In the genus Staphylococcus there are common parasites of humans and other animals.  S. aureus is the prime example that may occasionally cause serious infections.  Strains are known to be the common cause of suppurative infections such as pimples, boils, impetigo, etc.  Food poisoning, abscesses, endocarditis, scalded skin syndrome, toxic shock syndrome, pneumonia, and osteomyelitis are other diseases caused by this bacterium.  The organism is found primarily in the nose and the nasopharynx, but may also be found on the skin.

 Another commonly isolated member of the genus is S. epidermidis, a normal microbiota organism (as are others mentioned below), especially of the skin.  However, this species has been isolated from infection with increasing frequency due to body insults such as vascular grafts and prosthetic joints or heart valves, and from wounds, urinary tract infections, and endocarditis.  S. saprophyticus has been found to cause urinary tract infection in women, but is a rare disease agent.  Finally, S. haemolyticus, S. hominis, and S. simulans are somewhat rare and each represent 5% of isolates from infections due to what is commonly known as coagulase-negative staphylococci.

 Isolation and identification of pathogenic staphylococci and their antibiotic sensitivity patterns have assumed great importance in medical and public health laboratories.  Phage typing of isolates is considered by many workers in epidemiology to be of importance in tracing a source of infection.  Through the efforts of basic research over the years, a wide variety of characteristics of the genus and its members has accumulated (e.g., cellular and colonial morphologies, physiology, growth requirements, virulence, antigenicity, etc.).  A number of these are presently used in the diagnostic laboratory to identify a particular species of staphylococcus or differentiate it form other morphologically-related cocci such as the genus Streptococcus.  A good example of the latter is the catalase activity possessed by the staphylococci.

 Blood agar.  The most useful nonselective medium used for the primary isolation of staphylococci from clinical materials probably is blood agar.  A sugar-free blood agar base is recommended with the addition of 5% sheep or rabbit blood.  Hemolytic activity on blood agar is the way we detect the production of certain toxins, hemolysins, or lysins (all of these names refer to the same material) by staphylococci.  Hemolysis by the important alpha-lysin is detected by the use of sheep or rabbit erythrocytes.  The delta-lysin also hemolyzes rabbit erythrocytes.  Human red cells are not lysed by the alpha-lysin. The Hot-cold hemolysis is exhibited by a beta-lysin produced by certain staphylococci only on sheep red blood cells.  It should be noted that any of the above toxins, hemolysins, or lysins produce only beta hemolysis (clear area around colony or growth) on the appropriate red blood cells.

 Mannitol Salts Agar contains  mannitol as a fermentable carbohydrate, the indicator phenol red for detection, and a high concentration of salt to inhibit undesired organisms.  It has been observed that coagulase positive staphylococci grew luxuriantly, producing yellow-gold colonies with yellow zones. Nonpathogenic staphylococci produce small colonies with no color change of the surrounding medium.  Other bacteria are generally inhibited, making possible the use of a heavy inoculum without danger of overgrowth.  Incubation for 24-36 hours at 37C is recommended.
 
 

Part 1  Wednesday

A.    Each table will be provided with stock cultures of S. aureusATCC#25923,
       S. aureus  ATCC#33591(MRSA= Methicillin Resistant Staph. Aureus),
       S. epidermidisATCC#12228, and S. saprophyticus.

 1.  Prepare a gram stain.  Microscopically observe under the oil immersion  objective.  Describe       your observations.

 2.  Transfer each species to the media listed below,  by streaking one quadrant of an “X” plate*  of each media listed below.  Try to get isolated colonies by using the three sector method  in each quadrant.

  *The “X” come from the fact that the quadrant divisions make an X on the back of the plate.

      a.   Mannitol salt agar (MSA).

      b.   Blood agar (BA).

B.   Each student attempt to isolate a Staphylococcus spp. from his or her (i) throat; (ii) nares of the nose; and (iii) forehead by the following procedures:

 1.   Moisten a sterile swab in a tube containing Mueller-Hinton broth.  Remove excess liquid form the swab by pressing and turning against the inner surface of the tube.

