The Staphylococci and the Streptococci: Isolation and Identification
BackgroundontheStaphylococciand Streptococci:
Members of these two genera arecapableofcausingveryserious infectionsin humans andassuchtheirproper identification in the clinicallaboratoryis important.
Inthis laboratory exercise youwillbeworking with two of the most common members of the genus Staphylococcus,Staphylococcus aureus andStaphylococcus epidermidis.Both ofthese organisms have the followingcharacteristics:
1.Grampositive nonmotile cocci/ occursingly,inpairs or asclusters
2.Aerobicor facultative anaerobe
3.Allare catalase positive
4.They inhabitskin,hairfollicles,mucousmembranes, widespread inthe environment
SinceS. aureusis a significantpathogenit is important to be ableto identify itand differentiate it fromS. epidermidis.Thesinglemostsignificant characteristicwhich identifiesS. aureusis its ability tocoagulate (clot) plasma. This isaccomplished by the release of theenzyme coagulase. S. aureus producesthe enzyme and S. epidermidisdoesnot.
The members of the genus Streptococcusare morenumerous and a bit morediversethanthoseofthegenus Staphylococcus. The majorcharacteristics of the Steptococci are:
1.Grampositive nonmotile cocci,sometimesresemble “little footballs”
occursingly,pairsandinchains(if grown in broth).
2.Facultativeanaerobes
3.Allarecatalasenegative
4.Inhabit the pharynx, teeth, saliva, skin,colonetc…
5.Are classified primarily bytheirabilitytolyseredbloodcellson bloodagarandbyagroupspecific cell wall component (Lancefield System).
6.Types of Hemolytic Streptococci
a.Alpha Hemolytic - someRBCs lysed/green zone surroundingcolony
b.Beta Hemolytic -ManyRBCs lysed/clear zone surroundingcolony
c.GammaHemolytic - Nohemolysis
7.Many human pathogensbelongtothegroup A beta-hemolyic strep.
Mediafor Growth ofStaphylococci
Most members ofthisgenusarenottoopicky about the mediumthey will growonaslongasitcontainspeptides.
General use lab mediumlike nutrientagar,BHIagarorTSAwillsupportthe growth of the staphylococci.
MannitolSalt Agar (MSA) us used for theisolation and differentiation of Staphylococci. High salt concentration inhibitsthegrowthof most other bacteria.
Most strains ofS. aureus fermentmannitol causing a yellow zone toappear around the colony. Most strains of S. epidermidis do not ferment the
sugar and themediumwill remainredaroundthecolonies.
BloodagarisoftenusedtoisolateS. aureus and many strainswill lyseredbloodcellsproducingaclearzone aroundthecolony.Thislysisisnot diagnostic forS. aureusasnotallstrains produce hemolysins.
One other characteristic that some strains of S. aureus exhibit is the production of a yellow-golden pigment.
Mediafor Growth ofStreptococci
Somemembers of this genus are a bitmore particular about their nutritional requirements andseemtogrowbestonslightly more enriched media, althoughmost will grow on BHI agar or TSA.
Most all species and strains of streptococci willgrowwellon bloodagar. The pattern of Hemolysis seen with the various species is useful for classification and identification.
BileEsculinagarisusefulisolationand identification of the group Dstreptococci.Thisgroupwillwillhydrolyzeesculincausingthe media toblacken.
Materials:
1)cultures ofS.aureus,S.epidermidis,
S. pyogenes, S. faecalis
2)1 MSA plates
3)2 Blood agar plates
4)1 Bile Esculin agar plates
5)1 TSA plates
6)3 tubes of plasma (for CoagulaseTest)
7)Hydrogen peroxide
8)Pasteur pipettes
9)Bacitracin disks (B disk)
10)Sterile swabs
Protocol:
Label the bottomof one TSA, MSA, Blood Agar and Bile Esculin Agar plate as shown in the diagrambelow.
Essentiallyyouaredividingtheplate into 4 regions.
See Figure 1. BelowFigure 1.
SWAB each organismonto theappropriateregionofthe plate. You are NOTtryingtogetisolatedcolonies.
NOTE - when you turn the plate upside-down to streak it, the letterswill
be inverted!!
3)Repeat for the other plates
4)On a Blood agar plate ONLY,Swab the half of the plate with Sf and half the plate with Spy.Place a bacitracin (B) on each organism’s side of the plate.
5)Gramstain all of the organisms andrecordyourobservationsinTable1.
Table1:GramStainReactions
OrganismsS. aureus / S.epidemidis / S. pyogenes / S. faecalis
Gram reaction
morphology
NOTES:
Protocol: Day 2
1)Observetheplatesforgrowthandrecordyour observations in Table 2 below.
Table 2: Growth of Gram Positive Cocci on Various Media
Bacteria / Mannitol Salt Agar / Blood Agar / Bile EsculinAgarS. aureus
S. epidermidis
S. pyogenes
S. faecalis
2)Performa Catalase test on all of the known organisms and your unknown
a.Use TWO loopfuls of the organismtaken fromthe TSA plate
b.put the bacteria onto a clean microscope slide
c.added several drops of hydrogen peroxide and observe for bubbling. Ifbubblingoccurs,thetestispositive.
3)Performa Coagulase Test on the two different species of Staphylococci as follows: (we will be using a commercial test to determine the presence of coagulase. The instructor will demonstrate the assay to ensure it is properly performed and the correct results obtained)
a.Add a drop of the commercial reagent onto the proper section of the paper test sheet
b.Using the provided plastic pick collect a couple of colonies and mix them with the reagent.
c.Gently rock the sheet and observe. Seeing the beads in the reagent clump together will indicate a positive reaction.
4)An alternative method is the following:
a.Add a loopful of the organismto a tube containing 0.5mlplasma.
b.Incubateat37oC for1-4hoursandcheckfor coagulation (clumping)
c.If negative, leave additional 18 hours and recheck
5)Recordtheresultsofthe biochemical testsinTable3below.
Table 3: Miscellaneous Chemical Tests onGramPositiveCocci.
Bacteria / Catalase / Coagulase / HemolysisonBlood Agar / Esculin Hydrolysis / Bacitracin Sensitivity
S. aureus
S. epidermidis
S. pyogenes
S. faecalis
Question: Whatisthemajor difference between theStaphylococciand theStreptococci? (i.e. what test would you use to differentiate between these twogenera?)
Howwould you differentiate between thevariousspeciesof Streptococciand
Staphylococci?