FAMILY STREPTOCOCCACEAE LABORATORY DIAGNOSIS

a. Streptococcus pyogenes
I. DIRECT DETECTION METHODS
 Beta-hemolytic, Pinpoint colonies
- Not required to culture in vitro
 Transparent/Translucent
Streptococcus pyogenes  Smooth/Matte surface
A. Antigen Detection Test  Convex
 Appears as minute beads
 Specimen: Throat swab
 Sensitivity: 60-95% b. Streptococcus agalactiae
 Specificity: Very specific  Beta hemolytic
: Large false negative  Larger than Group A
 Methods:  Grayish-white
a. Latex Agglutination c. Streptococcus pneumoniae
b. Coagulation  Alpha-hemolytic
c. Enzyme-Linked Immunosorbent Assay (ELISA)  Small, grayish, mucoid(for prominent capsules)
d. ASTO Test (Antistreptolysin O)
 Tend to form doughnut colonies
B. Schultz-Charlton Test  Umbilicate; Dip in the center
 Diagnostic test for Scarlet fever 2. Chocolate Agar Medium
 Intradermal injection of anti-EGT (Erythrogenic Toxin) - Less utilized
 Detects presence of EGT in the system of the patient
 Results: B. Selective Culture Media
 Positive
1. Columbia Agar with Colistin & Nalidixic Acid (CNA)
 Disappearance of rashes
- Selective for Gram(+) organisms
 Neutralization of EGT
2. Phenylethyl Alcohol Agar (PEA)
C. Dick’s Test - Selective for Gram(+) organisms
 Susceptibility test 3. 5% Sheep Blood Agar with Sulfamethoxazole-trimethoprim
 Immunity of individual to EGT - Selective for S. agalactiae
 Intradermal injection of EGT
4. Todd Hewitt Broth with Antibiotics
 Results:
- Gentamycin, Nalidixic Acid & Colistin
 Positive
5. Streptococcus Selective Agar
 Absence of rashes
6. Strep A Isolation Agar
 Patient has anti-EGT
 Immunity/ Resistance to EGT *New Granada Medium
- Selective for S. agalactiae
Streptococcus agalactiae o Colistin: Selective agent
o Sodium methotrexate: Pigment enhancer
A. Latex Agglutination Test
- Incubated anaerobically
 Detects capsule - Red-orange carotenoid colonies
 Screening purposes
 Specimen Incubation Condition & Duration
 Serum, urine, CSF, vaginal swabs  5-10% CO2
 Preferred for S. pneumoniae
From neonate Mother(Carrier)  Acceptable for other Streptococcus spp.
 48 hours of incubation
Streptococcus pneumoniae
SECONDARY GRAM STAIN
A. Neufeld Quellung Reaction
 Gram-positive cocci in chains/pairs
 Detection of polysaccharide capsule
 Anti-capsular Antibodies BIOCHEMICAL TESTING
- Causes change in the refractive index in the IMAGE of the capsule 1. Catalase Test
- Capsule: Swollen appearance  Micrococcaceae, Staphylococcaceae vs. Streptococcaceae
 India Ink Stain  Negative Result: Streptococcus spp.
- Stains the background and other parts of the cell  Check hemolytic pattern in 5% Sheep Blood Agar
 Results 2. Bacitracin-SXT Susceptibility Test
 Positive  Purpose
 Colorless halo in a dark background - Differentiates Beta-hemolytic streptococci
II. LABORATORY DIAGNOSIS BY CULTURE GROUP Bacitracin SXT
A Susceptible Resistant
SPECIMENS
B&D Resistant Resistant
S. pyogenes S. agalactiae Enterococci S. Viridans C, F, G Variable Susceptible
pneumoniae
 Principle
Throat specimen Blood Blood Sputum Blood
- Different resistance pattern of microbes against antibiotics
**Less common: CSF Urine Blood
Skin Abscess CSF  Reagents
Blood  5% Sheep Blood Agar
CSF  0.04 U Bacitracin Disk (Taxo A)
 1.25µg/23.75 µg Trimethoprim-sulfamethoxazole
MICROSCOPIC EXAMINATION  Procedure
 Gram-positive cocci  Prepare 5% BAM
 Chains  Transfer 3 to 4 well-isolated colonies of the isolate
 Diplococci (S. pneumoniae)  Streak inoculum down the center half of the plate
 Gram-variable if old  Spread inoculum
 May appear elongated (Enterococci) - Lawn over entire plate
 Place Taxo A & SXT disc
CULTIVATION  Incubate
A. Enriched Culture Media - 35oC-37oC
1. 5% Sheep Blood Agar - Ambient Air
- 18-24 hours
 Hemolysis for Streptococcus pyogenes
 Enhanced in anaerobic incubation  Results
o Actions of Antistreptolysin O Susceptible Any zones of inhibition
 Streak & Stab Method Resistant No zones of inhibition
 Differentiates microbes accdg. to Smith Brown’s Classification  Considerations
 - Small % of Groups C, F, G are Bacitracin-susceptible
3. CAMP Test  Procedure
* CAMP Factor  Streak of a well-isolated colony into the Agar-Slant
- Christie, Atkins and Munch-Peterson  Incubate
- Synergistic hemolysis - 35oC-37oC
 Group B streptococci and S. aureus - Ambient Air
 Purpose - 24 to 48 hours
- Presumptive Identification of Group B Streptococci  Interpret results
Positive S. agalactiae  Results
Negative Other Beta-hemolytic Streptococci (+) Formation of brown-black precipitate
 Principle (-) No brown-black precipitate
- Group B Streptococci produce CAMP factor  Considerations
 Diffusible, protein-like factor - 3% Viridance Streptococci = Positive (+)
 Enhances the beta-hemolysis produced by S. aureus  S. bovis group
