Cultivation Of Parasites

gray-black gray-black

The cultivation of parasites has proven to be a beneficial technique in the study of human parasitology. The time, effort, and money spent in these techniques have prevented the methodology from being utilized in the routine medical parasitology laboratory. Teaching institutions have a constant need for study material, and therefore, have more of a need for the procedures enumerated below.

a. Boeck and Drbohlav's Medium (modified). This is a semi-solid egg-based medium that requires an overlay with Locke's solution. It is used for most intestinal protozoans (excluded: Balantidium coli and Giardia lamblia).

(a) Locke's solution (modified).

Sodium chloride (NaCl) 8.0 gm

Calcium chloride (CaCl22H20) 0.2 gm

Potassium chloride (KCl) 0.2 gm

Magnesium chloride (MgCh6H20) 0.01 gm

Disodium hydrogen phosphate (Na2HPO4) 2.0 gm

Sodium bicarbonate (NaHCO3) 0.4 gm

Potassium dihydrogen phosphate (KH2PO4) 0.3 gm

Distilled water 1,000.0 ml

1 Add the salts in the order listed to 500 milliliters of distilled water in a 2,000 milliliter Erlenmeyer flask and mix well. Slowly add the remaining 500 milliliters of water and mix until all the salts are dissolved.

2 Boil the solution for 10 minutes (a white precipitate should be formed).

3 Allow to cool at room temperature, filter, and autoclave for 15 minutes at 121° C (15 pounds).

Fresh eggs (medium-large) Locke's solution

4 ea 50 ml

1 Emulsify the mixture in a large Erlenmeyer flask containing glass beads or in a blender.

2 Filter through a double layer of gauze and dispense into 16 by 125 millimeter screw cap test tubes in seven to eight milliliter aliquots.

3 Place the tubes in slant racks and inspissate or autoclave (with all exhaust valves closed) for 15 minutes at 15 pounds of pressure until the media gels.

NOTE: The pressure must be allowed to escape from the autoclave at a very low rate. A sudden drop of pressure causes bubbles and unsettles the media.

4 Allow slants to cool and overlay with two or three milliliters of sterile Locke's solution under aseptic technique.

5 Incubate overnight at 35° C and check for sterility. Discard all tubes that show a turbidity in the overlay layer of liquid.

b. Egg Yolk-Infusion: (Balamuth, 1946; modified). The liquid Egg Yolk-Infusion medium has liver added and it is excellent for the cultivation of lumen protozoans. This medium has the added advantage of slow deterioration when stored for up to two months.

(a) Liver Extract Solution.

1 Dissolve 5.0 gm of Dry Extract of Liver (Wilson's) in 1000 ml of distilled water and heat to boiling.

6 Store in the refrigerator. Discard the unused media after a month of storage.

2 Filter through filter paper and sterilize at 121_for 15 minutes.

(b) Buffer solutions.

1 Stock solutions (1M).

2 Dihydrogen Phosphate solution (SOLUTION A)--dissolve 13.6 gm of KH2PO4 in 100 ml of distilled water and store in a dark glass bottle.

3 Dibasic Phosphate Solution (SOLUTION B)--dissolve 87.1 gm of K2HPO4 in 500 ml of distilled water and store in a dark glass bottle.

4 Working Solution (M/15 buffer)--mix 7.0 ml of solution "A" with 43.0 ml of solution "B" and add to 700 ml of distilled water. This solution should be prepared just prior to use.

(2) Preparation.

(a) Into 250 ml of Normal Saline, crumble the yolks of 8 hard boiled eggs and mix with a beater, mixer, or blender; or add 144 gm of dehydrated egg yolk to 144 ml of distilled water and 500 ml of Normal Saline, and mix with a beater, mixer, or blender.

(b) Heat in a double boiler for 20 minutes, and add 60 ml of distilled water (compensation for evaporation).

(c) Filter: for the fresh egg solution use a Buchner funnel with reduced pressure and two layers of #2 Whatman filter paper; for the dehydrated egg solution use a muslin bag and squeeze gently (this solution is too pulpy to filter through a Buchner funnel).

(e) Sterilize at 121, for 15 minutes.

(f) Refrigerate fresh egg solution until it reaches a temperature of 8° C. Refrigerate the dehydrated egg solution for 24 hours, until a yellow-colored sediment is formed.

(g) Filter through a Buchner funnel with reduced pressure and two layers of #2 Whatman filter paper.

(h) Add an equal amount of the Working Buffer Solution (M/15 Buffer) to complete the egg infusion, and measure the total amount.

(i) Add 0.5 ml of liver extract for every 100 ml of infusion.

(j) Dispense into 16 x 125 mm screw cap test tubes in 7 to 8 ml aliquots, and sterilize for 15 minutes at 121° C.

(k) Store in the refrigerator.

(3) Inoculation, Incubation and Processing-use the same procedure as for Boeck and Drbohlav's Medium.

c. Liver Infusion Solid Medium: (Cleveland and Collier, Cleveland and Sanders, 1930). Entamoeba histolytica grows well in this medium while the other intestinal protozoans do not. Therefore, it is useful as a selective medium and frequently used in metabolic research laboratories.

d. Charcoal: (McQuay, 1956). This is a modified mycobacterial medium which is excellent for the isolation and storage of amoebic organisms (e.g., Entamoeba hartmanii, Dientamoeba fragilis, and to some extent Entamoeba histolytica). The charcoal medium can be prepared as semi-solid slanted medium or in a liquid form.

+1 0

Post a comment