Question 1
I identified organism A as Staphylococcus Aureus. Upon looking at the culture on the blood agar, they were flat, and white in colour, showing beta haemolysis of the blood agar. When I further looked at them down the microscope, they showed branched cocci structures, following this, I performed a gram stain, where they came out as gram positive, and showed a purple colour, the next test I performed was the catalase test, this test came out as positive, shown by the bubbling. This confirmed that it was a species of Staphylococcus, to further confirm it was staphylococcus, I carried out a coagulase test which came out as positive, to work out its identity, I then carried out a staph latex test, to detect protein complexes produced by Staphylococcus Aureus, the clumping in organism A confirmed this test, as a positive result for Staphylococcus Aureus.
I identified organism B as Streptococcus Pyogenes, observation of this organism showed a white colour, which was raised with circular colonies. This showed beta haemolysis. Upon carrying out the gram stain, the bacterium turned a purple colour, where it was cocci in shape, and in chains. The gram stain showed that it was gram negative. Following this, I carried out the catalase test, where it showed a negative result, confirming a streptococcus organism. I then sub cultured onto a blood agar plate to isolate the colonies. I the carried out the staph latex test where it showed a negative result. The final test I carried out was the Lancefield group test, where it showed that the latex particles clumped when added to organism B, this showed that it was a group A streptococcus, therefore the final identity was Streptococcus Pyogenes
I identified organism C as Candida Albicans. First observations of colony C showed white raised colonies, roughly 3mm, round in shape. Upon carrying out a gram stain test, the organism came up as neither positive nor negative. I then incubated the organism into a germ tube, containing serum, and incubated it for two hours, when I observed organism C under the microscope, after these 2 hours, some branching structures were visible. Showing positive yeast presence. I then placed the yeast culture into corn meal agar, and left it for a week, where I then observed it under a microscope again. Branched structures and small chlamydospores were visible, showing that it is Candida Albicans.
I identified organism D as Escherichia Coli. First observation of the colonies showed yellow and green coloured colonies, where they were raised and circular in shape, with a smell of rotting meat. Upon observation under the microscope, there were free-floating bacilli in shape, after carrying out the gram stain; they turned pink in colour, showing that they were gram negative. The next test that I carried out was the oxidase test. Organism D showed up as negative, this shows no presence of cytochrome C, since E-coli lives in the gut, which is an anaerobic environment. After this, I subcultured the organism onto chromogenic coliform agar, in order to determine what the organism was, after a week, the colour of the colonies were pink/red. This showed a positive test for E-coli.
I identified organism E as Pseudomonas Aeruginosa. Upon first observations, on the agar plate, the colonies were a dark green colour, with a flat round shape. No haemolysis was noted, as they weren’t on blood agar. Upon looking under the microscope, the bacteria was arranged in single colonies in a bacilli shape, and dyed pink, after a gram stain, showing that they were gram negative. Following this, I carried out the oxidase test, where the organism showed a positive reading. Once done, I then subcultured E onto blood agar, in order to isolate single colonies. I then carried out the rapID NF plus on organism E, where the computer confirmed the identity to be Pseudomonas Aeruginosa.
Question 2
Patient A-
Question 4
A gram stain test is used to differentiate between bacterial organisms that have a peptidoglycan layer. The gram stain test consists of the use of the dye crystal violet, which is purple/violet in colour, and a counter stain, which is known as safranine. The gram staining process works so well, because gram-positive organisms contain one single membrane, alongside a thick mesh like cell wall that is made up of peptidoglycan. This peptidoglycan is cross-linked in its structure. This structure allows for them to retain the crystal violet dye, unlike gram-negative bacteria, as their cross-linked peptidoglycan layer is much thicker than the gram-negative constituent. Gram-negative bacteria have a much thinner peptidoglycan layer, and two membranes, of where the space between them is known as the periplasm. This is where the peptidoglycan layer is located, within the periplasm. It is much thinner than that of the peptidoglycan layer of gram-positive bacteria. This means that it has a much lower absorption rating of the crystal violet stain, than the gram-positive bacteria. This is why gram-negative organisms stain pink, rather than purple. Due to their failure to absorb lots of the dye. Some limitations of gram staining is that it is not able to differentiate between bacteria with different cell membrane structures, such as tuberculosis, in addition to this, it is also unable to identify what the organism is, and how pathogenic it is, further testing is required for that, furthermore, the gram staining test all comes down to perception of what colour the cells have actually turned. To one student the colour of the cell could be pink, meaning the bacterium is gram negative, and to another, the bacterium could be purple, meaning the organism is gram positive. This is a major limitation, as wrongly interpreting the gram stain test could potentially lead to a misdiagnosis in a clinical setting.
Question 5
The main principle of the Lancefield group test is to identify and divide the different groups of haemolytic streptococci species. It does this by identifying the different antigens, which are present on the cell walls, allowing for differentiations between the groups.
Question 6
The chromogenic UTI agar we used in the practical was used to detect species of E-Coli. If a species of E-coli is present, the colonies will be dyed a bright red/pink colour. This is due to a selective property. One of the selective properties of CUTI media is that they contain certain substrates, which are used for the detection of two enzymes, which are produced in common UTI causing species, such as E-coli and Enterobacter species. The two enzymes are beta-galactosidase and beta glucosidase. These two enzymes hydrolyse the chromogenic substrate. This causes the colony to turn a bright pink/red colour. Enterobacter species do not contain the enzyme beta galactosidase, but the do contain beta glucosidase. In this case, if an Enterobacter species is present, the colonies will turn a blue/green colour due to the different chromogenic substrates being broken down. This allows for differentiation between two common UTI causing bacteria.
Question 7
Corn meal agar is mainly used to provide ideal growth conditions for fungi. Once a colony of fungi is subcultured onto corn meal agar, the fungi will begin to grow quickly. This is because it provides the ideal growth conditions for it to grow. In this test, you are looking for chlamydospores and germ tubes. They are generally found and produced by Candida Albicans, which is a type of yeast. This allows for the identification of Candida Albicans in vitro. One additive which is in or