agar
• Page’s saline: 100 ml
• Difco Agar: 1.5 gm
• Dissolve agar in Pages saline with gentle heating stir or swirl.
• Autoclave at 15 psi for 15 minutes, label deeps with 12 months
expiration date and store in refrigerator.
Plate Preparation
• Melt agar deeps and pour into petridishes as needed. Plates may be
stored in the refrigerator up to 3 months.

16. Culture
• Warm 2 gar plates per specimen in a 37ᴼC incubator for 30 minutes.
• Add 0.5 ml Page’s saline to a slant culture of E. coli.
• Gently scrape surface of a slant and uniformly suspended the bacteria.
• Add 2 to 3 drops of suspension to middle of warm agar plate .
• Spread the bacteria on the agar surface with a bacteriological loop.
• Inoculate 2 to 3 drops of specimen sediment (ground tissue in Page’s
saline or centrifuged CSF) in the center of the agar plate.
• Incubate one plate at 37ᴼC and one at 42ᴼC.

17. Culture examination
• Examine plates every 7 days with 10X objectives; cysts will be visible
within 4 to m days and trophozoites earlier.
• Positive areas of agar can be cutout and placed face down on new
plate coated with bacteria (Culture transfer).

18. Media for cultivation of intestinal Protozoa

19. Flow diagram illustrating the stages in establishing intestinal
prtozoal in culture

20. Media used for cultivating free living amoebae

21. Cultivation of Haemoflagellates

22. Leishmania and Trypanosomes
• NNN medium
• NNN medium was developed by Novy, McNeal and modified by
Nicolle in 1904 for cultivation of Leishmania, is equally satisfactory
for trypanosomes also.
• This is a defibrinated rabbit blood agar medium. Several modifications
of this medium have been introduced.
• NNN Modified Medium is a modification of the original medium and
consists of two phases, blood agar (Part A) and Lockes solution (Part
B).

23. Principle:
• This medium consists of a blood agar base and an overlay medium.
• The blood agar base is a highly nutritious medium that supports the
growth of fastidious organisms like Leishmania and Trypanosoma.
• The specimens are inoculated into the liquid phase of the diphasic
medium and incubated.
• This favours the development of organisms in the insect vector.
• The amastigotes transform to promastigotes in about 24 hours

24. Composition of NNN medium
Part A (Ingredients gms / litre)
• Meat extracts: 3.000
• Peptic digest of animal tissue: 5.000
• Sodium chloride: 8.000
• Agar: 15.000
• Final pH ( at 25°C) 7.3 ± 0.2
Part B (Ingredients gms / litre)
• Sodium chloride: 8.000
• Potassium chloride: 0.200
• Calcium chloride: 0.200
• Monopotassium dihydrogen
phosphate: 0.300
• Dextrose: 2.500
• Final pH ( at 25°C) 7.0 ± 0.2

25. Procedure
Part A:
• Suspend 31 grams in 1000 ml distilled water.
• Heat to boiling to dissolve the medium completely.
• Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
• Cool to 45°C and aseptically add 10% sterile defibrinated rabbit or human blood.
• Mix well and dispense in 5 ml amounts in test tubes or 25 ml amounts in flasks.
• Allow tubed media to cool in slanted position.
Part B :
• Suspend 11.2 grams of Part B in 1000 ml distilled water.
• Heat to boiling to dissolve the medium completely.
• Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
• Cool and add approximately 2 ml in tubes or 10-15 ml in flasks over solidified Part A medium.
Storage:
• Keep the prepared media refrigerated until it is used (stable for 2-4 weeks) and bring it to room
temperature right before inoculation.

26. • Inoculation:
• Inoculate the specimen (spleen aspiration or bone marrow biopsy or
lymph node aspiration) into the liquid phase of the biphasic medium.
• Incubate at 21-26°C for 4 weeks. Check for the growth of the organism
and for possible fungal contamination after 4 days and continue by 4
days intervals for next 4 weeks.
• Result:
• If positive, luxuriant growth of the organism is seen in the media.
Giemsa stain the culture filtrate and observe under microscope; free,
flagellated, Leishmania promastigote.

28. 2. Schneider’s insect tissue culture medium:
• It is recommended in vitro culture of Leishmania. This medium is said to the more
sensitive than NNN medium.
• Schneider's insect medium has been specially formulated for the in vitro culture of
Drosophila melanogaster cells and tissues.
• A number of cell lines derived from Drosophila melanogaster are now available
and are extensively used in genetic and molecular biology research. Drosophila
cells are also used to study recombinant protein expression.
Composition of Schneider’s insect tissue culture medium
• Schneider’s Drosopihila tissue culture medium: 80 mL
• Fetal calf serum: 20 mL
• Antibiotic-antimycotic solution: 1.2 mL

29. Media for cultivation of haemoflagellates parasites

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