Kelman-tetrazolium (KM-TTC) medium [1] contained 1.0 g l^-1 casein hydrolysate (enzymic digest), 10 g l^-1 bacteriological peptone, 5 g l^-1 glucose, and 15 g l^-1 bacteriological agar and was fortified with 0.005% 2,3,5-triphenyl tetrazolium chloride (TTC). All the media ingredients were sourced from Hi Media Biosciences, Mumbai (referred to as HM hereafter), and the constituents normally employed in KM-TTC included bacteriological peptone (Cat. no. RM 001-500G), casein enzyme hydrolysate Type II (CR 028-500G), dextrose - anhydrous (RM077-500G) and bacteriological agar (RM 006-500G). TTC was sourced from Sigma-Aldrich, St. Louis, MO, USA (referred to as Sigma hereafter), and the stock (10 mg ml^-1) after filter-sterilization was stored as aliquots of 1.25 ml at -20°C, and incorporated to 250 ml of autoclaved (20 min at 1.1 Kg cm^-2) and cooled medium just before pouring into plates. Single-use pre-sterilized 9 cm plates (HM, Mumbai) with 20 ml medium were used in all the studies. The media plates were used on the same day 1-2 h after pouring or were stored at 4°C for up to 1-2 weeks. Other media employed included CPGA and SMSA [18]. The experiments were conducted during the period August 2013 to January 2014.

The isolate ‘NH-01’, originally isolated after plating the bacterial ooze from a wilted ‘Namdhari Hybrid’ tomato plant at the Institute farm on KM-TTC [16] formed the original experimental isolate (NCBI KJ399970). Unless mentioned differently, all the experiments were set up with the re-isolate ‘NH-Av01’ (race 1, biovar 3) from the susceptible tomato cv. Arka Vikas. The identity of this isolate (NCBI KJ412034) was confirmed through 16S rRNA seqmatch analysis at the Ribosomal Database Project of the Michigan State University with the maximum homology to the Type Strain LMG2299 (EF06361). The closest matches (99% homology) from the NCBI included the strain GRs-Asm isolated from Zingiber officinale (Jorhat, Assam, India; JQ359677) and strain HNEPRS1 from eggplant (Hainan province of China; KF030881) as on July 2014.

For the preparation of bulk glycerol stock, inoculum from 2-day old colony growth on CPGA was employed. For this the pooled inoculum from 4-5 typical fluidy-red colonies formed on KM-TTC following the streaking of -80°C glycerol stock was spotted on CPGA and the inoculum of 1.0 OD was prepared in autoclaved and filtered (0.2 µm) 20% glycerol, dispensed in 400 µl aliquots in 1.5 ml microfuge tubes (300 nos) and stored in three different -80°C freezer units to safeguard from unexpected instrument failure. Other isolates of R. solanacearum used in this experiment, which were obtained from different laboratories / geographic locations / crops, are described below under the section ‘KM-TTC peptone effects on different isolates of R. solanacearum’.

The effect due to different KM-TTC media treatments on colony characteristics of R. solanacearum was assessed by spotting 20 times of 5 µl stock culture of -80°C glycerol stock of ‘NH-Av01’ (100 µl per plate) after pooling the 1.0 OD inoculum from the requisite number of tubes. Post spotting, the inoculum was allowed to dry by open incubation in the laminar air flow cabinet for 10-12 min and the plates were incubated upside-down at 30°C after sealing individually in polypropylene (PP) bags. The colony characteristics were recorded based on visual assessment, colony diameter and scoring on a 0-5 scale for pigmentation (0, no pigmentation; 5, intense red-maroon color) and photographic documentation after 24-48 h in initial trials. In the subsequent trials, the extent of growth (which was also a reflection of the colony fluidity) was assessed per colony basis after retrieving the inoculum individually from three-six replicate spots (depending on the number of treatments) in 1 ml water by repeated rinsing (of the spot) until recovering all evident colony growth. This was followed by A₆₀₀ assessment employing a uv/vis spectrophotometer (Genesys 10uv, Thermo Scientific, New Hampshire, USA). An estimate of the extent of pigment production per colony was obtained by spinning down the sample at 14000 ×g for 3 min and OD estimation of the supernatant at 500 nm.

