-

1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by
aspiration directly into sterile flasks, under strictly aseptic
precautions.

2. Mix the serum with twice its bulk of sterile nutrient bouillon
(_vide_ page 163).

3. If considered necessary (on account of the presence of blood,
crystals, etc.), filter the serum bouillon through porcelain filter
candle.

4. Tube, and sterilise in the water bath at 56 deg. C. for half an hour
on each of five consecutive days.

5. Incubate at 37 deg. C. for forty-eight hours and eliminate
contaminated tubes. Store the remainder for future use.

~Serum Agar (Heiman).~--

1. Prepare nutrient agar (_vide_ page 167), to following formula:

Agar 2.0 per cent.
Peptone 1.5 per cent.
Salt 0.5 per cent.
Meat extract _quantum sufficit._

2. Make reaction of medium + 10.

3. Filter; tube in quantities of 6 c.c.

4. Sterilise as for nutrient agar.

5. After the third sterilisation cool the tubes to 42 deg. C., and add
to each 3 c.c. of sterile hydrocele fluid, ascitic fluid, or pleuritic
effusion (previously sterilised, if necessary, by the fractional
method); allow the tubes to solidify in a sloping position.

6. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
any contaminated tubes. Store the remainder for future use.

~Serum Agar (Wertheimer).~--

1. Prepare nutrient agar (_vide_ page 167), to the following formula:

Agar 2.0 per cent.
Peptone 2.0 per cent.
Salt 0.5 per cent.
Meat extract _quantum sufficit._

2. Make reaction of medium +10.

3. Filter; tube in quantities of 5 c.c.

4. Sterilise as for nutrient agar.

5. After the last sterilisation cool to 42 deg. C., then add 5 c.c.
sterile blood-serum from human placenta (sterilised, if necessary, by
the fractional method) to each tube; slope the tubes.

6. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
any contaminated tubes. Store the remainder for future use.

~Serum Agar (Kanthack and Stevens).~--

1. Collect ascitic, pleuritic, or hydrocele fluid in sterile flasks and
allow to stand in the ice-chest for twelve hours to sediment.

2. Decant 1000 c.c. of the clear fluid into a measuring cylinder and
transfer to sterile litre flask.

3. Add 0.5 c.c. dekanormal NaOH solution for every 100 c.c. serum (_i.
e._, 5.0 c.c.