Week 10 and 11 Lab UT Culture Unknown(1) 2.pdf

Lab Week 10 and 11 Identification of an Unknown Bacteria of the Urinary Tract We gratefully acknowledge Mary K. Ortiz, Ph.D. Professor of Biological Sciences KBCC, who made many of these slides. Lab Goals To distinguish between E...

Lab Week 10 and 11 Identification of an Unknown Bacteria of the Urinary Tract We gratefully acknowledge Mary K. Ortiz, Ph.D. Professor of Biological Sciences KBCC, who made many of these slides. Lab Goals To distinguish between Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis and Enterococcus faecalis on the basis of metabolic and exoenzyme testing The tests used include: The Gram stain MacConkey Agar Eosin-Methylene Blue Agar Indole Production Phenylalanine Deamination Nitrate Reduction Catalase Lab Format For each test this slide set provides Background Biochemistry Example Observations Expected Results (Controls) To write your report (5% of your lab grade) Review the lab manual and this slide deck (click on videos and links) You will receive a set of observations to interpret into results Compare these results with those of the controls and complete the table Write your report according to the template provided Filling Out the Table Your observations (what you would see) will be provided For example: Gram Stain à Observation: Purple, round, clusters Your results are the interpretation of those observations Example: Result: Gram-positive staphylococci The explanation of observations must include an explanation of the biochemical/enzymatic reaction that causes that result in your own words Example Explanation: The bacteria have many layers of peptidoglycan in their cell wall that retain the crystal violet dye. The organism/organisms indicated should include ALL species (in this case S. epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris) that would have that result. Organisms Indicated: Staphylococcus epidermidis AND Enterococcus faecalis The Gram Stain Gram Positive and Gram Negative Cell Walls Gram Staining Primary Stain: Crystal Violet (+ charge) Iodine: Mordant Binds to dye, making it too large to pass through thick peptidoglycan Alcohol: Decolorizer Rinses away CV from Gram Negative Cells Too much will cause the CV to leave even Gram Positive Secondary Stain: Safranin (+ charge) Remember, basic (+) dyes stain within the cell! Gram Stain: Gram-positive streptobacilli Photo by Joe Rubin Gram Stain: Gram-negative individual bacilli Gram Stain: Gram-positive staphylococci Results Species Results Gram-negative, bacilli, Escherichia coli random/individual Gram-negative, bacilli, individual, Proteus vulgaris pairs or short chains Gram-negative, bacilli, individual Pseudomonas aeruginosa or pairs Staphylococcus epidermidis Gram-positive staphylococci Gram-positive Enterococcus faecalis Pairs, short chains, clumps  MacConkey Selective: Only Gram negatives will grow Bile salts and Crystal Violet inhibit Gram positive growth Differential: Lactose fermenting bacteria will be pink Lactose: carbohydrate broken down for energy (fermentation) Neutral Red: A pH indicator Lactose fermentation produces acid, causing the neutral red to turn colonies pink/purple Grace Axler-DiPerte Ph.D. MacConkey agar Species Observation Interpretation Gram negative, lactose E. coli Pink growth fermenter Gram negative, non- P. vulgaris Colorless growth lactose fermenter Gram negative, non- P. aeruginosa Colorless growth lactose fermenter S. epidermidis No Growth Gram positive organism Gram positive organism E. faecalis No Growth  Eosin Methylene-Blue Agar Selective: Eosin and Methylene Blue are dyes which inhibit the growth of Gram positive organisms Differential: Lactose: carbohydrate broken down to make energy Acid is produced lowering the pH and causes the dues to precipitate Lactose positive colonies will be metallic green Joe Rubin Grace Axler-DiPerte Ph.D. (CC BY-NC-SA 2.0) Eosin-Methylene Blue Agar Species Observation Interpretation Gram negative, lactose E. coli Metallic green growth fermenter Gram negative, non- P. vulgaris Colorless growth lactose fermenter Colorless growth Gram negative, non- P. aeruginosa lactose fermenter S. epidermidis No Growth Gram positive organism Gram positive organism E. faecalis No Growth Indole Production/Tryptophan Hydrolysis test  Tryptophan Hydrolysis (Indole Production) Background and Biochemistry Tryptophan Hydrolysis