Considering that the red pigmentation in KM-TTC is due to formazan development from TTC, initial trials were planned around this constituent. TTC from two sources (HM and Sigma) was tried in KM-TTC employing freshly prepared and filter-sterilized stocks. Further, to assess whether the freezing and thawing of TTC stock could contribute to variations in colony characteristics, 1.25 ml aliquots for single-use in 250 ml medium, or the stock stored in 10 ml lots with recurrent thawing and re-freezing were assessed in comparison with freshly prepared TTC stock employing the Sigma and HM supplies. The basal medium included HM bacteriological peptone, casein enzyme hydrolysate type II (CH), dextrose and bacteriological agar. The peptone used here was selected from a lot that displayed fluidy- red colonies on KM-TTC.

In the initial trial, two peptone types from HM, namely (A), bacteriological peptone (RM 001; enzymatic hydrolysate of animal tissues) and (B), type-1 bacteriological peptone (RM 667; mixed enzymic hydrolysate of lean meat tissues) were tried. As per the outcome of the above trial, peptone belonging to two lots from each type (identified as A1, A2, B1 and B2) were tested. Based on the emerging information, eight different lots from HM including four each of bacteriological peptone (A1 to A4) and type-1 peptone (B1 to B4) obtained from different laboratories (Table 1) were tried.

In the next trial, different types of peptone (bacteriological, type-1, veg-bacteriological, microbiological or mycological as per the description given by different vendors) including varying batches / lots from one source (HM) or different brands (BD Biosciences, Sparks, MD, USA; Sigma-Aldrich, St. Loius, MO, USA; Biomatik Corporation, Cambridge, Ontario, Canada; Finar Limited, Ahmedabad, India; Sd-fine Chemicals, Mumbai, India) sourced from different laboratories were tested in KM-TTC medium. There were 20 media treatments altogether including KM-TTC with peptone from 19 distinct sources (P1 to P19) plus CPGA with P1 peptone as control (Table 2). The composite basal formulation constituted CH and agar from HM, and TTC from Sigma. The P1 medium used in this trial had a new lot of HM bacteriological peptone (06/13 manufacturing). P1-P7 constituted HM bacteriological peptone of different lots while P14-P16 included HM type-1 peptone from three lots. Some of the peptone lots used in this trial did not mention the manufacturing or the expiry date while a few had crossed the expiry, but were chosen with a view to test the effects due to ageing or hydration. Pooled and thoroughly mixed inoculum from six ampoules of glycerol stock was used for spotting (5 µl per spot ( 20) followed by incubation at 30°C and the assessment of colony characteristics and growth visually as well as through A₆₀₀ and A₅₀₀ estimations after 24-48 h.

Additional isolates from different geographical locations or crop sources were evaluated in comparison with ‘NH-Av01’ employing different KM-TTC (peptone) formulations. These included (i) ‘Rs-01-11’ isolate from eggplant, (ii), ‘Rs-09-94’ from chilli (race 1, biovar 3) sourced from Dr. R. Ramesh, ICAR Research Complex, Goa, South-West India, (iii), ‘GRs-Mnt’ (race 4, biovar 3), isolated from ginger (provided by Dr. Aundy Kumar, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi) and (iv), ‘KAU-Av01’ (race 1, biovar 3), isolated at the host laboratory from a wilted ‘Arka Vikas’ fruiting-stage tomato plant from the Agricultural Research Station, Kerala Agricultural University, Mannuthy, Kerala, South India, a hot-spot for bacterial wilt in solanaceaous vegetables. Glycerol stocks of these four isolates prepared and stored similar to the ‘NH-Av01’ isolate were spotted on the 20 media formulations (KM-TTC P1 to P19 and CPGA) followed by colony growth and pigmentation assessments.

Assessment of the virulence of cultures derived from the 19 KM-TTC (peptone) sources was made together with the culture from CPGA through seedling-challenge assay under glasshouse conditions employing the native ‘NH-Av01’ isolate. In a preliminary trial, inoculation of two week old seedlings of tomato (Solanum lycopersicum) cv. Arka Vikas was undertaken employing 2 ml of 0.1 A₆₀₀ inoculum (in water) per seedling. The experiment was repeated with root-injury-inoculation on two week old seedlings as per Thomas et al. [16]. The trials were laid out in completely randomized design with seedlings raised in organic coco-peat in protrays employing 10 replications (7 seedlings / replication). The extent of disease incidence (irreversible wilting or seedling mortality) was recorded weekly for four weeks. The experiment was repeated once. Correlation coefficient (r) between the extent of colony growth (A₆₀₀) or the pigmentation of colony supernatant in water (A₅₀₀) and percent seedling mortality was worked out. Similarly, r between mortality (%) and pigment levels in the inoculum used for seedling inoculation (0.1 at A₆₀₀) was determined to see if the pigment levels had any effect on culture virulence.

