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55 Cards in this Set
- Front
- Back
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what effect do fermentable carbohydrates in the BAP have on the growth of Streptococcus? |
fermentation of carbohydrates by Streptococcus may produce acid that could alter the acide labile Streptolysin S. Results in a loss of beta hemolysis |
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why should blood treated with dextrose, a citrate phosphate used as an anticoagulant, be avoided for use in blood agar? |
dextrose contains fermentable carbohydrate |
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why is sheep blood favoured for blood agar used in throat cultures? |
readily available good hemolytic reactions inhibits growth of Haemophilus haemolyticus |
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what is the optimum concentration of blood in agar plates? |
5% |
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what is the optimum depth of blood in agar plates? |
4-6mm |
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some strains of Streptococcus pyogenes produce beta hemolysis only as a result of _______________, which is destroyed by exposure to oxygen |
Streptolysin O |
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If Streptolysin O is destroyed in the presence of _______________, the strain of Streptococcus pyogenes will appear ________________ |
oxygen, nonhemolytic |
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what are some ways to reduce oxygen tension during incubation to give optimum hemolysis? |
pour plates to obtain subsurface colonies cutting the inoculum into the medium Anaerobic incubation Group A Streptococcus DNA Probe |
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describe a Pour Plate. why is this done? |
agar is inoculated before pouring into a plate produces subsurface colonies that form without the influence of oxygen gold standard for determining hemolysis too time consuming for routine use |
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why would you cut the inoculum into the medium or place a coverslip over a portion of the inoculum? |
reduce contact with oxygen to determine hemolysis |
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what is the prefered method of detecting Streptococcus pyogenes in throat swabs? why? |
anaerobic incubation. Produces good beta hemolysis and is not as time consuming as producing a pour plate |
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what method of detecting Streptococcus pyogenes might be in use in a high volume lab? describe it. |
Group A Streptococcus DNA Probe throat swabs are place in lysis reagent, DNA is extracted and amplified. DNA is detected. Positive or negative results are generated in about 4 hours. |
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what is the Bacitracin Susceptibility test used for? |
presumptive identification of Group A Streptococci.
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Outline the procedure for Bacitracin Susceptibility |
-growth is spread evenly over a segment of blood agar -0.04 bacitracin disc placed firmly on the surface of the blood agar plate -plates incubated within 15 minutes of disc application to prevent false susceptible results -incubated overnight at 35C -any zone of inhibition considered susceptible |
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what inoculum must be used for a bacitracin susceptibility test? |
-pure culture of beta hemolytic streptococcus -overnight broth culture or isolated colonies from primary plates |
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what are the acceptable controls for the bacitracin susceptibility test? |
susceptible control: Group A Streptococcus (S. pyogenes) resistant control: Group B Streptococcus (S. agalactiae) |
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is it acceptable to place a bacitracin disc directly on primary blood agar plates of throat cultures when doing a bacitracin susceptibility test? |
yes, must be applied to 1st or 2nd quadrant accuracy of testing reduced to about 70% beta hemolytic streptococci should be retested with a pure culture |
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What should bacitracin testing be performed on? |
beta hemolytic Streptococci alpha and nonhemolytic Streptococci can be susceptible to bacitracin and could be incorrectly reported as S. pyogenes |
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the PYR test is a colourimetric test for what enzyme? |
L-pyrrolidone aminopeptidase |
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what does the PYR test primarily used for? |
differentiate group D Enterococci from group D Nonenterococci. Also used in presumptive ID of S. pyogenes |
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what with the PYR results be for: Group D Enterococci? Group D Nonenterococci? Streptococcus pyogenes? Other beta Streptococci? |
Group D Enterococci: positive Group D Nonenterococci: negative S. pyogenes: positive Other beta Streptococci: negative |
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describe the principle behind the PYR test |
peptidase enzyme, found in some bacteria, hydrolyzes PyR substrate to a beta-naphthylamine by-product. A colour development reagent reacts with by-products to give a pink to red colour (positive reaction) |
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describe the procedure behind the PYR test |
reagent provided on paper discs or strips colonies are rubbed on strip and a drop of cinnamaldehyde reagent added pink to red colour indicates a positive test |
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should discs or strips used in PYR test be handled with bare hands? why or why not? |
no, reagent is harmful. contamination may result in false positive |
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what are acceptable controls for the PYR test? |
Positive control: Enterococcus faecalis, or Group A Streptococcus Negative control: Group D Nonenterococcus (S. bovis), Group B, C, F, G, Streptococci |
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describe the Co-agglutination Test for Streptococcal Grouping |
utilizes group specific Streptococcal IgG antibody with killed S. aureus cells attached to the Fc portion. When Fab end reacts with Streptococcal antigen, visible macroscopic agglutination results. agglutination within 15 seconds to 2 minutes considered positive. available for groups A, B, C, D, F, G |
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Describe Latex Agglutination for Streptococcal Grouping |
uses group specific IgG antibodies with latex particles attached to Fc portion. When antigen-antibody reaction takes place, latex antibody particles are bound together resulting in macroscopic agglutination. Extraction of Streptococcal antigen with enzyme (10 mins) or acid (~1 mins) may be required before adding latex reagent. This affects group D identification. macroscopic agglutination indicates a positive test. |
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what does the CAMP test determine? |
