GENUS STAPHYLOCOCCUS: Isolation and Identification

Staphylococcus is a genus of Gram +, nonspore-forming cocci belonging to the familyMicrococcaceae that are often found as normal human microbiota of the skin and nasalcavity. There are five organisms to consider as potential human pathogens in this genus: S.aureus, S. epidermidis, S. saprophyticus, S. haemolyticus, and S. hominis but the first threeare the most common isolates. S. aureus is often considered to be the most problematic ofthe three pathogens and is distinguished from the other two by being the only one able tocoagulate plasma. S. aureus is able to cause many superficial pyogenic (pus-forming)infections of the dermis and underlying tissues as well as serious systemic infections. Itcan produce a range of toxins including enterotoxins (food poisoning), cytotoxins(general systemic toxins), and toxic shock superantigens. The other coagulase- negativestaphylococci (S. epidermidis and S. saprophiticus) are much less frequently found aspathogens but are occasionally associated with endocarditis, prosthetic joint infections, andwound infections , just to name a few.

This exercise gives you the opportunity to use selective media, in this case based on highsodium chloride (MSA and SM1 10 are both selective media for the isolation ofStaphylococci- 7.5% NaCl). A selective medium has an inhibitory agent which favors thegrowth of certain bacteria by inhibiting others. MSA contains an additional indicator formonitoring mannitol fermentation, which makes it a differential media also. Of the bacteriawhich can grow in the presence of high NaCl, some are halophilic (requiring a certainconcentration of salt to grow) while other are haloduric (do not use the salt, but can tolerateit). Staphylococcus is not halophilic, but rather haloduric, in that it can live in or endure high NaClconcentrations. The high salt content in SM1 10 and MSA inhibits other common skinmicroorganisms. The other media being used in this exercise are for differentiatingpathogenic Staphylococcus from nonpathogenic, and for identification of the species.

Not only salt resistant, Staphylococcus is always facultatively anaerobic. When stained, it willbe seen in small clusters (staphylo = cluster). Staphylococcus is usually either betahemolytic or not hemolytic at all (called gamma hemolysis). Pathogenic Staphylococci canproduce a variety of virulence factors, including toxins, coagulase, leucocidins, and hydrolyticenzymes that can damage host tissues.

Blood agar (BAP) is a common medium used to culture bacteria because 1) it is a greatenrichment medium for fastidious bacteria, and 2) hemolysis of blood cells can be very usefulas an identification test. Blood agar is made with 5% sheep blood. CNA agar is a type ofblood agar: the only difference is that CNA has an antibiotic, naladixic acid, that inhibits gram -bacteria.

Hemolysis is the breakdown of red blood cells: hemolysins are enzymes produced by somebacteria and are released into the medium around the bacterial colony. It can be a completebreakdown of the cells, with the release of hemoglobin and a clearing of the red from thesurrounding medium around the colony. Or the hemolysis can be a partial breakdown,resulting in a greenish or green-yellow zone around the colony.

Fall 2011 – Jackie Reynolds, Richland College, BIOL 2420

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OBJECTIVES:Become familiar with the speciation of the genus StaphylococcusGrow and identify different staphylococcal species using selective and differential agar

MATERIALS NEEDED:

Sterile swabsSterile saline solution2 Mannitol salt agar (MSA) plates (for 2 sessions)Containers of alcohol + forcepsColumbia naladixic acid blood agar plate (CNA)Staphylococcus medium 110 (SM110) agar plateDNase agar plateNovobiocin (5 microgram) antibiotic disksMetric rulersRabbit plasma (frozen) for coagulase testStaphylococcus identification tables

SCHEMATIC OF IDENTIFICATION PROCEDURE1st period 2nd period 3rd period 4th period 5th periodMSA from CNA plate gram stain coagulase finish test readingNasal MSAplate catalase optional tests prepare report

SM110 TSA (Staphyloslide)DNAse

THE PROCEDURES:

Be sure to keep a list of all test results for your isolates.

1st Session1. The table will perform nasal swabs (2 per table), each specimen streaked onto a mannitol

salt agar plate.2. Aseptically remove a cotton tipped swab from its wrapper and dip into a tube of sterile

saline. Remove excess saline by pressing against the tube wall and rotating the swab.3. Use the moistened swab to collect bacteria from the anterior opening of the nostrils.4. Use the swab for the first section of an isolation streak plate (see exercise on PURE

CULTURE) using a MSA plate. Switch to your inoculating loop to do the 2nd and 3rd

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sections aseptically as you have done on other streak plates. Incubate at 37C or roomtemperature (if over the weekend).

