March
1952]
H. LAXMINARAYANA et al.
33
When agar or purple lactose agar, fortified with one per cent yeast autolysate,
was generally employed for pouring plates. Almost all organisms grew well on
these media and formed clear colonies which were easy for picking. In later series
of samples, yeast dextrose agar containing one per cent sodium citrate was employed
for encouraging the growth of heterofermentative streptococci while tomato juice
agar or wort agar was used for cultivating heterofermentative types of lactobacilli.
The various media were prepared according to standard methods [Levine and
Schoenlein, 1930 and Tanner, 1944]. The plates were incubated at 30°C., 37°C.,
and 45°C., for 3 to 4 days or longer until well developed colonies appeared. Small,
discreet colonies with entire or irregular margins growing below the surface and show-
ing acid production were recognized to be lactic acid organisms. Under the low
power (x 40) of a compound microscope they appeared either as smooth, rough
or filamentous colonies.
Well isolated representative colonies from each of the plates were picked into
yeast dextrose litmus milk tubes and incubated at temperatures at which the colonies
had been cultivated. After the cultures showed evidence of growth they were
examined microscopically and purified by further plating and repeated sub-culturing
in milk. In all cases, the organisms were tested for catalase production (recognized
by the evolution of gas bubbles when.1 per cent solution of hydrogen peroxide was
added on to the agar colonies) in order to eliminate the catalase positive micrococci
and other contaminants which might be mistaken for lactic acid bacteria. Only
such cultures as were found to be catalase negative and as did not show appreciable
surface growth on agar slants were presumed to be lactic acid bacteria [Orla Jensen,
1942] and taken up for further observations.
A total of 720 isolates, including 290 streptococci and 430 lactobacilli, were
obtained in the above manner. The isolates were maintained in milk at a low
temperature (10° to 15°C.) and transferred once every ten days.
Classification and identification of the organisms
As the number of isolates to be examined was very large it was found convenient
first to make a preliminary classification of the organisms based on well-recognized
group characteristics and then select a limited number of representative strains
for further study and identification.
Preliminary grouping of the organisms
All the isolates were subjected to the following tests for their preliminary classi-
fication.
Microscopic appearance. Microscopic appearance and arrangement of the cells
of organisms in freshly curdled milk cultures were examined.
Action on litmus milk. The cultures (soon after curdling) were inoculated into
plain litmus milk and incubated at 37°C. Observations were made regarding acid
production, clotting, reduction of litmus, gas production and proteolysis.
Growth at different temperatures. Young cultures of the organisms were inoculated
into plain litmus milk and yeast litmus milk and incubated at 10°C., 30°C., 37°C.,
45°C., and 50°C. The rate of acid production (indicated by colour of litmus) and
he time of curdling were noted. ...