20 MIC 206 Differential Tests 2 Results S24 PDF

11/5/2024 Differential Tests 2 Results Extracellular Enzymatic Activities of Microorganisms...

11/5/2024 Differential Tests 2 Results Extracellular Enzymatic Activities of Microorganisms Urease Test Nitrate Reduction Test Catalase Test Oxidase Test Utilization of Amino Acids (Phenylalanine) Agglutination Reaction: The Febrile Antibody Test For EACH assay, know the following: Name of the Assay: Example: Catalase Production 1. Input(s) & Output(s): Example : Input: Hydrogen Peroxide and Output: Oxygen + Water 2. Specific enzyme(s): Example : Catalase 3. Reagents/ pH Indicator: Example : Hydrogen Peroxide 4. Principle of the test: Example : Organisms with catalase can render hydrogen peroxide harmless 5. What does a positive and negative results look like: Example : + Bubble/ - No Bubbles Biochemistry 2 (PHEN) Phenylalanine slant (NO3 ) Nitrate Broth Escherichia coli Control (Uninoculated) Proteus vulgaris Escherichia coli Orphan Enterococcus faecalis (STR) Starch plate Pseudomonas aeruginosa (Straight line Inoculation) E.coli Escherichia coli Orphan (DNA ) DNA Hydrolysis (DNase plate) Bacillus subtilis (Straight line Inoculation) Orphan Escherichia coli Staphylococcus aureus B. subtilis Your orphan (PHAT) Lipid Hydrolysis (Spirit blue plate) (U) Urea broth Orphan (Straight line Inoculation) Escherichia coli Escherichia coli Proteus vulgaris Bacillus subtilis Orphan Your orphan (GEL) Gelatin Hydrolysis Stab (Blood) Blood Agar Plate Heavy inoculation 2 or 3 loops (Mini Q-streak’s) Escherichia coli Escherichia coli Bacillus subtilis Staphylococcus aureus Your orphan Your orphan Your IA will pick these up for incubation at room temperature. (OX) Oxidase (TSA Plate) (Mini Q Streak) (CAT) Catalase (NA Slant) Enterococcus faecalis Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Orphan Orphan 1  11/5/2024 (31) Phenylalanine Deaminase Test Deamination reactions remove the amine (N) groups, producing acidic compounds. Phenylalanase removes phenylalanine’s amine group to form phenylpyruvic acid and NH2+ Reagent: add 5 drops of 10% FeCl3 FeCl3 reacts with the phenylpyruvic acid, turning slant turns avocado green Identifies organisms that can Positive Negative synthesis the enzyme Phenylalanine Deaminase Positive control: Proteus vulgaris (21) Starch Hydrolysis Some bacteria hydrolyze starch by use of the enzyme amylase Amylase will break down starch (amylose: long chains of glucose) into individual glucose units Reagent: 4 – 5 drops of Gram’s iodine Iodine will complex with any remaining starch and turn blue/black Positive: A clear halo around colonies Negative: blue/black around colonies is negative for starch hydrolysis Positive control: Bacillus subtilis (26) Urea Hydrolysis Identifies organisms that can synthesize the enzyme Urease In the presence of urease, urea is broken down into ammonia and carbon dioxide. Ammonia increases the pH of the solution- ALKALINE environment! Positive control: Proteus vulgaris A pH indicator is phenol red If the pH of the media is Less than pH of 8.4, the indicator is yellow or orange Greater than pH of 8.4, the indicator is red Urease Urease Phenol red is fuchsia in the presence of Positive Negative ammonia 2  11/5/2024 (21) Gelatin Hydrolysis Identifies organisms that can synthesize and secrete the enzyme Gelatinase Gelatin is a protein of connective tissue and is also Gelatinase the solidifying agent in Jello Negative Organisms that can secrete the enzyme gelatinase will liquefy the medium. Organisms that are not able to synthesize gelatinase will not liquefy the nutrient gelatin agar, so the media will remain solid Gelatinase Incubated at room temp, test read cold Positive Positive control: Bacillus subtilis (21) DNA Hydrolysis Identifies organisms that can synthesize and secrete the DNase enzyme Deoxyribonuclease Positive (DNase) DNase catalyzes the DNase depolymerization Negative (breakdown) of DNA into nucleotides and will cause a clearing around the growth of DNase positive organisms Positive control: Staphylococcus