Biochemical Identification and Qualitative determination of Bifidobacteria directly on Petri plate.

The amplification of the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) gene and a biochemical assay to
detect fructose-6-phosphate phosphoketolase (F6PPK) have been extensively used to identify Bifidobacterium spp. In conven�tional assays, after isolation and harvesting of the stationary phase, cultures were treated with different reagents sequentially,
finally leading to the formation of ferric hydroxamate (reddish-violet color) which was measured spectrophotometrically. The
existing methods (F6PPK based) for identifying Bifidobacterium spp. were developed based on the availability of pure bacterial
cultures and were not suitable for mass screening. Therefore, the simultaneous isolation and identification of Bifidobacterium
spp. directly from agar plates is needed. Here, we have developed an on-plate method for the rapid, simultaneous isolation
and identification of Bifidobacterium spp. from a variety of cultures, in which the isolation, harvesting and washing of pure
cultures have been bypassed. Assay begins with making test plate and a master plate. This process assists in obtaining the
F6PPK positive, viable colonies from the master plate. The effects of the addition of minimal concentrations of cetyltrime�thylammonium bromide (0.20–0.45 mg/mL) on cell lysis, and the subsequent color development in the plate assays, were as�sessed. Mixed cultures of E. coli. Lactobacillus, and Bifidobacterium spp. were used. No phosphoketolase activity was observed
when the plates contained E. coli, and Lactobacillus spp, and activity were observed only with disrupted Bifidobacterium spp.
The on-plate F6PPK assay developed here was an efficient alternative to the currently available (F6PPK based) methods that
was only used after the isolation of pure Bifidobacterium spp. The detection of target bacteria using this on-plate F6PPK assay
will aid future studies looking at the composition and dynamics of intestinal microbiota.