PROTECTION AGAINST EVAPORATION 127 



and might change their reaction sufficiently to interfere with bacterial 

 growth. 



Flasks, test-tubes, etc., before being sterilized in the hot-air steril- 

 izer must be stoppered by plugs of clean cotton. A good surgical 

 cotton gives the best service. Pieces of it are rolled up fairly tightly 

 into a conical plug, which is forced into the mouth, so that some of 

 the cotton projects in mushroom-shape over the rim of the glass. 

 When the test-tubes are to be filled with culture media the cotton 

 plug is removed and afterward replaced. The filled tubes are then 

 sterilized by one of the methods adapted for the particular medium. 



It is, of course, evident that some air will enter the tubes through 

 the cotton after they are taken out of the sterilizer and cooled. Air 

 contains microorganisms, but since it is filtered through the dense 

 mass of cotton all bacteria are efficiently prevented from entering. 

 It is particularly necessary to sterilize all glassware in which blood 

 serum, blood-serum mixtures, gelatin and gelatin mixtures are to be 

 kept, because these media must be sterilized by fractional sterili- 

 zation and this method is not as safe as a long continuous or autoclave 

 sterilization. 



Filling of Test-tubes. The best method for filling test-tubes 

 with agar or gelatin is" as follows: A piece of rubber tubing is 

 connected with the stem of a glass funnel and the free end of the 

 hose is provided with a glass tube drawn out into a narrow outlet; 

 the hose should also have a burette clamp. The melted agar or 

 gelatin is poured en masse into the funnel which has previously been 

 warmed with hot water and small amounts can be let out conveniently 

 into each test-tube. A special filling apparatus has also been devised. 

 It is constructed somewhat like a chemical separatory funnel and 

 from it a definite amount of the melted medium (5 or 10 c.c.) can be 

 let out into the test-tubes. In filling test-tubes, it is important not to 

 allow any of the medium to. adhere to the mouth of the tube, as this 

 would make the cotton plug stick to the tube. 



Protection against Evaporation. When media distributed in test- 

 tubes are to be kept for some time or are to be placed in the incubator 

 for days and weeks, they must be protected against evaporation. 

 For this purpose rubber caps are used. Before being applied to the 

 mouth of the test-tube the caps must be soaked for a number of hours 

 in a solution of bichlorid of mercury (1 to 500 or 1000). This kills 

 the bacteria and moulds which might adhere to the rubber, and by 

 growing through the moist cotton, drop into the culture medium, thus 

 contaminating it. Another method is to seal the mouth of the test-tube 

 with paraffin or sealing wax after the cotton has been pushed down 

 to some extent. Perhaps the best method, of preserving culture media 

 in test-tubes for a longer period of time, and yet having them always ready 

 and easily accessible, is the following: Take an anatomical jar of the 

 Whitall-Tatum type or an ordinary Mason jar, place some cotton 

 on the bottom, moisten with a strong formalin solution, introduce 



