نلجأ لزراعتها " ولكن في حالالت نادرة " للتالي:
1- عندما تفشل الطرق الروتينية العادية لتشخيص الطفيل وما تزال الاعراض ظاهرة
2- لتأكيد الفحص الروتيني.
3- لدراسة ايض الطفيليات او القيمة المرضية للطفيل
4- لتخزين الطفيلي الى وقت الحاجة اليه.
5- لتحضير عينات نقية من نوع معين من الطفيل
*بعض الاوساط الزراعية المستخدمة لاستنباط الطفيليات:
A- Schneiders enriched medium for the invitro culture of lieshmania:
Materials:
Schneider's Drosphila medium.....80 ml
Fetal calf serum.....20 ml
Antibiotic-antimycotic solution.....1.2 ml
the antibiotic -antimycotic solutin contains pencillin, streptomycin and amphotercin.
Prearation:
a. Inactivate the fetal calf serum for 30 minutes at 56 C and allow to cool.
b. Mix 20 ml of inactivated fetal calf serum with 80 ml of Schnieder's medium.
c. Add 1.2 ml of the antibiotic - antimycotic solution , mix , aspecially dispense the medium in 3 ml amounts into sterile 16X 100 mm tubes and stopper.
, d. Label and freeze at -20C . The medium can be kept for up to 1 year at -20 C
it can be kept for up to 6 weeks at 4-6 C .
NOTE:The medium should be prepared in aseptic
working comditions..
Use: A-Warm 2 tubes of medium to room teperator .
B- Incubate eash tube with 0.1 ml of the specimen.
C- INCUBATE THE CULTURES at 24 C (_+2C) in the dark for up to 14 days . Room temporator is usually suitable.
D. Examine daily for promastigotes. Transfer a drop of the culture to a slide for examination using a wire loop. Cover with a coverslide and look for motile , flagellated promastigotes.
Note: Negative cultures must be subcultered after 4 days into fresh medium and examined daily for a
further 10 days.
B- Novy nicolle-McNeal (NNN) culture medium for the invitro culture of leishmania:
Materials:
Difco blood agar base...... 8 mg
Distelled water ......200 ml
Defibrinated rabbit blood*......0.6 ml in each 5 ml of medium
Preparation of defibrinated rabbit blood:*
Collect 20 ml of rabbit blood into a sterile flask containing about 100 glass beads of 4 mm diameter . Defibrinate the blood by rotating the flask for 5 minutes. Add 200 IU of pencillin , 200 mg of gentamycin , and 2 mg of streptomycin per ml of defibrinated blood.
Preparation:
a.POUR 200 ml of water into a flask,add the agar to the water , mix and warm the flask in boiling water
until the agar is completly dissolved.
b. Dispense the medium in 5 ml amounts into screw-cap bottles (20ml capacity).
Sterile by autoclaving with caps loosened at 121Cْ FOR 15 minuts and allow the agar to cool to 45-50Cْ.
c. Add 0.6 ml of sterile defibernated rabbit blood to each bottle and mix gently . Allow the medium to solidify with the bottles in sloped position.
d. Leave the bottles in an upright position at room temp. for 24 hours to allow fluid of condensation to form .
The bottles should be stored at 4-6 Cْ until required.
NOTE:The medium should be prepared in aseptic working conditions.
USE:
A- Incubate about 0.1 ml of specimen aseptically into the fluid of condensation of each of 2 bottles.at room temp.
B- Incubate the cultures at 24 Cْ (-+ 2Cْْ) in the dark .
C- Examine every 4 days . Transfer a drop of culture using a sterile wireloop to a slide for examination for promastigote.
NOTE: Negative cultures must be subcultured after 8 days into fresh medium and examined every 4 days for a further 20 days..
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