To assess if the colony characteristics observed on different peptone media influenced the response of pathogen in biotype classifications, biovar assay was set up with ‘NH-Av01’as per Huang et al. [15]. KM-TTC prepared with peptone from 11 different lots / types / brands that displayed considerable variation in colony characteristics or pigment levels (P1, P2, P7, P9, P10, P11, P12, P13, P15 and P18 with CPGA control; see Table 2) was used as the source of inoculum. The experiment was repeated once.

Effect on bacterial lawn development towards organizing antagonistic assays [19] was investigated by plating the suspension of ‘NH-Av01’ grown in CPG broth on 20 KM-TTC peptone formulations as above. Plates (10 cm diameter; HM, Mumbai; cat. No. PW002-1(500N0) with 50 ml medium were applied with 125 µl of 0.1 A₆₀₀ inoculum. After 4 h incubation at 30°C, 50 µl of 0.1 A₆₀₀ inoculum of two test organisms that had shown antagonistic activity against the pathogen (Enterobacter sp. and Pseudomonas sp.; Thomas unpublished results) were applied in marked wells. The plates were observed for lawn formation and inhibition-zone development for 2-4 days [19].

This included casein hydrolysate (CH) and glucose. Casein hydrolysate from two sources, namely, enzyme hydrolysate type II from HM (CR 028; enzymic digest of casein type II) and that from Sigma (Fluka 22090; pancreatic hydrolysate of casein) were tried in KM-TTC. Other constituents (bacteriological peptone, glucose and bacteriological agar) were from Hi Media. The source of glucose included HM, Fischer Scientific, Mumbai; Sisco Research Laboratories, Mumbai; Sd-fine Chemicals, Mumbai and Loba Chemicals, Mumbai. In addition, substitution of glucose in the medium with sucrose was tried employing HM source.

The experiments were laid out in completely randomized design [20] and the data were subjected to single factor ANOVA employing the data analysis tool of Microsoft Excel 2007. The mean ± SD values are presented in the results.

The extent of colony growth, fluidity and red pigmentation were relatively more with Sigma TTC compared with HM TTC. This was reflected in the whole colony growth (A₆₀₀ of 2.058 ± 0.110 and 1.548 ± 0.015, respectively) and the pigmentation of supernatant (A₅₀₀ of 0.069 ± 0.0254 and 0.012 ± 0.002, respectively) documented on day 2. Thus, Sigma TTC stock was selected for use in all further experiments. No significant differences were observed in the extent of colony growth with the use of fresh, one-time thawed or three-times thawed stocks of TTC, either from Sigma or HM source, but the pigmentation levels showed some variation, which however was not statistically significant (Fig. 1a, b; Fig. 2). In general, freshly prepared Sigma TTC showed relatively more pigmentation than fresh HM TTC consistent with the observations in the initial trial. For the sake of convenience, TTC aliquoted and stored at -20°C was preferred.

Fig. (1). Colony characteristics of Ralstonia solanacearum ‘NH-Av01’ on KM-TTC medium with the TTC from two sources. Colony growth as per OD at A600 (a) and pigmentation of supernatant at A500 (b) with the use of fresh, one-time thawed or three-times thawed stocks of TTC from HM and Sigma sources.

Fig. (2). Effect due to freshly prepared TTC versus frozen and thawed TTC employed in KM-TTC on colony characteristics of Ralstonia solanacearum ‘NH-Av01’ isolate. Medium prepared with fresh (a), frozen and one-time thawed (b) or three-times thawed (c) stocks of TTC from Sigma (upper row) or HM source (lower row).

Fig. (3). Comparing the colony growth of Ralstonia solanacearum ‘NH-Av01’ on KM-TTC with different types of peptone. New stock of Hi Media bacteriological peptone (A1) and an older lot (A2) and two lots of type-1 peptone including a recent (B1) and an older lot (B2) used in KM-TTC and documented photographically on day 1 (a) and day 2 (b). In grey scale, high red pigmentation appears dark in color and low pigmentation whitish.

