Biochemical Tests on Microbial Samples
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OBJECTIVES: To learn and understand various biochemical tests.
To culture and observe results on both selective and differential media.
To differentiate between differential and selective media.
- USE OF SELECTIVE AND DIFFERENTIAL MEDIA
Selective media is a type of media that only allows the growth of a particular microbe and, in turn, stopping growth or possibly killing of any other microorganism. Selective only allows the growth of a desired type of microorganism and, in turn, kills or inhibits the growth of any other microorganism in the medium. In our case, Mac Conkey agar is a selective medium that allows only the growth of Gram -ve and inhibits the growth of many Gram-positive bacteria. Phenylethyl agar (PEA) is also a selective media that only allows Gram +ve cocci bacteria’s growth and inhibits most Gram-negative bacteria (Tille, 2014).
Differential media is a type of media that is mostly used as an indicator media to distinguish different bacteria growing on the same media. Differential media basically utilizes the biochemical behaviors of a certain bacteria growing in the presence of specific nutrients or dyes such as neutral red, phenol red, or methylene blue, which is added to the medium to indicate the particular characteristics of a bacteria visibly. Blood agar is differential media that contains bovine heart blood, which distinguishes microbes on their ability to lyse blood. Positive tests on blood agar are from the microorganisms which are able to haemolyse blood. The results of blood agar are normally alpha-hemolytic, beta-hemolytic, or gamma hemolytic (non –hemolytic).
Mac Conkey agar can also be termed as a differential medium since it distinguishes microbes by their ability to ferment the lactose. (Hossain et al., 2006). Microorganisms that are Gram –ve and have the ability to ferment lactose can be distinguished on the Mac Conkey agar. From our list of microorganisms from the laboratory practice, the following results were observed after all of them were cultured on the three media, which were available. The media were Mac Conkey agar, PEA agar, and blood agar and had qualities of either being selective media or differential media.
| Microorganism
|
Mac Conkey agar | PEA agar | Blood agar |
| E. coli | GROWTH | NO GROWTH | GROWTH BUT BETA HAEMOLYTIC |
| Enterobacter aerogenes | GROWTH | NO GROWTH | GROWTH |
| Serratia marcescens | GROWTH | NO GROWTH | GROWTH |
| Pseudomonas aeruginosa | GROWTH | NO GROWTH | GROWTH |
| Proteus Vulgaris | GROWTH | NO GROWTH | GROWTH |
| Staphylococcus epidermidis | NO GROWTH | GROWTH | GROWTH |
| Staphylococcus aureus | NO GROWTH | GROWTH | GROWTH |
| Micrococcus luteus | NO GROWTH | GROWTH | GROWTH BUT NON-HAEMOLYTIC |
| Enterococcus faecalis | NO GROWTH | GROWTH | GROWTH BUT NON-HAEMOLYTIC |
- BIOCHEMICAL TESTS
Biochemical tests are done in microbiology to distinguish bacteria based on the biochemical characteristics. These biochemical characteristics include fat and protein metabolism, carbohydrate metabolism, enzyme production, compound utilization, growth on different Ph values, temperatures, salt concentrations, among others (Becton, 2006).
From our practice, different organisms were inoculated on the triple sugar iron agar and were incubated overnight at 37 degrees Celsius. The results were observed as the following.
- A TSI agar slant’s reaction is “yellow slant, yellow butt, and cracks in the media.”
Yellow slant and yellow butt are seen as a result of the production of a large amount of acid due to lactose fermentation (or sucrose). The acid produced turns the red phenol indicator into yellow in both butts and in slant. Crack in the media is a result of the production of gases during the process of fermentation of lactose. The bacteria which showed these results were E.coli and Enterobacter aerogenes.
- The reaction on a TSI agar slant is “red slant, yellow butt, and cracks in the media.”
The yellow butt is due to lack of fermentation but with small amount of glucose as compared to a slant but on the slant, the acid produced which is less as media in slant is also minimal will be oxidized to CO2 and water thus the slant was red( neutral to alkaline ph). Cracks in the media were due to the production of gases during fermentation. (Dowell, 1964). The fermentation process in which acid is produced is usually accompanied by the production of gases.
The bacteria which produced this result were Proteus Vulgaris and Serratia marcescens.
- A TSI agar slant’s reaction is “yellow slant, yellow butt, and blackening of the butt.”
The yellow slant and yellow butt were due to the production of a large amount of acid, which turns the red phenol indicator to yellow during the process of fermentation. The blackening of the butt was due to the production of hydrogen sulfide gas.
This result was observed on the Proteus Vulgaris plate.
- The reaction on a TSI agar slant is “red slant, red butt.”
Red slant and read but was due to lack of fermentation of either sucrose or lactose. The results were due to the inability of bacteria to ferment lactose or sucrose, and therefore no acid was produced, and thus the red color of the red phenol indicator was not changed. There were no cracked media as there was a fermentation process that produces the gas, which would in turn, lead to cracked media.
This result was seen on the Pseudomonas aeruginosa plate as the bacteria have no ability to ferment lactose or sucrose.
- BIOCHEMICAL REACTIONS OF SELECTED ORGANISMS
| Organism | Gram reaction | citrate | Starch hydrolysis | Catalase | Oxidase | coagulase | urea |
| E. coli | -ve | -ve | +ve | +ve | -ve | -ve | -ve |
| Enterobacter aerogenes | -ve | +ve | -ve | +ve | -ve | -ve | -ve |
| Serratia marcescens | -ve | +ve | -ve | +ve | -ve | -ve | +ve |
| Pseudomonas aeruginosa | -ve | +ve | -ve | +ve | +ve | -ve | -ve |
| Proteus Vulgaris | -ve | +ve | -ve | +ve | -ve | -ve | +ve |
| Staphylococcus epidermidis | +ve | -ve | -ve | +ve | -ve | +ve | +ve |
| Staphylococcus aureus | +ve | +ve | +ve | +ve | -ve | +ve | -ve |
| Micrococcus luteus | +ve | +ve | -ve | +ve | +ve | +ve | +ve |
| Enterococcus faecalis | +ve | -ve | -ve | -ve | -ve | +ve | -ve |
The three most important biochemical tests that can easily distinguish these organisms are Gram reaction, TSI agar reactions, and production of hydrogen sulfide gas.
References
Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen editions. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
Becton, Dickenson, and Company. 2006. BBL phenyl ethyl alcohol agar with 5% sheep blood formula package insert. http://www.bd.com/ds/technicalCenter/inserts/L007401(07)(0506).pdf.
Dowell, V. R., Jr., E. O. Hill, and W. A. Altemeier. 1964. Use of phenyl ethyl alcohol in media for isolation of anaerobic bacteria. J. Bacteriol. 88:1811–1813.
Hossain, M. S., Chowdhury, E. H., Islam, M. M., Haider, M. G., & Hossain, M. M. (2006). Avian salmonella infection: isolation and identification of organisms and histopathological study. Bangladesh Journal of Veterinary Medicine, 4(1), 7-12.
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