This is a carbohydrate fermentation test I did with several bacterial species. I used Phenol Red as an indicator and added 1% of different carbohydrate in each tube. This is after 23 hours incubation at 25 degrees Celcius. We incubate at this temperature, as my bacteria grow better at this temp. Some of the samples turned out like this. I don't really know how to interpret these? Can anyone help?
Dear Doroteja,
Although using a simple pH-sensitive indicator dye to determine whether various organisms are capable of fermentation is easy to understand, the assays themselves can be complex. Overall, any colour change will depend on the growth rate and cell numbers of the organism, plus their ability to up-take the substrate etc., and if the balance between growing cells, sample volume and dye concentration is not ideal, you can get some intermediate results (you should always shake your culture before assay as sometimes colour gradients can be confusing as in the middle of the bottom three images).
One way forward is to use a colour chart to record the colour of each culture at set time points, and you can arbitrarily say that once a culture has reached a particular colour it will be recorded as being positive or not - and once checked, simply allow the cultures to grow on and recheck until you are satisfied that sufficient cells have had a chance to ferment (or not). You could also follow a strict protocol that has been published for isolate indentification and simply accept that some strains cannot be characterised because of indeterminate results).
Regards, Andrew
Hello, Doroteja Vaitiekunaite
The picture is more or less typical for bacterial carbohydrate fermentation test
Phenol red is yellow at a pH < 6.8 and red at a pH of > 7.4, therefore if a bacterium ferments a sugar to acid a yellow color will develop.
On picture
A) Formation of acid and gas.
(B) Formation of acid,
(C) inoculated control,
D) alkaline byproducts,
(E) no acid or gas formation.
So you have acid and low alkaline formation in your tubes.
The orange color in the tube, this represents a pH > 6.8 and should not be considered as positive. The yellow tubes are positive.
Hope my explanation is clear.
Good luck
Andrew J Spiers thank you for your help. I was just thinking that the bacteria might not have reached the necessary concentration. However a lot of protocols say that you shouldn't wait for more than 24h because the protein digestive byproducts may change the results. Then again, I'm working with endophytes, they don't always grow as fast. So thank you again.
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