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Lab Exercise 19 Gram Positive Cocci 1 Lab Exercise 19 Gram Positive Cocci Staphylococci © Gloria Delisle, author. Licensed for use, ASM MicrobeLibrary (linked to http://www.microbelibrary.org) Objectives: 1. Perform, interpret and define the relevance of the catalase test. 2. Perform, interpret and define the relevance of the coagulase test. 3. Perform, interpret & define the relevance of the test for hemolysis on BAP. 4. Describe the Gram stain & arrangement of major GPC families. 5. Determine the identification of an unknown GPC organism using the above tests. 6. Relate the medical significance of each of the GPC covered in the lab. Medically significant Gram Positive cocci are represented by two main families: Micrococcaceae ( including the genera Staphylococcus and Micrococcus) and Streptococcaceae (including the genera Streptococcus and Enterococcus). I. Micrococcaceae - Catalase +, Gram positive cocci in clusters A. Genus Micrococcus – These bacteria are rarely associated with disease and are common environmental contaminants. They Gram stain as GPC in tetrads and produce yellow (Micrococcus luteus) or rose (M. roseus) colored pigments on enriched media. B. Genus Staphylococci - Salt tolerant GPC 1. Staph. aureus - These are pathogenic bacteria causing wound infections, abscesses, carbuncles, bacteremia, septicemia, osteomyelitis. Associated with purulent discharges and capable of producing a wide range of exotoxins (including hemolysins and DNAse), characterized as highly invasive. Causes food poisoning by the production of a heat resistant toxin. Important in nosocomial infections especially MRSA (Methicillin or Multiple Resistant Staph. aureus). Nasal carriers are important. 2. Staph. epidermidis - Normal skin flora, nonhemolytic, coagulase negative. Opportunistic pathogen isolated from catheters and I.V. lines and associated with transplant and immunosuppressed patients. 3. Staph hemolyticus - Normal skin inhabitant, beta hemolytic but coagulase negative. Opportunistic pathogen associated with UTI's. Lab Exercise 19 Gram Positive Cocci 2 II. Streptococcaceae - GPC in chain and pairs, easily over decolorized. This family produces a large number of exotoxins including hemolysins, erythrogenic toxins, nephrotoxins, and cardiohepatic toxins. Pathogenesis depends on species, strain, portal of entry and immune response. The Streptococcaceae are fastidious requiring blood agar for growth and producing typical characteristics as the blood cells are destroyed (hemolysis) for nutrients. A. Streptococci - chains and pairs, some encapsulated, bile esculin and salt negative 1. Strep. pyogenes - Group A strep, beta hemolytic, causing strep throat, scarlet fever, peurperal fever (post natal sepsis), skin infections such as impetigo, and pneumonia. Post infection sequela such as glomerulonephritis and rheumatic fever represent serious syndromes if infections are not treated immediately. Streptococci © Gloria Delisle, author. Licensed for use, ASM MicrobeLibrary (linked to http://www.microbelibrary.org) 2. Strep. agalactiae - Group B beta hemolytic strep causing neonatal meningitis thought to be associated with asympotomatic vaginal carriers,. Recently reported in AIDS patients. 3. Strep. pneumoniae -Alpha hemolytic, mucoid, and lancet- shaped. Virulent strains are encapsulated. Causes classis pneumonia, ear and eye infections. 4. Alpha strep - variety of nonpathogenic normal flora found on the skin and in the mouth. Occasionally associated with bacterial endocarditis. Many of these alpha strep are found on the respiratory tract as normal flora. Strep mutans is one of these strep associated with dental caries. Strep. sanguinis and Strep. parasanguinis are normal oral flora. B. Enterococci - Salt tolerant bile esculin positive strep. Normal flora of the G.I. tract. Opportunistic pathogens infecting decubiti (bedsores), causing UTI'S and associated with IV contamination. The enterococci are medically significant due to growing antibiotic resistance, they are referred to as VRE (Vancomycin Resistant Enterococci). Many species make up this group of strep including Enterococcus faecalis and Enterococcus faecium. More information can be found at http://www.cdc.gov/ncidod/eid/vol4no2/adobe/huy.pdf Lab Exercise 19 Gram Positive Cocci 3 The table below represents a simple differentiation between these genera of bacteria. Refer to the Atlas for sample test results. Family Micrococcaceae Streptococcaceae Test results Staphylococcus Micrococcus Streptococcus Enterococcus Arrangement in broth grape-like clusters tetrads sarcina chains (some pairs) paired (occasional chains) hemolysis on Blood agar Staph. epidermidis negative alpha, beta, gamma alpha or gamma Staph. aureus + growth on nutrient agar good good poor or none good Catalase positive positive negative negative MSA - Mannitol Salt Agar NaCl tolerance growth Staph. epidermidis negative negative growth negative negative negative Staph. aureus Coagulase + Staph. epidermidis Staph. aureus Materials: Hydrogen peroxide dropper slide Coagulase tube MSA plate Blood Agar plate + Stock cultures: Labeled 1-4 Staphylococcus aureus Staphylococcus epidermidis Micrococcus luteus Streptococcus sanguinis or Strep. parasanguinis Lab Exercise 19 Gram Positive Cocci 4 Procedure: 1. Gram stain each of the stock cultures and record the results. 2. Divide a Blood Agar plate into 4 sections and label them. Inoculate each section with a streak from each stock culture. 3. Perform a catalase test as described in the Atlas, by transferring a loopful of bacteria from each stock culture to a glass slide. Using a dropper apply a drop of hydrogen peroxide to each bacterial smear and record the results. Bubbling = positive 4. Divide an MSA plate into quadrants and inoculate it in the same fashion that the BAP was inoculated. 5. Perform a coagulase test, using the pre-made tubes and inoculating a tube with each of the stock cultures. Cover the tube with parafilm and incubate for 24-48 hours. 6. Incubate the BAP plate, MSA plate and coagulase at 37C. During the next lab period read and record the results. 7. Determine from the identification chart on page 1 of this lab, the identity of each stock culture. Lab Exercise 19 Gram Positive Cocci Lab Report Exercise 19 Bacterial Identification of Gram Positive Cocci Name____________________ 1. Use your book and the atlas as a reference. Define the following terms and describe a positive test. beta hemolysis - alpha hemolysis - gamma hemolysis - 2. Describe a positive coagulase test and identify the species of organism that would produce a positive test. 3. What advantage might coagulase give to a pathogen as it invades the human body? 5 Lab Exercise 19 Gram Positive Cocci 6 4. Fill in the chart below with your results and determine the identity of each of your stock cultures as a lab team. stock # Gram stain hemolysis - BAP MSA plate catalase test 1 2 3 4 Last Updated 6/30/2017 ©Janet Fulks coagulase Identification