bacteria species selection

Selective Media for Bacterial Growth

Selective media are media that are used to promote the growth of specific bacteria species while inhibiting the growth of other bacteria (Hardy Diagnostics 2014, n.p). Among the selective media are phenyl ethyl (PEA) agar, eosin Methylene blue (EMB), mannitol salt agar, and MacConkey agar. To extract Bacillus subtilis, Enterococcus feacalis, Staphylococcus epidermis, Proteus mirabilis, Enterobacter cloacae, and Escherichia coli, first inoculate the organisms aseptically in trypsic soy agar (TSA) dishes, stabs, broths, and slope colonies. Notably, TSA is a growth medium that is used to cultivate the vast majority of bacteria species. Different bacteria grow differently on TSA cultures (Dickson 2000, n.p). Incubate the cultures in an aerobic condition for 24 hours at a temperature of 35 degree Celsius to 45 degree Celsius. After 24 hours, make observation of the colonies.

Differentiation of Bacterial Species

Plates, stabs, broths, and slope cultures with the bacteria species show growth. Secondly, use selective media to differentiate the bacterial species using Kovak’s reagent, EMB plates, PEA plates, and fermentation assays. Incubate the resultant cultures aseptically on Kovak’s reagent (Highlands Edu 2015, n.p). Kovak’s reagent will encourage the growth of gram-negative bacteria based on the production of sulfide, formation of indole, and the state of mobility. Additionally, while using the cultures from TSA cultures, inoculate them with the SIM media through stabbing to a half an inch. After that, incubate the inoculums for 24 hours at 35 degree Celsius in aerobic conditions. After incubation, observe for the production of hydrogen sulfide and the bacterial movement. Hydrogen sulfide results from the medium turning black. Diffusion of the inoculation line shows motility. After the observation, apply Kovak’s reagent drop wise to the medium. Gram-negative bacteria with indole shows color change from pink to red on the surface.

Incubation on EMB Plates

Incubate the inoculated cultures on EMB plates in aerobic condition for a day. The plates may undergo incubation for more than a day when no growth appears. EMB is used to select lactose-fermenting bacteria, which appears blue-black in color with black centers. Escherichia coli, Proteus mirabilis, and Enterobacter cloacae will grow while Bacillus subtilis, Enterobacter feacalis, Staphylococcus epidermis, and Proteus mirabilis, which are gram positive, will not grow. Phenylethyl Alcohol Agar (PEA) selects for gram-positive bacteria while inhibiting gram-negative bacteria and Proteus species. In the fermentation process, incubate the inoculated tubes for 24 hours at 35 – 45 degree Celsius (Tankeshwar 2016, n.p). Color change and gas production confirm a positive test. The next step is gram staining techniques which differentiate the species in gram-positive and gram-negative groups. The gram-negative species are Escherichia coli, Proteus mirabilis, and Enterobacter cloacae while gram-positive bacteria are Bacillus subtilis, Enterobacter feacalis, Staphylococcus epidermis, and Proteus mirabilis. Immediate production of gases indicates a positive catalase activity while the absence of bubbles indicates negative catalase activity.

Identification of Proteus mirabilis

The unknown bacterium is Proteus mirabilis. This is because Proteus species are gram-negative non-lactose fermenting bacteria which are rods. Conduct a sucrose fermentation test where Proteus species penetrate the sucrose thereby degrading it, since it is the property of Proteus to degrade sucrose with the production of gas and acid. Koch postulate stage includes isolation of the bacterial species, infection of a healthy pear with the bacteria strain, and observation of the symptoms (Latshaw and Brunings 2010, n.p). The disease should be in both the healthy and the diseased pear. Rhizopus stolonifer causes pear rot disease (Kwon and Jung Lee 2000, p.11).

Bibliography

Dickson, J. S. (2000). Re: what bacterial results can grow out of (TSA) (CNA) & MacConkey Pet.dishes? [Online] Available at: ID: 950900743.Mi [Accessed Aug. 5, 2017]

Hardy Diagnostics. (2014). Sulfide, indole, and motility (SIM) medium - for gram negative enteric bacteria. [Online] Available at: [Accessed Aug. 5, 2017]

Highlands Edu. (2015). Use of selective and differential media. [Online] Available at: [Accessed Aug. 5, 2017]

Kwon, J.-H., and Lee, C.-J. (2006). Pear soft rot (Pyrus Serotina) by Rhizopus Stolonifer in Korea. [Online] Available at: [Accessed Aug. 5, 2017]

Latshaw S., and Brunings, A. (2010). Center for precollegiate education and training. [Online] Available at: [Accessed Aug. 5, 2017]

Tankeshwar, A. (2016). Carbohydrate fermentation test: uses, principle, procedure, and results. [Online] Available at: [Accessed Aug. 5, 2017]