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Review Labs 7-11, Including Take Home Lab Review Growth Patterns, Gram Stain Reactions, and Biochemical Reactions (Including API 20E) for: a. Pseudomonas aeruginosa i. Growth Patterns: Large, irregularly shaped, metallic sheen, produce pyocyanin (blue pigment), grape-like odor. ii. Gram Stain Reaction: Nonfermentative Gram-negative bacilli. Grow on MAC agar, oxidize glucose, reduce nitrate, and are oxidase positive. iii. Biochemical Reactions: They are also pyocyanin positive. Also have an alkaline/no change TSI reaction and grows when incubated at 42 degrees. b. Klebsiella pneumoniae i. Growth Patterns: Moist, grey mucoid colonies on MAC and EMB with a jelly-like appearance. Polysaccharide capsule. ii. Gram Stain Reaction: Gram-negative rod. String is formed when a colony is lifted with an inoculating loop. iii. Biochemical Reactions: Urease (+), Citrate (+), Lactose (+), Lysine decarboxylase (+), H2S (-), ONPG (+), VP (+), Motility (-). A/A in KIA test. c. Pasteurella multocida i. Growth Patterns: Appear as oval shaped rods, grow on BAP and CHOC, No growth on MAC, glucose fermented ii. Gram Stain Reaction: Gram-negative pleomorphic coccobacilli. iii. Biochemical Reactions: Oxidase (+), Catalase (+), Indole (+), Urease (-), Penicillin (S). Brownish halo, non-hemolytic, mucoid colonies with a musty smell. d. Campylobacter i. Growth Patterns: Uses special Campy blood agar ii. Gram Stain Reaction: Curved, gram (-) rods, non-spore forming, non-fermentative, Sshape or seagull wing appearance and may form spirals. iii. Biochemical Reaction: Darting motility, Oxidase (+), Catalase (+), Nitrate (+), H2S (-). Stain faintly with gram stain. Grow on CHOC, BAP, MAC. Grows at 42 degrees. Cephalothin (R). Difference Between MIC, MBC, KB, and E-Test for Susceptibility Testing. Be Able to Assign an MIC or MBC to a Presented Tube Dilution or Plated Method a. MIC (Minimum Inhibitory Concentration) i. Lowest drug concentration that inhibits bacterial growth. First tube thats not turbid. b. MBC (Minimum Bactericidal Concentration) i. Lowest drug concentration that kills 99.9% of the inoculum. Growth on plate. c. Kirby-Bauer Test i. Uses one large Mueller-Hinton Plate, disks are placed around the plate and shows zones of inhibition around disks that are susceptible to the antibiotic disk and no zone around disks that are resistant. d. E-Test i. Similar to the disk diffusion test and produces MIC results. Agar plates are inoculated in the same manner as those for disk diffusion tests. A plastic strip containing a gradient of the antimicrobial agent is then placed on the inoculated surface. The plates are incubated and then examined for an elliptical zone of inhibition. ii. Assign MIC based on picture Understand Concept of Antibiotic Infused Media (Screen Plates) and their Utilization in the Laboratory Including the Ability to Read the Plate as Growth/No Growth and What That Means for the Tested Bacteria (We Used MH w/ Oxacillin and BHI w/ Vancomycin) a. Media is infused with certain antibiotics. R = growth, S = no growth. McFarland Standard and How Its Used in the Lab a. Number of bacteria needed to be standardized. If theres too few, its false susceptibility. If theres too many, its false resistance. Turbidity standards used as references when preparing suspensions of microorganisms. Standards are numbered 0.5, 1, 2, 3, etc. Beta-Lactamase and How Its Detected in Lab a. Beta-Lactamase: Enzyme produced by certain bacteria to cleave the B-lactam ring in certain antibiotics. It can be detected by a Nitrocefin disk test, which is a chromogenic cephalosporin.

