Identification of Gram Positive Cocci Staphylococcus

Introduction The genus Staphylococcus contains both pathogenic and non-pathogenic organisms. They do not produce endospores but are highly resistant to drying, especially when associated with organic matter such as blood, pus, and other tissue fluids. Most staphylococci are found routinely on the surface of the skin. Breaks in skin and mucous membranes allow entrance of these organisms into the body where they may cause disease. The three major species include Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The latter two are rarely implicated in disease, but have been isolated in cases of endocarditis and urinary tract infections under certain circumstances. Staph. aureus is considered the pathogenic strain, causing abscesses, boils, carbuncles, acne and impetigo. Less commonly, pneumonia, osteomyelitis, endocarditis, cystitis, pyelonephritis, and food poisoning have been attributed to this organism. These three strains of staphylococci can be distinguished from each other by a number of biochemical tests. Principle The identification of organisms is based on cellular, cultural and biochemical characteristics. All species of Staphylococcus are Gram positive cocci. On nutrient agar they tend to be white, circular, entire, convex colonies. On blood agar Staphylococcus aureus may show hemolysis of the agar in the area around the colony. Additional biochemical tests that are useful in separating the Staphylococcus species include catalase, coagulase, growth and fermentation of mannitol salt, and resistance or susceptibility to the antibiotic novobiocin. The catalase test determines if the organism produces the enzyme catalase that breaks down hydrogen peroxide to water and oxygen. 2 H2O2
__catalase__

> 2 H2 O + O2

This enzyme allows organisms to breakdown harmful metabolites of aerobic respiration and may be seen in aerobic and facultatively anaerobic organisms. There are other enzymes that some organisms produce to handle toxic endproducts of metabolism so not all aerobes or facultative anaerobes produce catalase. Pathogenic organisms require mechanisms to help them overcome host defense mechanisms. One mechanism involves coating the bacterial cells in a body substance, such as fibrin, to fool the immune system. The coating of a natural body substance will

not trigger an immune response. The enzyme coagulase causes fibrin to be deposited on bacterial cells. Some organisms can not tolerate a high osmotic pressure. Media containing higher than normal salt concentrations may inhibit the growth of these non-tolerant organisms. Mannitol salt agar contains a high salt concentration so only salt tolerant organisms will grow on it. Additionally, mannitol salt agar contains the sugar mannitol. Some organisms can utilize mannitol as a food source and will produce acid endproducts from this metabolism. Since this process is invisible an indicator is added to the media to detect changes in pH. Phenol red is the indicator used in mannitol salt agar. It is red at a neutral pH but turns yellow if conditions in the media become acidic. Antibiotic susceptibility is another test that can be used to identify organisms. A filter paper disc is impregnated with an antibiotic, in this case novobiocin. When the disc is placed on agar, the antibiotic diffuses through the agar. An organism susceptible to the antibiotic will be unable to grow on the media containing the antibiotic. A zone of inhibition (no growth) will be seen around the disc. The size of the zone indicates the resistance or susceptibility of the organism to the antibiotic. Procedure Catalase 1. 2. Place a drop of 3% H2 O2 on a glass slide. Touch a sterile loop to a culture of the organism to be tested and pick up a visible mass of cells. Mix the organism in the drop of hydrogen peroxide. Observe for immediate and vigorous bubbling. Dispose of slide in the contaminated slide container.

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Interpretation: Bubbling indicates a positive (+) test and scant or no bubbling indicates a negative (-) test. Coagulase 1. Dispense 1 drop of Test Latex onto one of the circles on the reaction card and 1 drop of Control Latex onto another circle. Touch a sterile loop to a culture of the organism to be tested and pick up a visible mass of cells. Mix the cells in the drop of Test Latex. Repeat Step 2 for the Control Latex.

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Pick up and hand rock the card for up to 20 seconds and observe for agglutination or clumping of the latex particles. Dispose of the reaction card in the biohazard container.

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Interpretation: Agglutination of the Test Latex with no agglutination of the Control Latex is considered a positive (+) test for coagulase. No agglutination in either the Text Latex or Control Latex is considered negative (-) for coagulase. All reactions occurring after 20 seconds should be ignored. If agglutination occurs in the Control Latex the agglutination is due to some factor other than the enzyme coagulase and the test results are invalid. Mannitol Salt Agar 1. 2. Label a tube of mannitol salt agar with the organism to be tested and your initials. Using a sterile loop transfer the organism to be tested to the surface of the mannitol salt agar slant. Incubate the tube at 35o C. for a minimum of 18 hours. Examine the tube for evidence of growth on the slant and for a color change from red to yellow. Remove the markings from the tube using Grams decolorizer on a paper towel and place the tube in the designated area for disposal.

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Interpretation: Two different characteristics of the organism are determined with this agar. The first is the organisms ability to tolerate a high salt environment. Evidence of growth on the slant indicates the organism can grow in a high salt environment. Organisms that can ferment the sugar mannitol produce an acid end product that changes the red pH indicator in the media to yellow. Any yellow in the media is considered a positive test for mannitol fermentation. It is possible for organisms to grow on the media and not ferment mannitol. Novobiocin Susceptibility 1. 2. 3. Divide a nutrient agar plate into three sections. Label a section with the name of the organism to be tested. Using a sterile loop transfer the test organism to the plate and streak the section for confluent growth. Aseptically transfer a novobiocin antibiotic disc to the center of each streaked area. Gently press the disc to the surface of the agar.

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Invert the plate and place in the incubator for a minimum of 18 hours. Examine the plate for a zone of inhibition of growth around the antibiotic disc. Using a metric ruler, measure the diameter of the zone of inhibition and record the measurement in millimeters (mm). Discard the plate in the biohazard container.

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Interpretation: A zone of growth inhibition 17 mm or less in diameter indicates resistance (R) to novobiocin. If the zone is greater than 17 mm the organism is susceptible (S) to novobiocin. LABORATORY INSTRUCTIONS Cultures provided: Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Students work individually unless otherwise noted. 1. Make a smear of one of the organisms provided. (See page 18) Complete the remainder of the laboratory work before heat fixing, staining and examining the smear. Perform a catalase test on all organisms. Select one of the three organisms and perform a coagulase test. Allow the other members of your group to observe your results. Observe the results of the other 2 organisms. Select one of the three organisms and inoculate a mannitol salt agar slant. As in step 3, observe the results of all three organisms Test each organism for novobiocin susceptibility. Each person should test all three organisms. Record all results on the Laboratory Record Sheet. (Page 37) As time permits, Gram stain the smear prepared in Step 1 (Page 22).

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MCB 1000L Identification of Staphylococcus Test Gram Stain Catalase Coagulase Growth on mannitol salt Mannitol fermentation Novobiocin susceptibility All species of Staphylococcus are Gram ___________________ ____________________ and positive for the ___________________________ test. Also, all Staphylococcus species tolerate ___________________________ as indicated by their growth on mannitol salt agar. Which test differentiates Staph. aureus from the other species of Staphylococcus? Staph. aureus Staph. epidermidis Staph. saprophyticus

How can you differentiate Staph. epidermidis from Staph. saprophyticus?