 Procedure
 Prepare 5% BAM 6. PYRase Test
 Streak Beta-hemolytic S. aureus in the middle  Purpose
 Streak the test organism perpendicularly - Differentiate Group D Streptococci
- 3-4 cm long Positive Enterococcus
- Should not intersect with S. aureus Negative Streptococcus bovis
 Incubate
- 35oC-37oC  Principle
- Ambient Air L-pyrrolidonyl-B-naphthylamide
- 18-24 hours Pyrrolidonylamidase (PYRase)
 Result
Positive Zone of enhanced Arrow-head hemolysis Beta-naphthylamide + N,N-dimethylaminocinnamaldehyde
(Color Developer)
Negative No enhanced hemolysis
 Considerations Red Color
- Some Group A Streptococci: Intermediate positive result  Procedure
- Do not incubate anaerobically  Bacteria is streak on top of the disk
o Streptolysin O  A Color Developer is applied
 Interpret results
4. Hippurate Hydrolysis Test  Result
 Purpose
Positive Red color
- Presumptive identification of Group B Streptococci
Negative No Red color
Positive Streptococcus agalactiae
Negative Other Beta-hemolytic streptococci 7. 6.5% NaCl Test
- Aka Salt Tolerance Test
 Principle  Purpose
Hippuricase - Differentiate Group D Streptococci
Hippuric acid -----------------> Glycine + Benzoic acid Positive Enterococcus spp
Negative Non-enterococcus spp
Benzoate + FeCl3 -----------------> Ferric benzoate (precipitate)
 Principle
Oxidation - Based on the ability of the microorganism to survive in the presence of 6.5 % NaCl
Glycine + Ninhydrin -------------------> Purple color  Procedure
 Procedure  Inoculate 6.5% NaCl with 2-3 colonies of test organism
 Transfer colonies to Sodium hippurate broth  Incubate
 Incubate - 35oC-37oC
- 35oC-37oC - Ambient Air
- Ambient Air - 24-48 hours
- 24 hours  Result
 Centrifuge broth for 3-5 minutes (+) Positive: Turbidity
 Pipet 0.8mL of supernatant into small tube * Turbidity is hard to observe
 BENZOATE TEST  Addition of Bromcresol Purple
 Add 0.2mL of FeCl3 to the 0.8mL of supernatant \
 Change from PURPLE to YELLOW color (+)
 Observe for PRECIPITATION up to 10 minutes after addition of reagent  Reading of Results
 GLYCINE TEST NB/ TSB 6.5% NaCl
 Add 0.2 mL of Ninhydrin reagent Positive Equal Turbidity Equal Turbidity
 Incubate for 15-30 minutes Negative Good Very weak/None
 Results & Interpretation  Considerations
BENZOATE TEST GLYCINE TEST  80% of Group B Streptococci: Positive (+)
Leavy precipitation that  Occasional isolates of Group A: Positive (+)
(+)
persists for 10 minutes
Deep Blue/Purple color  False Positive: Heavy incubation
 False Negative: Growth may have settled out
Precipitation that clears
(-) Colorless result * Always agitate before reading
within 10 minutes
8. Optochin test
5. Bile Esculin Hydrolysis Test  Purpose
- Aka Bile Esculin Agar Test  Differentiate S. pneumoniae from other Alpha-hemolytic Streptococci
 Purpose Susceptible S. pneumoniae
- Presumptive identification of Group D Streptococcus Resistant Other Alpha-hemolytic Streptococci
Positive Group D Streptococci  Principle
Negative Non-Group D  Presence of Optochin (Ethylhydrocupreine hydrochloride)
 Selective lysis of S. pneumoniae
 Principle  Procedure
- Ability of the microorganism to grow in 40% Bile  Inoculate organism onto 5% SBA
- Ability of the microbe to hydrolyze ESCULIN  Place an Optochin disk (Taxo P) onto the inoculated plate
Group D streptococcus  Incubate (35-37oC ; 5-10% CO2 ; 18-24 hours)
Esculin ------------------------------> Esculitin + Dextrose  Read results
Ferric citrate
Esculitin -----------------> Brown- Black precipitate
  Result
≥ 14 mm for 10 µg Optochin
Susceptible
≥ 10 mm for 6 µg Optochin
Resistant No zone of inhibition
< 14 mm ZOI
Equivocal Result Confirm with Bile Solubility Test
Identified as S. pneumoniae if it is bile-susceptible

 Considerations
 Viridans Streptococci: Small zones of inhibition
 S. pseudopneumoniae: False Susceptible result under aerobic incubation
≤ 14 mm at Capnoic
≥ 14mm at Ambient

9. Bile Solubility Test

 Purpose
 Differentiates S. pneumoniae from other alpha-hemolytic streptococci
Positive/Bile-soluble S. pneumoniae
Negative/Bile-insoluble Other alpha-hemolytic
streptococci

 Principle
 Bile or solution of a bile salt such as Sodium desoxycholate  Rapidly lysis
pneumococcal colonies
 Procedure (2 Methods)
A. Broth Method
 Place 1-2 drops of 2% Sodium desoxycholate to the broth culture
 Gently shake
 Incubate
- 35oC
- Ambient Air
- 30 minutes
 Interpret results
B. Plate Method
 Place 1-2 drops of 5% Sodium desoxycholate to a colony
 Gently wash the liquid to the colony
 Incubate
- 35oC
- Ambient Air
- 30 minutes
 Interpret results
 Results
A. Broth Method
 Positive: Visible clearing
 Negative: No change in turbidity
B. Plate Method
 Positive: Lysed area where the colony had been
 Negative: No lysis