LaMotte EZ 3-0034 - Manual (Page 7)

Differentiating Coliforms and E. coli
• Colonies that have a blue-green teal color are not exhibiting any ß-
galactosidase activity (which is evidenced by a pink color). Because of this, they
are not considered to be either coliforms or E. coli and should be ignored
when counting coliform or E. coli colonies. Similarly, colonies that are white
are exhibiting neither color-causing enzyme and should not be counted.
• Colonies on the surface of the plate are exposed to the medium only on the
underside of the colony. This causes these colonies to appear with much less of
the indicator color. E. coli colonies may have only a slight purple tinge to them.
The purple color may appear only in the center of the colony with the
remainder of the colony being white. Similarly, coliforms on the surface may be
light pink or white with a pink center.
• When there are a lot of colonies of different colors to count, mark the plate
into quadrants and count one quadrant at a time. If the bacteria are evenly
distributed over the plate, count one quadrant and multiply by four to get the
total count.
In cu ba tion Hints
• Incubate dishes with lid side down to prevent condensation from dripping onto
the plate from the lid.
• There is no one standard to define room temperature. Most would consider
normal room temperature to vary from 68-74°F, but even within this range the
growth of bacteria will be varied. Members of the bacterial family
Enterobacteriaceae (which include coliforms and E. coli) are generally hardy
growers that prefer higher than room temperatures, but will grow more slowly
at lower temperatures.
• An adequate incubator can be made from a box with a 40 - 60 watt bulb in it
to provide heat at an even rate. If plates are incubated in an incubator with a
light bulb as the heat source, shade the plates from direct light from the bulb. It
is also possible to use heat tape that is used to prevent frozen pipes as a heat
source. Always consult your building or fire inspector for advice on the safe
handling and operation of homemade incubators.
• At elevated temperatures (90 – 98°F) incubation times for coliforms are
generally 24 – 48 hours. At elevated temperatures, no counts should be made
after 48 hours as any coliforms will be quite evident by that time. If new
colonies form after 48 hours they are most likely not coliforms, but some other
type of slow growing organism that should not be included in the data.
• At room temperature incubation times for coliforms are generally 48 hours or
more. At room temperature, the best procedure is to check the plates every 10
– 12 hours until some pink or purple colonies start to form. Allow another 24 –
30 hours for these colonies to mature. Do not count anything after 72 hours.
Since the coliforms (including E. coli) are generally the fastest growing
organisms, these will be the first to grow and be counted. Colonies that may
show up at a later time are probably not coliforms.
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