is a deamination reaction Tryptophanase (enzyme) hydrolyzes tryptophan (aa) to produce indole (not used by the cell), pyruvic acid and ammonia (NH3) (both used by the cell) We detect indole using Kovac’s reagent, which reacts with indole to give a red compound We use this test to distinguish E. coli from E. aerogenes Mary K. Ortiz, Ph.D. Tryptophan Hydrolysis (Indole Production) Biochemistry – continued (from lab manual page 152) tryptophanase Tryptophan Indole + Pyruvic acid + NH3 | |ß add Kovac’s reagent to detect indole | \/ No red compound Red compound (+ reaction if indole present) (- reaction) Forms a red ring at the top of the medium Mary K. Ortiz, Ph.D. Tryptophan Hydrolysis (Indole Production) Procedure (page 153 Lab Manual) In this exercise five bacteria are used: Escherichia coli (EC) Proteus vulgaris (PV) Pseudomonas aeruginosa (PA) Staphylococcus epidermidis (SE) Enterococcus faecalis (EF) Each bacterium is inoculated into 1 tube of tryptone broth Contains a high amount of tryptophan The tubes are incubated at 37oC (98.6oF) for 24-48 hours After incubation, add 10 drops of Kovac’s reagent and look for color change If tryptophanase present, then red ring forms at top of broth (+ result) If no red ring, no tryptophanase present (- result) Tryptophan Hydrolysis (Indole Production) + - Ex. 24 Part I – Tryptophan Hydrolysis (Indole Production) Results Interpretation Species Appearance (Result) E. coli Red ring + P. vulgaris Red ring + P. aeruginosa No red ring - S. epidermidis No red ring - E. faecalis No red ring - Ex. 24 Part II – Phenylalanine Deaminase Background and Biochemistry Phenylalanine (aa), with the enzyme phenylalanine deaminase (PAD) present gives phenylpyruvic acid and NH3 Some microbes use organic acids in reactions We detect phenylpyruvic acid with ferric chloride (FeCl3) FeCl3 reacts with phenylpyruvic acids to give a green color Bacteria of the genus Proteus are all positive for PAD, so this is a useful test to test for this group of bacteria. Mary K. Ortiz, Ph.D. Ex. 24 Part II Phenylalanine Deaminase- Biochemistry (from page 154 Lab Manual) PAD Phenylalanine Phenylpyruvic acid + NH3 7.0= pink/magenta If urease is present à NH3 is made à pH increases à medium turns hot pink/magenta (+ test result) If the medium remains orange, urease is absent (- test result) We use this test to test for the genus Proteus, a cause of UTIs Mary K. Ortiz, Ph.D. Ex. 24 Part IV – Urea Hydrolysis Biochemistry urease Urea CO2 + H2O + NH3 (orange | Broth) | \/ Hot pink/magenta (+ test result) (Remaining orange is a – test result) Mary K. Ortiz, Ph.D. Ex. 24 Part IV – Urea Hydrolysis: Procedure In this exercise five bacteria are used: E. coli (EC) Proteus vulgaris (PV) Pseudomonas aeruginosa (PA) Staphylococcus epidermidis (SE) Enterococcus faecalis (EF) Each bacterium is inoculated into 1 tube of Urea broth The tubes are incubated at 37oC (98.6oF) for 5 days In a positive test the broth turns hot pink/magenta In a negative test, the broth remains orange Ex. 24 Part IV – Urea Hydrolysis - Results https://www.proprofs.com/flashcards/upload-images/large/jlalpa/86848.jpg Ex. 24 Part IV – Urea Hydrolysis - Results - continued Bacterium Appearance Interpretation (Result) E. coli orange - P. vulgaris Hot pink/magenta + P. aeruginosa orange - S. epidermidis Hot pink/magenta + E. faecalis orange - Ex. 25 Part VI – Oxidase Activity Background and Biochemistry Cytochrome c oxidase has a main role in aerobic respirarion It helps transport electrons from cytochrome c to O2 (final e- acceptor) Oxidase reagent is used to test for oxidase activity Oxidase reagent is poured onto the solid medium If a microbe produces oxidase, the reagent turns from pink to black Mary K. Ortiz, Ph.D. Ex. 25 Part VI – Oxidase Activity Biochemistry – continued Cytochrome oxidase c Cytochrome c O2 (oxidized cytochrome c) | |ß Oxidase reagent (pink) | \/ Black color (+ test result) If the color remains pink, it is a – test result Mary K. Ortiz, Ph.D.  