 2.  The throat swab is done by inserting the swab so as not to make contact with the tongue or uvula, which are laden with indigenous flora. Touch the swab to the tonsils and pharynx, then withdraw it, again using care to avoid contact with other tissue.  Nose samples can be obtained by insertion of the swab into the anterior nares.  Forehead samples can be swabbed by a wiping motion over a small area.  Use a separate swab for each sampling.

 3.  Roll the swab over a small portion of the quadrant  and then streak with loop across the surface of each quadrant of an “X” plate of MSA.,

 4.   Discard swabs into disinfectant.

 5.  The fourth quadrant should be used for your isolated from a previous exercise.  Streak for isolation.

 6.  Properly label each plate and incubate at 37C.
 
 

Part 2   Thursday and/or Friday
 

A.   The Known Staphylococcal Cultures

 1.  All plates:  Macroscopically observe and compare colony characteristics of  S . aureus,  S. epidermidis,  and S. saprophyticus.  Use a dissecting microscope to observe the configuration of the colony.  Describe your findings, using proper terminology; a description of these terms is found in the first lab handout.

  a.  Mannitol Salt agar:  In many instances it is better to allow MSA incubate for 48 hours before reading the results.  In addition to colony characteristics, observe for the presence of mannitol fermentation.  (What combination causes the yellow color?)

  b.  Blood agar:   Observe the type of hemolysis produced.   Remember, staph produces only beta hemolysis or none at all.  Beta hemolysis is seen as a zone (clear)of complete red cell lysis characterized by transparency of the medium in that area.  Gamma hemolysis is the term applied when no visible red cells lysis has occurred.  It is a misnomer, and such colonies are best characterized as non-hemolytic. It should be noted that alpha hemolysis will not be observed with staphylococci.

NOTE:Alpha-hemolytic organisms (such as certain streps) will produce a zone of green, olive, or brown discoloration around their colonies, due to oxidative effects of peroxide wastes on heme (pigmented portion of hemoglobin)  from partially lysed red cells around the colony.

 2.  Catalase test:  Add a drop of 3% H2O2 to colonies on mannitol salt agar.  Quickly transfer a bit of a colony (with your inoculating loop) that shows bubbling to a sector of milk agar(see section B-2 below  for milk agar information) .  The catalase test should not be performed on blood agar plates-- why?  What is the reaction that is taking place to cause bubble formation?

 3.  Consequently, you should have a radial streak on milk agar of each of the known organisms.  This will take up four sectors.
 

B.   The Staphylococcal Isolates:

 Observe cultures obtained from the throat, nose,  and the forehead.  Describe the appearance of the colonies.  Select isolated colonies that appear to be typical Staphylococcus spp. for examination.  With these, carry out the following steps:

 1.  Do a gram stain and observe.

 2.  Radial streak onto a milk agar plate (30% evaporated milk in a nutrient medium such as heart infusion) to observe pigment production. Divide the milk agar plate into at least 8 sectors.   The white background enhances the determination of pigment.  Most coagulase-positive strains produce a creamy-yellow to a dark golden pigment on these media after incubation at 37^O C.  Occasionally, coagulase-negative strains may be pigmented.

 3.  Incubate the milk agar plate overnight at 37 C.  Thereafter store at room temperature until Tuesday when it will be used as your stock cultures for inoculation purposes.  See below Part 3.
 
 

Part 3   Tuesday the day before lab
 

 1.  You will need to prepare a 5 ml overnight culture (Mueller-Hinton) of  any pigmented S. aureus isolates.

 2.  Also radial streak all the isolates plus the four known staph    cultures onto a (one) blood agar plate.
 
 

Part 4  Wednesday (the following week)
 
 
 

A. Hot-Cold Hemolysis on Blood Agar

 NOTE: The Hot-cold hemolysis is exhibited by a beta-lysin produced by certain staphylococci only on sheep red blood cells.

 1.  This observation is performed on the radially streaked blood agar plate with the knowns and the isolates.

 2.  After an overnight incubation at 37^O C allow the plate to incubate another overnight at room temperature for an enhanced beta hemolysis that appears as a halo effect.

