Jane Allen writes:
> I maintain the microbiology lab for a small college. This week I
> made a modified Snyder Test Agar and after two days of incubation
> we have not seen much change of color. I didn"t have the dye
> that was in the Difco recipe so I substituted Brom-Phenol Blue.
> The list of pH indicators showed it as changing color at a pH
> comparable to the recommended dye. Can anyone tell me if my
> substitution should work? The students will be checking the
> tubes again on Tuesday.
The Snyder Test is time and pH dependent. It is read over a 4 day
period and the result is based on the speed of changing the pH
over that 4 day period. The Snyder Test pH indicator, Brom
Cresol Green has a pH range of 3.8 to 5.4 with a pK at 4.67.
Brom Phenol Blue has a pH range of 3.0 to 4.9 with a pK at
3.98. Thus the pH has to go much lower before any color change
is apparent and the standard 4 day incubation period would not
be applicable with Brom Phenol Blue. Will it work? Since this is
for a class, accuracy isn't as important but you may need to
incubate the tubes longer. How much longer I don't know. And it
may not work at all if the lactobacilli don't survive the lower
pH necessary to get a positive result.
> Also, I have been buying sheep's blood
> agar plates for the classes but I would like to start pouring
> them myself. Is there anything special I should know about this
> or is it just another media to make?
Nothing special other than cooling the medium to about 45 C before
aseptically adding the blood. There is also sometimes a problem of
bubbles in the medium. These can be removed after pouring by playing
a bunsen burner flame quickly over the surface of the agar after
pouring. The pouring and debubling need to be done somewhat quickly
because the plates solidify rather quickly at the lower temperature.
It is best to pour the plates in stacks, for this reason, rather than
having them laid out individually on the bench top. You can also add
the blood agar to the plates via some device that draws the agar from
the bottom of the flask via a tube and this will avoid the air bubble
problem. I hope this is clear. I would also suggest pouring a
small batch of 5-10 plates the first time before scaling up to 50+
plates.
Good Luck
Dick Murphy
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