Medicinal Microbiology Lab Report

Many infectious diseases caused by infectious agents, including bacteria, fungi, viruses and parasites, have plagued human existence. Thus the identification of bacteria becomes all the more important in the search for medicines and cure. Identification of bacteria is a multistep process because while some preliminary guesses can be made from the morphology of microbes on various differential agars, various other tests need to be done to differentiate and confirm their identity. RESULTS

Bacteriology – When grown on MacConkey agar, A had abundant growth of pink punctiform colonies that are circular and moist. Microbes A are gram-negative cocci. B had moderate growth of yellow punctiform colonies that are circular and moist. Microbes B cells are gram-positive cocci. C had abundant 2mm growth of pink colonies that are circular and have a mucoid consistency. Microbes C are gram-negative rods bacilli. D had abundant growth of 2mm yellow colonies that are circular and have a mucoid consistency. Microbes D are gram-negative bacilli that gave off a fishy smell. Swarming was observed on plate D. E has an abundant growth of gram-negative, 2mm circular blue-green colonies with diffused green pigments. E gave off a pungent odour and have a mucoid consistency. No growth was observed for F. When grown on blood agar under aerobic conditions, A had abundant growth of 1mm white, circular, moist colonies that exhibited gamma hemolysis.

B had moderate growth of 1mm white, circular, moist colonies that exhibited gamma hemolysis. C had abundant growth of 2mm white, circular, mucoid colonies that exhibited alpha hemolysis. D had abundant growth of 2mm white, circular, mucoid colonies that exhibited alpha hemolysis. Swarming was observed in plate D and colonies gave off a fishy smell. E has an abundant growth of 6mm blue-green colonies of indistinguishable shape with diffused green pigments. E exhibited beta hemolysis. Microbes E gave off a pungent odour and have a mucoid consistency. No growth was observed for F. When grown on blood agar under anaerobic conditions, A had moderate growth of 1mm white, circular, moist colonies that exhibited gamma hemolysis. B had moderate growth of punctiform white, circular, moist colonies that exhibited gamma hemolysis. C had moderate growth of punctiform white, circular, mucoid colonies that exhibited gamma hemolysis.

D had scanty growth of punctiform white, circular, mucoid colonies that exhibited gamma hemolysis. Colonies of D gave off a fishy smell. E has scanty growth of white, circular colonies that exhibited gamma hemolysis. Microbes E gave off a pungent odour and have a mucoid consistency. F had moderate growth of 1mm, white, circular, moist colonies that gave off a pungent odour and exhibited alpha hemolysis. When grown on nutrient agar under anaerobic conditions, no growth was observed for E. However, under aerobic conditions, E had moderate growth of 1mm blue-green pigmented colonies that were circular, mucoid in consistency and gave of a pungent odour. Catalase Test – No effervescence observed when hydrogen peroxide was added to A (i.e. negative test) but effervescence observed when added to B (i.e. positive test).

Oxidase Test – No colour change (i.e. negative test) for C, D and F but purple colouration observed for E (i.e. positive test). Oxidation-fermentation tubes – Under both anaerobic and aerobic conditions, agar in C were uniformly yellow, indicating positive tests. Under aerobic conditions, E has uniformed yellow agar indicating a positive test, while under anaerobic conditions had uniformed green agar, indicating a negative test. Triple sugar iron tubes – The yellow agar in C was cracked and had a column of air at the butt of the TSI tube. Majority of the agar in D was red but black precipitate was observed at the butt of the TSI tube. Slant of agar in E is red but agar at the butt is yellow. Oxygen requirement and metronidazole susceptibility – Under aerobic conditions, no growth was expected in F. However, under anaerobic conditions, there was a clear plaque around MZ disk but white colonies that exhibited alpha hemolysis observed at the edges of the plate furthest away from the MZ disk. Urea Slope – Uniformed yellow agar indicated negative result for E. Bright pink agar indicates positive result for D. Mycology – G was gram-positive, indicated by the purple colouration observed after gram-staining. When H was observed under the microscope, germ tubes were observed. DISCUSSION

Catalase test – This test is used to differentiate between many gram-positive microbes, as it detects the presence of catalase enzyme by observation of effervescence during oxygen gas production. A does not contain catalase but B does. Oxidase test –This test is used to differentiate between groups of gram-negative bacteria, as it detects cytochrome oxidase activity in the electron transport and nitrate metabolic pathways of certain microbes. C, D, and F do not contain oxidase enzyme while E does. Oxygen requirement and metronidazole (MZ) susceptibility – Conversion of MZ to its active form requires environments with low redox potential, thus anaerobes convert MZ to its active form. After which MZ binds covalently to DNA, disrupting its helical structure and inhibiting bacterial nucleic acid synthesis and resulting in cell death.