 NOTE:  S. aureusATCC 25923 should give a positive response.
 
 

B.   The Coagulase Test

 A key test for the determination of staphylococcal pathogenicity is the coagulase test.  Species which elaborate a substance which causes fibrin formation in citrated or oxalated blood plasma (tube test)  are considered coagulase positive and generally capable of causing infection.

 1.  Each student will provide an overnight broth culture of any pigmented S. aureus isolates.

 2.  Transfer 0.2 ml of each to 0.5 ml of freshly rehydrated coagulase plasma.  Mix.  Repeat with each of your isolates.

 3.  Each table will be provided with a known S. aureusATCC 25923 coagulase positive broth culture to be tested.  Perform this control test as a group so that each table group has known positive coagulase test to observe.

 4.  Incubate all tubes in a 37^OC water bath.

 5.  Observe for coagulation of the plasma over a period of two hours.  Any degree of clotting considered a positive test.

 6.  Record your results.

  Question:  What is the significance of coagulase to the Staphylococcus  aureusorganisms ?  Specifically, how does coagulase enzyme carry out the clotting process?  What specific components are involved?
 

C. Beta Lactamase--Dry Slide Beta Lactamase Method

 DrySlide Beta-Lactamase is used for detecting beta-lactamase (or what we commonly call penicillinase) production by bacteria, particularly Neisseria gonorrhoeae,  Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, and Staphylococcus aureus .

 All penicillins share the same 6-aminopenicillanic acid nucleus, e.g. a thiazolidine ring attached to a beta-lactam ring carrying a free amino group.  This basic structure is essential to the biological activity of penicillin compounds.  Organisms that produce beta-lactamase, an enzyme first described in microbial cultures by Abraham and Chain, enzymatically cleave the beta-lactam ring, producing penicilloic acid and rendering the antibiotic inactive.

 DrySlide Beta-Lactamase uses an acidometric methodology in which a pH indicator (chlorophenol red) is combined with benzylpenicillin (penicillin G) at a pH that maintains the integrity of benzylpenicillin.  If an organism that produces beta-lactamase is applied to the DrySlide reaction area, it cleaves the benzylpenicillin beta-lactam ring, producing penicilloic acid, lowering the pH, and causing chlorophenol red to change color.

 Organisms that produce beta-lactamase (beta-lactamase positive) change the color of chlorophenol red from purple to yellow; organisms not producing beta-lactamase do not alter the purple color of chlorophenol red within the time limits of the test.

 NOTE:  This test will be preformed initially by table as a group.   Use the S. aureus ATCC 25923 growing on  a blood agar plate as a positive control for the group test.  Afterwards,  individuals should test any identified S. aureus.

 1. Open the pouch and remove the slide.  Each 2” X 2” slide has four filter paper   reaction areas.

 NOTE:  Try not to open any more pouches than necessary to preform the test within a given group.

 2. Using a Pasteur pipette, dispense a drop of distilled or deionized water onto a reaction area of DrySlide Beta-Lactamase.  Avoid adding excess water.

 4. Using an appropriate inoculation device such as a loop, pick 5-6 isolated colonies or a sweep of confluent growth from the culture to be tested.

 5. Generously smear the organism onto the moistened reaction area of the DrySlide Beta-Lactamase.

 6. Examine the reaction area for appearance of a color change from purple to yellow within 20 minutes.

 7. If a definite color change from purple to yellow of any intensity occurs,  the test is completed.

 NOTE:  Beta-lactamase-positive organisms change the color of the reaction area from purple to yellow within 20 minutes.  A yellow color of any intensity where the organism was applied is considered a positive test.   S. aureus  ATCC #33591(MRSA) is a positive quality control culture.

  8.  Discard the used DrySlide into an autoclave bag.

 9.  Record you results.
 
 

D. ImmunoSCAN Staph Latex Test

 The demonstration of protein A in over 95% of the human strains of S. aureus  has provided a basis for a procedure using latex particles coated with human plasma.  The high affinity of protein A to the Fc portion of blood plasma immunoglobulin G (IgG), and of the clumping factor (bound coagulase) for fibrinogen in plasma, allows rapid agglutination of the coated latex particles in the presence of S. aureus.

