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 C. for half an hour on each of five consecutive days.

5. Incubate at 37 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 quant.i.ties of 6 c.c.

4. Sterilise as for nutrient agar.

5. After the third sterilisation cool the tubes to 42C., 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 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 quant.i.ties of 5 c.c.

4. Sterilise as for nutrient agar.

5. After the last sterilisation cool to 42C., 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 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.), and mix thoroughly.

4. Heat in the steamer for twenty minutes.

5. Weigh out 15 grammes agar, emulsify in a separate vessel with 200 c.c. of the alkaline fluid previously cooled to about 20C., and then add to the remainder of the fluid in the flask.

6. Bubble live steam through the mixture for twenty minutes to dissolve the agar.

7. Filter through papier Chardin, using a hot-water funnel.

8. Weigh out glucose 10 grammes (= 1 per cent.), and dissolve it in the clear agar.

8a. If desired, add glycerine, 5 per cent., to the clear agar.

9. Tube, and sterilise as for nutrient agar.

~Serum Agar (Libman).~--

1. Prepare nutrient agar (_vide_, page 167) using, however, 1.5 per cent. peptone (that is 15 grammes per litre instead of 10 grammes).

2. Adjust the reaction to 0 (i. e., neutral to phenolphthalein).

3. Filter and transfer 1000 c.c. liquefied medium to a sterile flask.

4. Weigh out dextrose 20 grammes and dissolve in the fluid agar.

5. Tube in quant.i.ties of 6 c.c.; and sterilise in the steamer at 100 C.

for thirty minutes on each of three consecutive days.

6. After the third sterilisation cool to 42 C. and add to each tube 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.

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

~Egg-alb.u.men, Insp.i.s.sated.~--

1. Break several fresh eggs (hens', ducks', or turkeys' eggs), and collect the "whites" in a graduated cylinder, taking care to avoid admixture with the yolks.

2. Add 40 per cent. distilled water, and incorporate the mixture thoroughly by the aid of an egg-whisk.

3. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid (or add the amount of dekanormal caustic soda solution calculated to yield the required percentage of soda in the total bulk of the fluid--i. e., 0.375 c.c. of dekanormal NaOH solution per 100 c.c. of the mixture).

_3a._ Glucose to the extent of 1 to 2 per cent. may now be added, if desired.

4. Strain the mixture through b.u.t.ter muslin and filter through a porcelain filter candle into a sterile filter flask.