Ex. 25 Part VI – Oxidase Activity: Procedure In this exercise five bacteria are used: E. coli (EC) Proteus vulgaris (PV) Pseudomonas aeruginosa (PA) Staphylococcus epidermidis (SE) Enterococcus faecalis (EF) Each bacterium is inoculated onto a TSA (Tryptic Soy Agar) Plate The tubes are incubated at 37oC (98.6oF) for 24-48 hours Add the oxidase reagent to the growth on the plate or on a swab of the sample If the plate turns blue-black, the microbe produces oxidase (+ test Result) If the plate/swab remains pink, the microbe does not produce oxidase (- test result) Ex. 24 part VI – Oxidase Activity: Results This shows the pos as blue. CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=1968827 Mary K. Ortiz, Ph.D. Ex. 24 Part VI – Oxidase Activity Results - continued Bacterium Appearance Interpretation (Result) E. coli No black - P. vulgaris No black - P. aeruginosa Black colonies + S. epidermidis No black - E. faecalis No black - Ex. 25 Part VII – Nitrate Reduction: Background and Biochemistry Some microbes use inorganic nitrate (NO3) as a final e- acceptor When NO3 is used, it is reduced to nitrite (NO2) by nitratase (enzyme) Some microbes further reduce NO2 to nitrogen gas (N2) by nitritase (enzyme) in a reaction called denitrification This video does not show total denitrification Note the difference in spelling between nitratase and nitritase as they are two separate enzymes Mary K. Ortiz, Ph.D. Ex. 25 Part VII – Nitrate Reduction Background and Biochemistry – continued We use NO3 broth to test for NO3 reduction There are 3 possible results: Nitratase only is produced Nitratase and Nitritase are produced (i.e. both enzymes are produced) Neither enzyme is produced (i.e no enzyme is produced) Nitrate reagents are added to the incubated NO3 broth tubes in this test If red color is observed, then nitratase reduced NO3 to NO2 If no red is seen, then both enzymes or no enzymes were produced To decide, zinc (Zn) dust is added to the tube as a reducing agent. If red color appears, then Zn reduced NO3 to NO2 and the microbe produced no enzymes If no red color appears, then N2 is present and both enzymes were produced by the microbe Mary K. Ortiz, Ph.D.  Ex. 25 Part VII – Nitrate Reduction – Biochemistry - continued nitratase nitritase NO3 NO2 N2 | |ß Nitrate reagents \|/ -------------------------------------------> | Red (NO2 present, \|/ nitratase present) No red (NO3 or N2) present | __________________________ | ßZn added \|/ \|/ Red color (NO3 present; no enz) No red color (N2 present; both enz) Ex. 25 Part VII – Nitrate Reduction Procedure In this exercise five bacteria are used: E. coli (EC) Proteus vulgaris (PV) Pseudomonas aeruginosa (PA) Staphylococcus epidermidis (SE) Enterococcus faecalis (EF) Each bacterium is inoculated into a Nitrate broth tube The tubes are incubated at 37oC (98.6oF) for 24-48 hours Add the Nitrate reagents to the tubes and observe: If red color is observed, record results (only nitratase present) If no red color is observed add Zn If red color is observed, no enzymes present If no red color is observed, both enzymes present (ntratase and nitritase present) Ex. 25 Part VII – Nitrate Reduction From http://1.bp.blogspot.com/-jp9k8ngWCCY/U35YkTTGgwI/AAAAAAAAAJo/SUBZ7igLLtM/s1600/IMG_0872.jpg Results Ex. 25 Part VII – Nitrate Reduction – Results Bacterium Appearance Interpretation (Result) Red after Nitrate reagents E. coli + for Nitratase only added Red after Nitrate reagents + for Nitratase only P. vulgaris added No red after Nitrate + for both enzymes P. aeruginosa reagents added and no (Nitratase and Nitritase) red after Zn added Weak red color after Weakly + for Nitratase S. epidermidis Nitrate reagents added only No red after Nitrate - for both enzymes E. faecalis reagents added and red (Nitratase and Nitritase) after Zn added Catalase Testing Catalase H202 is a toxic by-product of aerobic respiration Bacteria enzymatically break down hydrogen peroxide to non-toxic water and oxygen gas Oxygen gas is observed as bubbles after dropping hydrogen peroxide onto growth on the plate Producing catalase means the bacteria is capable of detoxifying by-products of aerobic respiration (for example aerobes and facultative anaerobes). From Brancaccio-Taras Laboratory manual Catalase Simulation Positive Result A negative result would not have bubbles. By The original uploader was Philippinjl at French Wikipedia. - Transferred from fr.wikipedia to Commons by Bloody- libu using CommonsHelper., CC BY-SA 2.0 fr, https://commons.wikimedia.org/w/index.php?curid=17397920 Results Species Observation Results Positive for E. coli Bubbles catalase Positive for P. vulgaris Bubbles catalase Positive for P. aeruginosa Bubbles catalase Positive for S. epidermidis Bubbles catalase Negative for E. faecalis No bubbles catalase