Lab 6 Biochemical Tests for Microorganism Identification PDF

Basic Microbiology Practical No.: 6 & 7 Biochemical Tests for Identification of Microorganisms Intended Learning Objectives (ILOs): By the end of this lab, the student is expected to: 1. Introduced to the selective and differential media and able to define both...

Basic Microbiology Practical No.: 6 & 7 Biochemical Tests for Identification of Microorganisms Intended Learning Objectives (ILOs): By the end of this lab, the student is expected to: 1. Introduced to the selective and differential media and able to define both types. 2. Able to differentiate between lactose and non-lactose fermenter bacteria on MacConkey agar 3. Understand the importance of catalase and coagulase tests and their principle. 4. Perform and interpret the results of a catalase and coagulase tests. 5. Understand the importance of Oxidase and Urease tests and their principle. 6. Perform and interpret the results of Oxidase and Urease tests. 7. Introduced to the API-20E test, how to interpret the results. Precaution: 1. Wear appropriate personal protective equipment for the laboratory procedure. 2. All specimens should be considered infectious. 3. All samples must be processed in line with SOP designed for the test. 4. Result must be recorded immediately. Page 1 Introduction/ Background: v Catalase Catalase is an enzyme that breaks hydrogen peroxide (H2O2) into oxygen (O2) and water (H2O). Excluding the Streptococci, most aerobic and facultative anaerobic bacteria possess catalytic activity. Testing the presence of this enzyme help us to identify species of the unknown bacteria. Reaction Mechanism: Aerobic organisms that have the enzyme catalase break down hydrogen peroxide in the following reaction. H2O2 catalase H2O + O2 NOTE: Do not use a metal loop or needle with H2O2; it will give a false positive and degrade the metal. Do not take colonies from Blood agar. v Coagulase Staphylococcus aureus is known to produce coagulase, which can clot plasma into gel in tube or agglutinate cocci on slide (convert fibrinogen to fibrin). This test is very useful to differentiate S. aureus from other coagulase-negative staphylococci. Most strains of S. aureus produce two types of coagulase, free coagulase and bound coagulase. While free coagulase is an enzyme that is secreted extracellularly, bound coagulase is a cell wall associated protein. Free coagulase is detected in tube coagulase test and bound coagulase is detected in slide coagulase test. Slide coagulase test may be used to screen isolates of S. aureus as well as tube coagulase may be used for confirmation Page 2 Principle: The bound coagulase is also known as clumping factor. It cross-links the α and β chain of fibrinogen in plasma to form fibrin clot that deposits on the cell wall. As a result, individual coccus sticks to each other and clumping is observed. v Oxidase The oxidase test identifies organisms that produce the enzyme cytochrome c oxidase. The oxidase reagent contains a chromogenic reducing agent, which is a compound that changes colour when it becomes oxidized. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds. Reaction Principle: The cytochrome c oxidase enzyme reduces the colourless oxidase reagent (1% tetramethyl p-phenylene diamine dihydrochloride) to dark blue-indigo-purple (indophenols) immediately or in 10-30 seconds. Page 3 Precautionary note: Ø This test must be done with cultures growing on solid media. Ø When obtaining results, look for a blue-purple colour change concentrated where the cells were added to the filter paper. As the reagent sits on the filter paper, it forms a blue ring, which DOES NOT indicate the presence of cytochrome c oxidase. v Urease Urea Agar was developed by Christensen in 1946 for the differentiation of enteric bacilli. The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease. Reaction principle: Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours. Weakly positive organisms may take several days, and negative organisms produce no color change or yellow as a result of acid production. v API test In clinical laboratories, time, space, and expense are all important factors. They do not use individual media as we do in our educational lab. Instead, many tests are done at the same time in one place in order to identify bacteria from patient samples. This involves using a multi-test system. API 20 E is a standardized identification system for Enterobacter and other non-fastidious, Gram-negative rods which uses 21 miniaturized biochemical tests. Page 4 Principle: The API 20 E strip consists of 20 micro-tubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension that reconstitutes the media. During incubation, metabolism produces colour changes that are either spontaneous or revealed by the addition of reagents. Precautionary note: Ø Before use, check that the packaging of the various components is intact. Ø Do not use strips which have been damaged: cupules deformed, desiccant sachet open. Ø Do not use reagents past the expiration date. Practical session 6 PART-A: Catalase Test PROCEDURE: Equipment: Safety cabinet, Flame Materials: Dropper, Pointed glass rods, clean microscope slide Reagents: Hydrogen peroxide (H2O2). Specimens: Well isolated colonies of bacteria (Sample A & B). 