386 BACTERIA. 



calibre into the indiarubber tube that connects the two flasks. When the 

 operation is complete the tube with the contained organisms is disconnected, 

 the imperforate indiarubber membrane is again tied in position, and the 

 whole is set aside until the organisms begin to develop ; they may then be 

 counted in situ> or with a long platinum needle special points may be 

 removed for microscopic examination, or for the purpose of making pure 

 cultures. 



In place of Hesse's tubes I have, with Mr. Coghill's assistance, devised 

 a flat glass bottle with a large central opening at the top and two side 

 openings just at the level of the gelatine surface. These orifices are placed 

 at the opposite sides of the jar. They are used just as are Hesse's tubes, 

 but possess several very obvious advantages over them. 



Another method used in estimating the number of bacteria in the air 

 was that of filtering the air through tubes containing sterilized glass 

 wool, asbestos, or sand ; these are now seldom used, as they all interfere 

 more or less with the transparency of the gelatine. Indeed, it is often a 

 very difficult matter to distinguish grains of sand from young colonies of 

 organisms. In place of these substances Miquel recommends that sterilized 

 cane sugar should be used. Here the method of procedure is as follows : 

 Loaf sugar is carefully ground in a mortar and passed through a couple of 

 metal sieves, the latter of which allows grains of not more than half a milli- 

 metre in diameter to pass ; this sifted sugar is packed into a glass tube about 

 eight inches long and the sixth of an inch in diameter. Near one end of 

 this tube is a slight constriction, on each side of which is placed a little 

 sterilized cotton wadding or glass wool, one of which serves to prevent the 

 sugar from escaping at the lower end, the other acting as a sterilized plug. 

 At the other end of the tube is a glass or indiarubber cap (see description 

 of Pasteur-Chamberland flask in Appendix) carefully sterilized and plugged 

 with cotton wadding. The tube is sterilized for an hour at 150 C., and 

 allowed to cool ; the cap is then removed and the sugar, which has been 

 previously well dried, is poured into the tube, which should be filled to a 

 depth of about 4 to 4^ inches. The whole is again sterilized at a tempera- 

 ture of 150 C. When the filter is to be used the sugar is packed against 

 the plug by tapping the tube gently, and the tube is held vertically whilst 

 the air is being drawn through the filter ; this may be done slowly for 

 twenty-four hours, or very rapid aspiration may be carried on where it is 

 required to ascertain the number of micro-organisms present in the air at 

 any definite period of the twenty-four hours. When the process is com- 

 pleted, the plug nearer the aspirator is carefully removed with forceps and 

 placed in a sterile glass box, which should be got ready beforehand, the 

 inner plug is then removed and is put into a test tube containing a small 

 quantity of liquefied nutrient gelatine ; this test tube is carefully plugged and 

 laid aside, the outer plug is immediately replaced, the cap is removed and 

 the sugar is poured out into a flask filled with sterilized water. A short 

 indiarubber tube is then slipped over the plugged end of the glass, the 

 upper end, from which the sugar has been emptied, being placed in a test 

 tube containing sterilized water ; this water is alternately sucked up into the 

 filter tube and then expelled, until the whole of the sugar with its contained 

 germs has been dissolved and driven into the test tube. This water is added 

 to that of the flask and well shaken until the whole of the sugar is dissolved. 

 The number and character of germs so obtained from the air are determined 

 by making plate cultures. One portion should be utilized for the demon- 