the ability of an organism to produce CAMP factor, used for presumptive identification of Group B Streptococci |
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what does the CAMP factor do? |
acts with Staphylococcal beta hemolysin to produce a lytic reaction at the point where the two toxins meet (sheep blood) |
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what is needed for the CAMP test? |
Sheep Blood Agar Staphylococcus aureus producing Beta Hemolysin
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describe the procedure for a CAMP test |
S. aureus is struck down the surface of warm, dry BAP. Pure culture of Streptococcus is struck perpendicular to Staph streak incubated for 6 (CO2) or 18 (air) hours arrowhead of complete hemolysis is positive (presumptive Group B Streptococcus) |
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what are acceptable controls for the CAMP test? |
positive control: Group B Streptococcus (S. agalactiae) negative control: Group A Streptococcus (S. pyogenes) |
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what are some precautions for the CAMP test? |
test organism must be confirmed to be a Streptococcus, other combinations of bacteria may give a false positive as many as 10% of Group A Streptococcus are CAMP positive. Combine CAMP test and Bacitracin to increase accuracy in ID |
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what does the hippurate hydrolysis test determine? |
ability of an organism to hydrolyze sodium hippurate. Final end product is benzoic acid, earlier hydrolysis product glycine is usually tested for (Rapid hippurate hydrolysis test) Presumptive ID for Group B Streptococci |
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in the Rapid Hippurate Hydrolysis test, __________ is detected by the addition of _____________. what colour is produced? |
glycine ninhydrin ammonia produced from deamination produces a deep purple colour |
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how is sodium hippurate stored? |
-20C |
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describe the procedure for a rapid hippurate hydrolysis test. |
hippurate solution warmed to room temperature and inoculated with enough colonies to give cloudy suspension. incubated at 35C for 2-4 hours 0.2mL ninhydrin reagent is added after the initial incubation period and the test is incubated for 10 mins positive = deep purple colour negative = no colour or pale purple |
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what are acceptable controls for the rapid hippurate hydrolysis test? |
positive control: Group B Streptococcus (S. agalactiae), Campylobacter jejuni negative control: Group A Streptococcus (S. pyogenes), Campylobacter coli |
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what are some precautions for the rapid hippurate hydrolysis test? |
contamination of the test medium with amino acids with give false positives Only pure cultures of Streptococci can be used, other bacteria may cause false positives difficulties deciding between light and dark purple |
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What does the bile Esculin Test determine? |
the ability of an organism to grow in the presence of bile salts and hydrolyze esculin |
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what is esculin? what happens when bacteria hydrolyze esculin in the BEA test? |
esculin is a glycoside composed of glucose and esculetin linked together by an oxygen bond. When bacteria hydrolyze esculin they leave glucose and esculetin in the medium, esculetin reacts with ferric salts in the medium to give a dark brown black colour. |
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what is the BEA mainly used for? |
identification of Group D Streptococci Non enterococci and Enterococci |
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what concentration of bile salts is recommended for testing group D Streptococci (BEA test) |
40% bile salts |
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Outline the procedure for the BEA test |
medium is inoculated with a pure culture of Streptococcus tests are incubated at 35C and examined periodically for up to 48 hours Positive = growth, brown black colour Negative = usually no growth, no change in colour |
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What are acceptable controls for the BEA test? |
Positive control: Group D (Enterococcus faecalis) Negative control: Group B (Streptococcus agalactiae), Group A Streptococcus, S. pneumoniae, S. viridans |
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What Precautions are associated with the BEA test? |
use a pure culture of Streptococcus to avoid false positives
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What does the Bile Solubility test determine? |
if bacterial cells will be lysed in the presence of bile salts. Primarily used to differentiate bile soluble Streptococcus pneumoniae from other bile insoluble alpha hemolytic Streptococci (Streptococcus viridans) |
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what are the reagents used for the bile solubility test? |
variety of bile salts can be used, sodium deoxycholate and sodium taurocholate are the most active. reagent prepared as a 10% aqueous solution |
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describe the procedure for the Bile solubility test |
an isolated colony on BAP is selected. drop of bile salt solution is added to the colony plate is allowed to sit for up to 30 mins uncovered
bile soluble: colony dissolves completely or partially leaving a zone of hemolysis (usually alpha) on the agar bile insoluble: colony remains intact |
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what are acceptable controls for the Bile solubility test? |
bile soluble: Streptococcus pneumoniae bile insoluble: Streptococcus viridans |
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what are some precautions for the bile solubility test? |
use fresh overnight cultures, older colonies might not be lysed by bile salts (false negative) |
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what does the optochin susceptibility test determine? |
used to differentiate Streptococcus pneumoniae (susceptible) from other alpha hemolytic Streptococci (resistant) such as Streptococcus viridans |
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What is the procedure for the optochin susceptibility test? |
single colony of alpha hemolytic streptococcus is picked and streaked evenly over a BAP. optochin disc pressed firmly against the surface of the plate. incubated within 15 minutes incubated 18-24 h in CO2 Susceptible: large zone of inhibition, >14mm Resistant: small zone of inhibition <14mm |
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what are acceptable controls for the optochin susceptibility test? |
Susceptible control: Streptococcus pneumoniae Resistant control: Streptococcus viridans |
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what are some precautions for the Optochin susceptibility test? |
some strains of Streptococcus viridans give small zones of inhibition. questionable results may be confirmed with bile solubility testing. |
