Streptococci (F) Flashcards
What are the 3 types of hemolysis?
1) Gamma-hemolysis
2) Alpha-hemolysis
3) Beta-hemolysis
What is gamma-hemolysis?
No hemolysis
What is alpha-hemolysis?
Appears as a greening of the agar around the bacterial colony
What is beta-hemolysis?
The RCs surrounding the colony are completely lysed, w/c results in a clear zone around the colony
What is the purpose of bacitracin susceptibility?
To differentiate Strep pyogenes from other beta(sign)-hemolytic streptococci
What is the sx used for bacitracin susceptibility?
Isolated colonies of test organism on SBA
What is the medium used in bacitracin susceptibility?
5% SBA plate
What is the rgnt used in bacitracin susceptibility?
Bacitracin disk, 0.04 unit
What is the principle in bacitracin susceptibility?
Grp A streptococci are susceptible to low lvls (0.04 units) of bacitracin, whereas other grps of beta(sign)-hemolytic streptococci are resistant. Rare strains of grp A streptococci are resistant (approx 1%), whereas some strains of grps B, C, and G streptococci are sensitive (5% - 10%).
Sensitivity to bacitracin presumptively identifies an isolate as Strep pyogenes. This procedure was designed for use only w/ pure cultures; however, some clinical microbiologists will add a bacitracin disk to a primary throat culture to screen for Strep pyogenes
What is the procedure (or steps) of bacitracin susceptibility?
1) Streak surface of agar plate to obtain isolated colonies
2) Aseptically place bacitracin disk onto inoculated surface. Press down gently on the disk to ensure complete contact w/ the agar surface
3) Incubate the plate at 35 DC for 18 - 24 hrs in a CO2 incubator
What is the temp needed for incubation in bacitracin susceptibility?
35 DC
What is the time duration for incubation in bacitracin susceptibility?
18 - 24 hrs
What type of incubator is used in bacitracin susceptibility?
CO2 incubator
What are the interpretations for bacitracin susceptibility?
1) Susceptible: any ZOI around the bacitracin disk
2) Resistant: uniform lawn of growth up to the edge of the disk
What is the principle (including the purpose) of bile solubility test?
The bile solubility test differentiates Strep pneumoniae (+) from alpha-hemolytic streptococci (-). Bile or a solution of a bile salt, such as sodium desoxycholate rapidly lyses pneumococcal colonies.
Lysis depends on the presence of an intracellular autolytic enzyme. Bile salts lower the surface tension bet the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process
Grp D streptococci and enterococci grow in the presence of bile and also hydrolyze esculin to esculetin and glucose
Esculetin diffuses into the agar and combines w/ ferric citrate in the medium to produce a black complex
What is the purpose of sodium desoxycholate?
To rapidly lyse pneumococcal colonies
What is the purpose of bile salts?
It lowers the surface tension bet the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process
What bacteria grow in the presence of bile and also hydrolyze esculin to esculetin and glucose?
1) Grp D streptococci
2) Enterococci
What is the action of esculetin?
It diffuses into the agar and combine w/ ferric citrate in the medium
What is the product when esculetin diffused into the agar and combined w/ ferric citrate?
Black complex
What is the procedure (or steps) of bile solubility test?
1) Place 1 - 2 drops of 10% sodium desoxycholate to the side of a young (13 - 24 hr), well-isolated colony growing on 5% SBA
* note: a tube test is performed w/ 2% sodium desoxycholate
2) Gently wash liquid over colony, w/out dislodging the colony from the agar
3) Incubate the plate at 35 DC in ambient air for 30 mins
4) Examine for lysis of colony
What is the temp needed for incubation in bile solubility test?
35 DC
What is the time duration of incubation needed in bile solubility test?
30 mins
What are the results (expected results) of bile solubility test?
1) (+): colony disintegrates; an imprint of the lysed colony may remain within the zone
2) (-): intact colonies
What is the principle of hippurate test?
The end products of hydrolysis of hippuric acid by hippuricase include glycine and benzoic acid. Glycine is deaminated by the oxidizing agent ninhydrin, w/c is reduced during the process. The end products of the ninhydrin oxidation react to form a purple-colored product. The test medium must contain only hippurate, because ninhydrin might react w/ any free amino acids present in growth media or other broths
What is the process (or steps) of hippurate test?
1) Add 0.1 mL of sterile H2O to a 12 X 75 mm plastic test tube
2) Make a heavy suspension of the organism to be tested
3) Using heated forceps, place a rapid hippurate disk in the mixture
4) Cap and incubate the tube for 2 hrs at 35 DC; use of a H2O bath is preferred
5) Add 0.2 mL ninhydrin rgnt and reincubate for an additional 15 - 30 mins. Observe the solution for development of a deep purple color
What is the time duration needed for incubation in hippurate test?
2 hrs
What is the temp needed for hippurate test?
35 DC
What are the results (or expected results) for hippurate test?
1) (+): deep purple
2) (-): colorless or slightly yellow pink color
What is the principle of PYR test?
The PYR test is predominately used in identification schemes for gram-(+) cocci. Presence of the enzyme L-pyrroglutamylaminopeptidase that hydrolyzes the L-pyrrolidonyl-β- naphthylamide (PYR) substrate to produce a e a β-naphthylamine. The β-naphthylamine is detected in the presence of of N, N-methylaminocinnamaldehyde reagent by the production of a bright red-colored product
What is the procedure (or steps) of PYR test?
