CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



555 



solution to 100.0 cc, 0.5% aqueous 

 solution of acid fuchsin. 



(6) Cool (4) and add beaten egg white. 



(7) Boil and filter. 



(8) Distribute in 100.0 cc. lots in 

 bottles. 



(9) Autoclave for 30 minutes at 15 

 pounds pressure. 



(10) When ready for use melt (9) and 

 add to each 100.0 cc, 1.0 cc. of 

 (5), 1.0 g. lactose (or 5.0 cc. of 

 20.0% solution, sterilized in the 

 Arnold), 0.1 g. glucose (or 5.0 cc. 

 of a 2.0% solution, sterilized in 

 the Arnold sterilizer), and 0.5, 

 0.3 or 0.2 cc. of a 0.1%, stock 

 solution in distilled water of 

 brilliant green. 

 <b) Kligler prepared the medium as 

 indicated above. The reaction was 

 adjusted between pH 7.0 and 7.2. 

 When adding the brilliant green (see 

 step (7) above) add 0.25 cc. of a 1.0% 

 solution of neutral red to each 100.0 

 cc. of agar. 

 <c) Park, Williams and Krumwiede pre- 

 pared the medium as follows: 



(1) Dissolve 30.0 g. of agar in 1000.0 cc. 

 of water in the autoclave. 



(2) Dissolve 6.0 g. meat extract, 10.0 

 g. NaCl and 20.0 g. peptone in 

 1000.0 cc. of water by heating in 

 the Arnold. 



(3) Mi.x (1) and (2). 



(4) Add normal soda so that the final 

 reaction will be neutral to Andrade 

 indicator (pH 6.9). 



(5) Boil 30 minutes. 



(6) Cool and clear with egg. 



(7) Filter until clear. 



(8) Bottle in 100.0 cc. quantities. 



(9) Autoclave. 



<10) When ready for use melt the agar 

 and to each 100.0 cc. add 1.0 cc. of 

 Andrade indicator, 5.0 cc. of a 

 sterile solution in distilled water 

 of 20.0% lactose and 2 0% glucose, 

 (1.0% lactose and 0.1% glucose) 

 and the proper amount of a 0.1% 

 solution of brilliant green in 

 distilled water. (See (13)). 



(11) Mix well. 



(12) Pour thick plates. Use porous 

 tops as the plates must be dry. 



(13) To determine the amount of dye 

 to add prepare four dilutions of 

 dye: 1 to 500,000, 1 to 330,000, 1 to 

 250,000 and 1 to 200,000, which 

 corresponds to 0.2, 0.3, 0.4 and 0.5 

 cc. of a 0.1% solution of dye to 

 100.0 cc. Inoculate these plates 

 with a freshly isolated strain of 

 B. tj/phosus or a positive stool. 

 Choose the dilutions of dye that 

 (1) at which the typhoid colonies 

 are of good size and undiminished 

 in number compared with the 

 control plate, but many other fecal 

 types are excluded, (2) a lower 

 dilution where the typhoid colo- 

 nies are reduced in size and num- 

 ber, but almost all the other 

 flora have disappeared. 

 References: Krumwiede, Pratt and Mc- 

 Williams (1916 p. 4), Krumwiede, Kohn, 

 Kuttner and Schumm (1918 p. 275), 

 Kligler (1918 p. 320), Giltner (1921 p. 

 366), Park, Williams and Krumwiede 

 (1924 p. 128). 



1779. Aronson's Fuchsin Sulphite Agar 



Constituents : 



1. Water 1000.0 cc. 



2. Agar 35.0 g. 



3. Meat extract 10.0 g. 



4. Peptone 100 g. 



5. NaCl 5.0 g. 



6. Na2C03 (10 0%) 60.0 cc. 



7. Sucrose (20.0%) 50.0 cc. 



8. Glucose (20.0%) 50.0 cc. 



9. Fuchsin (saturated) 4.0 cc. 



10. Sodium sulfite (10.0%) .... 20.0 cc. 



Preparation : 



(1) Add 2 to 1 and allow to soak over 

 night. 



(2) Add 3, 4 and 5 to (1) and boil in 

 streaming steam for 4 to 5 hours. 

 Reaction slightly acid. 



(3) Slant and remove the insoluble 

 material. 



(4) Distribute in 100.0 cc. lots by means 

 of a graduated flask into sterile 

 200-250.0 cc. Erlenmeyer flasks. 



(5) Prepare a 10.0% solution of Na2C03 

 (siccum). 



(6) Prepare a 20.0% solution of sucrose. 



(7) Prepare a 20.0% solution of glucose. 



