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Here's a special treat for all my visual learners! Check out these infographics I created to DDx between all the Gram positive Cocci...

Laboratory workup of Gram POS Cocci

Staphylococcus species

Streptococcus species

1. Determine the morphology/grouping of the gram-pos organism

GRAM POSITIVE COCCI IN...
CHAINS
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Answer

Streptococcus 

​​​

  • S. pneumonoiae

  • Viridans streptococci (S. mutans)

  • S. pyogenes

  • S. agalactiae 

  • Enterococcus faecalis

  • Peptostreptococcus

  • Gram pos cocci in CHAINS

CLUSTERS
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Answer

Staphylococcus 

 

  • S. aureus, S. epidermidis, S. saprophyticus

  • Gram pos cocci in CLUSTERS

2. Perform a Catalase Test 

Catalase: an enzyme that catalyzes the release of oxygen from hydrogen peroxide (H2O2)​​

CATALASE NEGATIVE:

No bubbles are formed

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Answer

Streptococcus 

 

  • S. pneumonoiae, Viridans streptococci (S. mutans), S. pyogenes, S. agalactiae, Enterococcus faecalis, Peptostreptococcus

  • Gram pos cocci in CHAINS

  • Catalase NEG

CATALASE POSITIVE:

Bubbles are formed

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Answer

Staphylococcus 

 

  • S. aureus, S. epidermidis, S. saprophyticus

  • Gram pos cocci in CLUSTERS

  • Catalase POS

Staphylococcus Species Workup

Staphylococcus aureus

Staphylococcus epidermidis

Staphylococcus saprophyticus

If catalase is POSITIVE, then perform a Coagulase Test 

COAGULASE NEGATIVE:

No clot formed on slide (left) or tube (right) tests

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Answer

S. epidermidis 

or

S. saprophyticus

 

  • S. epidermidis vs. S. saprophyticus

  • Gram pos cocci in clusters

  • Catalase POSITIVE

  • Coagulase NEGATIVE

COAGULASE POSITIVE:

Clots/clumps formed on slide (left) or tube (right) tests

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Answer

S. aureus

 

  • S. aureus

  • Gram pos cocci in clusters

  • Catalase POSITIVE

  • Coagulase POSITIVE

If Coagulase Test is NEGATIVE, determine Novobiocin sensitivity

NOVOBIOCIN RESISTANT

NOVOBIOCIN SESNSITIVE

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Answer

S. saprophyticus

 

  • S. saprophyticus

  • Gram pos cocci in clusters

  • Catalase POSITIVE

  • Coagulase NEGATIVE

  • Novobiocin RESISTANT

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Answer

S. epidermidis 

​

  • S. epidermidis

  • Gram pos cocci in clusters

  • Catalase POSITIVE

  • Coagulase NEGATIVE

  • Novobiocin SENSITIVE

Perform a Clindamycin Induction Test (D Test)

  • What you need to know:

    • Since clindamycin and streptogramin are among the few drugs of choice in the treatment of methicillin resistant S. aureus (MRSA) infections, knowing the resistance to these antibiotics is imperative.

    • The D test is used to study the macrolide lincosamide streptogramin resistance (MLSB), both constitutive and inducible as well as macrolide streptogramin resistance (MSB) in Staphylococcus aureus.

    • Basically, the D test is performed to determine if S. aureus is susceptible to Clindamycin.

 

  • How testing is performed:

    • Erythromycin (macrolide) and clindamycin (lincosamide derivative) discs are placed adjacent to each other over the Mueller Hinton agar medium inoculated with the test organism.

    • The test is performed in the same MHA plate in which the antibiotic sensitivity test is being done, taking into consideration that the discs are placed adjacent to each other maintaining the distance.

    • Clindamycin resistance can be induced by Erythromycin which causes the Kirby Bauer zone around the Clindamycin disc to be blunted and form a "D" if clindamycin can be induced by erythromycin to be resistant. So this is called INDUCABLE RESISTANCE. 

