Modification of the phosphoketolase assay for rapid identification of bifidobacteria

Abstract

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.

Introduction

The identification of a bacterial isolate as a member of the genus Bifidobacterium is difficult and labor intensive. The most reliable non-molecular test for identification of bifidobacteria is fructose-6-phosphate phosphoketolase activity (Scardovi and Trovatelli, 1965, Scardovi and Trovatelli, 1969). Since the fructose-6-phosphate phosphoketolase enzyme is intracellular, harvested cells are first disrupted by sonication to release cellular extracts for the assay. Cultures are identified as belonging to the genus Bifidobacterium by visual observation of the formation of a reddish–violet color.

The most time consuming aspect of the assay is cell disruption, whether by sonication or use of a French pressure cell. The equipment is expensive and cell disruption requires 5 to 10 min for each culture tested. Thus, only a small number of samples can be processed at a given time. The present procedure describes a modification of the phosphoketolase test for identification of bifidobacteria. The modified procedure does not require expensive cell disruption equipment and allows one to assay more cultures within a given amount of time.