'2SS Journal of Applied Microscopy. 



The filtrate should be : (1) perfectly clear ; (2) slightly alkaline in reaction ; 

 (S) should not become cloudy or coagulate when boiled in a test-tube for one to 

 two minutes ; and (4) should solidify when cooled. 



If the filtered gelatin answers the above requirements, it is ready to be 

 "tubed."' that is to say. filled into clean plugged tubes. The latter are fre- 

 quently sterilized by heating in a dr)--heat sterilizer for one hour at 150 degrees. 

 This procedure, however, is not necessar}-, since sterilization of the cotton plug 

 will be obtained by subsequent exposure to steam. This medium should be filled 

 into the tubes by means of a small funnel, to a depth of one and a half inches. 

 The utmost care should be taken to prevent touching the neck of the tube with 

 the gelatin, since othen\ise the cotton will adhere to the tube. The tubes of 

 gelatin are then sterilized in steam for fifteen minutes on each of three consecu- 

 tive days. 



NUTRIENT BOUILLON. 



A liter of meat extract is prepared according to the directions given above 

 under gelatin. To this blood-red fluid ten grams of Witte's pepton and five 

 grams of salt are added, and the mixture is warmed at 60 degrees till solution 

 results. The liquid is then titrated according to the directions given. Portions 

 of 10 cc. placed in each of four test-tubes receive U.4, 0.6, 0.8, and l.UU cc. of 

 deci-normal alkali. After boiling the contents of each tube, red and blue litmus 

 papers are immersed in the hot liquid. The neutral point is thus readily ascer- 

 tained. It is usually reached by the addition of about ('..5 cc. of the alkali. 

 The remaining liquid is measured, and the amount of normal alkali necessar)- to 

 neutralize this entire quantit}- is then calculated. This calculated amount, plus 

 10 cc. of normal alkali per liter, is then added to the liquid. 



The liquid is then placed in a flask, or in the enamelled jar, and immersed in 

 a water-bath, which is then heated to boiling for about one-half hour. The 

 liquid may be boiled directly over the flame, in which case, owing to the loss of 

 water by evaporation, it is advisable to mark the level of the liquid at the begin- 

 ning of the operation. A better procedure is to weigh the jar and contents 

 before and after boiling, and for the number of grams lost in weight a corres- 

 ponding number of cubic centimeters of distilled water are added. 



The liquid is then filtered through a wet filter. Although the filtrate may be 

 perfectly clear, it not infrequently happens that on subsequent sterilization a 

 cloudiness or deposit forms. This can be avoided if the filtered liquid is con 

 centrated by boiling to about one-half its original volume and then replacing the 

 water lost by evaporation and filtering a second time. The clear bouillon is then 

 filled into tubes and sterilized by steaming for fifteen minutes on each of three 

 consecutive days. 



NUTRIENT AGAR-AGAR. 



A liter of bouillon is prepared as above, except that the second heating or 

 concentration is omitted. The liquid is placed in a clean beaker or jar, and 

 twenty grams of finely cut up agar are added. The jar and contents are weighed 

 and placed over a flame and boiled. Agar dissolves better in clear bouillon than 

 in a meat extract : moreover, the subsequent filtration is rendered easier because 



