Cautions Hugh & Leifson's medium is often used as a basal medium. This medium normally contains bromothymol blue which may be toxic to some microbes.

If the microbe cannot grow in Hugh & Leifson's medium use a different basal medium enriched with either 2% serum of 0.1% yeast extract.

Some microbes only show acid production after several days of incubation because the sugar fails to reach the inside of the cell of these 'late lactose fermenters'. The ONPG test can be used to determine the fermentative ability of these microbes.

Method The O-F test is performed by inocculation of two tubes of media, one of which is covered with a layer of sterile oil. The carbohydrate source is usually glucose but the basal medium should be supplemented with maltose or pentoses for those microbes that do not attack glucose. .
Results Oxidizers show acid production in the open tube only while fermenters show acid in the oil covered tube and often in the open tube. Some microbes may grow but may not produce acid in either tube. Confirm this result on a different medium.
Positive control  
Negative control