MicrobiologicalJournal of Microbiological Methods 40 (2000) 221224Journal of Methodswww.elsevier.com/locate/jmicmethModification of the phosphoketolase assay for rapid identification ofbifidobacteriaJ.I. Orban, J.A. Patterson*Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-1151, USAReceived 13 October 1999; received in revised form 4 February 2000; accepted 6 February 2000AbstractThe 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. 2000 Elsevier Science B.V. All rights reserved.Keywords: Bifidobacteria; Bifidobacterium; cell disruption; CTAB; identification; phosphoketolase1. IntroductionThe identification of a bacterial isolate as amember of the genus Bifidobacterium is difficult and labor intensive. The most reliable non-molecular testfor identification of bifidobacteria is fructose-6-phos- phatephosphoketolaseactivity(Scardoviand Trovatelli, 1965, 1969). Since the fructose-6-phos- phate phosphoketolase enzyme is intracellular, har-*Corresponding author. Tel.: 1 1-764-494-4826; fax: 1 1-764- 494-9346. E-mail address: jpatterspurdue.edu (J.A. Patterson)vested cells are first disrupted by sonication to release cellular extracts for the assay. Cultures areidentified 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 phos- phoketolase test for identification of bifidobacteria. The modified procedure does not require expensive0167-7012/00/$ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 00 )00133-0222J.I. Orban, J.A. Patterson / Journal of Microbiological Methods 40 (2000) 221 224cell disruption equipment and allows one to assay more cultures within a given amount of time.2. Materials and methods2.1. ProcedureThe reagents and procedure are as described by Scardovi (1981) for detecting fructose-6-phosphate phosphoketolase activity, except that CTAB was used for cell disruption. CTAB or cetrimonium bromide (hexadecyltrimethylammonium bromide) is a cationic detergent used as an antiseptic or cleaning agent. It is freely soluble in alcohol and also soluble in water at 1:10 ratio (CTAB:water) (Stecher et al., 1976). It has also been used to disrupt cells for whole cell enzyme assays (Patterson and Hespell, 1985). All reagents were obtained from Sigma (St. Louis, MO) and were made up in distilled water unless indicated otherwise. Trichloroacetic acid (TCA) and fructose-6-phosphate solutions were made up fresh daily.2.2. Bacterial culturesBacterial cultures tested included strains ofbifidobacteria and lactobacilli (obtained from Dr. Peter Muriano, Colorado State University, Stillwater,CO, Table 1), and 18 fecal isolates from human subjects. Fecal cultures had been isolated from anaerobic agar plates selective for bifidobacteria(Munoa and Pares, 1988). Cultures were maintained on Reinforced Clostridial broth (Difco Laboratories, Detroit, MI) and were grown overnight at 378C in 10 to 20 ml of broth.2.3. Cell preparationCells were washed twice (10 000 3 g, 48C, 15 min) with phosphate buffer (KH2PO4, 0.05 M, and cysteine ? HCl, 500 mg / liter, mixed 1:1 (V/ V), ad- justed to pH 6.5 with fresh NaOH) and resuspended in 1.0 ml of phosphate buffer. Washed bacterial cells either underwent no pretreatment, sonication for 6 min in ice, or were incubated with CTAB for 5 min prior to the assay. CTAB was added in graded amounts of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 ml (450 mg / ml stock solution) to determine the level of CTAB that would be effective for