92 MICROSCOPIC METHODS 



and covered with a cover-glass. 1 It is more usual, however, to 

 employ hanging-drop preparations. The technique of making 

 these has already been described (p. 69). In examining them 

 microscopically, it is necessary to use a very small diaphragm. 

 It is best to focus the edge of the drop with a low-power 

 objective, and, arranging the slide so that part of the edge 

 crosses the centre of the field, to clamp the preparation in this 

 position. A high-power lens is then turned into position, and 

 lowered by the coarse adjustment to a short distance above its 

 focal distance ; it is now carefully screwed down by the fine 

 adjustment, the eye being kept at the tube meanwhile. The 

 shadow of the edge will be first recognised, and then the bacteria 

 must be carefully looked for. Often a dry lens is sufficient, but 

 for some purposes the oil immersion is required. If the bacteria 

 are small and motile, a beginner may have great difficulty in 

 seeing them, and it is well to practise at first on some large non- 

 motile form, such as anthrax. In fluid preparations the natural 

 appearance of bacteria may be studied, and their rate of growth 

 determined. The great use of such preparations, however, is to 

 find whether or not the bacteria are motile, and for determining 

 this point it is advisable to use either broth or agar cultures not 

 more than twenty-four hours old. In the latter case a small 

 fragment of growth is broken down in broth or in sterile w r ater. 

 Sometimes it is an advantage to colour the solution in which 

 the hanging-drop is made up with a minute quantity of an 

 aniline dye, say a small crystal of gentian violet to 100 c.c. of 

 bouillon. Such a degree of dilution wdll not have any effect on 

 the vitality of the bacteria. Ordinarily, living bacteria will not 

 take up a stain, but even though they do not, the contrast 

 between the unstained bacteria and the tinted fluid will enable 

 the observer more easily to recognise them. In determining 

 whether or not a bacterium is motile, great difficulty is often 

 experienced in distinguishing between true motion and Brownian 

 movement, especially if the organism be small. The essential 

 criterion to be fulfilled is that the bacteria shall be moving in 

 all directions, the observation of individuals lying close together 

 starting to move in opposite directions being important. The 

 observation of hanging-drop preparations must be correlated 

 with the results of staining for the presence of flagella which, so 

 far as is known, are present in all motile forms. 



Within recent years the method of observing living micro- 



1 In bacteriological work it is essential that cover-glasses of No. 1 thickness 

 (i.e. '14 mm. thick) should be used, as those of greater thickness are not 

 suitable for a jViuch lens. 



