Question

What can cause a phenol red indicator of carbohydrate fermentation to give a false positive with gas?

I heavily incubated my organism in the test tube and it is producing a huge gas bubble in the Durham tube and a clear yellow (acid) color. I incubated my tube at my organism's optimism growth conditions (oxygen and temperature wise) for 48 hrs exactly.. i was supposed to get a red no gas result ( -) what might have gone wrong?

Answer 1

The carbohydrate fermentation test is useful for the differentiation of bacteria, based o their ability to ferment a specific carbohydrate. The basal media is incorporated with a single specific carbohydrate source, Glucose/ Lactose/ Sucrose. During fermentation acid production or the acid/alkalinity is tested using pH indicator like Bromo Cresol Blue (BCB), Bromo Cresol Purple (BCP) or Phenol Red. Phenol red, is red colour in alkaline pH (at >7.4) changes to yellow in acidic pH (at <6.8), and gas production is indicated using gas accumulation inside Durham’s tube.

However, the basal media may also contain basic protein sources, like peptone.

If the tubes are heavily inoculated, or over incubated, the carbohydrate source get exhausted. The bacteria then switch to oxidative metabolism instead of fermentative metabolism. Due to oxidative breakdown of protein source, nitrogenous products are produced, giving false readings.

Further, lack of specificity or lack of sensitivity in the test, may give false results:

a) Lack of Sensitivity: Inability to detect small amounts of chemical or the organism in question. This would lead to false negative result, due to lack of adequate sensitivity while test conductance.

b) Lack of Specificity: Inability to discriminate between chemical and organism in question, similarity in chemical and organism, leads to false positive result, due to lack of specificity during test.

Sensitivity = True positives / (true positives + false negatives)

Specificity = True negatives / (true negatives + false positives)

The closer value of sensitivity or specificity near to one indicates more accuracy.