Modification of the phosphoketolase assay for rapid identification of bifidobacteria.
The phosphoketolase assay is commonly used as a definitive criterion for identification of bifidobacteria. A limitation of the assay is the time-consuming process of cell disruption, either by use of the French Pressure Cell or by sonication. We have replaced the time consuming cell disruption process with a more rapid cell membrane disruption process by pretreating cells with the detergent hexadecyltrimethylammonium bromide (cetrimonium bromide, CTAB). The effect of no pretreatment, sonication or the addition of CTAB (0.45 mg/ml) on color development in the phosphoketolase assay was tested using pure cultures of bifidobacteria and lactobacilli. No phosphoketolase activity was observed with bifidobacterial cultures without cell disruption or with lactobicilli that had undergone cell disruption. All bifidobacterial cultures gave a similar color formation whether sonication or CTAB addition was used to disrupt cells. Use of CTAB to disrupt cell membranes is an effective alternative to the time consuming traditional cell disruption procedures and increases the number of cultures that can be simultaneously assayed and presumptively identified using the phosphoketolase assay.
Orban
J I
JI
Department of Animal Sciences, Purdue University, West Lafayette, IN, USA.
Patterson
J A
JA
D000446:Aldehyde-Lyases / Q000378:metabolism*; D015373:Bacterial Typing Techniques; D001644:Bifidobacterium / Q000145:classification* / Q000201:enzymology; D002458:Cell Fractionation; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D013010:Sonication
D002593:Cetrimonium Compounds; D000446:Aldehyde-Lyases; C065556:fructose-6-phosphate phosphoketolase; D000077286:Cetrimonium