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The Genus 19 Staphylococcus

embers of the genus Staphylococcus are gram-positive spherical organisms about 1 micrometer in diameter. They M occur singly, in pairs, and in irregular clusters, and form yel- low, orange, or white colonies on media. They are salt tolerant and grow on ordinary bacteriological media as well as on the selective media used in this exercise. Up to three species of Staphylococcus are studied in this exercise. Cer- tain strains of Staphylococcus aureus are the cause of food poisoning and toxic shock syndrome. They also are the cause of boils and carbuncles. A second species, S. epidermidis, usually is a saprobe of the skin that is rarely involved in human infection. The third species, S. saprophyticus, is an opportunistic species that may cause urinary tract infections in women of childbearing years. In this exercise, staphylococcal species will be isolated from the body’s environment and their properties examined.

A. of Staphylococci

Species of Staphylococcus are tolerant to salt and, therefore, they can be PURPOSE: to isolate and selected out from a mixture of in a high-salt medium. In addition, identify staphylococcal species from the nasal cavity S. aureus ferments mannitol, an alcoholic derivative of the hexose mannose, and other environments. while S. epidermidis and S. saprophyticus do not. Therefore, if the differential medium contains mannitol, the two species may be differentiated from one another. In this section, we will use mannitol salt agar, a medium that is both selective and differential. The salt concentration is a high 7% to inhibit other organisms, and mannitol is available for fermentation to acid. The indicator phenol red is included in the medium to detect acid pro- duction. It will change from red to yellow in the presence of acid-producing S. aureus. No color change will occur with the other two species.

S pecial Materials • Plates of mannitol salt agar • Sterile swabs • Tubes of sterile saline • reagents • slants 43038_CH19_0171.qxd 1/3/07 3:53 PM Page 172

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P rocedure 1. Select or prepare two plates of mannitol salt agar and, on the bottom sides, label them in the prescribed manner with your name, the date, and the name of the medium. Label one plate for a “nose swab” and the second for an environment of your choice.

2. Moisten a sterile swab in sterile saline solution, and swab the internal perimeter of the nose cavity. Apply the swab to one area of the appropriate Do not swab the nose too vigorously, to avoid mannitol salt , and place the swab in disinfectant. Perform the breaking one or more streak plate technique: Using a sterile loop, pass it a couple of times blood vessels. across the swabbed area and then streak into a second area of the plate to obtain isolated colonies, as described in Exercise 2A and directed by the ! instructor. Continue streaking into the third and fourth areas of the plate. Incubate the plate in the inverted position at 37° C for 24 to 48 hours, then refrigerate it.

3. Moisten a second swab, and attempt to isolate staphylococci from another environment. Consider a telephone receiver, a doorknob, the surface of a dollar bill, the area under your fingernail or behind your ear, or other source. If you wish to test a food sample, use the custard filling from a donut or a sample of hamburger meat or something similar. (If food is left out to “ripen” for a day or two, the chances of obtaining staphylococci will be greater.) Apply the swab to the second plate as in step 2, and con- tinue streaking with the loop to obtain isolated colonies. Incubate the plate as above.

4. Observe the plates for the presence of round opaque colonies that are white or pigmented. These are probably colonies of Staphylococcus species. S. aureus produces golden-yellow pigmented colonies, while the other species produce white colonies. Also, colonies surrounded by a yellow halo contain bacteria that fermented the mannitol and probably contain S. aureus. Where no color change to yellow has taken place, the colonies are possibly S. epidermidis or S. saprophyticus, since these species do not fer- ment mannitol. Another possibility is Micrococcus luteus, as noted below. A labeled representation of the plate indicating colony color and any color changes from mannitol fermentation may be entered in the Results section and Table 19.2.

5. The presence of staphylococci may be verified by performing the Gram stain technique on samples from isolated colonies. Typical clusters of gram-positive cocci should appear. Illustrations and your notes should be placed in the Results section. On occasion, Micrococcus luteus will be iso- lated on the medium. These bacteria also are gram-positive cocci but they occur in characteristic tetrads (clusters of four cells) or in cubical packets of eight cells (the sarcina configuration). The colonies on agar are bright yellow.

6. Other characteristics can be used to identify or differentiate between spe- cific species of Staphylococcus. However, to do this requires pure cultures of the isolated species. 43038_CH19_0171.qxd 1/3/07 3:53 PM Page 173

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7. Obtain three nutrient agar slant tubes and label each tube with your Extra caution should be name. Also, label one tube A, another B, and the third C. These will be used observed when working to produce three pure cultures of Staphylococcus. with potential pathogens. 8. Based on colony color (pigmentation), mannitol fermentation results, and the purity of the colony based on Gram , select three separate ! colonies to pure culture. One colony should be positive for mannitol fer- mentation (yellow halo). Identify this colony as isolate A. The other two should be white colony isolates that cannot ferment mannitol (no yellow halo). Identify these colonies as isolates B and C.

9. Sterilize your and transfer a loopful of colony isolate A to the agar slant labeled A. Streak the loop up the length of the agar, being care- ful not to gouge the agar (Exercise 1). Repeat the process with a sterile loop from colonies B and C to slants B and C. Incubate the three slants at 37ºC for 24 to 48 hours. If they will not be used until the following week, they can be refrigerated after 48 hours.

