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.
(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
(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
(d) Q.S. to 500 ml with Normal Saline.
(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
(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.
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.
Store in the refrigerator.