Thawing of deep-frozen parasites

Preparation of thawing solutions

Solution A: Add 12g of Nacl in 100ml millipore water

Solution B : Add 1.6 gram of Nacl in 100ml Millipore water

Solution C : mix solutions of 0.9% Nacl and 0.2% gram of dextrose

Sterilise by filteration

Sample is transferred to sterile centrifuge tube

Measure the volume of blood present in the sample

Add 0.2 ml of solution A for every 1 ml of thawed blood drop by drop constantly

Allow the tube to stand for 5 minutes

Add solution B drop by drop using 10ml for each 1ml f thawed blood

Add solution as mentioned above

Centrifuge at 2000rpm for 5 minutes

Remove the supernatant

 Resuspend cells in RP-C to provide 3-5% Haematocrit

RP-InComplete medium
Dissolve16.2 gram of powdered RPMI1640(WITHOUT SODIUMBICARBONATE AND WITH L-GLUTAMINE)

In 900ml of culture grade water in 1000ml graduated beaker

Place it in magnetic stirrer for 20-30 minutes

Make the volume to 960ml with Millipore water

Sterilize through filtration

Aliquot into 250ml or 500ml medium bottles

RP-Complete medium

Add 4.2ml of 5% sodiumbicarbonate solution to RP-I 100ml

Add 5ml 8% albumax stock to RP-I 100ml

STOCK SOLUTIONS
Dissolve 5gram sodium bicarbonate in 100ml Millipore water

8% Albumax

Dissolve 8gram Albumax and 50mg Hypoxanthine in 100ml RPMI 1640

Filter sterilize the stock solutions

Aliquot in 5ml and 25ml Volume and store at -20C

Wash the erythrocytes 3 times in RP-I 1640 to remove CPD, serum, and
leukocytes if present.

Dilute to 5% hematocrit with cMCM in small flasks of 25

cm2 (0.2 mL of packed cells to 4 mL of cMCM) or in 75-cm2 flasks (1.0 mL to 20
mL).

Add parasites to an appropriate parasitemia (see below).

Put the flask in a candle jar and loosen the screw cap. Produce low oxygen by
burnt out candle and place the jar at 37 °C.(5%CO2 , 94%N2 , 1%O2)

Replace the MCM every day (not necessary the day after subcultivation).
Subculture the cultures 2 times/week.

SUBCULTURING

Parasites are grown in human erythrocytes in a settled layer of cells in RP-C at 37C in flat
bottomed containers under low O2, balanced N2 with 24 hour medium changes

Fresh erythrocytes should be added every 4-5 days regardless of parasitemia

If initial patient parasitemia is high (>0.4%), change the medium the next day,
otherwise leave it for 48 to 72 h before the first change of culture medium.

Subcultivate to keep parasitemia below 1 to 2% by adding fresh

uninfected RBC.

Washed uninfected RBC for subculture should be kept in the refrigerator for 24 h
before first usage (to discourage any remaining leukocytes)
 Split the cultures every 2 days . Remove the medium and divide the cellular
contents of plate to old and new 100-mm petridish

Add 100µL of freshly washed erythrocytes and 12mL of RP-C to each dish

Transfer erythrocytes to 15-mL centrifuge tube,spin 400g for 5 minutes

Discard plasma and buffy coat, wash cells twice in 10 Vol of RP-I

Suspend at ratio of 1:1 in RP-C

Add parasites from cultures to freshly washed erythrocytes to give a initial

parasitemia of 0.1-1.0%

( 100-mm petridish contain 200-250µL of packed cells and sufficient RP-C to give 3-
5%Hematocrit.parasitemia increases eight times during first 48 hours and 3-4 over second 48-h
interval)

For a parasitemia above 12% medium should be changed every 12-h.

Culture synchronization using sorbitol

Wash the culture twice with 5% aqueous sorbitol at 37C and once with RP-C

Suspend parsites from cultures in 3-4 volume of 15% D-sorbitol at 37C for 5 minutes

Dilute with 7 volume of 0.1% glucose to give a final concentration of 5% sorbitol

Vortex, incubate another 5 minutes and centrifuge at 1000g

Wash the cultures twice with RP-C

Sorbitol treatment for second time 27h later that will create young rings

GELATIN FLOATATION TREATMENT

Wash cultures once in RP-I then suspend either in 4 –volume of gelatin solution

Mix with 1:1 RP-C

The above procedures will eliminate ring infected erythrocytes

Uninfected and ring infected erythrocytes will settle in 20-30min at 37C leaving a suspension

Pipet the supernatant from the settled cells and recover parasitized erthrocyte by centrifuging at
300-400g

Second gelatin treatment of settled cells