Mfawa T. A. Eno-Williams
Biology 2310 Lab
Lab Report 3
Microbiological Media and Biochemical Test
October 22, 2018
Introduction:
Microbial media are any liquid or solid used as a growth medium to support bacterial growth—essentially, media serves as a home containing everything bacteria needs to grow under laboratory conditions (Britannica, 2017). However, certain microorganisms may only cultivate on a certain media, react to chemicals present in a certain media or simply produce a different result. It is for this reason that there are no all-encompassing media used in microbiology but different media each carrying specific purposes. Selective media, as implied by the name, is used for growth of only selected bacteria (Libretexts, 2018). For example, if a bacterium is resistant to a certain antibiotic, that antibiotic may be added to a medium in order to ensure other organisms lacing this resistance are unable to cultivate (Libretexts, 2018). Differential media enables general growth, but it also shows the metabolic differences between bacteria and allows an observer the opportunity to be able to clearly compare between bacteria (Lab Archive, 2016). Differential media uses the biochemical characteristics of a microorganism cultivating in the presence of certain added nutrients or indicators to be able to visibly differentiate bacteria strains (Libretexts, 2018). Growth medium however, contains substances required for the growth of microorganisms and thus cultivates and supports the progression and preservation of all cultures (Britannica, 2015). In this particular lab, different media were used namely the TSA, MHT, MSA, Blood Agar, EMB and SIM tubes.
The Tryptic soy agar (TSA), (along with Tryptic Soy broth tubes) are universal media supporting a wide array of microorganisms including both gram-positive and gram-negative bacteria (Acharya, 2018). TSA is primarily used as an initial growth medium for the purposes of colony morphology observations, isolating and developing a pure culture and achieving sufficient growth for further biochemical testing and culture storage (Acharya, 2018). Tryptic Soy Agar contains digests of casein and soybean meal; these combinations make the medium nutritious through the supply of organic nitrogen, particularly amino acids and longer-chained peptides (Acharya, 2018).
The Mueller-Hinton Tellurite (MHT) medium was created by Mueller and Hinton in 1941 with the purpose of isolating pathogenic Neisseria species (Aryal, 2015). Presently however, it is used more for the routine susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion technique (Aryal, 2015). When performing susceptibility testing on Streptococcus species, 5% of sheep blood and nicotinamide adenine dinucleotide can also be added (Aryal, 2015).
Mannitol Salt Agar (MSA), is both a selective and a differential medium used to isolate and identify Staphylococcus aureus (Acharya, 2017). MSA incorporates 7.5% of sodium chloride which aid in cultivating only bacteria that can tolerate high salt concentrations (Acharya, 2017). The concentration of MSA media inhibits the growth of most other gram-positive and gram-negative bacteria (Acharya, 2017).
The Blood agar is a differential growth medium enriched with 5% sheep blood. Microorganisms such as streptococci that are fastidious, do not grow well on other media but are encouraged to grow on blood agar (Acharya, 2017). Blood agar contains inhibitors for bacteria such as Neisseria and Haemophilus genera and must be warmed to inactivate these inhibitors and to release essential growth factors (Acharya, 2017). When this blood agar is heated it converts it into chocolate agar (due to its chocolate color) and supports the growth of these bacteria (Acharya, 2017). This plate was first used by James Brown in 1919 (Acharya, 2017).
Eosin methylene blue (EMB) agar is both a selective and differential medium. It is selective in that only gram-negative organisms grow on it, while it is differential in that it differentiates between gram-negative organisms that can use lactose as a car-bon source and with those that cannot (Lab Archive, 2016).
SIM (Sulfur, Indole, and Motility) tubes are differential media that are used to show whether a microorganism has the ability to reduce sulfur, produce indole from degraded tryptophan and if it has motility (Lab Archive, 2016).
In this lab, four bacterial samples of Escherichia coli, Staphylococcus epidermis, Enterobacter aerogenes and Citrobacter freundii were used. The objective was to determine how the samples react on the various media and to observe their response or lack thereof.