2nd Session1. Your aim is to isolate a Staph from your MSA plate, using salt resistance as a key indicator.

Staphylococcus is salt resistant, although not the only genus of bacterium that will grow inhigh salt. Another characteristic you should look for is any colony around which themedium has turned yellow (identifying the fermenters of mannitol, of which some species ofStaph are). Identify 2 colonies from the 2 MSA plate that fit this Staph profile.

2. Divide a CNA plate and another MSA plate in half. You will use a dense zig-zaginoculation to streak the 2 potential Staph colonies on opposite sidesof the CNA and MSA plates. Be SURE that you have only 1 colonyon the loop for this transfer.

3. After sterilizing forceps with alcohol and a flame, pick a novobiocindisc from the holder to place on the opposite sides of the CNA plate.

4. Incubate plates at 37C or room temperature (if over the weekend).

3rd Session1. Observe the MSA plates noting IF colonies grow, meaning that they

are salt resistant. If growth is present, then the second question iswhether the bacterium uses the sugar mannitol. If the medium surrounding the colonieshas changed from red to yellow, mannitol has been fermented and the phenol red pHindicator in the medium has changed colors as a result of the acid from sugar breakdown.

2. On the CNA plate, you are checking for 2 reactions---sensitivity to novobiocin antibioticAND hemolysis of blood.

a. Novobiocin sensitivity - A zone 16 mm or larger indicates sensitivityb. Hemolytic reactions

• alpha (α) hemolysis – green zone around colony, caused by leaking hemoglobinconverted to biliverdin, called a partial hemolysis

• beta (β) hemolysis – complete clearing around colony caused by breakdown ofRBCs by streptolysin enzymes

• gamma (γ) hemolysis - no hemolysins, no zoneStaphylococcus species are either beta hemolytic or gamma (not hemolytic). Staph

aureus produces a toxin which typically causes wide zones of beta (complete)hemolysis.

3. Gram stain the 2 colonies (from CNA plate) to verify that you have gram + cocci in clusters.If your isolate is not G+ cocci, that will be the end of the isolate’s use in this exercise.

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4. Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase + (allspecies of Staph are +). If your isolate is not this profile, that will be the end of the isolate’suse in this exercise.

a. You will carry through the rest of this exercise with your table’s 2 Staph isolates..b. IF NEITHER OF YOUR TABLE’S ISOLATES ARE STAPHYLOCOCCUS, ask your

instructor to give you an unknown Staph to work with for the rest of this exercise, oryou can “borrow” another table’s isolate.

5. Using your Staphylococcus isolates, streak 2 agar plates---SM110 and DNAse (divide eachin half if your table has 2 Staph isolates).

6. Incubate plates at 37C or room temperature (if over the weekend).

4th Session1. Check DNase plates for hydrolysis. Refer back to the DNAse exercise in the manual.2. Check the SM110 for growth and for pigment. Nutrients and vitamins in this medium

enhance the pigmentation of the pathogenic Staphylococcus, those colonies becoming alight yellow-orange colony.

3. What you do with your Staph isolate is now determined by its reactions on SM110and DNAse.

If DNAse- and SM110 –, no other tests will be run. If DNAse + and SM110 +, run the coagulase test on the isolate

4. Presumptive S. aureus can be confirmed using the coagulase test.THE TUBE COAGULATION TEST

Inoculate a tube with a ½ ml of rabbit plasma withthe bacterial inoculum. Place at 37C and check atthe next lab period. To interpret, tip the tube at anangle, looking for solidification of the plasma. Anydegree of coagulation is considered a positive test.

5. There are enough tests that have been performed toidentify your Staphylococcus isolates, assuming thatyour species are common ones. The following tablehas those 3 species listed with the corresponding testresults. Try to identify your isolates.

Differentiation of S. aureus from other common human staphylococci normal floraS. aureus S. epidermidis S.saprophyticus

Alpha toxin (-hemolysis) + (most strains) - -Growth on 7.5% salt (mannitol salt)Acid from mannitol + - + (most strains)Coagulase reaction + - -Pigment production (SM110) Usually golden Usually white Usually whiteDNase production + (usually) - -Sensitivity to novobiocin sensitive sensitive resistant* Novobiocin sensitivity = >17mm zone size

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LABORATORY REPORT SHEET

QUESTIONS:

1. Which type of hemolysis is often associated with pathogenicity?

2. What are the distinguishing features of the genus Staphylococcus?

3. Can you give the test reaction of S. aureus for each of the major tests run---MSA, SM110,

coagulase, catalase, oxidase, and DNAse?

4. What happens to RBCs in beta and alpha hemolysis?