2.3 Results and Discussion
2.3.2 Morphological, biochemical and physiological characterization of the isolate DM5 DM5
2.3.2.1 Gram staining and scanning electron microscopic analysis of isolate DM5 The appearance of violet colour cells after Gram staining, confirmed the Gram positive nature of the isolate DM5, a characteristic of lactic acid bacteria (Fig. 2.3.1).
It was also revealed through Gram staining that the cells were rod shaped.
Fig. 2.3.1 Gram staining of the isolate DM5 showing violet colour cells, indicating the Gram positive nature.
Scanning electron microscopy of the isolate DM5 showed phenotypically homogeneous short rod cells, arranged singly or in pairs with a width and length of 0.5-0.6 m and 1.2-1.4 m, respectively (Fig. 2.3.2). Moreover, when the isolate DM5 was grown in MRS agar (2%, w/v) plate at 37°C for 24 h, it showed the white, smooth and small circular colonies (Fig. 2.3.3).
Fig. 2.3.2 Scanning Electron Microscopic analysis of the isolate DM5 showing rod shape and random arrangement of cells.
Fig. 2.3.3 Colony morphology of the isolate DM5 showing white, small circular colonies on MRS agar plate at 37ºC.
2.3.2.2 Triple sugar iron and motility indole test of isolate DM5
The triple sugar iron (TSI) slant contains agar, a pH-sensitive dye (phenol red), lactose, sucrose, glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous
A B
ammonium sulfate. If the test organism is able to ferment any of the three sugars in the medium and produce the by-products, which are usually acids, which will change the colour of the phenol red to yellow. If the test organism is able to utilize thiosulphate anion as a terminal electron acceptor, reducing it to sulphide, the newly-formed hydrogen sulfide reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a black precipitate. The isolate DM5 was able to ferment all the three sugars and change the red colour of the medium to yellow but it did not utilize thiosulphate anion and hence was unable to produce any black precipitate. On the other hand, E. coli DH5α used as a positive control was able to change the colour of the medium to yellow producing black precipitate as shown in Fig. 2.3.4.
Fig. 2.3.4 Triple Sugar Iron Agar test of the isolate DM5. (A) Triple sugar iron agar slant inoculated with E. coli DH5α showing the black precipitate.
(B) Triple sugar iron agar slant inoculated with isolate DM5.
The motility Indole Lysine Agar Slant is a semisolid agar used for the identification of the members of the family Enterobacteriaceae by detecting motility, indole and lysine decarboxylation or deamination. The isolate DM5 grew along the stab line keeping the surrounding medium clear which indicated that isolate DM5 was
non-motile and did not belong to the family Enterobacteriaceae. Whereas, E. coli DH5α which was used as a positive control when stabbed into the semi-solid agar migrated by means of its flagella and produced turbidity throughout the medium thus giving a positive result for the motility test (Fig. 2.3.5).
Fig. 2.3.5 Motility Indole Lysine test of the isolate DM5. (A) Motility indole lysine agar stab inoculated with E. coli DH5α showing turbidity throughout the medium. (B) Motility iron agar stab inoculated with isolate DM5 with no turbidity.
2.3.2.3 Detection of nitrate production and catalase activity
The nitrate agar slant is composed of a medium that contains large amount of nitrate along with alpha-napthylamine and sulphanilic acid. These two compounds react with nitrite and turn red in colour. If the test organism is able to reduce nitrate to nitrite or another nitrogenous compound such as ammonia or nitrogen gas, the medium turns red in colour due to production of nitrite, indicating a positive nitrate reduction test. The isolate DM5 was unable to grow or produce any colour change in the medium indicating a negative nitrate reduction test. E. coli DH5α was used as a control which showed nitrate reducing ability (Fig. 2.3.6).
A B
Fig. 2.3.6 Nitrate reduction test of bacteria. (A) Nitrate agar slant inoculated with E.
coli DH5α showing red colour. (B) Nitrate agar slant inoculated with isolate DM5.
The catalase activity test proved the catalase negative nature of the isolate DM5 as it could not hydrolyse H2O2, whereas, E. coli DH5α was able to hydrolyse H2O2, which was evident from the bubbling that was observed when drops of H2O2
was added to E. coli DH5α culture (Fig. 2.3.7). The above all morphological and biochemical characteristic features of isolate DM5 are enlisted in Table 2.3.2. The results biochemical tests of isolate DM5 are compared with gram positive Pediococcus pentosaceous SPA and gram negative Escherichia coli DH5α.
Fig. 2.3.7 Catalase activity test of bacteria. (A) E. coli DH5α showing bubble in presence of 3% H2O2. (B) Isolate DM5 showing no bubble formation.
A B
A B
Table 2.3.2 Morphological and Biochemical Characteristic features of isolate DM5.
Parameter Isolate DM5 P. pentosaceus SPAa E. coli DH5α
Gram staining Gram positive Gram positive Gram negative Cell morphology Small rod shape Cocci shape Rod shape Colony characteristic White, smooth
small colony
White smooth circular colony
Small circular colony
Catalase test Negative Negative Positive
Motility test Non motile Non motile Motile
Indole test Negative Negative Positive
Nitrate test Negative Negative Positive
H2S gas production Negative Negative Positive
aPatel and Goyal, 2010.
2.3.2.4 Temperature, pH and salt tolerance of isolate DM5
The isolate DM5 showed growth between 10 to 40°C, but not beyond 40°C, indicating the mesophilic nature of the isolate DM5. This result indicated that the isolate DM5 could be associated with widespread member of genus Lactobacillus plantarum, which was unable to grow at 45°C like Lactobacillus helviticus and Lactobacillus delbrueckii (Briggs, 1953). The isolate DM5 could grow in presence of 6.0% and 6.5% (w/v) NaCl; however, further increase in salt concentration inhibited the growth of the isolate DM5. The isolate DM5 could grow efficiently within pH range of 4-8 but, the pH of 2 and 10 did not support the growth of the isolate at 37°C.
Among all the Lactobacillus species, Lactobacillus plantarum was able to grow in presence of 4% (w/v) NaCl and in low pH (Briggs, 1953), therefore the isolate DM5 could be cluster with Lactobacillus plantarum. The isolate DM5 also showed similar growth pattern with the isolate Lb. plantarum from Hamei (traditional alcoholic beverage in Manipur, India), which was able to grow in presence of 6.5% NaCl and at
pH 3.9 but was unable to grow in presence of 8% NaCl and pH 9.6 (Tamang et al., 2007).