Hippurate test
The figure shows the result of an hippurate test , where tube A is a negative control. Tube B contains Streptococcus agalactiae, which is hippurate positive and tube C contains Streptococcus dysgalactiae subsp. dysgalactiae, which is hippurate negative. Note that a colour change has occured in tube B because S. agalactiae is hippurate positive. Observe also that hippurate negative bacteria may give a faint colour change.
Image: Karl-Erik Johansson (BVF, SLU) och Lise-Lotte Fernström (BVF, SLU).
General
Some bacteria can hydrolyze hippurate to the amino acid glycine and benzoate by means of the enzyme hippuricase. Glycine can be detected with ninhydrin (2,2-Dihydroxyindane-1,3-dione), which reacts with free amino groups (-NH2) and a blue product is formed.
Method
- Suspend a loop-full of bacteria (for instance Campylobacter sp.) in 0.5 ml of sodium hippurate siolution.
- Incubate the suspensionen at 37ºC during 2 h in a water bath.
- Then carefully add 0.2 ml of ninhydrin solution without mixing.
- Incubete the dube during another 10 min. at 37ºC before reading the result.
- Positive test resultat: Deep blue colour.
- Negative test result: Pale blue colour.
Use
The hippurate test is primarely used to distinguish between Campylobacter jejuni (hip+) and Campylobacter coli (hip-) and to distinguish between different streptococci (see figure).The test is also used, in combination with other methods, to type Brachyspira spp.