LABORATORY #10 Non-Fermentative Gram-Negative Bacilli Laboratory #10 Non-Fermentative Gram-Negative Bacilli Skills= 7 points Objectives: At the completion of this lab, the student should be able to: 1. Select the appropriate culture media to optimize the laboratory recovery of nonfermentative gram- negative bacilli. 2. Demonstrate the following reactions of Pseudomonas aeruginosa and Escherichia coli: a. Growth characteristics on BAP, MacConkey and CNA agar b. Growth in TSI c. Oxidase reaction d. Pigment production e. Oxidative vs. fermentative capabilities Materials: Miscellaneous media (BAP, CNA, MacConkey) cultures of: Escherichia coli Pseudomonas aeruginosa 2 Triple Sugar iron (TSI) slants Oxidase test materials 4 O-F glucose tubes Mineral oil References: 1. Mahon and Manuselis, Textbook of Diagnostic Microbiology, Third Edition, Chapter 21 Overview: The gram-negative, nonfermentative bacilli differ from the Enterobacteriaceae, Vibrio, Aeromonas, and other aerobic, facultative gram-negative rods by not fermenting carbohydrates, and thereby producing no change on triple sugar iron (TSI) slants. Most nonfermentative bacilli (NFB) are opportunistic pathogens that primarily attack the debilitated host. Although these bacteria account for approximately 15% of the isolates seen in the clinical lab- by far, the most common NFB is Pseudomonas aeruginosa. Pseudomonas aeruginosa has been implicated in wound and hospital-acquired infections, cystic fibrosis patient respiratory infections, septicemia, and osteomyelitis. Procedures: 1. Determine amount of growth, colony characteristics, and hemolysis for Escherichia coli and Pseudomonas aeruginosa on BAP, MacConkey, and CNA agars and record results on MLAB 2534 – Laboratory 10 – Page 1 LABORATORY #10 Non-Fermentative Gram-Negative Bacilli report form. 2. Inoculate one TSI with Pseudomonas aeruginosa and one TSI with Escherichia coli and incubate at 35° C for 24 hours. Read and record results according to established protocol. 3. Perform an oxidase test on Escherichia coli and Pseudomonas aeruginosa and record results on report form, according to established protocol. 4. Pigment Production Discussion: Three types of pigment, pyocyanin, pyoverdin (also called fluorescein), and pyorubin, may be produced by various strains of Pseudomonas. Pyocyanin is bluish-green, non-fluorescing, water and chloroform soluble pigment which is a phenazone derivative. It constitutes a reversible oxidation-reduction system and acts as a respiratory catalyst, enabling the organism to use oxygen after the cytochrome system has been poisoned by cyanide. Pyoverdin (fluorescin) is a greenish-yellow, fluorescent, water soluble, chloroform-insoluble pigment which is formed in the presence of phosphate. Pyorubin is a pink soluble pigment. Procedure: A. Observe any pigment produced by Pseudomonas aeruginosa and Escherichia coli on any of the agars and record results by giving the color AND the name of the pigment. Pseudomonas species P. aeruginosa P. fluorescens P. putida P. cepacia P. stutzeri P. maltophilia P. pseudomallei Pyocyanin Pyoverdin (fluorescin) Pyorubin + - + + + - -(+) - 5. Oxidation-Fermentation (OF) Test Discussion: Bacteria produce acid from carbohydrates by two methods. One is an anaerobic process called fermentation; the second, designated oxidation, is an aerobic process. Since gram negative bacteria which attack carbohydrates usually do so exclusively by either fermentation or oxidation, the determination of the type of carbohydrate metabolism carried out by an organism is of taxonomic significance. Enteric fermentation broth carbohydrate media will detect acid production by fermentative organisms. Oxidative organisms do not usually produce a detectable amount of acid in enteric fermentation broth media since the MLAB 2534 – Laboratory 10 – Page 2 LABORATORY #10 Non-Fermentative Gram-Negative Bacilli acidity produced by oxidation is less than by fermentation and because sufficient alkaline products are frequently produced from the peptone in the medium to neutralize the acid produced by oxidative metabolism. Triple sugar iron agar (TSI) can be used to differentiate fermentative from oxidative bacteria. A TSI reaction of acid butt with or without an acid slant is indicative of a fermenter. No change