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Its yellow when the b-lactam ring is intact and it turns red when the B-lactam ring is hydrolyzed by B-lactamase. b. Nitrocefin Test is the most sensitive method, appropriate for testing: Haemophilus, N. gonorrhoeae, Staphylococci, M. catarrhalis, Enterococci, and Bacteroides species. How to Set Up Tube Dilution Test and KB Susceptibility Test a. Tube Dilution Test: Pipette 0.5 mL of MH broth in tubes 2-10, Pipette 0.5 mL antibiotic solution to tubes 1-2, Transfer 0.5 mL from tube 2 to tube 3 and continue with that pattern to tube 9. Discard 0.5 mL from tube 9. Add 0.5 mL of broth suspension (Same one used for KB test) to each tube. Incubate at 35 degrees for 24-48 hours. Methods of Isolating Anaerobic Bacteria a. Anaerobic Jars, Anaerobic Bags, Anaerobic Chamber Aerotolerance Test a. Determines if an isolate can grow aerobically, anaerobically, or both. The test is performed by inoculating a single colony onto anaBAP and CHOC agar. Aerobic organisms only grow on the CHOC agar, but most anaerobic bacteria grow only on the anaBAP. Some anaerobic bacteria are aerotolerance and may grow slightly on CHOC agar. They produce much heavier growth on the anaBAP. Facultative anaerobes grow on the CHOC agar and the anaBAP. b. If the specimen is received and it is not in liquid form, it is required to suspend it in a liquid before performing the test. Know Anaerobic Flow Chart How to Detect Mycoplasma and Ureaplasma a. Mycoplasma: Diene stain, Cannot be gram stained due to lack of cell walls, have to use serology b. Ureaplasma: U-9B broth media used, Cannot be gram stained due to lack of cell walls, have to use serology Diseases Caused by Chlamydia and How Its Isolated a. C. trachomatis: Pneumonia of newborns, nongonococcal urethritis in men, PID, salpingitis, cervicitis, urethritis, endometritis, conjunctivitis (adult and newborn). Isolated by media that requires epithelial cells because they are intracellular parasites. Serology required and reference laboratories. Direct Detection Methods: DFA, EIA, DNA probes, Giemsa stain for neonatal conjunctivitis, Nucleic acid amplification tests. b. C. pneumoniae: Pneumonia and other respiratory disease. Culture and Serology. c. C. psittaci: Ornithosis and psittacosis/parrot fever. Serology. AFB Staining Process Know (+)/(-) Result Appearance, Fluorescent Stain a. Ziehl-Neelsen stain b. Kinyoun stain c. Auramaine stain d. (+) Result: All Mycobacteria can be visualized using acid fast staining. Mycolic acid in cell wall allows acid fast staining to work on certain bacteria. Acid-fast bacteria will appear red. e. (-) Result: Non-acid fast bacteria will appear blue. Correlating Gram Stain with Culture Result a. Gram (+) bacteria are usually larger colonies and they grow faster than Gram (-) bacteria. Aerobic bacteria grow faster than anaerobic bacteria. QC Purpose, Example in Lab a. Purpose for QC is to ensure that your test results are accurate. b. Example: Running controls will ensure that your test results are correct and accurate. Normal Flora 6 body sites (Or mention if the site should be sterile) a. Skin: Propionibacterium acnes, Peptostreptococcus b. Respiratory Tract: Prevotella, Porphyromonas, Fusobacterium c. GI Tract: Bacteroides, Bifidobacterium, Clostridium, Eubacterium d. Genitourinary Tract: Fusobacterium, Provotella, Bacteroides, Lactobacillus, Anaerobic cocci e. Blood: Should be sterile in healthy individuals Schlichter test a. This test is used to determine the maximum dilution of the serum of body fluid. This is done when the patient has a serious infection. Peak and trough specimens are usually tested. Used to determine the antibacterial level of the serum or CSF of patients who are receiving

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antibiotic therapy. The test uses eight serial dilutions of the patients serum to determine what dilution is bactericidal to the infecting organism. Pasturella multocida will probably be one of the organisms to ID 2 Sputum, 2 Wound Slides Count the number of epithelial cells, neutrophils, and bacteria. Enumerate with Few, Moderate, Many. Given 3 anaerobes Gram stain, Aerotolerance plates, Disk test results (if applicable), Biochemical tests. Describe growth and identify organism. Tables will be provided. May need to perform an API strip test