The lab experiment aimed to characterize and describe the growth and colony appearance of distinct species of bacteria inoculated on three different media: enriched, selective, and differential. The selected bacterial strains were Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Streptococcus aureus, Enterococcus faecalis, and an unknown sample. Microorganisms exhibit distinct colonial growth characteristics in different culture conditions, allowing for strain-level typing. Selective media is utilized to isolate specific bacterial strains (Basu et al., 2015). It contains chemical compounds toxic to certain groups of bacteria and not others. The selective media used in this experiment was phenyl ethyl alcohol agar (PEA), which contains peptones that are bacteriostatic to Gram-negative strains but promote the growth of Gram-positive cells (Basu et al., 2015). Bacteria that cannot metabolize the peptone proteins cannot grow.
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Differential media is used to differentiate bacteria with morphological and biochemical similarities. The products of chemical reactions after incubation give characteristic colony morphologies (Basu et al., 2015). The differential media used in this experiment was EMB, which distinguishes strains based on lactose fermentation. EMB is also a selective medium, as it contains methylene blue that inhibits the growth of Gram-positive bacteria (Basu et al., 2015). Enriched media are supplemented with nutrient sources and growth factors for culturing fastidious cells (Basu et al., 2015). It lacks inhibitory substances. A blood sample was then enriched medium used in this experiment.
Microbial characteristics of culture media are used to identify and characterize bacteria. In this experiment, P. aeruginosa, E. aerogenes, S. aureus, and E. faecalis are Gram-positive strains, while E. coli is a Gram-negative cell (Murray, Rosenthal, & Pfaller, 2015). Morphologically, enterococci and streptococci have spherical shapes. P. aeruginosa, E. aerogenes, and E. coli are rod-shaped microorganisms (Murray et al., 2015). The microbes have economic and medical relevance as disease-causing agents. Some of these strains – P. aeruginosa and E. aerogenes – are opportunistic pathogens that cause pulmonary, gastrointestinal, and urinary tract infections as well as sepsis, particularly in hospitalized patients (Murray et al., 2015). Methicillin-resistant staphylococcal skin infections by S. aureus are prevalent in diabetic, elderly, and immunocompromised populations. E. faecalis is usually a nonpathogenic intestinal microorganism. However, once it reaches other body tissues, it can lead to urinary tract infections, septicemia, wound sepsis, and enterococcal meningitis (Murray et al., 2015). E. coli lives in the human gut, and it causes diarrhea and vomiting.
Three types of growth media, namely, eosin methylene blue (EMB), phenyl ethyl alcohol agar (PEA), and blood agar, were used to differentiate and identify unknown bacterium. Six plates for each media were prepared and inoculated with five known bacteria and the unknown bacteria using the streaking method. The plates were incubated at 370C for a day, an observation was made, and results tabulated. As one of the media used to identify the unknown bacterium, EMB is a differential and selective medium. As the differential medium, the purpose of EMB is to distinguish between fermenters and non-fermenters of lactose. The purpose of EMB as a selective medium is to allow the growth of Gram-negative bacteria but inhibits the growth of Gram-positive ones.
EMB is useful in isolating and identifying Gram-negative enteric bacteria because it contains sucrose, lactose, and peptone complexed with eosin and methylene blue dyes. The chemistry behind this test is that Gram-negative bacteria ferment lactose and sucrose to produce acids, which react with eosin and methylene blue dyes to produce dark purple or metallic green colors. Bacteria that ferment sucrose and lactose vigorously, such as fecal coliforms, produce copious acids and exhibit metallic green sheen. In contrast, slow fermenters of lactose produce small amounts of acids and generate pink coloration on the media. Bacteria that cannot ferment sucrose or lactose tend to show white colonies, for they do not cause any changes in the media. Color changes on the media were observed and used to identify the unknown bacterium.
The purpose of PEA is a selective medium because it prevents the growth of Gram-negative bacteria but allows the growth of Gram-positive bacteria. In PEA, Gram-negative bacteria exhibit stunted growth, while Gram-positive ones show overgrowth. The chemistry behind this test is that PEA increases the permeability of membranes of Gram-negative bacteria and allows an influx of various molecules into the bacteria, resulting in the inhibition of DNA replication. Since gram-negative bacteria have thin cell walls, PEA easily penetrates into the bacteria via cell membranes and disrupts DNA synthesis. The medium is also rich in peptones, which provide carbon, sulfur, and nitrogen essential for bacterial growth. Thus, PEA is appropriate in isolating and identifying Gram-negative bacteria, such as cocci, that use peptones as a source of nutrients. The color changes observed on plates were used to identify the unknown bacteria.
Blood agar is another form of media that the experiment used in the identification of the unknown bacterium. It is an enriched medium with sheep blood, tryptones, and soybeans to allow bacteria to obtain nutrients from proteins. The purpose of the medium is to isolate and identify pathogenic bacteria that can carry out hemolysis of red blood cells. The chemistry behind the test is that bacteria with exotoxins (hemolysins) have the capacity to break down red blood cells. Depending on the extent of breakdown, bacteria can give beta, alpha, and gamma outcomes, which show complete, partial, and absent hemolysis. Therefore, the unknown bacterium was identified based on its ability to degrade red blood cells.