Fig. (4). Effect due to different lots of bacteriological peptone (A 1-A4) or type-1 peptone (B1-B4) from Hi Media Biosciences source on colony characteristics and pigmentation of Ralstonia solanacearum ‘NH-Av01’ isolate recorded on day 2. Description of A1-A4 and B1-B4 as per Table 1.

Fig. (5).  Colony growth of Ralstonia solanacearum ‘NH-Av01’ on KM-TTC with peptone belonging to different brands, types or lots. P1-P19 represents 19 different KM-TTC (peptone) formulations with different brands and lots as in Table 2 and P20 represents CPGA control. Considerable differences in the extent of colony growth and pigmentation observed as documented on day 2 in rear view (a) or in front view (b).

Fig. (6). Colony growth as per OD at A600 (left panel) and extent of colony pigmentation as per A500 (right panel) of different isolates of Ralstonia solanacearum from various crop sources or geographic locations on KM-TTC documented on day 2. P1-P19 represents 19 KM-TTC peptone formulations with different brands and lots as in Table 2 and P20 represents CPGA control. The isolates included ‘NH-Av01’ from tomato, ‘Rs.01.11’ from eggplant, ‘Rs. 09-94’ from chilli, ‘GRs-Mnt’ from ginger and ‘KAU-Av01’ from tomato.

Fig. (7). The colony growth of Ralstonia solanacearum isolate ‘Rs.01.11’ from eggplant and ‘Rs. 09-94’ from chilli on KM-TTC (peptone) formulations with peptone belonging to different brands, types or lots. P1-P19 represents 19 different KM-TTC peptone formulations with different brands and lots as in Table 2 and P20 represents CPGA control. Upper two rows in a plate show ‘Rs.01.11’ and the lower two rows ‘Rs. 09-94’. Considerable differences in the extent of colony growth and pigmentation as documented on day 2 in rear view.

Fig. (8). The colony growth of Ralstonia solanacearum isolate ‘Rs.01.11’ from eggplant and ‘Rs. 09-94’ from chilli on KM-TTC (peptone) formulations with peptone belonging to different brands, types or lots. P1-P19 represents 19 different KM-TTC peptone formulations with different brands and lots as in Table 2 and P20 represents CPGA control. Upper two rows in a plate show ‘Rs.01.11’ and the lower two rows ‘Rs. 09-94’. Considerable differences in the extent of colony growth and pigmentation as documented on day 2 in front view of open plates.

Fig. (9). The colony growth of Ralstonia solanacearum isolate ‘GRs-Mnt’ from ginger (Race 4) and ‘KAU-Av01’ from tomato on KM-TTC (peptone) formulations with peptone belonging to different brands, types or lots. P1-P19 represents 19 different KM-TTC peptone formulations with different brands and lots as in Table 2 and P20 represents CPGA control. Upper two rows in a plate show ‘GRs-Mnt’ and the lower two rows ‘KAU-Av01’. Considerable differences in the extent of colony growth and pigmentation as documented on day 2 in rear view.

Fig. (10). The colony growth of Ralstonia solanacearum isolate ‘GRs-Mnt’ from ginger (Race 4) and ‘KAU-Av01’ from tomato on KM-TTC (peptone) formulations with peptone belonging to different brands, types or lots. P1-P19 represents 19 different KM-TTC peptone formulations with different brands and lots as in Table 2 and P20 represents CPGA control. Upper two rows in a plate show ‘GRs-Mnt’ and the lower two rows ‘KAU-Av01’. Considerable differences in the extent of colony growth and pigmentation as documented on day 2 in front view of open plates.

Fig. (11). Effect of peptone source in KM-TTC medium on the virulence of Ralstonia solanacearum during seedling challenge inoculation on tomato. Following root-injury and soaking-inoculation of cv. Arka Vikas (after two weeks of sowing), seedling mortality was recorded four weeks later (a). The extent of colony pigmentation in the supernatant of whole colony suspension and of the 0.1 A600 inoculum used for seedling challenge were recorded at A500 (b).

Fig. (12). Effect due to peptone from 12 different sources on colony growth of Ralstonia solanacearum ‘NH-Av01’ in KM-TTC medium (a) and the response of the culture during biovar assay (b). Peptone treatments from different brands as indicated in Table 2. The biovar assay was as per Huang et al. [15]. Treatments in the order control, glucose, trehalose, mannitol, sorbitol, dulcitol, maltose and cellobiose.

