14 THOMPSON YATES LABORATORIES REPORT 
Temperature of Incubation 
In Bombay it is found better not to use an incubator. The microbe grows best at the 
ordinary room temperature. By placing a large number of tubes to incubate, under varying 
conditions, I found the best growth on those incubated at 74° F. in a cupboard. In this country 
an incubator would be required. 
Growth on Agar 
The colonies appear in 24 to 48 hours. If numerous, they appear to the naked eye as 
small round points, colourless and translucent. Under Zeiss Obj., A they are translucent with 
slightly greenish tinge, granular, and have slightly ragged edges. 
If the colonies are scanty the individual ones may grow larger. The growth takes place in 
successive concentric circles round the original colony, which appears lieaped up in the centre and 
is more the colour of mother-of-pearl as it grows older. 
A plague colony slips about on the surface of the agar when touched with a platinum wire. 
If a dried agar slant tube is inseminated by introducing o.i to 0.2 c.cm. of a broth culture 
by means of a long thin glass pipette, and if the culture is smeared evenly over the surface with a 
thin glass rod or with the end of the pipette (care being taken not to dig into the agar), the 
so-called 'ground-glass' appearance is seen after one or two days' incubation. The layer of 
growth should be viewed from underneath through the substance of the agar with the light coming 
from opposite, the eye being almost level with the slanting surface of the agar tube held about a 
foot off. The layer of growth looks like bright ground-glass or like the back of a looking-glass. 
The agar must be dry so that it will absorb the broth leaving the bacilli on the surface. If there 
is any broth not absorbed, or any condensation water present, the growth in contact with the 
liquid is smeary and somewhat milky. After inseminating the tubes it is well to leave them on 
their sides for a few minutes and then incubate them in the upright position. In examining them 
care should be taken not to allow any liquid that may be at the bottom of the tubes to flow over 
the surface of the growth. 
This ' ground-glass ' appearance was first shown by Haffkine. It is a very useful method 
of testing the purity of a culture known to be plague, because a strange colony is generally detected 
at a glance. Some microbes, proteus for example, will grow in amongst the plague, when the 
film will no longer have a typical ' ground-glass ' appearance, but will appear somewhat slimy. 
Neither is this ' ground-glass ' appearance peculiar to the plague microbe alone, for I 
succeeded in separating a spore-forming micro-organism from the air that showed the 'ground-glass' 
appearance after 24 hours' growth ; but tlie growth became opaque after 48 hours' incubation, 
coincident with the formation of spores. 
Broth 
Haffkine considers the stalactitic growth of plague in broth to be characteristic of the 
microbe. He puts a drop or two of cocoa-nut oil or ghee (native clarified butter) in the flasks 
before they are sterilized ; the oil globules form a thin broken film on the surface of the broth. 
After inoculation the flask must be incubated on a firm shelf or table. In three to six days 
numerous thin threads of growth are seen hanging down from the surface into the otherwise clear 