aureus (21) Lipid Hydrolysis Test Determines whether a bacterium can synthesize and secrete the enzyme Lipase Lipase – Neutral triglyceride (fat) is hydrolyzed into fatty acids and glycerol – The release of fatty acids will lower the pH of the media – the pH indicator detects this ***Spirit blue agar plates*** – pH indicator Spirit Blue – It contains tributyrin (fat) – Positive result: Lavender color/dark blue halo around the colonies – Negative: light blue/whitish around the colonies Positive control: Bacillus subtilis Lipase Lipase Positive Negative 3  11/5/2024 (29) Catalase Production Test Catalase 2 H2O2 2 H2O + O2 (g) The enzyme catalase breaks down hydrogen peroxide (H2O2) into water and O2 – H2O2 is toxic to enzyme systems of microbes but is generally produced by aerobic and facultative microbes as a byproduct of aerobic metabolism. Catalase renders H2O2 harmless. Reagent: 3% H2O2 – 2H2O2 + catalase → O2 (g) + 2H2O + catalase Enzyme catalysts remain unchanged throughout the reaction. Add a 3-4 drops of hydrogen peroxide – Bubbling indicates the presence of catalase (positive Catalase Catalase result) Positive Negative Enterococcus faecalis is the negative control Staphylococcus aureus is the positive control (30) Oxidase Production Test Dropper This test is used to determine if cytochrome c oxidase is a Hole member of the electron transport chain of the organism. – Cytochrome c is a highly conserved protein across the spectrum of complex species, found in plants, animals, and many unicellular organisms. – The cytochrome c molecule allow one to study the evolutionary relationships of organisms. To open the oxidase reagent ….. – Squeeze the sides of the reagent tube to break the glass to active the reagent. You should hear glass Do not break! take this Add one drop of oxidase reagent (tetramethyl-p- top off phenylenediamine hydrochloride) to a colony, and one drop to quadrant one of the mini quadrant streak of each Squeeze organism on the plate. here, If cytochrome c oxidase is presence, then the oxidase Like a reagent will donate its electrons to cytochrome c and the glow stick reagent will turn purple/blue from clear. (30) Oxidase Production Results Once all the drops are applied – Watch for the Pseudomonas aeruginosa to change color. – Specifically watch the edges! Positive: Blue/Purple (Pseudomonas aeruginosa) Negative: Clear or No Reaction (Escherichia coli) TABLE OF RESULTS Result Interpretation Dark blue at the Cytochrome c same time as the oxidase is present positive control No color change Cytochrome c to blue same time oxidase is not as the positive present control 4  11/5/2024 (28) Nitrate Reduction Test Nitrate reduction tests for the presence of Nitrate Reductase – Nitrate (NO3) → Nitrite (NO2) Some organisms can further reduce nitrite to other reduced nitrogen compounds NH4 ammonium(-3) 2e- NO3 NO2 Nitrate (+5) Nitrite (+3) e- 2e- NO 1/2NO N2 Nitric oxide (+2) Nitrous oxide (+1) Dinitrogen (0) Nitrate Reductase Reduced Nitrogen Compounds (28) Nitrate Reduction Test NH4 ammonium(-3) Nitrate 2e- solution A+B NO3 NO2 only detects Nitrate (+5) nitrate reductase Nitrite (+3) e- 2e- NO2 NO 1/2NO N2 Nitric oxide (+2) Nitrous oxide (+1) Dinitrogen (0) Start with the E.coli (Positive Control) and the tube label “Control” tube. Add the two reagents are used to test for NO2 9 drops of Nitrate solution A (sulfanilic acid) 9 drops of Nitrate solution B (dimethyl- alpha- naphthalamine) – The E.coli will start off red, and may becomes clear, then add 5 drops of each reagent The addition of reagent should hold the red color in the positive control. The control tube will remain clear…. (28) Nitrate Reduction Test NH4 ammonium(-3) 2e- NO3 NO2 Nitrate (+5) nitrate reductase Nitrite (+3) e- 2e- NO 1/2NO N2 Nitric oxide (+2) Nitrous oxide (+1) Dinitrogen (0) If the media does not change color after adding the necessary amounts of reagents, one MUST confirm the negative result. Confirmation of a Negative To a negative result, add a pinch of zinc dust Zinc dust will reduce (NO3 → NO2 ) – The red color confirms