Fig. (13). Effect due to different peptone sources in KM-TTC medium during antagonistic assayagainst Ralstonia solanacearum in agar-well diffusion assay. KM-TTC / CPGA medium prepared with different peptone formulations, plated with the isolate ‘NH-Av01’, and inoculated with 50 µl A600 culture of Enterobacter sp. (lower left) and Pseudomonas sp. (lower right) showing differences in the evenness of lawn, fluidity of culture and the extent or the clarity of clear-zone. Upper row shows distilled water and null controls. P1 to P20 as per Table 2.

Fig. (14). Effect due to glucose from different sources or sucrose in KM-TTC medium on colony characteristics of Ralstonia solanacearum ‘NH-Av01’. Glucose from Hi Media (a), Fischer Scientific (b), Sisco Research Laboratories (c), Sd-fine Chemicals (d) and Loba Chemicals (e), or sucrose (f) from Hi Media Biosciences.

It was not possible to obtain a clear estimate of the extent of colony fluidity. In general, more the colony growth, the more was the culture fluidity and vice versa. Thus, the extent of colony growth documented by way of A₆₀₀ estimation was also a reflection of the extent of culture fluidity.

The chance observation that the failure of fluidy reddish colony development in KM-TTC coincided with the use of a new bottle of HM bacteriological peptone and that peptone formed the major constituent in KM-TTC medium prompted detailed experiments evaluating the effects due to peptone. As it was no more possible to evaluate the previous lot of peptone since the stock had run out, KM-TTC plates prepared with the earlier batch and stored at 4°C were used for validation which confirmed that the new lot differed from the previous one. This apparently suggested the chances of a defective new batch of peptone.

Comparing the new stock of bacteriological peptone (A1) with another older lot (A2) along with two lots of type-1 peptone including a recent (B1) and an older lot (B2) (all from HM) indicated that both the type and lot of peptone had prominent effects on colony characteristics of R. solanacearum. While the A1 lot showed low pigment formation, A2 displayed proper colour development by 16-24 h. Type-1 peptone showed an opposite trend where the newer B1 stock displayed intense red colony colour and the older lot showed lighter pigmentation (Fig. 3a). By second day, the difference in colony pigmentation and fluidity became more obvious (Fig. 3b). By this time, 3 weeks had elapsed after the opening of the ‘A1’ stock and an increase in the extent of color development with the same lot was observed suggesting an ageing or hydration effect with the ‘A1’ peptone.

Extending the observations to four lots of bacteriological peptone (A1- A4, where A4 was same as A1, but a sub-lot of it stored with loose capping under the ambient conditions for 2 weeks), or four lots of type 1 peptone (B1- B4) sourced from different laboratories endorsed that the extent of colony growth and colony color were significantly influenced by the peptone type and the lot (Fig. 4; Table 1). In general, it appeared that older the lot, more the colony fluidity and intensity of red coloration. The observations with the A1-derived sub-lot A4 also suggested the chances of such an ageing effect on growth and colony color. The time-span from the date of manufacturing and that after opening the bottle both appeared to influence the overall bacterial colony growth and the extent of pigmentation. The A2 and B4 lots which had crossed the expiry date showed good fluidy growth and high red color development.

The trial employing 19 different KM-TTC peptone formulations with different brands and lots of peptone (P1-P19; plus CPGA control) again showed considerable differences in the extent of colony growth and pigmentation as documented on day 1. The differences between different lots, brands and type of peptone in this trial became more obvious by the second day by which time some treatments (eg. P2, P3) showed the tendency for colony spreading (Fig. 5,a,b). The rear view of the plates displaying the distinct pattern of a red centre and white outline gave a clearer indication of the pigmentation pattern (Fig. 5a) while the front view indicated the extent of fluidity (Fig. 5b). The extent of colony growth and pigmentation as per the colony diameter and the OD estimates indicated significant differences with the source of peptone employed (Table 2). Overall, colony growth and fluidity ranked highest in P8 medium (BD Peptone) followed by P14 (HM type-1 new lot) and P18 (mycological peptone). Growth was very poor in the case of peptone from FINAR (P12) and Sd-fine (P13). Visibly, the peptone from Sigma (P10), HM veg-peptone (P17) and HM mycological peptone (P18) showed intense maroon pigmentation. Others displayed pink to intense red colour or light (P13) to no pigmentation (P12). As per A₅₀₀ assessment, P4, P5 and P7 ranked high in pigmentation (Table 2).

