Module 10: Laboratory Procedures - Hemolysis, Bacitracin, PYR, CAMP, Hippurate, Bile Esculin, Bile Solubility Flashcards
What is the first step of IDing Streptococcus?
Hemolysis reactions on blood agar
What would happen to strep growing on a BA that had fermentable carbohydrates in it?
Strep ferments that carbs, produces an acid, and may alter the acid labile Streptolysin S, which may cause a loss of beta hemolysis
What strep would show different hemolysis reactions if human or horse blood is used in BA?
Group D strep
What undesirable characteristic may human blood have in terms of strep hemolysis reactions
May contain antistreptolysin O which could inhibit hemolysis
What is the optimum concentration and depth of a BA plate?
5% sheep blood, 4-6mm depth
When would a Group A strep appear non-hemolytic?
If the strain only produces beta hemolysis due to Streptolysin O, which is destroyed by oxygen
What are the 4 strategies to reduce oxygen when working up GAS?
- Pour plates
- Cutting the inoculum into the plate
- Anaerobic incubation
- GAS DNA probe
What is involved in the pour plate GAS method?
Inoculate molten agar, pour into plate, incubate
Sub-surface colonies form and are examined under low magnification
What is involved in the inoculum cutting GAS method?
Gouging the agar with the inoculum or placing a coverslip overtop to reduce oxygen
What is involved in the anaerobic GAS method?
Used commonly in throat swabs
Anaerobic incubation used
What is involved in the DNA probe GAS method?
Throat swabs placed in lysis reagent, DNA is extracted, amplified, and DNA is detected
What is the bacitracin test used for?
Presumptive GAS identification
What is the bacitracin test procedure?
Apply bacitracin disk to primary or sub plate, incubate overnight at 35 degrees, read for ZOI
What are the results of the bacitracin test?
Sensitive = any ZOI = Not GAS, other beta-hemolytic Streptococcus Resistant = no ZOI = GAS
What does the PYR test detect?
L-pyrrolidone aminopeptidase enzyme
What is the PYR test used for?
Differentiates Group D Enterococcus from Non-Enterococcus, and presumptive ID of GAS
What is the PYR test principle?
PYR substrate disc is hydrolyzed by PYR enzyme to amine byproduct which is detected by PYR reagent causing a pink/red color
What is the PYR test procedure?
Rub bacterial colonies on dH2O moistened disk, add cinnamaldehyde reagent, observe for red color
What is the CAMP test used for?
Presumptive ID of GBS
What does the CAMP test detect?
The ability of the organism to produce CAMP factor
How does the CAMP test work?
CAMP factor produced by GBS with Staphylococcus beta-hemolysin produce a lytic reaction where the two meet, creating an arrowhead of hemolysis
What is the procedure for the CAMP test?
Streak S. aureus vertically down a BA plate, streak test/control organism perpendicular to the staph streak stopping just short
Incubate in CO2 or O2 (CO2 is faster) and inspect for arrowhead
What is a precaution about the CAMP test?
The organism used must be confirmed as strep before this test is done, because other organisms are CAMP positive and may give false results
What is the hippurate hydrolysis test? What does it demonstrate?
Demonstrates the organisms ability to hydrolyze sodium hippurate into glycine by the addition of ninhydrin producing a purple color from the ammonia produced from the glycine deamination
What is the hippurate hydrolysis test used for?
Group B Streptococcus
What is the procedure for the hippurate hydrolysis test?
Warm frozen (-20 degree) reagent up to RT and inoculate until cloudy
Incubate at 35 degrees for 2-4 hours
Add 0.2mL of ninhydrin and continue incubating for 10 minutes
Observe for color
What are the results of the hippurate test?
Deep purple = positive
No color/light purple = negative
What are some precautions with the hippurate test?
Ninhydrin reacts with all amino acids and will cause a false positive if the test is contaminated with media peptones
Controls must be done with each test to determine dark/light purple
What is the BEA test? What does it demonstrate?
Demonstrates the organisms ability to grow in the presence of bile salts (usually 40%) and hydrolyze esculin
Why does BEA turn brown when the test is positive?
Esculin is glucose and esculetin bound with oxygen, bacteria that hydrolyzes it breaks and the bond
Esculetin reacts with ferric salt in the media and turns it brown
What is the BEA test used for?
Group D Streptococcus
What is the procedure for the BEA test?
Streak tube slant or plate, incubate at 35 degrees for up to 48 hours, inspect for growth and brown color
What is the positive result for BEAs?
Growth on the slant/plate and the media being dark brown
What are some precautions with the BEA test?
Inoculum must be pure as this test is not specific for GDS other organisms can go positive
What is a rapid BEA test?
Buffered esculin in nutrient broth with ferric salts
May go positive in as little as 1 hour
What does the bile solubility test determine?
Determines if the bacterial cells will be lysed by the presence of bile salts, which attach to their cell membranes and alter the surface tension and activates autolytic enzymes
What is the reagent used for the bile solubility test?
10% bile salt solution
Either sodium deoxycholate or sodium taurocholate
What is the procedure of the bile solubility test?
Add 1 drop of bile salt solution to 18-24 hour alpha colonies and wait 30 minutes with the lid ajar
Observe for dissolution
What organisms does the bile solubility test differentiate?
S. pneumoniae and S. viridans
What are the positive and negative results for the bile solubility test?
Partial or complete colony dissolution = positive = S. pneumoniae
No dissolution = negative = S. viridans
What are some precautions for the bile solubility test?
Colonies must be fresh as older colonies may not be lysed, causing a false negative result
Some S. pneumoniae strains may be resistant to bile salts
What is the optochin susceptibility test? What does it differentiate?
