Microbiology Practical 3 PDF

LS4001 Practical 3 Basic Microbiology 2025 Alan Calder, Dagmara Krzysztofiak, Tom Vujakovic Caretaker Module Leader: Dr Simon Gould  AIMS To study different techniques that are used for the identification of bacteria....

LS4001 Practical 3 Basic Microbiology 2025 Alan Calder, Dagmara Krzysztofiak, Tom Vujakovic Caretaker Module Leader: Dr Simon Gould  AIMS To study different techniques that are used for the identification of bacteria. Gram staining Biochemical tests (Catalase / Oxidase). Note: You are expected to have studied the on-line resources to prepare for this class.  Learning Outcomes: On successful completion of this practical, attendance at microbiology lectures and use of the recommended resources, you should be able to recognize and discuss the characteristics of a variety of medically important microorganisms. SAFET Y  Regard EVERY microorganism as pathogenic, even if it is not one of the recognised pathogens. Cover any cuts on your skin with a waterproof dressing. Do not put ANYTHING in your mouth. ar the designated labcoat at ALL times. Take it off when leaving the laboratory. Tie Long hair back to avoid fire and contamination risks. Keep mobile phones turned off and do not be handle them. Do not expose cultures to the air for longer than is necessary. Do not place tube caps or other stoppers on the bench. Always flame the bacteriological loop before and after use. Do not place contaminated loops on the bench. Avoid the production of aerosols. Fully immerse pipettes and other contaminated waste in the disinfectant ja Q: Why is this important? Identifying bacterial species is required in order to make accurate clinical decisions. Gram Staining GRAM STAIN……. Species broadly classified into two major groups. Differentiation is based on cell wall properties. Often the first stain carried out for identification.  Gram stain for the Bacillusfollowing subtilis species. Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Microscopic appearance. Either: or Cocci Bacilli  Preparing Slides. Write on the frosted part of the slides using penc Add a drop of water to the slide using a sterile loop. Apply a thin bacterial smear to approx. 1/3 of slide. Allow the slides to air dry.  The loop flaming procedure. Avoiding aerosol production: Sterilise the loop using a blue Bunsen flame. Always cool the loop down touching the plate. Which of the following loop positions looks correct? A. A B. B C. C D. D E. B and D  Gram Staining Procedure:. am +ve Gram -ve Heat-fix the smear using a Bunsen burner Flood slides with crystal violet Wash Flood slides with iodine solution Wash Decolorize with Alcohol Wash Flood slides with counter-stain carbol fuchsin. Wash the slides and blot them dry using tissue  Viewing the stained slides. Use 100 x oil immersion - don’t try to guess the colour. Watch the video Focus on an area where there the cells are spread out (edge of smear). BIOCHEMICAL TESTS  WEAR GLOVES. Many species can be similar in shape, size and arrangements. Biochemical activity can vary from one species to the other. Differences in enzyme production or metabolism can help identification. Video on Canvas  Catalase Test Theory: Aerobic breakdown of sugars leads to Hydrogen peroxide (H2O2) formation. H2O2 broken down into water (H2O) and Oxygen (O2). Can differentiate between morphologically similar species eg. staphylococci (catalase-positive) from streptococci (catalase-negative). Are they catalase positive or negative? Catalase Test Procedure: INVER TAP T ELBOW Catalase reagent BUBBLES?  Catalase Test Result: Reagent capillar y Positive: Bubbling. Negative: No bubbling. Culture  Oxidase Test Theory: Differentiation through the presence / absence of the cytochrome oxidase enzyme. Enzyme involved in the reduction of oxygen to water in the respiratory electron transport chain. Positive species reduce Tetramethyl phenylenediamine dihydrochloride (TMPD) to a dark blue / purple end product.  Oxidase Test Results: Count 10 - 15 seconds. An Oxidase Negative Result.  COMPLETE YOUR RESULTS TABLE: Quick Gram Stain Oxidase test Catalase test Organism Shape +/- +/- +/- Unknown A Unknown A