In this trial, the treatments P1 to P7 constituted the same type of peptone (bacteriological) from a single source (HM). Lot to lot or batch to batch variation was evident in pigmentation, but this did not seem to correspond exactly with the duration since the date of manufacturing as observed with the P4 to P7 lots. The P1 lot, which was a newly acquired pack (10/13) with a recent date of manufacturing (06/13) displayed more pigmentation than most other lots while P2 and P3 sets belonging to the same lot but obtained from two different laboratories (02/13 manufacturing) lagged behind and varied in pigmentation. The same applied to P14 - P16 treatments all of which constituted type-1 peptone from HM. Several lots of bacteriological peptone from HM (P1, P4, P6, P7) showed similar colony characteristics as BD (P8), Difco (P9) or Sigma (P10) peptone.

Observations with the four isolates representing different crops or geographic locations and belonging to the same or different races (1 or 4) endorsed that the extent of colony growth, fluidity and the intensity / the pattern of pigmentation of R. solanacearum on KM-TTC were notably influenced by the source of peptone (Fig. 6). However, some variation was observed in the response of different isolates. Among the two isolates from Goa, the eggplant isolate (Rs-01-11) showed relatively less variation while the chilli isolate (Rs-09-94) displayed a clearer effect on the bacterial colony appearance as documented on day 1 and day 2 (Fig. 7, 8). Between the ‘GRs-Mnt’ and ‘KAU-Av01’ isolates, such effect was more striking in the former (Fig. 9, 10).

In a preliminary trial, where mere soil-drenching inoculation was practiced with the ‘NH-Av01’ isolate, some amount of variation in pathogenicity was observed where the cultures from P13, P17, P18, P19 and P20 appeared more virulent, P1, P3, P8 and P15 proved medium virulent while the rest were low in virulence (data not shown). In the next trial, where root-injury-inoculation was adopted, significant differences in the extent of disease incidence was observed depending on the source medium (Fig. 11a; P = 0.013). In this trial, which was set up during the cooler months of December-January (average day and night temperatures of 18°C and 26°C), the disease incidence was overall less compared with other times with the temperatures around 25-32°C. However, the disease incidence at 4 week stage was relatively high (≥ 40%) in P1, P3, P4, P5, P10, P11, P12, P13, P15, P16 and CPGA while it was low (< 30%) in P8, P9 and P19 and intermediate in the rest. The extent of disease incidence did not appear to be correlated with the overall growth of the culture on the KM-TTC medium (r between seedling mortality at 4 weeks and culture OD at 600 nm -0.323; df = 19, NS), or the pigmentation (r between seedling mortality and the A₅₀₀ of the supernatant -0.063; df = 19, NS). Further, the pigment levels in the inoculum used for seedling challenge (0.1 at A₆₀₀) was quite low (Fig. 11b). However, it appeared that some of the treatments where the pigment production was very negligible (P3, P11, P12, P13 and CPGA), the disease incidence was relatively high.

No effect due to the peptone source in KM-TTC medium on biovar classification was observed in spite of the considerable variations observed in the colony characteristics and pigment levels in the 11 different KM-TTC formulations and CPGA (Fig. 12). The ‘NH-Av01’ isolate, irrespective of the medium-source, showed the utilization of all the sugars qualifying to be biovar 3.

The development of lawn, its uniformity/evenness, thickness and colour as well as the clear-zone formation were significantly modified by the peptone source used in the lawning-medium (Fig. 13). As per the observations on the second day, P6, P10, P14, P17, P18 and P19 media formulations showed notable colour development in the lawning medium and the clear-zone development was very obvious in these instances. Thus it appeared that the pigmentation in the lawning medium and the inhibition zone development were notably modified by the source of peptone used in the KM-TTC medium.

Fluidy red-white colonies were formed on KM-TTC with HM and Sigma CH with a slightly more reddish pigmentation in the latter. The difference, however, was not significant (colony growth at A₆₀₀ of 1.61725 ± 0.1035 and 1.7035 ± 0.12347, respectively, and the supernatant A₅₀₀ of 0.0725 ± 0.0095 and 0.0655 ± 0.020, respectively) as documented on day 2. Based on the above observations, CH (type II) from HM source was employed in the studies involving different lots and brands of peptone.

No obvious differences in colony growth or the pigmentation levels were observed with the source / brand of glucose as observed on day 2 (Fig. 14). Substitution of glucose with sucrose did not show any significant effect on growth or pigmentation as on day 1 or day 2 (data not shown).