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”
CAMP controls
pos: Group B streptococcus (S. agalactiae)
Neg: Group A Streptococcus (S. pyogenes)
CAMP test precautions
must be confirmed strep before doing test
10% of Group A strep are CAMP positive (susceptible to bacitracin while group B is generally resistant)
Anaerobic incubation may increase chance of Group A testing positive
CAMP discs containing beta toxin are available and may be used instead of the S. aureus streak
Rapid Hippurate Hydrolysis
determines ability of an organism to hydrolyze sodium hippurate
end product of hydrolysis is benzoic acid (after 48hr incubation) but glycine after rapid test.
Positive results in 4hrs or less
Glycine is detected by addition of Ninhydrin (deaminates glycine, Ammonia produced which reacts with ninhydrin to give purple color)
Group B Strep are positive; used for PRESUMPTIVE ID
Rapid Hippurate Hydrolysis test requirements
Na Hippurate as disc, or aqueous solution (stored at -20)
Ninhydrin solution may be purchased in solution
Rapid Hippurate Hydrolysis procedure
Hippurate solution warmed to Room temp and inoculated with enough colonies to give cloudy suspension
Incubate at 35DegC for 2-4hrs
Add 0.2mL of ninhydrin reagent and incubate for 10 min
Pos = deep purple color Neg = no color or pale purple
Rapid Hippurate Hydrolysis control
Pos = Group B Streptococcus (S. agalactiae), Campylobacter jejuni
Neg = Group A Streptococcus (S. pyogenes), Campylobacter coli
Rapid Hippurate Hydrolysis precautions
contamination of test medium with amino acids from media peptones = false positive
Only pure cultures of Strep can be used
False positive may be caused by other genera of bacteria (ex. Listeria monocytogenes, some Group D Streptococci and a few Streptococcus viridans)
Bile Esculin Test (BEA)
determines ability of organism to grow in the presence of bile salts and hydrolyze esculin (a glycoside composed of glucose and esculetin linked together by O2 bond)
Bacteria that can hydrolyze esculin break the bond leaving glucose and esculetin in the medium.
Esculetin reacts with ferric salts in medium to give DARK BROWN-BLACK color
Group D Streptococci Non Enterococci and Enterococci are bile esculin positive
Bile Esculin Test medium
40% bile salts recommended for testing group D Streptococci
May be plate or slant tube
Inoculate medium with pure Streptococcus Incubate at 35DegC Examine periodically for up to 48hrs POS= Growth, brown/black color NEG= no growth, no change in color of medium
Bile Esculin Test Control and precautions
Pos= Group D (Enterococcus faecalis) Neg= Group B (Streptococcus agalactiae), Group A Streptococcus, S. pneumoniae, S. viridans
Use pure culture to avoid false positive
Esculin hydrolysis may be done as rapid test using buffered solution of esculin in nutrient broth with ferric salts
Group D Streptococci often give a pos result in 1 hr
Bile Solubility test
determines if bacterial cells will by lysed in the presence of bile salts
Used to differentiate bile soluble Streptococcus pneumoniae from other bile insoluble alpha hemolytic streptococci (Streptococcus viridans)
Bile salts attach to bacterial cell membrane and alter the surface tension. Activates autolytic enzymes in cell causing lysis
Bile Solubility test reagent
bile salt - Sodium deoxycholate and sodium taurocholate are the most active
Reagent prepared as a 10% aqueous solution
Bile Solubility test procedure
select and isolated 18-24hr old colony on BAP
Add drop of bile salt solution to colony
Plate is allowed to sit for up to 30 min
Bile soluble - colony dissolves completely or partially leaving a zone of hemolysis (usually alpha) on the agar, no colony is seen on top of the hemolysis
Bile insoluble- colony remains intact
In test tubes: Soluble = clear, cells lysed
Insoluble = cloudy, cells intact
Bile solubility test control
NOT POS/NEG
Bile soluble control - Streptococcus pneumoniae
Bile insoluble control - Streptococcus viridans
Bile solubility precautions
use fresh overnight colonies to prevent false neg
Unusual for S. viridians to be soluble in bile salts but some strains of S. pneumoniae are insoluble in bile (opposites of expected control results)
Optochin susceptibility
used to differentiate Streptococcus pneumoniae (susceptible) from other alpha hemolytic Strep (resistant) such as S. viridans
Optochin (ethylhydrocupreine hydrochloride) is incorporated into discs
A zone of inhibition indicated susceptibility but must be measured
Optochin Susceptibility procedure
Single colony of alpha hem Strep is picked and streaked evenly over BAP
Optochin disc placed firmly against plate
Incubate within 15min to prevent pre diffusion
Incubate plate for 18-24hrs in CO2
*Original sputum blood agar plates can also have an optochin disc (6mm) added to them
susceptible: Large zone of inhibition, >14mm
Resistant: no zone or =14mm
Optochin Susceptibility control
do once a week and with new lots
Susceptible= Streptococcus pneumoniae Resistant = Streptococcus viridans
Optochin susceptibility precautions
some strains of S. viridans will give small zones of inhibition around optochin disc (less than 14mm and may be reported as resistant) Questionable results may be confirmed with bile solubility test
If optochin is incubated with ambient air, zone sizes will be LARGER than in CO2
S. pneumoniae takes longer to start growing without CO2, allowing pre-diffusion of optochin and larger zones of inhibition
