Module 10: Lab Procedures for Strep

Question 1

3 types of hemolysis

Answer 1

Beta: complete; clear zone around bacterial colony
Alpha: partial; greenish zone around colony
Gamma: non-hemolytic

Question 2

Blood agar base to support growth of Streptococci

Answer 2

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)

Question 3

Blood source

Answer 3

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

Question 4

Atmosphere for incubation

Answer 4

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.

Question 5

Some strains of S. pyogenes produce beta hemolysis only as a result of the action of

Answer 5

Streptolysin O

Question 6

Bacitracin Susceptibility

Answer 6

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)

Question 7

PYR test use and results

Answer 7

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

Question 8

PYR test principle

Answer 8

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

Question 9

PYR test procedure and controls

Answer 9

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

Question 10

Streptococcal Grouping or typing: DEFINITIVE ID

Answer 10

Co-agglutination test for Streptococcal Grouping

Latex Agglutination for Streptococcal Grouping

Question 11

Co-agglutination test for streptococcal grouping

Answer 11

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

Question 12

Latex Agglutination for Streptococcal Grouping

Answer 12

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

Question 13

CAMP test for Group B Stretococcus

Answer 13

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

Question 14

Materials and procedure for CAMP Test

Answer 14

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)

Question 15

CAMP test interpretation and reporting

Answer 15

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”

Question 16

CAMP controls

Answer 16

pos: Group B streptococcus (S. agalactiae)
Neg: Group A Streptococcus (S. pyogenes)

Question 17

CAMP test precautions

Answer 17

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

Question 18

Rapid Hippurate Hydrolysis

Answer 18

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

Question 19

Rapid Hippurate Hydrolysis test requirements

Answer 19

Na Hippurate as disc, or aqueous solution (stored at -20)

Ninhydrin solution may be purchased in solution

Question 20

Rapid Hippurate Hydrolysis procedure

Answer 20

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

Question 21

Rapid Hippurate Hydrolysis control

Answer 21

Pos = Group B Streptococcus (S. agalactiae), Campylobacter jejuni

Neg = Group A Streptococcus (S. pyogenes), Campylobacter coli

Question 22

Rapid Hippurate Hydrolysis precautions

Answer 22

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)

Question 23

Bile Esculin Test (BEA)

Answer 23

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

Question 24

Bile Esculin Test medium

Answer 24

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

Question 25

Bile Esculin Test Control and precautions

Answer 25

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

Question 26

Bile Solubility test

Answer 26

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

Question 27

Bile Solubility test reagent

Answer 27

bile salt - Sodium deoxycholate and sodium taurocholate are the most active
Reagent prepared as a 10% aqueous solution

Question 28

Bile Solubility test procedure

Answer 28

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

Question 29

Bile solubility test control

Answer 29

NOT POS/NEG

Bile soluble control - Streptococcus pneumoniae
Bile insoluble control - Streptococcus viridans

Question 30

Bile solubility precautions

Answer 30

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)

Question 31

Optochin susceptibility

Answer 31

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

Question 32

Optochin Susceptibility procedure

Answer 32

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

Question 33

Optochin Susceptibility control

Answer 33

do once a week and with new lots

Susceptible= Streptococcus pneumoniae
Resistant = Streptococcus viridans

Question 34

Optochin susceptibility precautions

Answer 34

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