Materials and Methods:
In this lab, each student was to work in pairs of two with each pair being given the appropriate materials. The experiment was performed over the time of two class days. On the first day, one MSA, EMB and Hektoen plate was used respectively. In addition, 2 SIM tubes, 2 Bile Esculin tubes, 2 Phenol red lactose tubes, 2 Phenol red glucose tubes, a bottle of hydrogen peroxide, a striker and Bunsen burner, a wire inoculating loop and Kovac’s Regent were used to perform this experiment.
Each of the 3 plates were separated into four quadrants and labeled. In each quadrant, Escherichia coli, Staphylococcus epidermis, Enterobacter aerogenes and Citrobacter freundii were lightly inoculated in their respective sections by sterilizing the inoculating loop over the flame of the Bunsen burner. After cooling, the loop was dipped into the bacteria culture and lightly transferred onto its respective quadrant in a zig zagged pattern. After the bacteria was spread to a quadrant, it was sterilized to remove all bacteria traces. Once this was done in each plate’s quadrants, the plate was inverted and inoculated at 37C for 48 hours.
Following this, E. coli and C. freundii samples were inoculated respectively into 8 tubes using the aseptic technique once more. First, the test tube containing C. freundii along with the Bile Esculin tube was obtained. The inoculating loop was sterilized and once cooled, was dipped into the tube containing C. freundii and inoculated into the Bile Esculin tube. As previously done, the loop was sterilized to remove bacteria. Furthermore, the C. freundii was transferred into a SIM tube by sterilizing the loop and once cooled, pressing the loop into the SIM 1cm from the bottom of the tube. It was sterilized after. Similar to the Bile Esculin tubes, C. freundii was aseptically inoculated into the Phenol Red Glucose and Lactose tubes respectively. The same process was repeated with the E. coli samples. Finally, each tube was labeled and incubated for 48 hours at 37C for future observation.
After 24-48 hours had passed, the plates and tubes were removed from incubation. Two to three drops of Kovac’s Regent were poured into the SIM tubes containing the C. freundii and the E.coli respectively. While a reaction was being awaited, the plate media were each given 2 drops of hydrogen peroxide on each of their quadrants. Following this, the results were observed.
Results:
It was hypothesized that the Staphylococcus epidermis would not have as much growth being that unlike the other 3 bacteria, it is gram-positive and is best cultivated at 30C; whereas Escherichia coli, Enterobacter aerogenes and Citrobacter freundii are all gram-negative bacteria and able best cultivated at 37C.
The results for the HEK plate indicated scattered colony growth in the S.epidermis quadrant along with the presence of a green/yellow color. Each of the other quadrants gave the plate a red/pink color. The quadrant containing the E.aerogenes had thick yellow growth, the C. freundii quadrant indicated smaller yellow colonies while the E.coli quadrant had even smaller yellow scattered colonies. In addition, the HEK plate was used for a catalyst test with hydrogen peroxide. Results of the test indicated heavy white bubbling on the E.aerogenes quadrant, while the C. freundii and E.coli quadrants showed lighter white bubbling. Thus, each three quadrants tested positive for the catalyst test.
HEK plate after incubation and catalyst test
E.coli, E.aerogenes, C.freundii, S.epidermis on Hektoen plate. Yellow growth colonies for E.coli, C.freundii, and E.aerogenes. Green/yellow growth for S.epidermis.
The results of the EMB plate indicated growth in all four quadrants. The E.coli, E.aerogenes, and C.freundii quadrants each possessed a metallic green color in the colonies cultivated.
EMB plate after incubation
S.epidermis, E.coli, C.freundii and E.aerogenes on EMB agar plate. Metallic dark green growth for E.coli, E.aerogenes, and C.freundii. Little growth in S.epidermis quadrant.
The MSA plate results showed growth (and no color change) in the quadrant containing S.epidermis. However, there appeared to be no growth nor color change on the other 3 quadrants, with the exception of the E.aerogenes quadrant that contained a little circle of yellow color change.
MSA plate after incubation
E.aerogenes, E.coli, S.epidermis and C.freundii on MSA agar plate. Presence of growth for S.epidermis; small circle of yellow coloration for E.aerogenes.
