AGAR MEDIA 43 



[11. Tube, and sterilize for 30 minutes at 100 C. on two successive 

 days. Slope (see B. 9 infra}. 



[This method always gives a perfectly clear, transparent and very slightly 

 opalescent medium. The yield is very approximately 100 per cent. that is 

 to say that from a litre of broth rather more than a litre of agar is obtained. 

 No trouble is ever experienced in getting the agar to adhere to the walls of 

 the tubes. 



[If the autoclave be used the medium is generally of a brownish colour 

 from over-heating, and it is sometimes difficult to get the medium to stand 

 up in the tubes.] 



B. Another method, which is also recommended, is as follows : 



1. Prepare a peptone-beef-broth, according to the instructions given on 

 p. 30, up to and including Stage 5. 



2. To this broth add 20 grams (2 per cent.) of agar cut up into small 

 pieces. The agar should be swollen by soaking in cold water for an hour 

 or two, and then wrung out in a cloth before being added to the 

 broth. 



3. Heat the mixture to 100 C. in an enamelled saucepan, and keep it at 

 this temperature until the agar is dissolved (about half an hour), stirring 

 all the time. 



4. Test the reaction, which should be neutral or faintly alkaline. If 

 heated in presence of acid, agar becomes converted into sugar. 



5. Cool to 55 or 60 C. and add the white of an egg beaten up in 100 grams 

 of water. Mix thoroughly. 



6. Autoclave for an hour at 120 C. The albumin is coagulated and 

 carries down the impurities with it. 



7. Pour the liquid while still hot on to a moistened Chardin paper arranged 

 in a hot water funnel. Cover the funnel with a glass plate. 



8. Collect the liquid as it niters in a previously sterilized flask, and tube 

 at once. This must be done as quickly as possible as agar sets about 40 C., 

 and a funnel as usual should be used in tubing it to prevent the medium 

 soiling the mouths of the tubes. Each tube should contain 8 to 10 c.c. 



9. Sterilize at 115 C. for 20 minutes. After sterilization and while the 

 medium is still hot, slope the tubes on some such piece of apparatus as that 

 pictured on p. 52, so that the agar solidifies with a sloped surface. Leave the 

 tubes in this position for 36 hours. 



Some bacteriologists recommend the addition of a small quantity of an aqueous 

 solution of gum arabic to the agar, to prevent the thin upper part becoming detached 

 from the wall of the tube when it is placed vertically. But gum arabic makes the 

 medium distinctly cloudy, and does not appear to effect the purpose for which it 

 is added. 



If the method of preparation described be followed step by step, the agar will 

 be found to adhere sufficiently well. Gelatin to the amount of 20 grams per litre 

 may be added as recommended by Nicolle. 



Modification. Filtration may be accomplished in the following manner, 

 even more easily than by the above method. 



Before adding the agar to the broth (Stage 2) leave it to soak in 6 per 

 cent, hydrochloric acid (water, 500 : HC1, 30) for 24 hours, and wash m a 

 large quantity of water. Then soak in a 5 per cent, solution of ammonia 

 (water, 500 : ammonia, 25) for some hours, wash in a large quantity of 

 water, and squeeze the agar dry in a cloth. The agar is now ready to add 

 to the broth, and the further steps are as described. The resulting jelly 

 does not adhere well to the walls of the tubes, and the process cannot be 

 recommended. 



