LABORATORY IDENTIFICATION OF STREPTOCOCCI III.

Bile Esculin Hydrolysis

*This test is used to differentiate group D Streptococcus and Enterococcus spp. from other
I. Bacitracin (Taxo A) and Trimethoprim Sulfamethoxazole (SXT, ST) Susceptibility Streptococcus spp. Group D Streptococcus and Enterococci are able to grow in 40% bile
Reagents and Materials: and hydrolyze esculin.
Blood Agar Plates *Bile Esculn Agar contains 40% bile (4% oxgall) that inhibits the growth of most organisms
0.04 U Bacitracin disks and esculin, a coumarin derivative. Esculin when hydrolyzed, is converted to esculetin that
1.25 μg Trimethoprim Sulfamethoxazole disks reacts with ferric citrate (indicator) forming a brown-black precipitate.
Procedure: Reagents and Materials:
1. Pick a few of the suspected colonies using a loop and inoculate unto half of a blood Bile Esculin Agar (Plate or Slant)
agar plate by multiple overlapping streak in three directions to obtain a uniform Procedure:
lawn of bacterial growth. 1. Pick a well isolated colony using a sterile loop and streak unto a bile esculin agar.
2. Stab the agar two or three times. 2. Incubate for 24-48° at 35-37°C. Observe for any growth and blackening of the
3. Using sterile forceps, place a bacitracin (A) disk and an SXT disk in the center of the medium.
streak. *Space the disks evenly so as to be able to observe zones of inhibition Intepretation:
4. Incubate for 18-24° at 35-37°C. Observe for any zone of inhibition. Positive: growth indicates tolerance to 40% bile;
Intepretation: Blackening of the medium indicates that the organism is able to hydrolyze esculin.
Any zone of inhibition around either disk indicates susceptibility. (+) Negative: no growth; no change in color of the medium.
Growth until the rim of the disks indicates resistance. (-)
Bacitracin SXT IV. Growth in 6.5% NaCl
Group A Streptococcus S R *6.5% salt is able to inhibit the growth of most organisms. Ability to grow in high salt
Group B Streptococcus R R concentrations is characteristic of some gram-positive cocci and gram-negative bacilli. The
Not Group A nor B R S test is important in differentiating between Group D Streptococcus (Non-enterococci) and
Enterococcus spp. Non-enterococci are intolerant to high salt concentrations while
II. CAMP (CHRISTIE, ATKINS, MUNCH-PETERSEN) Reaction Enterococcus spp are tolerant to 6.5% salt concentrations.
*This is a presumptive test for group B streptococci Reagents and Materials:
Reagents and Materials: 6.5% NaCl broth
Blood Agar Plates Procedure:
Beta-hemolytic S. aureus 1. Pick a 3-5 suspected colonies and inoculate unto a 6.5% NaCl broth.
Procedure: 2. Incubate for 24-73° at 35-37°C. Observe for any growth, indicated by turbidity.
1. Streak S. aureus down the center of a blood agar plate. Intepretation:
2. Streak 18-24°-old suspected colonies at a right angle to the S. aureus streak. Be Positive: turbidity in the broth after incubation.
careful not to hace the streaks touch each other. Negative: clear broth after incubation.
3. Incubate at 35-37°C for 24° under ambient conditions. Increased CO2 or anaerobic
conditions may cause false-positive results.
Intepretation:
Positive: arrowhead zone of hemolysis at the junction of the streak of S. aureus and
Streptococcus. Initial report may be “Group B Streptococcus, presumptively
identified by CAMP test.”
Negative: no arrowhead zone of hemolysis

i
Rene Ritche C Gorospe, RMT, MT(ASCP )
V. PYRase Test VI. Optochin (Taxo P)
*The test is based on the ability of the organism to hydrolyze the substrate, L- *The test is used to differentiate S. pneumoniae from other alpha-hemolytic Streptococci.
pyrrolidonylarylamidase. This test is useful in differentiating Enterococcus spp. and Group *in the presence of ethylenehydrocupreine hydrochloride, colonies of S. pneumoniae are
A Streptococcus, both of which are PYRase positive, from other streptococci, which are lysed. Other species of alpha-hemolytic streptococci are resistant.
PYRase negative. Reagents and Materials:
*A disk impregrnated with the substrate is used. After inoculation of the test organism, if Blood Agar Plates
hydrolysis occurs, the substrate is converted to ß-naphthylamide, which produces a red Optochin (P) disks, containing ethylenehydocupreine hydrochloride (6μg or 10μg)
color after the addition of a developer, p-dimethyl-aminoccinamaldehyde. candle jar or CO2 pack.
Reagents and Materials: Procedure:
PYR disks 1. Using a sterile loop, pick a few colonies of the test organism unto a Blood Agar
Color developer Plate using multiple overlapping streak in three directions.
Procedure: 2. Place an optochin disk at the center of the streak.
1. Slightly moisten the PYR disk using sterile water.. 3. Incubate under increased CO2 conditions for 24° at 35-37°C. observe for zones of
2. Pick a few colonies of the test organism and rub the inoculum gently over the inhibition.
surface of the moistened disk. Intepretation:
3. Allow for the inoculum to incubate with the substrate for 2mins at room Positive: presence of a zone of inhibition
temperature. Negative: absence of a zone of inhibition
4. Add one drop of color developer and observe for development of a pink to cherry
red color within 1 minute.
Intepretation:
Positive: appearance of a pink to cherry red color after the addition of the color
developer.
Negative: no change in color is observed.
*The test, in combination with Bile Esculin hydrolysis, is useful in differentiating
between members of Lancefield Group D:
BE PYRase
Group D Enterococcus + -
Group D non-enterococcus + -

i
Rene Ritche C Gorospe, RMT, MT(ASCP )