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Table 1. Tabulated Results that Identified the Unknown Bacterium.
|Agar Plate Tests|
|Types of Bacteria||EMB (Indicate Lactose Fermentation by Colony Coloration)||Phenylethyl Alcohol||Blood Agar|
|E. coli||Green||No Growth||Alpha|
|E. aerogenes||Dark purple||No growth||Alpha|
|P. aeruginosa||Pink||Light green||Beta|
|S. aureus||No Growth||White/clear||Gamma|
|E. faecalis||Growth/purple||No growth||Gamma|
|Unknown bacterium||No growth||White plus growth||Beta|
The unknown bacterium exhibit inhibited growth in EMB medium, white colonies in PEA medium, and beta hemolysis in blood agar. Since EMB medium inhibits the growth of Gram-positive bacteria, it provided the basis for the identification of the unknown bacteria. Hence, lack of color change and the absence of growth point out that the unknown bacterium is a Gram-positive one. Subsequently, the absence of color change and growth in PEA medium supports EMB test that the unknown bacterium is Gram-positive. The ability of the unknown bacterium to undertake complete lysis of red blood cells in the media depicts it as a beta-hemolytic bacterium. Comparatively, the unknown bacterium exhibit similar properties to S. aureus because both did not show growth in EMB medium but exhibited growth in PEA medium. However, its difference occurs in the degree of hemolysis as S. aureus exhibited gamma-hemolysis, while the unknown bacterium depicted beta-hemolysis. Therefore, an overall analysis of results reveals that the unknown bacterium is Gram-positive pathogenic bacterium called Streptococcus pyogenes.
Discussion and conclusions
From the results of the EMB test, Gram-positive strains (no growth) can be distinguished from Gram-negative cells. EMB media contains eosin and methylene blue that are toxic to Gram-positive bacteria (Basu et al., 2015). Therefore, these compounds inhibited the growth of P. aureus and S. pyrogenes (Unknown isolate). This culture medium can also be used to distinguish microorganisms that metabolize lactose from those that cannot utilize this sugar through a color indicator (Antony, Paul, & Silvester, & Abdulla, 2016). Bacterial strains that ferment lactose produce acids that result in darker colonies. Thus, in this experiment, lactose-users like E. faecalis, and E. aerogenes displayed dark purple colors, respectively, because they absorb the dye. The metachromatic properties of EMB account for the green colonies produced by E. coli, which also ferments lactose (Antony et al., 2016). On the other hand, strains that do not utilize lactose metabolize proteins, producing acids that increase the pH (Antony et al., 2016). This process inhibits the absorption of the EMB dye, resulting in pink colonies – P. aeruginosa.
Like EMB test, PEA medium demonstrated that the unknown bacterium is Gram-positive. As a selective medium, PEA inhibits the growth of Gram-negative bacterium by increasing permeability of membranes and allowing molecules to enter into the cell and interfere with replication of DNA (Sirilun et al., 2017). Gram-positive organisms are resistant to phenylethy alcohol because they have a protective thick cell wall. The test indicated that P. aureus, S. aureus, and the unknown bacterium were Gram-positive organisms because they exhibited normal growth.
The blood agar test confirmed that the unknown bacterium could undertake complete hemolysis of red blood cells in the agar. Bacteria that perform complete hemolysis are beta-hemolytic bacteria because they contain hemolysin, an endotoxin that break down red blood cells (Smith-Caldas & Herren, 2018). Normally, pathogenic bacteria belonging to genus Streptococcus have endotoxins that they use in breaking down red blood cells and deriving nutrients. While S. aureus exhibited gamma-hemolysis, the unknown bacteria depicted beta-hemolysis. Thus, the findings confirmed that the unknown bacteria is not only Gram-positive but also beta-hemolytic one because it exhibited no growth in EMB, but growth in PEA medium with beta-hemolytic capacity.
Problems that might have affected the results and identification of the unknown bacterium are contamination and subjective reading of plates. Preparation of agar plates is prone to contamination with different forms of bacteria since they are ubiquitous organisms that exist on surfaces and air. Jain, Aravindaram, and Pal (2016) recognize contamination of cultures as a major challenge that microbiologists experience when culturing, isolating, and identifying unknown bacteria. Since the study used pure cultures of E. coli, E. aerogenes, P. aeruginosa, S. aureus, E. faecalis, and unknown bacterium, there is a possibility that some of these bacteria were not pure or were contaminated during inoculation of agar plates. Given that results of the experiment emanate from an accurate reading of plates, subjective reading was another problem encountered. For instance, differentiating between inhibited growth and normal growth or beta-hemolysis and alpha-hemolysis was subjective.
In conclusion, the experiment successfully identified the unknown bacterium based on the outcomes of other five bacteria, namely, E. coli, E. aerogenes, P. aeruginosa, S. aureus, E. faecalis. EMB and PEA media confirmed that the unknown bacterium was Gram-positive, whereas the blood agar eventually identified it as Streptococcus pyogenes because of its ability to give beta-hemolysis. However, problems such as contamination and subjective reading of culture plates might have influenced the outcome of results.
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