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

288 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

identification by phase contrast microscopy (PCM).

or else "palisade" arrangements) (Scardovi, 1986).

the bifidobacteria strains.

*Bifidobacterium* strains are characterized phenotypically, including morphology

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"

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

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

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

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

can be photographed as well, using the images advanced software package if available.

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

(*Streptococcus thermophilus* & *Lactobacillus bulgaricus*) (see Figure 4.12).

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 discussed separately below.

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

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 for group formation (Figure 4.4).

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 contours (Reuter, 1963).

Phase Contrast Micrographs of *Bifidobacterium* reference strains: **Fig. 4.1**, *B. adolescentis* on modified MRS; **Fig. 4.2**, *B. adolescentis* on RB; **Fig. 4.3**, *B. infantis* on modified MRS and **Fig. 4.4**, *B. infantis* on RB, taken at 1000 magnifications.

*Bifidobacterium* in Human GI Tract:

Screening, Isolation, Survival and Growth Kinetics in Simulated Gastrointestinal Conditions 291

**Figure 4.** Phase Contrast Micrographs of *Bifidobacterium* strains: **Fig. 4.9**, *B. bifidum* WN-04 on unmodified MRS; **Fig. 4.10**, *B. longum* GB-03 on unmodified MRS; **Fig. 4.11**, *B. infantis* on unmodified MRS and **Fig. 4.12**, *B. longum* GB-03 and an assortment of other 2 Lactic Acid Bacteria (*Streptococcus* 

This was the most common type of morphology encountered among the *Bifidobacterium* isolates of *B. longum* GB-03 and *B. bifidum* WN-04 on the unmodified MRS agar (Figures 4.5, 4.7, 4.9 & 4.10). Since only the general cell structure was used to differentiate this species from the other bacteria, PCM proved sufficient for this purpose. Variations of morphology within these small groups were visible under PCM as indicated by the following examples. The isolate of *B. longum* GB-03 in Figure 4.12 exemplified the diversity of rods and coccus cells morphology including bifid structures also; with the absence of any coccus build cells when grown on RB agar in Figure 4.6. By comparison with the reference strains, the cells morphology of *B. longum* GB-03 isolate is more peculiar to that displayed by *B. infantis* (Figure 4.4) and the isolate of *B. bifidum* WN-04 (Figures 4.7 & 4.8). All the *Bifidobacterium* isolates displayed long and short club-shaped rods, most of which were long and thin with

F6PPK is certainly a key enzyme in the "bifidus pathway" and it allows the discrimination of the specific feature on expression of fructose-6 phosphate in cellular extracts that assigned

*thermophilus* & *Lactobacillus bulgaricus*), taken at 1000 magnification.

*3.2.2. Long and thin–rods with blunted ends cell morphology* 

blunted ends and of conventional "V" and/or "Y"-shaped cells.

**3.3. Confirmation of identity of** *Bifidobacterium* **strains** 

the bifidobacteria to the genus level (Sgorbati, 1979).

*3.3.1. Fructose-6-Phosphate Phosphoketolase (F6PPK) verification test* 

Phase Contrast Micrographs of the isolate strains: **Fig. 4.5**, *B. longum* GB-03 on modified MRS; **Fig. 4.6**, *B. longum* GB-03 on RB; **Fig. 4.7**, *B. bifidum* WN-04 on modified MRS and **Fig. 4.8**, *B. bifidum* WN-04 on RB, taken at 1000 magnifications.

**Figure 4.** Phase Contrast Micrographs of *Bifidobacterium* strains: **Fig. 4.9**, *B. bifidum* WN-04 on unmodified MRS; **Fig. 4.10**, *B. longum* GB-03 on unmodified MRS; **Fig. 4.11**, *B. infantis* on unmodified MRS and **Fig. 4.12**, *B. longum* GB-03 and an assortment of other 2 Lactic Acid Bacteria (*Streptococcus thermophilus* & *Lactobacillus bulgaricus*), taken at 1000 magnification.