D

  • Interpretation:

    • The organism susceptible to clindamycin without any flattening of the zone (D test negative) near clindamycin disc (resistant)  = Macrolide streptogramin resistance (MSB)

    • Susceptible to both antibiotics = No resistance 

    • Flattening of the clindamycin zone (D test positive) near the erythromycin disc (resistant)  = Inducible MLSB (IMLSB)

    • The growth of the organism up to the edges of the disc  = Constitutive MLSB (CMLSB) Resistance

Streptococcus Species Workup

S. pneumonoiae (encapsulated)

Viridans streptococci (S. mutans)

S. pyogenes (Group A Strep)

S. agalactiae  (Group B Strep)

Enterococcus faecalis (Group D Strep)

Peptostreptococcus (Anaerobe)

If catalase is NEGATIVE, then determine the pattern of hemolysis on sheep blood agar, then continue the appropriate testing algorithm to determine the bacterial organism.

Some bacteria produce extoxins that cause the lysis of RBCs which can be seen on a blood agar plate

GAMMA HEMOLYSIS

ALPHA HEMOLYSIS

BETA HEMOLYSIS

Partial clearing

Green color

Degrades hemoglobin into biliverdin

Complete clearing

Yellow color

Produces hemolysins

No clearing

White/transparent color

Negative for hemolysins

Quellung test

Bacitracin sensitivity

Aerobe vs. Anaerobe

Optochin sensitivty

Tests for presence of a capsule

"Quellung= SWELLING"

NEGATIVE

No swelling

No capsule

POSITIVE

Swelling around capsule

Encapsulated

RESISTANT

No clearing

SENSITIVE

Clearing around disk

BILE SOLUBLE

Clearing of broth in presence of bile

NOT SOLUBLE

Turbid solution

No lysis present

Bile Solubility testing

Some bugs make a self-lysing enzyme to depress the growth of old colonies. The presence of bile salt will accelerate this .

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Answer

Viridans Strep

S. mutans &

S. mitis 

 

  • Gram pos cocci in CHAINS

  • Catalase NEGATIVE

  • ALPHA (partial) hemolysis

  • NEG Quellung rxn

    • NO capsule

  • NOT bile soluble

  • Optochin RESISTANT

S. pneumo

 

​

​

​

  • Gram pos cocci in CHAINS

  • Catalase NEGATIVE

  • ALPHA (partial) hemolysis

  • POS Quellung rxn

    • ENCAPSULATED

  • BILE SOLUBLE

  • Optochin SENSITIVE

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Answer

Group A Strep:

S. pyogenes

 

  • Gram pos cocci in CHAINS

  • Catalase NEGATIVE

  • BETA (complete) hemolysis

  • BACITRACIN SENSITIVE

Group B Strep:

S. agalactiae

 

  • Gram pos cocci in CHAINS

  • Catalase NEGATIVE

  • BETA (complete) hemolysis

  • BACITRACIN RESISTANT

SENSITIVE

Clearing around disk

RESISTANT

No clearing

AEROBE

Growth near top of tube

(highest O2 concentration)

Grows in 6.5% NaCl

​

ANAEROBE

Growth at base of tube 

(lowest O2 concentration)

No growth in 6.5% NaCl

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Answer

Enterococcus

Group D Strep

 

​

  • Gram pos cocci in CHAINS

  • Catalase NEGATIVE

  • GAMMA (no) hemolysis

  • AEROBIC organism

  • Growth in 6.5% NaCl

Pepto-

streptococcus

S. bovis

 

  • Gram pos cocci in CHAINS

  • Catalase NEGATIVE

  • GAMMA (no) hemolysis

  • ANAEROBIC organism

  • No growth in 6.5% NaCl

SUMMARY

See below for how to differentiate between the various Streptococci bacteria

based on hemolytic patterns and other laboratory tests.

anaerobes

MICRO & IMMUNO 

INDEX

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