Isolation and Identification of Staphylococcus, Streptococcus, and Gram-Negative Pathogens: Laboratory Methods and Biochemical Testing

Isolation and Identification of Staphylococcus and Streptococcus

Staphylococcus Identification

Staphylococcus species, particularly Staphylococcus aureus, are important human pathogens. Laboratory identification involves selective and differential media, as well as confirmatory biochemical tests.

• Selective Media: Mannitol Salt Agar (MSA) and SM110 are used to isolate Staphylococcus species. S. aureus ferments mannitol, turning the medium yellow.

• Blood Agar: Used to assess hemolytic activity. Beta hemolysis (complete lysis of red blood cells) is characteristic of some S. aureus strains.

• Coagulase Test: Differentiates S. aureus (coagulase-positive) from other staphylococci. 97% of S. aureus strains can coagulate plasma.

• DNase Test: S. aureus produces DNase, which hydrolyzes DNA in DNase methyl green agar, resulting in a clear zone around growth.

Procedure for Coagulation (Coagulase Test)

1. Obtain three tubes of plasma and inoculate with colonies from different sources (nose, fomite, unknown).

2. Incubate tubes at 37°C and check for coagulation after 60 minutes. Results may take up to 24 hours; heavy inoculum yields faster results.

3. While waiting, perform a Gram stain to confirm Gram-positive cocci.

Interpretation: Coagulation (clot formation) indicates a positive result for S. aureus.

Beta Hemolysis

• Definition: Beta hemolysis is the complete lysis of red blood cells around bacterial colonies on blood agar, producing a clear zone.

• Significance: Indicates production of hemolysins, important for pathogenicity.

• Example: Staphylococcus aureus showing beta hemolysis on blood agar.

DNase Test

• Principle: DNase hydrolyzes DNA in the medium, causing a color change (clearing) around the streak.

• Interpretation: A clear zone indicates DNase production, characteristic of S. aureus.

Streptococcus Identification

Streptococcus species are classified by hemolytic patterns and further identified by biochemical tests.

• Alpha Hemolysis: Partial hemolysis, producing a greenish discoloration on blood agar (e.g., S. pneumoniae).

• Beta Hemolysis: Complete hemolysis, producing a clear zone (e.g., S. pyogenes, S. agalactiae).

Confirmation Tests for Streptococcus

Beta Hemolytic Tests	Alpha Hemolytic Tests
Bacitracin test	Optochin test
SXT test	Bile esculin test
CAMP reaction	6.5% NaCl test
Hippurate test

Beta Hemolysis Procedures

1. Streak blood agar and place bacitracin and SXT disks; streak S. aureus for CAMP test.

2. Inoculate Hippurate broth; beta strains hydrolyze hippurate, producing a blue color.

CAMP Test

• Purpose: Identifies group B beta-hemolytic streptococci (Streptococcus agalactiae).

• Principle: CAMP factor from S. agalactiae enhances hemolysis by S. aureus beta-hemolysin, forming an arrowhead-shaped zone of enhanced hemolysis.

• Procedure: Streak S. aureus perpendicular to the beta strain on sheep blood agar.

Alpha Hemolysis Procedures

1. Streak blood agar and place an Optochin disk (for S. pneumoniae sensitivity).

2. Inoculate Bile esculin agar; blackening indicates esculin hydrolysis (Group D streptococci and enterococci).

3. Inoculate 6.5% NaCl broth; growth indicates Group D enterococcus.

Gram-Negative Pathogen Isolation

Isolation of Salmonella and Shigella

Selective and differential media are used to isolate and differentiate Salmonella and Shigella from other enteric bacteria.

• MacConkey Agar: Non-lactose fermenters (e.g., Salmonella, Shigella) produce colorless colonies; lactose fermenters produce pink/red colonies.

• Hektoen Enteric Agar: Salmonella and Shigella form greenish-blue colonies. Salmonella may have black centers due to H2S production.

• Xylose Lysine Deoxycholate (XLD) Agar: Salmonella forms red colonies with black centers; Shigella forms red colonies; coliforms are yellow.

Further Biochemical Testing

• Pick colonies of Shigella or Salmonella and inoculate Iron Kligler tubes (Kligler Iron Agar) to test for glucose/lactose fermentation and H2S production.

• Colonies that do not ferment lactose are further tested with urea and SIM (Sulfide, Indole, Motility) tests.

EnteroPluri-Test System

Principle and Application

The EnteroPluri-Test is a miniaturized, multi-test system for the identification of Enterobacteriaceae and other Gram-negative rods. It allows simultaneous inoculation of 12 media and performance of 15 biochemical tests.

• Advantages: Saves time and reagents; provides rapid identification.

• Interpretation: Color changes in each compartment indicate positive or negative results for specific biochemical reactions.

• Example: Decarboxylation of lysine turns the medium purple; fermentation of glucose turns the medium yellow.

Representative Biochemical Reactions in EnteroPluri-Test

Test	Positive Result	Negative Result	Purpose
Glucose fermentation	Yellow color	Red/orange color	Detects glucose fermentation
Gas production	Cracks/bubbles	No bubbles	Detects gas from glucose fermentation
Lysine decarboxylation	Purple color	Yellow color	Detects lysine decarboxylase
Ornithine decarboxylation	Purple color	Yellow color	Detects ornithine decarboxylase
H2S production	Black precipitate	No blackening	Detects hydrogen sulfide production
Indole production	Red ring after reagent	No color change	Detects tryptophanase activity
Urea hydrolysis	Pink color	Yellow/orange color	Detects urease activity
Citrate utilization	Blue color	Green color	Detects citrate as sole carbon source
Voges-Proskauer	Red color after reagent	No color change	Detects acetoin production

Additional info: The EnteroPluri-Test is interpreted using a code system based on positive/negative results, which is then matched to a database for organism identification.

Summary Table: Key Biochemical Tests for Pathogen Identification

Test	Organism Detected	Positive Result	Negative Result
Coagulase	S. aureus	Clot formation	No clot
DNase	S. aureus	Clear zone	No clearing
Bacitracin Sensitivity	S. pyogenes	Zone of inhibition	No inhibition
Optochin Sensitivity	S. pneumoniae	Zone of inhibition	No inhibition
Bile Esculin	Group D Strep/Enterococcus	Blackening	No blackening
6.5% NaCl Growth	Enterococcus	Growth	No growth
CAMP Test	S. agalactiae	Arrowhead hemolysis	No enhancement
Hippurate Hydrolysis	S. agalactiae	Blue color	No color change
Iron Kligler	Salmonella, Shigella	Blackening (H2S)	No blackening
Urea Hydrolysis	Various	Pink color	No color change
SIM Test	Various	Motility, H2S, Indole	Negative for one or more