METHOD OF PREPARING AGAR 



determination must be of the same 

 diameter and of clear glass. 



Another indicator, brom cresol pur- 

 ple,* (0.04 per cent solution in 95 per 

 cent alcohol) may be used as an alter- 

 native for brom thymol blue. Its use 

 is especially desirable if the reaction of 

 the agar is more acid than pH = 6.4, 

 because brom thymol blue is not very 

 sensitive at this point. Brom cresol 

 purple, on the other hand, is not sensi- 

 tive at pH = 7.0 and therefore cannot 

 be used if the medium is of neutral 

 reaction. 



The pH values corresponding to the 

 color pairs (acid and alkali) prepared 

 by the method of Barnett and Chap- 

 man have been worked out by Medalia." 

 The color of brom cresol purple is a 

 deep shade of purple at pH = 6.8 

 with increasingly lighter shades to 

 pll^6.2. At pH = 6.0 the color is 

 of a grayish hue not easily confused 

 with that of pH = 6.2. 



Adiustment of reaction. 



If the correct color of the indicator 

 does not appear in the agar as tested, 

 add dilute NaOH (e. g. N/20) from 

 a burette until the shade is obtained 

 which represents the desired H-ion con- 

 centration, that is between pH = 6.8 

 and 7.0. Fifty times the amount of 

 N/20 NaOH added from the burette 

 equals the amount of normal NaOH 

 to be added to one liter of the medium 

 if 1 cc. of the agar is being tested. 

 When testing 2 cc. of agar, multiply 

 by 25 instead of 50. 



In this adjustment, it is permissable 

 to use any strength NaOH, but the 

 strength of that added to the medium 

 must be an exact multiple of the 

 strength of NaOH used in titration; 

 if the ratio is not 1:20 proper allow- 

 ances must be made. 



*Prepared by Hynson, Wescott & Dunning, Baltimore.Md. 



Method of preparing agar. 



The important point is to secure an 

 agar of the correct reaction and com- 

 position which contains no trouble- 

 some precipitates. Methods of cook- 

 ing and filtering to accomplish this 

 vary with the ingredients used. Those 

 suggested below have been found satis- 

 factory in practical use; but other 

 methods securing the same results are 

 allowed. White of egg, however, must 

 not be used for clarification. 



The finished medium may be tubed 

 or bottled, placing 10 cc. in each tube 

 or 55 cc. (enough for five plates) in, 

 each bottle. 



Sterilization shall be accomplished 

 by heating in the autoclave for 20 

 minutes after the pressure reaches 

 15 lbs.; or after the agar is completely 

 melted, heat in flowing steam on three 

 successive days for 20 minutes each day. 



All glassware and all apparatus such 

 as kettles, funnels, and filtration flasks, 

 must be kept scrupulously clean by 

 running hot water over or through them 

 after use before the agar has had time 

 to harden. There is danger otherwise 

 of dried particles of agar chipping off 

 and giving rise to sediment in future 

 batches of agar which in the poured 

 plates may be mistaken for colonies. 



Procedure No. 1. Mix all of the 

 ingredients together cold. Heat in an 

 autoclave at 15 lbs. pressure for 40 to 

 00 minutes according to the quantity 

 of medium being made in each batch. 

 Allow the autoclave to cool very slowly 

 so as not to disturb the sediment. 

 Decant through a cotton filter taking 

 care not to pour the sediment on the 

 cotton until the bulk of the liquid has 

 passed through. 



This simple procedure with certain 

 brands of peptone and grades of agar 

 gives excellent results. 