1) Before inoculation, moisten disk slightly w/ rgnt grade H2O. Do not flood disk
2) Using a wooden applicator stick, rub a small amt of several colonies of an 18 - 24 hr pure culture onto a small area of the PYR disk
3) Incubate at room temp for 2 mins
4) Add a drop of detector rgnt, N, N-dimethylaminocinnamaldehyde and observe for a red color within 1 min
What are the results (or expected results) for PYR test?
1) (+): bright red color within 5 mins
2) (-): no color change or an orange color
What is the principle of CAMP test?
Certain organisms (including grp B streptococci) produce a diffusable extra-cellular protein (CAMP factor) that acts synergistically w/ the beta-lysin of Staph aureus to cause enhanced lysis of RBCs
What is the procedure (or steps) of CAMP test?
1) Streak a beta-lysin-producing strain of Staph aureus down the center of a SBA plate
2) Streak test organisms across the plate perpendicular to the Staph aureus streak (multiple organisms can be tested on a single plate if they are 3 - 4 mm apart)
3) Incubate overnight at 35 DC in ambient air
What is the temp needed for incubation in CAMP test?
35 DC
What are the results (or expected results) in CAMP test?
1) (+): enhanced hemolysis is indicated by an arrowhead-shaped zone of beta-hemolysis at the juncture of the 2 organisms
2) (-): no enhancement of hemolysis
What is the other term for optochin test?
Taxo P
What is the principle (and purpose) of optochin test?
This test is used to determine the effect of optochin (ethylhydrocupreine hydrochloride) on an organism
Optochin lyses pneumococci (positive test), but alpha-streptococci are resistant (negative test)
What is the procedure (or steps) of optochin test?
1) Using an inoculating loop, streak 2 or 3 suspect colonies of a pure culture onto half of a 5% SBA plate
2) Using heated forceps, place an optochin disk in the upper third of the streaked area. Gently tap the disk to ensure adequate contact w/ the agar surface
3) Incubate plate for 18 - 24 hrs at 35 DC in 5% CO2; note: cultures do not grow as well in ambient air, and larger ZOIs occur
4) Measure ZOI in mm, including diameter of the disk
What is the time duration needed for incubation in optochin test?
18 - 24 hrs
What is the temp needed for optochin test?
35 DC
What are the results (or expected results) for optochin test?
1) (+): ZOI is 14 mm or greater in diameter, w/ 6 mm disk
2) (-): no ZOI
3) Equivocal: any ZOI < 14 mm is questionable for pneumococci; the strain is identified as a pneumococcus only if it is bile-soluble
What is the principle of bile esculin agar (test)?
Gram (+) bacteria other than some streptococci and enterococci are inhibited by the bile in this medium. If streptococci and enterococci can grow in the presence of 40% bile and hydrolyze esculin, they subseqently turn the indicator, ferric ammonium citrate, a dark brown color. This dark brown color results from the combination of esculetin (the end product of esculin hydrolysis) w/ ferric ions to form a phenolic iron complex
What is the end product of esculin hydrolysis?
Esculetin
What is the procedure (or steps) of bile esculin agar (test?)?
1) Inoculate 1 - 2 colonies from an 18 - 24 hr culture onto the surface of the slant
2) Incubate at 35 DC in ambient air for 48 hrs
What is the temp needed for incubation in bile esculin agar (test?)?
35 DC
What is the time duration of incubation needed in bile esculin agar (test?)?
48 hrs
What are the results (or expected results) in bile esculin agar (test?)?
1) (+): blackening of the agar slant
2) (-): no blackening of the medium
What is the principle of sodium chloride broth, 6.5% (test?)?
NaCl broth is prepared from heart infusion broth, a general purpose medium that already contains 0.5% NaCl. When 6? NaCl is added to this medium, the salt concentration becomes 6.5?%. NaCl broth also contains glucose as a carbohydrate source, and some formulations add bromcresol purple, a pH? indicator
If the organism can tolerate this high concentration of salt, it will grow in the medium and produce turbidity. Fermentation of glucose produces acid and can cause the medium to turn from purple to yellow if the pH indicator is present
What is the procedure (or steps) of sodium chloride broth, 6.5% (test?)?
1) Several colonies are inoculated into the broth
2) Incubate overnight at 35 DC
What is the temp needed for incubation in sodium chloride broth, 6.5% (test?)?
35 DC
What are the results (or expected results) for sodium chloride broth, 6.5% (test?)?
1) (+): any growth in the broth is considered (+) if the indicator does not change color
* note: to avoid a false (-) result, the broth should be gently mixed before interpretation. Inoculating the broth too heavily may give a false (+) result
2) (-): no growth in the broth
Can slide agglutination test be used for streptococci?
Yes
What is the purpose of granada agar?
Granada agar w/ an enrichment broth can be used to directly culture vaginal-anorectal swabs from pregnant females for detection of Strep agalactiae
How to report colonies as Strep agalactiae?
After overnight incubation, colonies that appear orange, can be reported as Strep agalactiae
What is the sx used for granada agar?
Vaginal-anorectal swabs from pregnant females
What is the purpose of carrot broth?
It can be used to to both enrich? for and identify Strep agalactiae (grp B strep)
What is the sx used for carrot broth and what is done to the sx?
Vaginal-anorectal swabs from pregnant females are the sx used
These swabs are placed in the broth and incubated overnight
What is the sx used for carrot broth and what is done to the sx?
Vaginal-anorectal swabs from pregnant females are the sx used
These swabs are placed in the broth and incubated overnight
How to demonstrate the presence of Strep agalactiae via the use of carrot broth?
Development of an orange color indicates the presence of Strep agalactiae
What is the interpretation (or meaning) if no orange color develops (in the carrot broth) yet there is growth in the broth?
It is sub-cultured to rule out the presence of Strep agalactiae
