130 LABORATORY COURSE IN SERUM STUDY 



vided with extracts already made which require only to be 

 properly diluted with saline before use, and with these extracts 

 will do fixation tests for Gonococcus and for Glanders. 



Bring to a boil. Strain through cheese cloth. 



Bring to original volume. Add agar 1$ per cent, Witte's peptone 2 per cent, 

 NaCl of 1 per cent. 



Titrate. Reaction should be neutral to phenolphthalein. 



Filter through a filter made with a layer of cotton, one of filter paper, another 

 layer of cotton. Filter several times. 



Autoclave \ hour at 15 Ib. pressure. 



Glucose 2 per cent. 



10 grams of glucose dissolved in 50 c.c. distilled H 2 0. Sterilize three days 

 for 20 minutes in Arnold. 



Ascitic fluid 20 per cent. 



Ascitic fluid is filtered through Berkefeld, sealed in sterile flasks, incubated 

 at 37.5 several days before using. Glucose solution and ascitic fluid 

 are mixed before adding to tubed and sterilized agar. Incubate tubes. 

 Keep two days before using. 



Transplant the stock cultures every 48 hours. The gonococcus must always 

 be kept at 37.5 C. Any inequality of temperature will cause a poor growth of 

 the organism. 



For the antigen transplants use veal agar prepared as above, except that salt, 

 glucose and ascitic fluid are omitted. The reaction should be very carefully 

 adjusted ; it should be neutral at the last titration before autoclaving, and when 

 ready to use 0.1 to 0.2 per cent acid, preferably 0.1 per cent. Medium is titrated 

 hot with N/20 NaOH. No change should take place when phenolphthalein is 

 added. One or two drops of NaOH produce a faint pink color which disappears 

 on adding one or two drops of N/20 HC1. The agar is then bottled and auto- 

 claved one hah' hour at 15 Ib. pressure. 



To prepare antigen take 24-hour stock cultures and transplant to potato 

 tubes (tube 6x1 inch) of salt free veal agar neutral to phenolphthalein. Incu- 

 bate 24 hours. Transfer all this growth by means of sterile cotton swabs to 

 wide-mouthed Blake bottles (one tube to a bottle) containing neutral salt free 

 veal agar, gently rubbing the swab over the entire surface of the agar. Incubate 

 for 24 hours. \Vash off all this growth, if good, with neutral sterile distilled water, 

 10 c.c. to a bottle. If the growth is poor, 5 c.c. is sufficient. The bottle should 

 be gently tipped back and forth two or three times after the water is added and 

 the growth scraped off lightly with a bent glass rod. Do not allow the water 

 to remain on the agar more than a few seconds. The resulting emulsion is auto- 

 lyzed for one hour in a water bath at 56 C. and at 80 C. for one hour. Filter 

 the autolyzed emulsion through a Buchner funnel which has been well packed 

 with paper pulp and then through a sterile Berkefeld filter of N or V porosity. 



As new niters are very alkaline they are taken to pieces before use and boiled 

 in distilled water at least three times, five minutes each time, being scrubbed 

 thoroughly with a small brush in fresh water after each boiling. Then the filter 

 is set up and hot distilled water allowed to stand in it for five minutes. Hot 

 neutral distilled water is then run through it under gentle pressure until fluid is 

 clear and neutral when tested with phenolphthalein. After a filter has been used 