of the butt and slant of TSI or slight alkalinization of the slant is indicative of an oxidizer or nonutilizer of sugars. Occasionally, an oxidizer will show slight acidity on the slant of TSI. In cases where the TSI reaction is doubtful, an oxidation-fermentation test can be carried out using OF medium with 1% glucose (dextrose) as described. An OF medium should be used to determine the pattern of carbohydrate metabolism of oxidative bacteria. OF base medium contains less peptone than do the fermentation bases and contains 0.3% agar. The agar prevents convection currents and allows the acid produced to remain concentrated in the medium adjacent to where it is produced and not be diluted by mixing throughout the medium. Procedure: 1. Two tubes are required for the OF test. Inoculate two, each with P. aeruginosa and E. coli by using a straight inoculating needle. Transfer a generous amount of the test organism to the OF medium by stabbing four (4) times approximately 1/3 the depth of the medium. 2. Overlay one inoculated tube from each pair with 1 mL of mineral oil. (The tubes overlayed with oil are called "closed" tubes). This overlay prevents the diffusion of oxygen into the medium and creates an anaerobic condition in the tube. 3. Incubate with caps loose at 35° C for 24 hours. 4. Following incubation, interpret the results based on the interpretive table below. 5. On the report form, record as Oxidative (O), fermentative (F), or negative (N). Negative indicates no fermentative or oxidative metabolism of the carbohydrate in the media. Reading and Interpretation: Open Tube Closed Tube Method of Glucose Utilization Yellow (A) Yellow (A) Fermentative Yellow (A) Blue-Green (-) Oxidative Blue-Green (-) Blue-Green (-) Non-utilizer or possible no growth MLAB 2534 – Laboratory 10 – Page 3 LABORATORY #10 Non-Fermentative Gram-Negative Bacilli MLAB 2534 – Laboratory 10 – Page 1 LABORATORY #10 Non-Fermentative Gram-Negative Bacilli NAME ___________________________________ DATE __________________________________ Laboratory #10: Non-Fermentative Gram-Negative Bacilli Report Form Points: 7 Organism BAP * CNA * Mac * TSI (A/K/G/H2S) Oxidase (P/N) Pigment Oxidation/Fermentation ( F/ O/ N) E. coli P. aeruginosa * Include amount of growth, colony morphology, and hemolysis. If no growth present, notate it. MLAB 2534 – Laboratory 10 – Page 1 LABORATORY #10 Non-Fermentative Gram-Negative Bacilli MLAB 2534 – Laboratory 10 – Page 1 LABORATORY #10 Non-Fermentative Gram-Negative Bacilli Name:_______________ Date:_______________ Lab #10: Non-Fermentative Gram-Negative Bacilli Study Questions Points= 12 1. Define “oxidation”. (1 pt.) 2. Explain how oxidation differs from fermentation. (1 pt.) 3. Explain the use of OF media in differentiating between oxidative and fermentative organisms. (1 pt.) 4. Why is it necessary to keep the caps loose on OF media after inoculation? (1 pt.) 5. How do the non-fermentative gram-negative bacilli different from the Enterobacteriaceae with respect to oxidase activity? (1 pt.) 6. Which non-fermentative gram-negative bacillus is most often implicated in infections? List at least three (3) common sites of infection for this organism. (2 pts.) 7. List as least four (4) characteristics of Pseudomonas aeruginosa. (2 pts.) MLAB 2534 – Laboratory 10 – Page 1 LABORATORY #10 Non-Fermentative Gram-Negative Bacilli 8. Nonfermenting gram-negative rods will typically produce the following reactions on KIA or TSI: (1 pt.) a. Acid/Acid (A/A) b. Acid/alkaline(A/Alk) c. Alkaline/acid (Alk/A) d. Alkaline/alkaline (Alk/Alk) 9. An organism producing a yellow color in the open tube and a blue-green color in the closed tube of O-F glucose medium is said to: (1 pt.) a. Ferment glucose b. Oxidize glucose c. Not utilize the carbohydrate d. Be nonviable 10. Quality control on O-F glucose tubes with ATCC strains of organisms gave the following reactions: (1 pt.) E. coli Ps. aeruginosa Open tube Yellow Yellow Closed tube Yellow Yellow The results are: a. Acceptable b. Unacceptable; they are consistent with mineral oil layered onto the open and closed tubes c. Unacceptable; they are consistent with mineral oil not layered onto the closed tubes d. Unacceptable; they are consistent with mineral oil layered onto the open MLAB 2534 – Laboratory 10 – Page 2