**3.1. Morphological characterization of** *Bifidobacterium* **reference strains**

The basic morphologies, namely short, regular, thin cells with pointed ends, coccoidal regular cells, and long cells with slight bends or protuberances are discernible among the 2 *Bifidobacterium* reference strains (*B. adolescentis* and *B. infantis)* shown in Figures 4.1, 4.2, 4.3, 4.4 and 4.11) on modified MRS and RB media. From these micrographs alone, it is obvious to validate that individual variations of the average phenotypic morphologies of bifidobacteria are present as described earlier. The PCM also provided a rapid and clear visualization of the basic bifidobacteria cell morphology, while at the same time, allowed only broad comparisons amongst the bifid structures within a mixture of 2 other LABs (*Streptococcus thermophilus* & *Lactobacillus bulgaricus*) (see Figure 4.12).

The typical colonies of bifidobacteria are altogether round and white on RB and modified MRS media. Colonies are usually picked off of a suitable plate and may be kept sub-cultured 2 – 3 times on a freshly prepared agar as to obtain pure culture without contamination. The morphologies of the 2 reference strains and their relationship to each will now be discussed separately. When the strain of *B. adolescentis* is resuscitated and cultured on modified MRS medium (Figure 4.1) or RB medium (Figure 4.2), it may be differentiated clearly from the *B. infantis* (Figures 4.3 & 4.4) on the basis of morphology. The *B. infantis* was also resuscitated and cultured under the similar conditions. As it can be observed from Figure 4.2, *B. adolescentis* on RB displayed long and thick rod-shaped and regular coccoidal cells. The cells of *B. adolescentis*  strain on RB were almost paired and assembled, a feature which was highlighted by PCM. The existence of distinct "V"- and/or "Y"-shapes and some long cells with protuberances or slight curvature of this isolates when grown on RB agar, is a powerful diagnostic feature, particularly when distinguishing this specie from closely related *B. minimum* when grown on Trypticase-Phytone-Yeast extract (TPY) agar stabs (Biavati *et al.,* 1982). In addition, curved cells with smooth and rounded ends are the most one dominating in the micrograph. These features were not compatible with descriptions of this particular species' morphology as described by Reuter (1963), but were common to other species of the genus.

The *B. infantis* strain displayed slender, often short rod-shaped and of the typical clubshaped extremities, which cells of these species are reported to exhibit (see Figures 4.3 & 4.4). The morphology of this strain is almost the same when grown on both the MRS and RB solid growth media. Furthermore, *B. infantis* showed a distinct tendency for chain formation on RB medium. These cells often occurred in "V" and "Y"-shapes and were similar to that of many other species of the genus. Nevertheless, it was also possible to differentiate between this strain and the closely related *B. longum* GB-03 (own isolate, Fig. 4.6) on the basis of small variety of club-shaped extreme morphology.