the negative result – If media remains clear, this indicates the other reduced nitrogen compounds, such as NH4, NO, or N2 5  11/5/2024 (28) Nitrate Reduction Test Summary Determines whether an organism is capable of reducing nitrate (NO3-) to nitrite (NO2-) or other nitrogenous compounds (such as N2) via the enzyme Nitrate Reductase Part A: Identification of NO2 Two reagents are used to test for nitrite: Nine drops of Nitrate solution A (sulfanilic acid) and Nine drops of Nitrate solution B (dimethyl- alphanaphthalamine) The red color is positive for nitrate or nitrite Clear or opaque is a negative result. Add Nine more drops before confirming the negative result. Part B: Confirmation of a Negative result To a negative result, add a pinch of Zinc Dust – Zinc dust will act to reduce any remaining nitrate to nitrite. – This produces a red color, which confirms that the enzyme nitrate reductase was not present and is, therefore a negative result If the color stays clear, this indicates the further reduction of NO2 to NH4, NO, or N2, which confirmed the positive result. (Sup) Blood Agar Blood agar plates contain 5% sheep blood agar with TSA This media allows the identifying Hemolysis of RBCs Three types – Alpha Hemolysis Partial lyses of Red Blood Cells and will contain a greenish tint around the stab region. – Beta hemolysis It causes the complete lysis of the red cells in the media. The area around and under the colonies are lightened and transparent. – Gamma hemolysis No lysis if the RBCs are detected (Sup) Coagulase Test Pick one colony from your blood agar plate. S. aureus (Positive Control) Orphan E. coli (Negative Control) Get your 3 slides prepared. Your I.A. will add the 30ul of plasma to each circle. Using a clean toothpick, transfer a colony from the blood agar plate to the slide and mix with the plasma. Mix the sample for 30- 90 seconds. Read results after 2 minutes. Clumping S. aureus will produce the enzyme coagulase Enzyme will coat the bacteria cell with plasma proteins. No Clumping 6  11/5/2024 (70) Slide Agglutination Terms Antibody Immunoglobulin that interacts with a specific target molecule Antigen Any molecule that initiates an antibody-mediated response Antiserum Part of the blood that contains antibodies Agglutination Clumping of particles, due to cross-linking of cells to antibodies Serotyping The ability to distinguish a group of closely related microorganisms by a characteristic set of antigens This test is used in multiple applications to detect antibody/ antigen interactions: 1. Characterization of bacterial strains based on the immunologic reaction of cell surface structures. Flagellin protein (H antigen) Lipopolysaccharide ( O antigen) Capsule (K antigen) 2. Blood typing, A+,A-,B+,B-,O, O- 3. An allergic reaction type (70) Slide Agglutination Pick one colony from your blood agar plate. S. aureus (Positive Control) Orphan E. coli (Negative Control) Get your card stock or 3 slides prepped Place one drop of antiserum per test circle. Using a clean tooth pick, transfer a colony from the blood agar plate to the slide and mix with the antiserum. Mix the sample for 30- 90 seconds. Read results after 2 minutes. S. aureus will possess protein A antigen, which is the molecule that the antibodies will recognize and bind to. This will cause the agglutination reaction that can be seen. Lab Report Questions for Differential Test 2 (Lab report questions are due at the start of the next lab period) #21 (Extracellular Enzymatic Activities of Microorganisms) Questions 1 and 3 #26 (Urease Test) Questions 1 and 3 #28 (Nitrate Reduction Test) Questions 2, 3, and 4 #29 (Catalase Test) Questions 1 and 2 #30 (Oxidase Test) Questions 1 and 2 #31 (Utilization of Amino Acids) Question 3 7  11/5/2024 Clean up/ Waste Plates in the biohazard waste bags. Oxidase reagent tube in the biohazard waste bag Test tubes in the rack in the biohazard area. – Please remove tape from the test tubes – Please organize the tubes by type in the racks Gloves biohazard waste bags. Pipettes in the biohazard waste bags. 8