B. Differentiation between Staphylococcal Species

Each of the three species of Staphylococcus has specific characteristics that PURPOSE: to distinguish can be used to separate it from the other two species. S. aureus has the abil- between species of Staphylococcus based on ity to produce , an enzyme that clots blood plasma and enhances biochemical characteristics. its virulence by providing resistance to phagocytosis. The species also produces DNase to digest DNA and is sensitive to the antibiotic novo- biocin. Of the three species, only S. saprophyticus is resistant to (Table 19.1).

In this exercise, the three samples of isolated staphylococci from Part A will be tested for coagulase and DNase activity as well as sensitivity to novobiocin.

TABLE

19.1 Characteristics of Staphylococcal Species

CHARACTERISTIC S. AUREUS S. EPIDERMIDIS S. SAPROPHYTICUS

Colony color Golden-yellow White White Acid production Yes No No from mannitol Coagulase production Yes No No DNase activity Yes No No Novobiocin sensitivity Yes Yes No 43038_CH19_0171.qxd 1/3/07 3:53 PM Page 174

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S pecial Materials • Isolated staphylococci from Part A • Tubes containing 0.5 ml of coagulase plasma • DNase test agar • 1 N HCl • Mueller-Hinton agar • 30 µg Novobiocin discs • Sterile forceps

P rocedure Obtain the three slant cultures (isolates A, B, and C) that you made in the previous lab period.

I. Coagulase Activity 1. Obtain three tubes of coagulase plasma and label them with your name. Then, label one tube A, one B, and one C. 2. Inoculate coagulase tube A with a heavy loopful of bacteria from slant A. Sterilize the loop and repeat the transfers for slants B and C to coagulase tubes B and C. 3. Observe the tubes at intervals designated by the instructor. Incubation can continue for up to 24 hours. Coagulase activity is determined by tilting each coagulase tube to the side and noting the presence of a solid immov- able clot (Figure 19.1). If the plasma still flows, clot formation and coagu- lase activity have not taken place. 4. Enter your results in the Results section, using (+) for coagulase activity and (–) for lack of activity. Include the time for clotting, if known.

II. DNA Digestion 1. Obtain a DNase test agar plate and divide the bottom of the plate into three sections. Label each section with a colony designation (A, B, or C), and “DNA test.” Make sure your name also is on the plate. 2. Aseptically make a single line of inoculation of each staph isolate (A, B, and C) in its respective sector on the DNase test agar plate. 3. Incubate the plate in the inverted position for 24 to 48 hours at 37ºC.

A The first species is unable to clot the plasma because it cannot produce coagulase.

FIGURE 19.1 B The second species produces The coagulase test performed with two species coagulase and clots the plasma. of staphylococci. 43038_CH19_0171.qxd 1/3/07 3:53 PM Page 175

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4. Test for DNA digestion by flooding the plate with 1N hydrochloric acid (HCl). DNA will normally react with HCl and form a very fine precipitate, which will give a cloudy appearance to the agar after several minutes. If DNA digestion has occurred, the area near the streak will remain clear. The remainder of the plate, however, becomes cloudy. A dark background is recommended, and observations should be made from an angle to see the cloudiness. Compare your results with the control plate containing no bacteria. 5. Determine whether DNA has been digested in each of the sectors of the plate and record each result in the Results section. Enter a (+) for DNase activity and (–) for lack of activity.

III. Novobiocin Sensitivity 1. Obtain three Mueller-Hinton agar plates and label the bottom side of each plate with your name and “novo sensitivity test.” Label one plate A, one B, and one C for each staph isolate. 2. Inoculate the plates with the respective staph isolate following the “3+1” procedure for making a as outlined in Exercise 8.C (Figure 8.2). 3. Using alcohol-dipped and flamed forceps, aseptically apply a novobiocin an- tibiotic disc to the center of each plate. Gently tap the forceps on each disc to ensure it is firmly in contact with the agar. 4. Incubate the plates in the inverted position for 24 to 48 hours at 37ºC. 5. Observe the novobiocin plates. Novobiocin sensitivity is determined by measuring the diameter of the halo (zone of inhibition) around the disc where no bacterial growth has occurred. If the zone diameter is less than 17 mm, the bacterium is considered resistant to novobiocin. If the zone diameter is greater than 17 mm, the bacterium is considered sensitive to the antibiotic. Use a metric ruler to measure each zone diameter. 6. Record the measurements and whether the bacterial isolate is resistant (R) or sensitive (S) to novobiocin in the appropriate Results section.

From the series of tests and observations made of the three Staphylo- coccus isolates, determine which is S. aureus. From the results, is one of the other isolates either S. epidermidis or S. saprophyticus—or are both present?

Q uestions 1. What is the relationship of coagulase activity to the pathogenicity of Staphy- lococcus aureus? 2. Why are species of Staphylococcus often found as contaminants of ham and ham products? 3. Why is mannitol salt agar considered a selective as well as a differential medium? 4. Suppose that a coagulase test were performed without a control tube. How would this omission decrease the reliability of the test? 5. Explain how the ability to produce DNase would benefit the pathogenicity of S. aureus. 43038_CH19_0171.qxd 1/3/07 3:53 PM Page 176 43038_CH19_0171.qxd 1/3/07 3:53 PM Page 177

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Name

Date Section

Exercise19 Results The Genus Staphylococcus

A. Isolation of Staphylococci

Mannitol Salt Agar Plates

Source:

Stained Smears of Staphylococci

Source: ______

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B. Differentiation between Staphylococcal Species

Table 19.2. Characteristics of Staphylococcal Isolates A, B, and C Characteristics A B C

Colony color

Acid production from mannitol

Coagulase production

DNase activity

Novobiocin sensitivity