26 CULTURE MEDIA 



NUTRIENT AGAR 



In making agar medium it is preferable to use powdered agar, as this goes into 

 solution more readily than the shredded agar. The reaction of agar is slightly alka- 

 line, so that if 1 3^ to 2% of agar is added to nutrient bouillon having a reaction 

 of +i the finished product will be found to be about +0.8. 



To make: Weigh out 15 to 20 grams of powdered agar and place in a mortar. 

 Make a paste by adding nutrient bouillon, little by little, and when a smooth even 

 mixture is made, pour it into the inner compartment of a rice cooker and add the 

 remainder of the 1000 c.c. of bouillon. The use of the balance is preferable. 



The outer compartment of the rice cooker should contain the 25% salt solution. 

 Bring to boll, and the agar will be found to have entirely gone into solution after 

 five to ten minutes of boiling. 



Then, using a funnel which has been heated in boiling water and which contains 

 a small pledget of absorbent cotton, or the cotton between two layers of gauze, we 

 filter the agar, tube it, and sterilize it in the autoclave or Arnold. One and one-half 

 percent agar can be readily filtered through filter-paper and gives a clearer medium. 



Some prefer to place the filter stand, funnel with gauze cotton filter and flask in 

 an Arnold sterilizer, for twenty minutes, so that when taken out the funnel will not 

 only be hot but the filter, being moist, will allow of more rapid filtration. 



By taking of meat extract 3 grams, peptone 10 grams, salt 5 grams, 

 powdered agar 15 grams, the white of one egg and 1000 c.c. of water, 

 making at first a paste of all the ingredients in a mortar, then gradually 

 adding the remainder of the 1000 c.c. of water,*putting in the rice cooker, 

 bringing to a boil without stirring, allowing to boil fifteen minutes and 

 then filtering through absorbent cotton placed between two layers of 

 gauze in a hot funnel, we obtain a satisfactory medium, the reaction of 

 which will be from +0.7 to +0.9. It is very important not to interfere 

 with the pultaceous coagulum which forms on the surface of the boiling 

 agar. 



Where very exact adjustment of the reaction of the finished product is desirable 

 the method of preparation of the Committee on Water Analysis of the American 

 Public Health Association is to be preferred. 



Dissolve 15 grams of agar in 500 c.c. of water in the inner compartment of the 

 rice cooker previously described. After the agar is in solution (after ten to fifteen 

 minutes boiling) remove the inner compartment, containing the 3 % agar solution, and 

 allow it to cool to about SSC. Mix in the mortar, as described in the directions for 

 making nutrient bouillon from Liebig's extract, 3 grams of Liebig's extract, 10 grams 

 of peptone and 5 grams of sodium chloride in 500 c.c. of water containing the whites of 

 one or two eggs. Heat this mixture to 50 to S5C^kid pour it into the agar solution, 

 in the inner compartment, which has been cooled to about 55C. Now titrate this 

 mixture containing 500 c.c. of double strength agar and 500 c.c. of double strength 

 peptone, meat extract and salt solution. The resulting 1000 c.c. gives i^% agar 

 and i% peptone solution. Having adjusted the reaction by the addition of the 