 The ImmunoSCAN Staph reagent detects protein A and/or clumping factor (bound coagulase) commonly used as an aid in the identification of S. aureus  colonies directly from culture media.  The reagent contains latex particles covalently bound to purified human fibrinogen and human IgG to produce specific and discernible agglutination of the latex particles when mixed with a suspension of S. aureus.

 NOTE:  This test will be preformed initially by table as a group.   Use the S. aureus ATCC 25923 growing on  a blood agar plate as a positive control for the group test.  Afterwards,  individuals should test any identified S. aureus.

 1.  Gently mix the latex reagent to get a homogeneous latex suspension.

 2.  Dispense one drop of the latex reagent directly onto one of the circles on the slide.

 3.  Collect enough bacterial growth equivalent to four 1 mm colonies with the    inoculating loop.

 4.  Transfer the growth directly to the latex reagent that was added in Step 2.

 5.  Using the loop, gently mix and emulsify the culture in the reagent.  Exercise caution not to damage the surface of the slide when mixing.

 6.  Rotate the slide in complete circular motions for a maximum of 60 seconds or until visible agglutination is observed.  The agglutination patterns are clear-cut and are recognized under normal lighting conditions.

 NOTE:  S. aureus  ATCC #25923   or ATCC #29213  may be used as a positive quality control.

 7.  Discard the slide into the autoclave bag.

 8.  Record your results.
 
 

E. DNase  Test Agar

 Bacto - DNase Test Agar with Methyl Green used for detecting nuclease activity in microorganisms such as Staphylococcus aureus  and Serratia  marcescens.

 It has been  demonstrated that the increased nuclease activity of S. aureus cultures isolated from clinical specimens can be correlated with coagulase production.  Consequently, this lead to the  suggestion that nuclease activity could be used to identify potentially pathogenic staphylococci.  Remember that the nuclease of S. aureus  is a phosphodiesterase which is capable of breaking both DNA and RNA.

 Microorganisms elaborating deoxyribonuclease, when streaked on the DNase Test
 Agar surface, and incubated, depolymerized the DNA.  The basis of the test medium is that Methyl Green combines only with highly polymerized DNA and at pH 7.5 and imparts a green color to the unaffected medium.  When combination does not take place or the DNA is broken down the color fades.  Therefore this  method detects DNase activity  by a clearing of the green color around DNase producing colonies or growth.

 1.  Divide a DNase test agar plate into the number of sectors to accommodate all of   the individual S. aureus  isolate cultures, plus the S. aureus  ATCC #25923   positive control, plus a S. epidermidis  negative control.

 2.  Label the individual sectors.

 3.  Radially  streak each organism to the appropriate sector.

 4.  Incubate overnight at 37^O C.

 5.  Record results.
 
 

F. Spirit Blue Agar

 Bacto - Spirit Blue Agar is a basal medium to which is added a lipoidal substrate for the detection, enumeration and study of lipolytic microorganisms.  It was originally shown that spirit blue is inert bacteriologically and ideally suited for use as an indicator in this medium for detecting fat-splitting microorganisms.

 The prepared complete medium containing the lipid is pale lavender in color.  Colonies of lipolytic organisms are recognized by development of a deep-blue color and clearing beneath and surrounding the colonies or growth (a kind of a halo effect).  No comparable color change will be detected around colonies of non-lipolytic organisms.
 

 1.  Divide a Spirit Blue agar plate into the number of sectors to accommodate all of   the individual S. aureus  isolate cultures, plus the S. aureusATCC #25923   positive control.

 2.  Label the individual sectors.

 3.  Radially  streak each organism to the appropriate sector.

 4.  Incubate overnight at 37^O C.

 5.  Record results.
 
 

G. Tabulate All of Your Results

 NOTE:  All of the results for the known cultures and your isolates should be put into an organized form .