GENUS STAPHYLOCOCCUS: Isolation and Identification Staphylococcus is a genus of Gram +, nonspore-forming cocci belonging to the family Micrococcaceae that are often found as normal human microbiota of the skin and nasal cavity. 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 three are the most common isolates. S. aureus is often considered to be the most problematic of the three pathogens and is distinguished from the other two by being the only one able to coagulate 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. It can produce a range of toxins including enterotoxins (food poisoning), cytotoxins (general systemic toxins), and toxic shock superantigens. The other coagulase- negative staphylococci (S. epidermidis and S. saprophiticus) are much less frequently found as pathogens but are occasionally associated with endocarditis, prosthetic joint infections, and wound infections, just to name a few. This exercise gives you the opportunity to use selective media, in this case based on high sodium chloride (MSA and SM1 10 are both selective media for the isolation of Staphylococci- 7.5% NaCl). A selective medium has an inhibitory agent which favors the growth of certain bacteria by inhibiting others. MSA contains an additional indicator for monitoring mannitol fermentation, which makes it a differential media also. Of the bacteria which can grow in the presence of high NaCl, some are halophilic (requiring a certain concentration of salt to grow) while other are haloduric (do not use the salt, but can tolerate it). Staphylococcus is not halophilic, but rather haloduric, in that it can live in or endure high NaCl concentrations. The high salt content in SM1 10 and MSA inhibits other common skin microorganisms. The other media being used in this exercise are for differentiating pathogenic Staphylococcus from nonpathogenic, and for identification of the species. Not only salt resistant, Staphylococcus is always facultatively anaerobic. When stained, it will be seen in small clusters (staphylo = cluster). Staphylococcus is usually either beta hemolytic or not hemolytic at all (called gamma hemolysis). Pathogenic Staphylococci can produce a variety of virulence factors, including toxins, coagulase, leucocidins, and hydrolytic enzymes that can damage host tissues. Blood agar (BAP) is a common medium used to culture bacteria because 1) it is a great enrichment medium for fastidious bacteria, and 2) hemolysis of blood cells can be very useful as an identification test. Blood agar is made with 5% sheep blood. CNA agar is a type of blood 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 some bacteria and are released into the medium around the bacterial colony. It can be a complete breakdown of the cells, with the release of hemoglobin and a clearing of the red from the surrounding 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
OBJECTIVES: Become familiar with the speciation of the genus Staphylococcus Grow and identify different staphylococcal species using selective and differential agar MATERIALS NEEDED: Sterile swabs Sterile saline solution 2 Mannitol salt agar (MSA) plates (for 2 sessions) Containers of alcohol + forceps Columbia naladixic acid blood agar plate (CNA) Staphylococcus medium 110 (SM110) agar plate DNase agar plate Novobiocin (5 microgram) antibiotic disks Metric rulers Rabbit plasma (frozen) for coagulase test Staphylococcus identification tables SCHEMATIC OF IDENTIFICATION PROCEDURE 1st period 2nd period 3rd period MSA from CNA plate gram stain Nasal MSAplate catalase SM110 DNAse 4th period 5th period coagulase finish test reading optional tests prepare report TSA (Staphyloslide) THE PROCEDURES: Be sure to keep a list of all test results for your isolates. 1st Session 1. 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 2
sections aseptically as you have done on other streak plates. Incubate at 37 C or room temperature (if over the weekend). 2nd Session 1. 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 in high salt. Another characteristic you should look for is any colony around which the medium has turned yellow (identifying the fermenters of mannitol, of which some species of Staph 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-zag inoculation to streak the 2 potential Staph colonies on opposite sides of the CNA and MSA plates. Be SURE that you have only 1 colony on the loop for this transfer. 3. After sterilizing forceps with alcohol and a flame, pick a novobiocin disc from the holder to place on the opposite sides of the CNA plate. 4. Incubate plates at 37 C or room temperature (if over the weekend). 3rd Session 1. Observe the MSA plates noting IF colonies grow, meaning that they are salt resistant. If growth is present, then the second question is whether the bacterium uses the sugar mannitol. If the medium surrounding the colonies has changed from red to yellow, mannitol has been fermented and the phenol red ph indicator 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 antibiotic AND hemolysis of blood. a. Novobiocin sensitivity - A zone 16 mm or larger indicates sensitivity b. Hemolytic reactions alpha (α) hemolysis green zone around colony, caused by leaking hemoglobin converted to biliverdin, called a partial hemolysis beta (β) hemolysis complete clearing around colony caused by breakdown of RBCs by streptolysin enzymes gamma (γ) hemolysis - no hemolysins, no zone Staphylococcus 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. 3
4. Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase + (all species of Staph are +). If your isolate is not this profile, that will be the end of the isolate s use 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, or you can borrow another table s isolate. 5. Using your Staphylococcus isolates, streak 2 agar plates---sm110 and DNAse (divide each in half if your table has 2 Staph isolates). 6. Incubate plates at 37 C or room temperature (if over the weekend). 4th Session 1. 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 a light yellow-orange colony. 3. What you do with your Staph isolate is now determined by its reactions on SM110 and 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 with the bacterial inoculum. Place at 37C and check at the next lab period. To interpret, tip the tube at an angle, looking for solidification of the plasma. Any degree of coagulation is considered a positive test. 5. There are enough tests that have been performed to identify your Staphylococcus isolates, assuming that your species are common ones. The following table has those 3 species listed with the corresponding test results. Try to identify your isolates. Differentiation of S. aureus from other common human staphylococci normal flora S. aureus S. epidermidis S.saprophyticus + (most strains) Alpha toxin ( -hemolysis) Growth on 7.5% salt (mannitol salt) Acid from mannitol + + (most strains) Coagulase reaction + Pigment production (SM110) Usually golden Usually white Usually white DNase production + (usually) Sensitivity to novobiocin sensitive sensitive resistant * Novobiocin sensitivity = >17mm zone size 4
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? 5