Staphylococcus Identification

Identification of Staphylococcus, mainly S. aureus, can cause a range of infections from boils, cellulitus, impetigo, toxic shock and food poisoning.  So it is important to be able to identify and differentitate between the different Staphylococcus spp for treatment.   After a sample has been isolate and a culture grown a Gram Stain is preformed. Gram stains are the first test performed and  Gram reaction and cell morphology dictates the next test performed.  Staphylococcus spp are Gram (+) and have a grape cluster like appearance.

Gram stain of Staphylococcus aureus.  Gram (+) (purple colored cells) coccus (round) cell can be seen in cluster arrangements.












If the Gram stain is Gram (+) coccus then the next test performed is the Catalase test


Organisms that have the catalase enzyme can detoxify the cell of hydrogen peroxide by converting it to oxygen gas. Bubbles indicate oxygen gas production after the addition of hydrogen peroxide directly to colonies growing on the TSA plate.


Catalase Test


Catalase test performed on a culture of S. aureus grown on TSA for 24 hours.  The catalase test can be performed several ways: drop reagent directly onto the plate (note:  if grown on blood agar then this method will not work since the hydrogen peroxide will react with the blood), placing a colony into a tube of hydrogen peroxided, or placing a colony onto a slide then dropping reagent onto the colony.   Caution:  bubbling can result in airisolizing of the bacteria so used caution and do not inhale.  In this picture hydrogen peroxide was dropped directly onto the TSA plate and bubble were observed indicating that the catalase enzyme is present and can detoxify the cell by converting the hydrogen peroxided, toxic to many cells, converting it into free oxygen gas:
2H2O2 (hydrogen peroxide) ------->catalase enzyme ----->2H2O + O2 (free oxygen gas)

  Notice the morphology of the S. aureus colonies:  round, mucoid, yellow (due to the production of the pigment yellow pigment staphyloxanthin).  The yellow pigment is characteristic of S. aureus only while other Staphylococcus spp grow white.  
Since all Strep spp are Gram (+)  coccus but catalase (-) you would proceed to Streptolococcus identification if you had a (-) catalase test.

Oxidase Test for Gram Negative

Had the organism been Gram (-) then you would do the oxidase test:


The oxidase test is performed using a reducing agent to detect bacteria’s ability to produce cytochrome c oxidase or inophenol oxidase(iron containing heamoprotein), enzymes in the electron transport chain.  The reducing agent (N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD)) changes or produces a purple color as it become oxidized. Pseudomoonas aeruginosa: purple color formed indicating the presence of cytochrome c oxidase on the oxidase strip impregnated with TMPD. Escherichia coli: no purple color formed indicating there is no cytochrome c oxidase. Organisms that are oxidase positive are aerobic and can use hydrogen as the final electron acceptor in respiration with an end product of water and hydrogen peroxide which is broken down by catalase.  Most Enterobacteriacae are oxidase negative.
 


There are numerous test that can be done after the Catalase test to select and identify S. aureus:

Coagulase Test



Coagulase is a virulance factor produced by Staphylococcus aureus.  It converts soulable fibrinogen, found in rabbit plasma, into insoulable fibrin to form a clot.   Coagulase tubes are inoculated with the sample and can be check in about an hour, clot formations may be visible, or it could take up to 24 hours.  The clot will start to break down after 24 hrs of incubation.
Top Tube:  S. epidermidis, (-) no clot formation seen and media remains liquid.

Bottom Tube:  S. aureus, (+) clot formation seen as a solid clump in the bottom of the tube.





Mannitol Salt Agar (MSA)

•Importatn Ingredients:  Phenol red (color indicator), mannitol (sugar), 7.5% NaCl (inhibits most bacterail growth.
•IDifferential:  Between pathogenic S. aureus and S. epidermidis.
•ISelective: NaCl inhibits non-halophylic organisms.  Organism that can grow are considered halophylic and are usually Gram (+) organisms.
•Mannitol:  fermentation of acid causes a drop in pH of the media and the indicator (phenol red) turns yellow.
Top culture:  S. aureus (+) for growth and mannitol fermentation, yellow halo around growth.
Right:  S. epidermidis (+) for growth (-) for fermentation, no color change
Bottom:  S. saprophyticus (+) for growth and fermentation, yellow halo
Left:  E. coli (-) for growth
                                    

Novobiocin (antibiotic disc)


(A) S. saprophyticus: No zone of inhibition

Novobiocin, antibiotic disc, sensitivity is used to identify Staphlococcus saprophyticus.  S. saprophyticus (A) is resistant (no zone of inhibition) to novobiocin, while Staphlococcus aureus (B) and Staphlococcus epidermidis ( C ) and are both sensitive.
(B) S. aureus ~37 mm

(C) S. epidermidis ~41 mm

Measuring a Zone of Inhibition:


• Radius:  Measure half the distance of the zone, ie measure from the edge of the clear zone to the middle of the antibiotic disc, then multiply by 2.  Use this method  when part of the zone is not clear or has grown into another zone.
• Diameter:  Measure the entire length of the zone, from one edge of the clear zone directly across to the other edge of the clear zone.

DNase Test


 

DNase agar can be used to differentiate between potentially pathogenic S. aureus and other Staphlococcus by looking for the presense of the exoenzyeme DNase. After growth for 24-48 hrs 1N HCL is added to the agar plate. Addition of HCL precipitates DNA in the media causing cloudiness unless the DNA has been hydrolyzed creating a clear zone around the growing colony.
Top culture Staphylococcus aureus (coagulase +): zone of clearing around the growth after addition of 1N HCL indicating the DNA in the agar has been hydrolyzed. Note: the yellow color of the streak.
Lower left:  Staphylococcus epidermidis: growth with no zone of clearing
Lower Right:   Staphylococcus saprophyticus: growth with no zone of clearing
 

All Images Copyright T. Sturm

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