Module 10: Lab Procedures for Strep Flashcards
3 types of hemolysis
Beta: complete; clear zone around bacterial colony
Alpha: partial; greenish zone around colony
Gamma: non-hemolytic
Blood agar base to support growth of Streptococci
free of fermentable carbohydrates (carbs in medium are fermented by strep, producing acid in medium, may alter acid liable Streptolysin S. Results in loss of beta hemolysis)
Recommended: Trypticase Soy Agar (TSA)
Avoid: Citrate phosphate dextrose used as anticoagulant in blood bank blood (contains fermentable carbs)
Blood source
sheep blood
gives good hemolytic reactions
inhibits growth of Haemophilus haemolytic (common upper respiratory tract with beta hemolytic colonies that could resemble strep)
5% concentration of blood in plate
4-6mm depth for best growth results
Atmosphere for incubation
Pour plates used to obtain subsurface colonies that form without influence of oxygen
Cutting the inoculum into the medium will reduce contact with oxygen
***Anaerobic incubation is preferred to detect S. pyogenes in throat swabs. Yields good beta hemolysis. Reduces growth of aerobic and facultative bacteria making beta hemolytic strep more evident
Group A Streptococcus DNA Probe: throat swabs placed in lysis reagent. DNA is extracted and amplified. DNA detected and results in 4 hrs.
Some strains of S. pyogenes produce beta hemolysis only as a result of the action of
Streptolysin O
Bacitracin Susceptibility
used for Presumptive ID of Group A Streptococci
Almost all Group A strep are susceptible
Disc diffusion test
*Must use a PURE culture of beta hemolytic Streptococcus
Original planted throat BAP must also have bacitracin disc applied
Bacitracin A Disct placed on surface of BAP
Incubate within 15 min of disc placement
Incubate overnight 35Deg.
*Any zone of inhibition is considered susceptible
Susceptible Reported as “Presumptive Group A Streptococcus”
Resistant reported as “beta hemolytic Streptococcus not Group A”
Susceptible control: Group A Streptococcus (S. pyogenes)
Resistant control: Group B Streptococcus (S. agalactiae)
PYR test use and results
colourimetric test for enzyme L-pyrrolidone aminopeptidase
Primarily used to differentiate group D Enterococci from Group D Nonenterococci
Also used for presumptive ID of S. pyogenes
Group D Enterococci: pos
Group D Nonenterococci: Neg
S. pyogenes: Pos
Other Beta streptococci: Neg
PYR test principle
peptidase enzyme is found in some bacteria
It hydrolyzes the substrate to a beta-naphthylamine byproduct
Substrate is L-pyrrolidone-beta-naphthylamide (PYR substrate)
A color development reagent, p-dimethylamino cinnamaldehyde reacts with the -amine to give pink/red color (pos result)
Summary:
PYR (substrate) + peptidase (enzyme in bacteria) = amine + colour reagent = pink-red colour
PYR test procedure and controls
Colonies rubbed onto a commercial strip or disc with a drop of cinnamaldyhyde reagent added
Almost instant pink-red color = pos test
Observe strip for 10 min before recording as negative
**use pure culture of streptococcus for the test
Pos control: Group D Enterococcus species (enterococcus faecalis or faecium), or Group A Streptococcus
Neg control: Group D Nonenterococcus species (Streptococcus bovis), Group B, C, F, G Streptococci
Streptococcal Grouping or typing: DEFINITIVE ID
Co-agglutination test for Streptococcal Grouping
Latex Agglutination for Streptococcal Grouping
Co-agglutination test for streptococcal grouping
Definitive ID
Uses Streptococcal IgG antibody with killed S. aureus cells attached to the Fc portion
Leaves the Fab ends of the antibody free to react with streptococcal antigen
Antigen-antibody reaction takes place = visible macroscopic agglutination (within 15sec-2min)
Commercial kit; reagents for groups A, B, C, D, F and G available
Colonies must be taken directly from primary isolation plate if enough colonies are present
Latex Agglutination for Streptococcal Grouping
Definitive ID
uses group specific IgG antibodies with latex particles attached to the Fc portion
When antigen-antibody reaction takes place, latex antibody particles are bound together by cell wall antigen resulting in macroscopic agglutination
Extraction of the streptococcal antigen may be required before adding the latex reagent
- done using enzymes, 10min incubation or by acid (1min)
- extraction does not affect ID of group A but DOES affect group D ID
- following extraction, drop of latex rgt is added to extract with macroscopic agglutination indicating a pos test
CAMP test for Group B Stretococcus
determines ability of an organism to produce CAMP factor (camp factor with Staphylococcal beta hemolysin acts on sheep blood cells to produce a lytic reaction at the point where two toxins meet)
Used as PRESUMPTIVE ID of Group B Strep
Materials and procedure for CAMP Test
Sheep blood agar (1.5mm depth, warmed to room temp)
S. aureus producing Beta Hemolysins (ATCC 25923 S.aureus)
-S. aureus may be stock cultured on nutrient agar slants but will occasionally stop producing B-hemolysins and need to be replaced with new strain
-S. aureus struck down surface of BAP, pure culture of Streptococcus is struck perpendicular to staph streak
-Pos and neg controls may be done on same plate
-Incubate in increased CO2(6hrs) or in air(18hrs) but NOT anaerobically
Inspect plate for an arrowhead (or bowtie) of complete hemolysis at the point where the two toxins meet (pos result)
CAMP test interpretation and reporting
CAMP pos = an arrowhead of hemolysis where staph and strep meet
Pos test result as “presumptive Group B Streptococcus”
Neg test result as “not group B Streptococcus”