1. Control organisms: a. Positive control (Staphylococcus aureus) b. Negative control (Streptococcus sps) Method: 1. Take a drop of H2O2 on clean glass slide. 2. Pick up a colony to be tested from nutrient agar plate with the help of a disposable loop. 3. Dip the loop in the drop of H2O2. Observation: Ø Rapid appearance of sustained gas bubbles- Positive Ø No gas bubble production - Negative Page 5 Record your results PART-B: Coagulase Test PROCEDURE: Equipment: Safety cabinet, Bunsen burner Materials: Pipettes, Small test tubes, Glass slide, pointed glass rod. Reagents: Citrated mammalian plasma. Specimens: 1. Well isolated colonies of culture (Sample A& B) 2. Control organisms: a. Positive control (Staphylococcus aureus) b. Negative control (Streptococcus sps. Or S. epidermidis) Method: Two methods: 1. Slide coagulase 2. Tube coagulase Slide coagulase test: 1. Place a small amount of growth from culture plate onto a clean microscope slide. 2. Add one drops of mammalian plasma onto the smear and mixed well with inoculating needle. 3. Agglutination or clumping of cocci within 5-10 seconds is taken as positive. Page 6 Observation/ Results and Interpretations: Positive: Agglutination or clumping of cocci within 5-10 seconds. - Staphylococcus aureus Negative: No agglutination or clumping - Staphylococcus epidermidis 2. TUBE COAGULASE TEST: Principle: The free coagulase secreted by S. aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma. Procedure: 1. Heavily inoculate a tube containing 0.5 ml citrated mammalian plasma with unknown organism. 2. Incubate the mixture at 370C. 3. Examine the plasma tube for coagulation by gently tilting the tube. 4. Observe tube (s) at 30 minutes, 1 hr, 2hr, and up to 4-hour of intervals. Page 7 Interpretation of result: Positive: Gelling (clotting) of the plasma – S.aureus Negative: No Gelling (No clotting) – S. epidermidis. PART-C: Oxidase Test PROCEDURE: Equipment: Safety cabinet, Bunsen burner. Materials: Pointed glass rods & sterile filter paper Reagents: Oxidase reagent - 1% tetramethyl-p-phenylenediamine dihydrochloride. Specimens: Well isolated colonies of bacteria (to be identified). Control organisms: Positive control (Pseudomonas aeruginosa) & Negative control (Proteus sp) Method: There are various methods of performing this test 1. Dry filter paper method 2. Wet filter paper method 3. Cotton-tipped stick method 4. Plate method Page 8 Dry Filter Methods: 1. With a sterile swab, obtain a small amount of organism from an agar slant or plate. 2. Place one drop of reagent onto the culture on the swab. 3. Positive reactions turn the bacteria violet to purple immediately or within 10 to 30 seconds. Note: Delayed reactions should be ignored. Observation: Positive: Development of dark purple colour (indophenols) within 10 seconds Negative: Absence of colour Results and Interpretations: Controls: Sample A: Sample B: PART-D: Urease Test PROCEDURE: Equipment: Safety cabinet, Bunsen burner Materials: Sterile loop, Christensen`s urea agar medium. Specimens: 1. Well isolated colonies of culture (Sample A& B) Page 9  2. Control organisms: a. Positive control (Proteus sps ) b. Negative control ( salmonella sps) Method: (slant method) 1. Inoculate the slant of Christensen`s urea medium with the test organism. 2. Incubate at 37ºC (or the appropriate temperature for the organism) for 24 hours to four days. Observe results next day Interpretation of result: Positive: Pink to red colour Negative: No change in colour RECORD YOUR RESULTS Controls: Sample A: Sample B: Page 10 References: 1. Bharti Arora, D. R. Arora (2015): Practical Microbiology. ISBN: 81-239- 14095-9. Page 1 – 218. 2. Steven Obenauf, Susan Finazzo (2016): Microbiology Fundamentals. A Clinical Approach. Page 1 – 318. Page 11 Page 12  FLOW CHART FOR IDENTIFICATION ////////////////////////////////// Gram Negative Bacilli Aerobic Motility Anaerobic Bacilli Lactose Klebsilla fermentation Shigella / EMB Agar Oxidase /// Urease Oxidase Pseudomonas Proteus Spp. aeruginosa / Escherichia coli Page 13