Differentiates S. pneumoniae from S. viridans and other alpha hemolytic streps
Optochin is a disc test
What is the procedure for the optochin test?
Streak an alpha Streptococcus colony on a BA, add an optochin disk, incubate overnight at 35 degrees in CO2
Measure the ZOI
What are the positive and negative results of the optochin test?
ZOI >/= 14mm = sensitive = S. pneumoniae
ZOI <14mm = resistant = S. viridans
What are some precautions of the optochin test?
Plates must have disk added and incubated within 15 minutes to allow growth and diffusion at the same rate otherwise false results occur
If incubated in O2 zone sizes will be different as S. pneumoniae takes longer to grow allowing larger ZOI
The reagent used for the color development in the PYR test is
a. ferric salts
b. triphenyltetrazolium chloride
c. esculetin
d. cinnamaldehyde
d. cinnamaldehyde
The CAMP reaction is seen as
a. hemolysis
b. agglutination
c. color change
d. none of the above
a. hemolysis
In a positive esculin hydrolysis test, esculin is hydrolyzed to
a. ferric salts
b. bile salts
c. glycine and glucose
d. esculetin and glucose
d. esculetin and glucose
The end product for the rapid hippurate hydrolysis test is
a. ninhydrin
b. benzoic acid
c. glycine
d. cinnamaldehyde
c. glycine
A positive hippurate hydrolysis test is
a. deep purple
b. pale purple
c. deep red
d. pink
a. deep purple
A positive bile esculin test is
a. yellow
b. purple
c. red
d. black
d. black
Using three day old colonies for the bile solubility test
a. does not affect the results
b. gives faster results
c. may give false insoluble results
d. may give false soluble results
c. may give false insoluble results
This question may have more than 1 correct answer:
Sources of fermentable carbohydrates in blood agar are
a. yeast extract
b. blood bank anticoagulant
c. meat extract
d. agar
a, b, and c
yeast extract, blood bank anticoagulant, and meat extract
This question may have more than 1 correct answer:
Bacitracin susceptibility
a. uses an antibiotic disc
b. definitively identifies GAS
c. requires use of a pure culture
d. provides results in 4 hours
a and c
uses and antibiotic disc and requires use of a pure culture
This question may have more than 1 correct answer:
The PYR test
a. is red when positive
b. detects a peptidase enzyme
c. gives results within 10 minutes
d. may be performed on mixed cultures
a, b, and c
is red when positive, detects a peptidase enzyme, and gives results within 10 minutes
This question may have more than 1 correct answer:
The S. aureus used for CAMP testing
a. must produce beta hemolysin
b. may be obtained from the ATCC
c. produces double zone hemolysis
d. occasionally needs to be replaced with a new strain
a, b, c, and d
must produce beta hemolysin, may be obtained from the ATCC, produces a double zone of hemolysis, and occasionally needs to be replaced with a new strain
This question may have more than 1 correct answer:
Reagents used for the bile solubility test are
a. hydrogen peroxide
b. sodium taurocholate
c. sodium hydroxide
d. sodium deoxycholate
b and d
sodium taurocholate and sodium deoxycholate
This question may have more than 1 correct answer:
In optochin susceptibility testing
a. no zone is interpreted as resistant
b. small zones are interpreted as resistant
c. large zones are interpreted as susceptible
d. small zones indicate S. pneumoniae
a, b, and c
no zone is interpreted as resistant, small zones are interpreted as resistant, and large zones are interpreted as susceptible
In the co-agglutination test for streptococcal grouping
a. antibody is coated on red blood cells
b. antibody is attached to staphylococcal cells
c. antibody is attached to latex particles
d. antigen must be extracted from streptococcal cells
b. antibody is attached to staphylococcal cells
In the latex agglutination test for streptococcal antigens, the method of antigen extraction affects results for
a. GBS
b. GAS
c. GDS
d. all of the above
c. GDS
The medium recommended for detection of hemolysis in streptococci is
a. 2% sheep blood
b. 5% sheep blood
c. chocolate agar
d. 5% human blood
b 5% sheep blood
The most accurate method of determining hemolysis is
a. examination of blood agar incubated in CO2
b. examination of blood agar incubated anaerobically
c. microscopic examination of colonies in a pour plate
d. examination of colonies formed under a coverslip
b. examination of blood agar incubated anaerobically
The recommended atmosphere for incubating blood agar plates inoculated with throat swabs is
a. ambient
b. 5% CO2
c. anaerobic
d. 10% CO2
c. anaerobic
What may cause fermentable carbohydrates to be present in media?
Use of yeast extract, meat extract, blood bank anticoagulant (CPD). and some peptones
What base may be used for BA plates?
TSA (trypticase soy agar)
Why is sheep blood used for BA plates for throat cultures?
Good hemolytic reactions, inhibits H. haemolyticus which is a non-pathogen in the upper respiratory tract
What are the 2 tests that can definitively identify which group of Streptococcus is being tested?
Co-agglutination tests and latex agglutination tests
How does the co-agglutinating test work?
Streptococcal IgG antibodies are attached to dead S. aureus cells by the Fc portion
The Fab portion reacts with the strep and causes agglutination
How does the latex agglutination test work?
Streptococcal IgG antibodies are attached by the Fc portion to latex particles, which are then bound to other particles by cell wall antigen
The Fab portion reacts with the strep and causes agglutination
When is extraction required in strep grouping? How does it work? What is a precaution?
May be necessary for the latex test
Works by enzymes or acid (quicker)
Affects Group D identification
