Abstract
The main purpose of this experiment was to identify organisms that cause infection of the urinary tract and to prescribe effective antibiotics that the organism could not resistant. The selected methods in this experiment include Gram stain, spot test, biochemical testing, sensitive plate set up, reading biochemical testing and reading sensitive plate set up. The results of the experiment indicate presence of pathogens responsible for urinary tract infection mainly staphylococcus aureus as well asserting the patient’s sensibility certain antibiotics. These antibiotics included Flucloxicillin Fox 10, Vancomycin Va 5, PencillinP.05 and Clindamycin.
Aim
The aim of this experiment is to identify the pathogen responsible for Urinary Tract Infection in the patient and to prescribe suitable medication for the condition. Standard microbiology techniques will be employed to establish the most appropriate measure to address the patient’s condition.
Introduction
According to Mayo Clinic Staff (2010), urinary tract infection starts in the urinary system. The Kidneys, bladder and urethra form the urinary system. This system can become infected, but most infections involve the bladder and the urethra, which form the lower urinary system. Microbiologists can confirm a urinary tract infection by testing a patient’s tissue sample to identify the microorganism responsible.
This experiment will employ some tests including agar streaking and differential split agar plate, which isolates and differentiate the bacteria responsible for urinary tract infection (Friedman et al. 1991). Strain testing that involves performing biochemical testing and colony morphology and gram stain to identify responsible organism (Mortensen 2009). Antibiotic sensitivity testing that includes setting up of sensitivity plates (Dennis Guttmann 1963).These tests are done to ascertain the degree of sensitivity or resistance of organism isolated from patient’s tissue sample to an appropriate variety of antibiotic drugs.
Method
The first test conducted was the Gram stain test. A single drop of saline was dropped onto a slide, after which a single colony was selected and immersified in saline then spread over the 1.5-centimeter slide. The slide was air dried, then heated and finally allowed to cool. Three to four drops of 1% crystal violet was added to the smear and left to stand for 30 seconds. Before the smear could dry, it was rinsed with distilled water and Gram’s iodine added. After 30 seconds, it was rinsed with 95% ethanol until the run-off was colorless. Safranin, was added and left to stand for 30 seconds. The slide was dried by gently blotting it on paper towel. It was examined under a compound microscope using 100X oil immersion objective to identify individual microorganisms.
Two biochemical tests were conducted to validate bacterial identification. First, was the test to validate oxidase positive bacteria, where an oxidase strip was touched to a loopful of bacteria. The other test was the microbact 12A strip, where a bacterial suspension was made by picking 1 to 3 isolated colonies from culture plate re-suspended in 2.5 milliliters of sterile saline. After opening the microbsct strip, 4 drops of bacterial suspension were added to each of the 12 wells. Three wells( 1,2,3) were overlaid with mineral oil using a sterile Pasteur pipette, resealed and incubated for 24 hours. After the 24 hours, 2 drops of Indole reagent was added to the eighth well and evaluated within 2 minutes. In well 10, 1 drop each of VPI and VPII reagent were added and evaluated after 15 minutes. In well 12, 1 drop of TDA reagent was added and results recorded immediately. The reaction in each well was recorded as either positive or negative by comparing to a color chart.
For the Sensitivity plate setup, nutrient agar plate was divided into 4 sections. Using a sterile swab, a single colony was transferred into a 2.5 milliliters sterile bottle. It was mixed gently but thoroughly and poured over the entire plate then allowed to dry for 15 minutes. A different antibiotic disk was placed into each quadrant using forceps. The plates were sealed with parafilm, inverted and incubated for 24 hours for later observation.
Results
Specimen used was from the wound of the patient and several tests ran. As recorded in the figure below (i), Colony morphology test turned grey and many small colonies were observed. They were circular in shape with a flat margin elevation. The mannitol salt test revealed a yellow coloring while the gram test turned purple an indication of positive result. Bergey et al. (1994) outline gram stain as the separation of all bacteria into either primary dye, which is gram-positive or secondary dye, which is gram-negative. In this test, when the bacteria was treated with iodine they assumed the crystal violet in their thick layer of peptidoglycan. The catalase test produced bubbles and the coagulae test showed positive revealing the presence of the suspected organism.
Figure i. Results of the wound swab tests.
Figure ii. Results of the antibiotic sensitivity tests.
The sensitivity test was done on four antibiotics. Flucloxicillin Fox 10 and Clindamycin did not show sensitivity as indicated in figure (ii).penicilin showed no results as the antibiotic disk was moved away. The only sensitive antibiotic was Vancomycin Va 5.
Discussion
This practical was to identify microorganisms that are responsible for the urinary tract infection in the patient and to establish suitable remedy for the condition using antibiotics. The gram stain test revealed existence of staphylococcus- aureous. The specimen showed sensitivity to antibiotics.
Using the microbact 12A system to identify the pathogenic bacteria, a positive reaction in glucose and mannitol tests, but a negative reaction in Xylose test indicates the presence of Staphylococcus species, which live in areas of high salt concentration.
Oxidase test shows organisms that secretes cytochrome oxiase enzyme. Cytochrome oxidase is involved in an electron movement link as it transfers electrons to oxygen from donor molecule. Oxydase reagent possesses a chromogenic agent, a compound that when oxidized, changes color.The reagent turns blue within 15 seconds if the organism tested produces cytochrome oxidase. It is a test used to determine if an organism cytochrome oxidase (MacFaddin (ed.) 2000).
References
Allen, ME 2010, ‘MacConkey Agar Plates Protocols’, American Society for Microbiology.
Bergey, D, Holt, JG, Krieg, N & Sneath, P 1994, Bergey’s Manual of Determinative Bacteriology, 9th edn, Lippincott Williams & Wilkins.
Beveridge, T & Davies, JA 1983, ‘Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain’, Journal of bacteriology , p. 156.
Dennis Guttmann 1963, ‘Diagnosis of Urinary infection:Comparison of a Pour-Plate Counting Method with a Routine Method’, British Journal of Medicine.
Friedman, MP, Danielski, JM, Day, T, Dunne, J, Evangelista, A & Freeman, T 1991, ‘Rapid Isolation and Presumptive Diagnosis of Uropathogens by Using Membrane Filtration and Differential Media’, JOURNAL of Clinical Microbiology, vol 29, no. 11, p. 2385.
MacFaddin, J (ed.) 2000, ‘Biochemical Tests for Identification of Medical Bacteria’, 3rd edn, Lippincott Williams and Wilkins, Philadelphia.
Mayo Clinic Staff 2010, Urinary tract infection, Web.
Mortensen, N 2009, ‘STUDIES IN URINARY TRACT INFECTIONS:Biochemical Characteristics of Coagulase-negative Staphylococci Associated with Urinary Tract Infections’, Journal of Internal Medicine, vol 186, no. 6.
Swenson, JM, Facklam, RR & Thornsberry, C 1990, ‘Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus and Lactobacillus species’, in Antimicrob Agents Chemother.