The blue capped Bile Esculin tube contining the incoulated C.freundii was changed from a clear/yellowy color to a cloudy browm/black color. Similarly, the E.coli incubated Bile Esculin tube changed from its clear/yellowy color into a brown color. The Phenol Red Glucose and Lactose tubes however, were both changed from a red color to a bright yellow for both the E.coli and C.freundii bacteria. Finally, the SIM tube containing C.freundii experienced a color change from its previously clear/yellow gel color to a dark purple/black color with a white film on the top. Once the Kovac’s reagent was applied, the top white film turned a pink color. The E.coli SIM tube on the other hand, showed no changes from its original clear/yellow color. However, once the Kovac’s reagent was applied, the top layer turned a pink color as well.
C.freundii, Phenol Red Lactose (white cap), Glucose (Green cap), Bile esculin (blue cap and SIM (smaller white cap) after incubation
C.freundii reaction to Phenol Red, Glucose, Bile esculin and SIM agars.
E.coli, Phenol Red Lactose (white cap), Glucose (Green cap), Bile esculin (blue cap and SIM (smaller white cap) after incubation
E.coli reaction to Phenol Red, Glucose, Bile esculin and SIM agars.
Conclusion:
First and foremost, the original hypothesis theorizing little growth in Staphylococcus epidermis was proven wrong as there was the presence of growth in most plates despite the bacteria’s classifications. However, with the presence of growth by S.epidermis alone and no growth in the other gram-negative bacteria, the MSA plate can be concluded to be selective to gram-positive bacteria. The growth of S.epidermis on the MSA plate further indicates its ability to grow in salty enviroments, classifying it as a halophile.
In the HEK plate, the change of the media to a pinkish color implies that the pH of the media was changed. However, with the green color of S.epidermis it was concluded that the bacteria contains the ability to reduce sulfur to hydrogen sulfide.
The predominant growth of the gram-negative bacteria on the EMB plate indicated that gram-positive bacteria was not supported on this medium. In conclusion, these implied gram-negative bacteria contain the ability to use lactose as a carbon source. The metallic color of the E.coli, C.freundii and E.aerogenes quadrants confirmed their ability to ferment lactose.
The black/purple coloring of the Bile Esculin inoculated with C.freundii implied that it is positive for hydrolysis of esculin. Contrarily, E.coli had no color change and thus tested negative for for hydrolyzed esculin. In the SIM tubes, the C.freundii bacteria showed positive results. The response of the C.freundii black/purple color change indicated that the bacteria is a sulfur reducer. The white film indicated the presence of motility while the change of color from white to pink indicated it to be indole positive. E.coli on the other hand, tested negatively to being a sulfur reducer as it did not have any color change change its pigmentation, but tested positive to the indole and motility testing.
In conclusion, the unique results of each bacteria brought a wider picture to the importance of media and biological testing in microbiology and the way in which they allow us to accurately classify and group microorganisms as similar or different according to their proven results.
References
Britannica, T. E. (2017, June 15). Growth medium. Retrieved October 20, 2018, from https://www.britannica.com/science/growth-medium
Libretexts. (2018, January 03). 6.3C: Selective and Differential Media. Retrieved October 20, 2018, from https://bio.libretexts.org/TextMaps/Microbiology/Book:_Microbiology_(Boundless)/6:_C ulturing_Microorganisms/6.3:_Culturing_Bacteria/6.3C:_Selective_and_Differential_Me dia
Britannica, T. E. (2017, June 15). Growth medium. Retrieved October 20, 2018, from https://www.britannica.com/science/growth-medium
Acharya, T. (2017, May 19). Mannitol Salt Agar (MSA): Composition, uses and colony characteristics -. Retrieved October 22, 2018, from https://microbeonline.com/mannitol- salt-agar-msa-composition-uses-and-colony-characteristics/
Acharya, T. (2018, February 09). Tryptic Soy Agar (TSA): Composition, Preparation and Uses -. Retrieved October 23, 2018, from https://microbeonline.com/tryptic-soy-agar-tsa- composition-preparation-uses/
Acharya, T. (2017, May 10). Blood Agar: Composition, Preparation, Uses and Types of Hemolysis -. Retrieved October 22, 2018, from https://microbeonline.com/blood-agar- composition-preparation-uses-and-types-of-hemolysis/
Aryal, S. (2015, August 24). Mueller Hinton Agar (MHA) – Composition, Principle, Uses and Preparation. Retrieved October 23, 2018, from https://microbiologyinfo.com/mueller- hinton-agar-mha-composition-principle-uses-and-preparation/