2. Record the number of additional fermentation tubes which show 10
per cent. (10%) or more of gas.
3. Make a lactose-litmus-agar or Endo’s medium plate from the
smallest portion of each sample showing gas. Incubate plate at 37°
C.
Note:—In case the smallest portion in which gas has been formed shows
less than 10 per cent. (10%) of gas, it is well to make a plate also
from the next larger portion, so that, in case the smallest portion
gives a negative end result it may still be possible to demonstrate B.
coli in the next larger dilution.
_Fourth Day_:
1. Examine Endo’s medium or lactose-litmus-agar plates. If typical
colonies have developed, select two and transfer each to a lactose
broth fermentation tube and an agar slant, both of which are to be
incubated at 37° C.
2. If no typical B. coli colonies are found, incubate the plates
another 24 hours.
_Fifth Day_:
1. Select at least two colonies, whether typical or not, from the
Endo’s medium or lactose-litmus-agar plates which have been
incubated an additional 24 hours; transfer each to a lactose broth
fermentation tube and an agar slant, and complete the test as for
typical colonies.
2. Examine lactose broth fermentation tubes inoculated from plates
on the previous day. Tubes in which gas has been formed may be
discarded after the result has been recorded. Those in which no
gas has formed should be incubated an additional 24 hours.
_Sixth Day_:
1. Examine lactose broth fermentation tubes reincubated the previous
day.
2. Examine microscopically agar slants corresponding to lactose
fermentation tubes inoculated from plate colonies and showing
gas-formation.