182 



Appendices to Fourth Annual Report 



The method adopted is as follows. A bread paste flask in which are 

 growing three or four different species is taken. Four sterilised plates are 

 prepared ; * at the same time eight quarter litre flasks are carefully 

 cleaned, tilled with distilled water, plugged, and then boiled for at least 

 one hour. 'I'hese are allowed to cool, and from one of the growths in the 

 bread paste flask a small particle is transferred by means of a sterilised 

 platinum needle to the distilled water. This particle is broken up and 

 diffused through the water by shaking the flask thoroughly ; 5 c.c. of this 

 water is then removed with a sterilised glass pipette, and [daced in a 

 second flask, which is also thoroughly shaken ; a test tube containing 

 warm nutrient gelatine is then inoculated with five drops of this solu- 

 tion ; the tube is carefully agitatsd until the water and gelatine are 

 thoroughly mixed, care being taken that no air bubbles are included, and 

 also that none of the fluid passes into the plug, and so to the mouth of 

 the test tube. This mixture is then poured out on to the prepared 

 glass plate, labelled and left in the sterilised moist air chamber for a 

 day, after which it should be carefully examined every twelve hours. 

 Some of- the various species grow so rapidly, or liquefy the gelatine so 

 suddenly, that unless this be done the growths in their immediate neigh- 

 bourhood may be overlooked. The mode of growth is then noted, both 

 as seen with the naked eye and under a glass of low magnifying power. 

 Some species, as we have seen, liquefy the gelatine very rapidly, others but 

 slowly, whilst others again grow in the gelatine without even softening 

 it. Certain forms appear to bring about an evolution of gas, which may 

 be seen forming a bubble when the organism is growing at some little 

 depth in the substance of the nutrient jelly, and if the examination is 

 being made near a window the image of the window may be seen 

 reflected from the surface of the bubble. Other species occur as 

 perfectly solid growths, the margins of which are sharply defined and 

 smooth, or beautifully ' scolloped ' or crenated, or there may be distinct 

 rays shooting out from the central mass. Cultivations into nutrient 

 jelly in test tubes are taken from these various centres of growth, and if 

 time is an object bread paste flasks are also inoculated at once. Similar 

 plate cultivations are made of the other points in the original flask, the 

 different plates are compared, the preponderating forms of each noted, 

 and from the data so obtained it might be stated almost precisely which 

 forms on the plate correspond with the various forms in the flask. But, 

 in order to ensure greater precision, the secondary cultivations, both in 

 bread paste and gelatine, should be carefully observed and compared with 

 the originals. After taking secondary cultivations, but not before, im- 

 pressions of the points of growth on the plate surface may be taken by 

 laying on a thin cover glass and then removing it carefully with a pair 

 of forceps. t It will be found that the small groups of bacteria adhere to 

 the glass, leaving depressions in the gelatine from which the bacteria 

 have been removed. These are stained and set aside for examination. 

 Other single points are also transferred from the gelatine to the glass cover- 

 slip with a platinum needle, a hand lens or dissecting microscope being 

 used to help in isolating the individual points of growth. By means of 

 the above methods it has been possible to separate numerous distinct 

 species, but it must be remembered that slight differences of colour are 

 observed where masses of the same species of organism are growing in 

 thin or thick layers, on different nutrient materials, or at different 



* Third Annual Report of Scottish Fishery Board, p. 75. {JV.B, — Somewhat 

 larger plates, 4^ inches square, are now used). 



t See Lancet, August 22, 1885, p. 336, "Notes from a Bacteriological Labora- 

 tory," by Edgar M. Crooksliank, M.B., Lond. 



