The objective of this lab was to determine if different cultures would successfully grow on diverse agar medias and to distinguish the appearance of growth on the mediums. The experiment involved three different types of growth medias – selective, differential and enriched. The experiment also involved four broth cultures – Escherichia Coli, Enterobacter Aerogenes, Staphylococcus Epidermidis and Streptococcus Faecalis.
Selective media was a growth medium that enhanced and inhibited the growth of certain organisms due to an added media component (Madigan et al., 2012). The selective medium used in this experiment was KF Streptococcal Agar. Differential media allowed the identification of microorganisms based on observable properties (Madigan et al., 2012). In this experiment, the differential media used was Eosin Methylene Blue (EMB) Agar. The methylene blue in EMB inhibited the growth of gram-positive bacteria and only allowed gram-negative organisms to be grown. EMB had a pH of 7.2 and used lactose and sucrose for energy. (Madigan et al., 2012)
Enriched media allowed metabolically fastidious organisms to be grown because of the added specific growth factors (Madigan et al., 2012). The Tryptic Soy Agar was used as the enriched media in this experiment. TSA was mainly used as a control for growth medium. It was also used to observe colony morphology, to develop a pure culture and to achieve adequate growth for further testing (Liu, 2005)
In this experiment, we transferred the four liquid broth cultures onto each of the Agar plates using a sterile cotton swab. We dipped the swab into the broth once and created a stroke, 2 cm long, on each of the agar plates in a specific order. We started with the least enriched media, EMB (differentiable), and ended with the most enriched media, TSA (enriched); with KF being swabbed in between.
After completing the experiment, we can note that each medium used produced different results. The results showcase the differences between enriched, differential and selective media. The enriched media, TSA, had growth for all four different cultures. In this experiment, TSA acted as a growth medium because the most common and even fastidious forms will grow on this medium. TSA is also known as Soybean-Casein Digest agar (Dickson, 2002). It can be considered a defined medium because it can be easily replicated. It contains tryptone (digest of casein), soytone (digest of soybeans), sodium chloride, agar and water (Dickson, 2002). The reason why a good majority of bacteria can grow on TSA is because casein, and soytone provide amino acids to the bacterial culture (Dickson, 2002). Since all of these cultures grew on TSA it proves that they are indeed fastidious and require the addition of amino acids to support their growth.
Eosin Methylene Blue (EMB) was our differential medium. Only two of our cultures produced any type of growth. Escherichia Coli produced green metallic colonies and Enterobacter Aerogenes produced pink colonies. EMB is used specifically for the detection of gram-negative bacteria (Madigan et al., 2012). EMB contains lactose, eosin, methylene blue dyes and a few other ingredients (Madigan et al, 2012).
The Eosin and methylene blue act as indicators between gram-positive and gram-negative organisms. Since EMB contains lactose, gram-negative organisms like Escherichia Coli ferment the lactose and produce dark green metallic colonies (Madigan et al, 2012) (Wellesley, 2010). They turn green due to the dyes and the acid by-products of fermentation (Wellesley, 2010). Enterobacter Aerogenes must then be gram-positive because its colonies did not turn green but turned pink instead. The other cultures, Staphylococcus Epidermidis and Enterococcus Faecalis, did not produce any growth and therefore must have reacted with ingredients of the EMB to suppress their growth.
Lastly, the KF Streptococcal Agar only had one culture produce growth which makes it easily identifiable as a selective media. The only culture that produced any growth was Streptococcus Faecalis. Just by comparing the names of the broth culture and the agar we find a similarity, the word Streptococcus. The KF media, being a selective media, promotes growth of only one type of organism and discourages the growth of any others (Madigan et al, 2012). It makes sense that the medium was selective in producing the growth for its own relative organism.
In conclusion, enriched, differential and selective mediums produced different growth results for each culture. The enriched media, Tryptic Soy Agar, produced identical looking growth observations for all four organisms. The differential media, EMB, showcased the difference in gram positive and gram-negative medias by turning them different colours; pink for E. Aerogenes and green for E.Coli respectively. Lastly, the selective medium, KF, promoted the growth of one culture, Streptococcus Faecalis, and discouraged the other cultures.
Resources:
Department of Biology, 2012, Biology 240 Fundamentals of Microbiology Laboratory Manual. University of Waterloo. pp. 45-47. Dickson, J. (2002) “MadSci” Faculty, Microbiology, Iowa State University. Oct http://www.madsci.org