Thus the clear zone of inhibition was observed around the MZ disks confirms that F is an anaerobe, which is consistent with the lack of growth of F when inoculated on blood agar aerobically. Oxidation-fermentation (OF) tubes – OF test is important in differentiating members of the family Enterobacteriaceae, glucose fermenters, from the aerobic pseudomonads and similar gram-negative negative bacteria, because the latter are non-fermenters (Mahon et al., 2011). Glucose fermentation undergoes the glycolytic pathway, resulting in a colour change in the medium from yellow to green. C fermented glucose both in the absence and presence of air, resulting in yellow agar. E was only able to ferment glucose in the presence of air, so green agar was observed under anaerobic conditions. Triple sugar iron tubes – This test is a used to determine if a gram-negative rod is a glucose-fermenter or not, in addition to testing if the microbe can ferment sucrose and/or lactose, gas production during glucose fermentation and H2S production – all of which are useful in differentiating gram-negative rods from the Enterobacteriaceae family (Mahon et al., 2011). Acid is produced when sugars are fermented, resulting in a drop in pH.

Phenol red turns yellow once the pH drops below 6.8. Moreover, because of the slanted shape of the agar, microbes are exposed to anaerobic and aerobic environments in varying degrees. Since only the butt of E is yellow while the slant is red, it can be deduced that E only ferments glucose such that the acid produced is not enough to turn the slant yellow. If the tube had been observed between the first 8-24 hours of inoculation, one would find that E had yellow slant and yellow butt; but after 24 hours, the yellow slant changes colour to red as alkaline amines are formed from the oxidative decarboxylation of peptides derived from proteins in the medium. The black precipitate observed at the butt of tube D indicates that microbe D can use thiosulfate anion as a terminal electron acceptor, reducing it to sulfide. The ferrous sulfate in the agar medium then reacts with hydrogen sulfide to form ferrous sulfide, which is observed as black precipitate. C is a lactose- and glucose-fermenter because complete permanent acidification of both the butt and the slant of the tube were observed. The volume of air at the butt that pushed the agar upwards is hydrogen gas. Urea Slope – This test helps to identify certain species of Enterobacteriaceae, such as Proteus spp. Microbes that posses urease will hydrolyze urea, releasing ammonia.

The agar turns a bright pink to indicate the presence of an increase in pH, which can be attributed to the presence of ammonia. Bacteriology – MacConkey agar is only moderately inhibitory and is designed to prevent growth of gram-positive bacteria. Thus, A and B being gram-positive bacteria, only managed to survive as punctiforms instead of 1mm-sized colonies that they are when grown in optimal conditions (i.e. aerobically on blood agar). Sheep blood agar is the optimal agar for inoculating most bacteriology specimens because this medium supports growth for all except the most fastidious clinically significant bacteria. Under aerobic conditions, β-hemolysis was observed in E, indicating the secretion of α-toxin, which is a lecithinase that dissolves membrane lipids, resulting in cell lysis. The presence of β-hemolysis indicates secretion of the protein β-hemolysin that breaks down red blood cells, forming a clear zone around colonies (Madigan et.al, 2014). α-hemolysis was observed in C and D, producing greenish pigment due to incomplete lysis of red blood cells. F did not survive on aerobic conditions, indicating that F is an obligate anaerobe. This means that F does not have enzymes, namely superoxide dismutase and/or catalase, which convert reactive oxygen species to non-damaging species (Mahon et al., 2011).

REFERENCES

Koneman, E. W. (1997). Koneman’s Color Atlas and Textbook of Diagnostic
Microbiology. USA: Lippincott Williams & Wilkins. Madigan, et. al (2014). Brock Biology of Microorganisms (14th ed., p. 78). Boston: Pearson. Mahon, C., Lehman, D., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology. Missouri: Elsevier. Tille, P. M. (2014). Bailey & Scott’s Diagnostic Microbiology. Missouri: Elsevier. APPENDIX