**3. Morphological identification of bifidobacteria by phase contrast microscopy (PCM)**

In the morphological analysis of bifidobacteria population, *in situ,* in human faeces and/or other foods products, microscopes have been used to determine the degree of heterogeneity of these probiotic's populations. The morphology of bifidobacteria determined microscopically has been used as an aid to phenotypic differentiation within the group, while the effect of medium type, low pH and high bile salt concentrations on the bifidobacterial cell morphology has also been studied by this method. Individual

*Bifidobacterium* strains are characterized phenotypically, including morphology identification by phase contrast microscopy (PCM).

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 289

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

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

Morphological consistency is greater among the *Bifidobacterium* isolate (*B. longum* GB-03 and *B. bifidum* WN-04) as shown in Figures 4.5 to 4.11) than the *Bifidobacterium* reference strains. Cell shapes ranged from long and thick–rods with protuberances to long and thin–rods with blunted ends and slightly bifurcated club-shaped extremities, with a number of variations on these basic shapes. Two morphological groups and their potential significance are

Figures 4.6 and 4.8 display both isolates of *B. longum* GB-03 and *B. bifidum* WN-04 on RB medium, which consisted of long and thick cells with slight bends. The regular morphology of these cells and the star-like aggregates arrangement (Figure 4.6) was evident under the PCM when grown on RB agar. Also, the presences of sparsely distributed single cells were also evident under the PCM (Figure 4.8). The morphology of these cells was consistent with any of the *Bifidobacterium* reference strains discussed previously. The isolates' morphologies resembled the reference strain of *B. infantis* which are never elongated but have a penchant

Although no conclusions could be drawn on the basis of morphology alone, the presence of "V"-shaped rods, protuberances with a large variety of bending in *B. bifidum* WN-04 isolate appeared to resemble the reference strains of *B. bifidum*, especially the "amphora-like" cells that are characteristic (Sundman & Bjorksten, 1959). On the RB media, PCM analysis allowed a better correlation of the natural isolates to the reference strains. Speciation of *B. longum* GB-03 (in Figure 4.6) conversely appeared to favour the reference strain of *B. longum*, especially the ultra-elongated and relatively thin cellular elements with slightly irregular

Reuter (1963), but were common to other species of the genus.

**3.2. Morphological differentiation of isolates of bifidobacteria** 

*3.2.1. Long and thick–rods with protuberances cell morphology* 

variety of club-shaped extreme morphology.

discussed separately below.

for group formation (Figure 4.4).

contours (Reuter, 1963).

Bifidobacteria are gram-positive, anaerobic, rods of various shapes (short, regular, thin cells with pointed ends, coccoidal regular cells, long cells with slight bends or protuberances) or a variety of branching (pointed, slightly bifurcated, club-shaped or spatulated extremities), single or chains of various arrangements (in star-like aggregates or disposed in "V" or "Y" or else "palisade" arrangements) (Scardovi, 1986).

As a pattern to characterize the heterogeneous population of bifidobacteria associated with human origin and other sources, the PCM examinations and two different media (RB & modified MRS) were used to demonstrate a better phenotypical correlation of the natural isolates to the reference strains on RB, MRS and modified MRS media as shown in Figures 4.1 – 4.12). These media are unique and appear to be still the most predominant in culturing the bifidobacteria strains.

Isolates of bifidobacteria are normally cultured anaerobically on appropriate agars at 38 ºC for 3 – 4 days. For gram-staining, a loopful of the culture is streaked on microscope slides (46 × 25 mm) and the staining technique followed thoroughly. Subsequently, the slide is observed under phase contrast microscopy, preferably at 1000 magnification by oil immersion and can be photographed as well, using the images advanced software package if available.
