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Influence of Temperature on the Growth of Pseudomonas Putida

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

Zebunnesa Zeba
Department of Natural Science, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217, Bangladesh.

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

Abstract/ Executive Summary:

Lots of reports on the generation of stress by increase in temperature in the bacterial cells especially in Escherichia coli has been observed so far. Current study further emphasized such effect on the cells of Pseudomonas putida (SUBP03). Conventional methods relating growth assessment of bacteria were employed. The optical density of bacterial cells at 600 nm (OD₆₀₀) in the minimal broth along with the culturable cells were assessed in the form of colony forming units (CFUs) in the minimal agar media at different temperatures (27 °C, 30 °C, 33 °C, 37 °C and 40 °C). Morphological observations were made to further clarify the bacterial physiology and the spot tests were performed to examine the cell viability. Cells of P. putida (SUBP03) were found to grow vigorously at 30 °C, while the growth was found to decline at lower temperature (27 °C) and along with the increase in temperature (at 33 °C, 37 °C and 40 °C). However, the morphological changes were insignificant. Furthermore, cells were noticed to completely lose culturability at 40 °C after 48 hours.

Keywords: Critical growth temperature, High temperature stress, Optimum growth temperature, Pseudomonas putida,Viable but non culturable (VBNC) cells

Location: Department of Microbiology, Stamford University Bangladesh.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Insects

Objectives:

To assess the optimum and critical growth temperature of our laboratory stock culture of Pseudomonas putida (SUBP03).

Methodology:

Conventional experiments measuring the bacterial growth were conducted. Laboratory stock cultures of Pseudomonas putida (SUBP03) was used in this study. Minimal media (dextrose 1.0 g/L, dipotassium phosphate 7.0 g/L, monopotassium 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 (19). After 24 hour incubation on minimal agar plates at 37 °C, one loopful of each of the bacterial culture was introduced into 5 ml minimal broth followed by 100 rpm (rotation per minute) at 37 °C for 4–6 hours (pre–culture). After adjusting optical density of the pre–culture at 600 nm (OD₆₀₀) to 0.1, 30 µL each was introduced into 2 different sets of 30 ml of minimal broth and incubated at 27 °C, 30 °C, 33 °C, 37 °C and 40 °C at shaking condition (100 rpm). At every 12 hours cell growth was monitored by measuring OD600, and the formation of colony forming units (CFUs) were estimated by counting the colonies up to 72 hours at every 24 hour intervals For the observation of cell morphology and arrangements, simple staining (Crystal Violet, Hucker’s Solution) was applied as previously done (21-23). An aliquot of 10 µL from each bacterial culture suspension was removed by 12 hours intervals and the shape and organization of cells were observed under light microscope (Optima Biological Microscope G206, manufactured in Taiwan) at 1000 × magnification (21). Finally spot tests were conducted to further confirm the bacterial viability under temperature stress. As described previously, each of the bacterial culture suspensions was serially diluted in 9 ml nutrient broth to obtain up to 10^–4 fold dilution (19, 21-23). 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 every 12 hours of growth (19, 21-23).

Source of Information: Stamford Journal of Microbiology, 2015, Vol. 5, Issue 1, 9-12, ISSN: 2074-5346 (Print); 2408-8846 (Online)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The findings of the current study revealed the heat shock state in case of P. putida (SUBP03), which may further increment the existing knowledge on the stress response in Pseuomonas spp. However, the limitation of this study underlies the lack of study in genetic level with the presentation of preliminary data. However, detection of the optimal and critical growth temperatures of P. putida (SUBP03) may draw interest within the closely related fields. Nevertheless, the expressional analysis of certain heat shock genes are worth to understand the detailed scenario as well as to complete the current investigation.

Knowledge Management: Journal