Enterobacter Cloacae Sowing of the Material

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Table of Contents

Isolation of a Pure Culture and Its Primary Identification
Gram Method Staining
Identification Media Sowing
Conclusion
Reference

In the first stage of research, the test material contents of the intestines is diluted with saline in proportion of 1 to 10, so that the solutions in dilution range from 10^-1 to 10^-8 are prepared. From each dilution, inoculations of different mediums are made, such as Endo plate culture media, and bismuth-sulfite agar. Next, the preparations should be incubated at 37°C for 18-24 hours.

To quantitatively characterize the aerobic microflora, a dosed inoculation on plate media from four diagnostically significant dilutions 10^-4, 10^-5, 10^-6, 10^-8 of the starting material is carried out. Dilutions are prepared in five bacteriological test tubes: 9.9 ml of isotonic sodium chloride solution for 10^-3, 10^-8 dilutions are poured into two test tubes, and 4.5 ml for 10^-4, 10^-5 dilutions into three tubes. The suspension should be sequentially transferred from the previous dilution into the subsequent one. Before work, lamellar media should be dried at a temperature of 60±1°C for 20 minutes, and at 40±1°C for 45 minutes. The microorganisms are sown on a dense canted nutrient medium. After successful sowing, the culture needs to be placed in a thermostat, where its development is monitored closely in order to properly describe the quantitative and qualitative nature of its growth.

Isolation of a Pure Culture and Its Primary Identification

In the next stage of research, isolated colonies should be selected from plate culture media and inoculated separately for the primary identification of microorganisms. García-González et al. (2018) state that Enterobacter cloacae is a ubiquitous Gram-negative, facultative anaerobic, rod-shaped bacterium belonging to the Enterobacteriaceae family (p.1). All crops are incubated at 37ºC for 18 20 hours to let the isolated colonies grow, otherwise identification of bacteria is impossible. To determine the qualitative composition of bacteria, the colonies on the plate are grouped according to the nature characteristics of each colony. This is the most significant feature of the microorganisms growth on a solid nutrient medium. The color differentiation of isolated colonies, which depends on the bacteria to differentiating components of the nutrient medium ratio, is of decisive importance in the assessment of Enterobacteriaceae on the plate media.

Gram Method Staining

In this stage of research, the results of the primary identification of bacteria are recorded and analyzed. Sowing has been already done on the medium of the minimum differentiating series to determine the genus. Most importantly, the Gram method has to be used to determine the staining ability of the cultures cells. The methods coloring is carried out as follows:

A drop of saline is applied to the defatted glass, on which the culture is suspended.
The preparation is dried and fixed over the flame of the burner, where it is held 2-3 times.
A piece of filter paper soaked in gentian violet is applied to the smear for 1-2 minutes.
The dyed filter paper is removed, without rinsing it with water.
Lugols iodine is applied to the paper for 1-2 minutes, until the smear is blackened.
Lugols iodine is removed without applying water.
A 96° alcohol solution is poured on the preparation for 20 seconds. Additionally, to exclude excessive discoloration of bacteria, iodine should be added to the solution.
The preparation is rinsed with water.
A piece of filter paper soaked in an aqueous solution of fuchsine is put on the stain for 1-2 minutes.
The dyed filter paper is removed, and the smear is rinsed with water.
The preparation is dried, then put in the microscope using the immersion oil.

The preparation is stained correctly, and the color of the culture reveals to be in the range from pink to red, thus implying that the bacteria are gram-negative, which aligns with the Enterobacteriaceae genus.

Identification Media Sowing

After the Gram method staining, the colonies are sown onto the specific identification media the combined Olkenitskys or Kliglers media. With these media, several cultures characteristics are simultaneously revealed: the ability to form hydrogen sulfide, and the relationship to glucose and lactose. Thus, the colonies are sown on both media with a bacteriological needle first along the beveled part with a stroke, and then with a prick into the thickness of the agar column. To let the cultures grow, crops are incubated at 37±1°C for 18-20 hours. Next, based on the totality of biochemical properties revealed on the combined medium, a conclusion can be made about the possible species of the culture.

In Olkenitskys medium, glucose has fermented to acid and gas, which gives the culture a yellow color with multiple discontinuities. By the combination of biochemical signs on the combined medium, several genera of Enterobacteria can be suspected simultaneously. Therefore, to establish the genus and species belonging of bacteria, the minimum differentiating series should be used. Intraspecific differentiation, as well as differentiation into biological groups, is based on differences in the fermentation of carbohydrates and the presence of amino acid decarboxylases. For example, in some cases, O- and H-antigens are serotyped.

Conclusion

The presence of Enterobacter cloacae in the substrate is indicated by hydrolysis of gelatin, as well as arginine, which is highly specific to this Enterobacteria species. On Endo medium, the cultures obtain colors in range from raspberry to pink, while on bismuth-sulfite agar they demonstrate the color range from pale green to greenish-brown. The cultures are transparent, shiny, and can be easily removed from the medium, the latter is not stained under the colonies. These qualitative characteristics indicate that the colonies present are from Enterobacteriaceae genus. All isolated strains had typical Enterobacteriaceae morphology, represented by gram-negative rods with peritrichous flagella, and grew well at 37°C on different media, which further establishes the studied culture as Enterobacter cloacae.

Reference

García-González, T., Sáenz-Hidalgo, H. K., Silva-Rojas, H. V., Morales-Nieto, C., Vancheva, T., Koebnik, R., & Ávila-Quezada, G. D. (2018). Enterobacter cloacae, an emerging plant-pathogenic bacterium affecting chili pepper seedlings. The Plant Pathology Journal, 34(1), 110.

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Enterobacter Cloacae Sowing of the Material

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