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Impact of Different Carbon Sources on the in Vitro Growth and Viability of Escherichia Coli (sube01) and Salmonella Spp. (subs01) Cells

Ifra Tun Nur
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh.

Jannatun Tahera
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh.

Md. Sakil Munna
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh.

M. Majibur Rahman
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh.

Rashed Noor
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh.

Abstract/ Executive Summary:

With a previous observation of Escherichia coli growth cessation along with temperature variation within three different bacteriological culture media (nutrient agar, Luria-Bertani agar and minimal agar), current investigation further depicted on the possible growth dynamics of Escherichia coli (SUBE01) and Salmonella (SUBS01) growth and viability upon supplementation of different carbon sources (dextrose, sucrose, lactose, glycerol and tween 20) at 37°C under the aeration of 100 rpm. Viability of the tested bacterial species was assessed through the enumeration of the colony forming unit (cfu) appeared upon prescribed incubation for 12-24 hours on different agar plates consisting of the above mentioned carbon sources. Besides, to inspect the cellular phenotypic changes, morphological observations were conducted under the light microscope. Variations in bacterial growth (either growth acceleration or cessation) were further noticed through the spot tests on the agar plates. Considerable shortfalls in the culturable cells of E. coli and Salmonella spp. were noted in the minimal media separately consisting of sucrose, lactose, glycerol or tween 20 while an opposite impact of accelerated growth was noticed in the media supplied with dextrose. The data revealed a hierarchy of consequence of carbon sources as nutrient generators whereby the favourable bacterial growth and survival order of the carbon sources was estimated as dextrose > glycerol > lactose > tween 20 > sucrose.

Keywords: Carbon sources, Colony forming unit (cfu), Escherichia coli, Nutrient diversity

Location: Department of Microbiology, Stamford University Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

1) To distinguish among the growth patterns of E. coli (SUBE01) and Salmonella spp. (SUBS01) on culture media with variations in carbon sources (dextrose, glycerol, lactose, tween 20 and sucrose),

(2) To observe the retention within the cell viability in response to the diversity within carbon sources as major nutrients for in vitro cultivation.

Methodology:

Laboratory stock culture of Escherichia coli (SUBE01) and Salmonella spp. (SUBS01) were used in this study. Experiments demonstrating the bacterial growth in terms of cell turbidity (optical density at 600 nm; i.e., OD₆₀₀ ) and colony forming units (CFUs) were conducted. Minimal media (dextrose 1.0 g/l, dipotassium phosphate 7.0 g/l, mono potassium phosphate 2.0 g/l, sodium citrate 0.5 g/l, magnesium sulfate 0.1 g/l and ammonium sulfate 1.0 g/l) for both agar (MA) and broth (MB) were used for the assay of the bacterial culturability. D-Sucrose, D-lactose, glycerol and tween 20 (Sigma-Aldrich Corporation, USA) were used separately as carbon source, which were included in minimal media (both agar and broth) instead of dextrose (D-glucose) (Sigma-Aldrich Corporation, USA). After 24 hour incubation on MA plates at 37°C, one loopful of the bacterial culture was introduced into 5 ml MB followed by incubation at 37°C for 4-6 hours at 100 rpm to form the pre-culture. After adjusting the optical density of the pre-culture at OD₆₀₀ to 0.1, 30 µl each was introduced into 5 different sets of 30 ml of MB and incubated at 37°C at shaking condition (100 rpm). At the time points of 12, 36 and 72 hours, the cell growth was monitored by measuring OD₆₀₀ and by counting the CFUs on agar plates. Manifestation of culturable cells was further confirmed by the supportive spot tests. As described previously, each the culture suspension was serially diluted in 9 ml minimal broth to obtain up to 10 -4 fold dilution. From each dilution, an aliquot of 5 µl was dropped on to the minimal agar, dried off for 15 minutes, and finally the plates were incubated at 37°C for 24 hours. Spotting on the agar was accomplished at 24 hours of growth. Results were statistically analyzed by determining the p value through t test. Standard deviations were also measured through statistical hypothesis testing 21 . All the data found in this study were estimated as significant (p = 0.05). Simple staining (Crystal violet, Hucker’s solution) was conducted to assess the cellular morphology as was done previously. An aliquot of 10 µl from the bacterial culture suspension was removed at 24 hours of growth, and the cellular morphology, shape and organization were observed under the light microscope (Optima Biological Microscope G206, Taiwan) at 1000x magnification.

Source of Information: Bangladesh J Microbiol, Volume 32, Number 1&2, June-December 2015, pp 39-44

Article Link (Online Journal): DOI: http://dx.doi.org/10.3329/bjm.v32i0.28476

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

With a consecution of those works, the presented study revealed the prelusive nutritional diversity and proposed that there is a hierarchy that abide by the carbon source consequence (dextrose > glycerol > lactose > tween 20 > sucrose) and it would be useful during the enrichment techniques. Furthermore, tween 20, even being into the category of detergent with a possible role in despairing the cell membrane; yet the molecule has been found to be better utilized compared to that of sucrose. Further studies on tween molecule degradation by bacteria would be of greater interest to understand the overall bacterial catabolic strategies for diversified substrate utilization.

Knowledge Management: Journal