Basic Gram Positive

Bench-top Evaluation
Kevin Dieckhaus MD FIDS

Directions for this

module:

Gram Positive
Growth
Patterns on Media

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Negative

Streptococc
us

Catala
se
Test

Positive

Staphylococ
cus
Ne
g

Hemolysi
s
Pattern

Latex
Agglutinati
on

Pos

And/or
Coagula
se

BetaAlpha-hemolyticGammahemolytic
Streptococcus hemolytic
Streptococcus
Streptococcus
Bacitrac
in
Disk
YES

Strep.
pyogenes

YES

Positive

?
Enterococcus

Optochi
n
Disk
Zone of
inhibition
?

Negative

No

Strep.
pneumoniae

Viridans strep
(multiple
species)

Coagulasenegative
staph

Staph.
aureus

Staphylococcus on blood
agar

Staph epidermidis
White colony
No hemolysis (colonies
are connected)

Staph aureus
Golden color (aureus = gold)
Beta hemolysis (zone of clear
color around the colonies)

Catalase Test
Blood agar both gram positive and negative bacteria
can grow
MacConkey no growth (not a gram negative)
If gram positive (no growth on MacConkey) then must
know:
Is it staph or strep?
Staph is catalase +
Strep is catalase Catalase test place hydrogen peroxide on a slide
Introduce colonies from the blood agar plate into the
peroxide
If bubbles catalase +
Catalase + is staphylococcus

Which Staphylococcus?
Catalase +, grow on blood agar

Options:
S. aureus (coagulase +)
S. epidermis (coagulase -, novobiocin
sensitive)
S. saprophyticus (coagulase -, novobiocin
resistant)

Next test is Coagulase

Coagulase Test
Latex Agglutination
Add staphlyococcus (catalase +) colony from blood
agar plate to antibodies
If clump when mixing then coagulase + (most likely S
aureus)
On left coagulase +
Staph aureus
On right coagulase
S. Epidermis or
saprophyticus

Coagulase Test (formal method, higher sensitivity)

Inoculate staphylococcus (catalase +) with calf serum
and incubate for one hour
If it solidifies then coagulase + (most likely Staph
aureus)

Which Streptococcus?
Catalase -, grow on blood agar

Options:
S. pyogenes (, bacitracin sensitive)
S. agalacticae (, bacitracin resistant)
Group D ( or or )
E. faecalis (grows in 6.5% NaCl)
S. bovis (susceptible to 6.5% NaCl)

S. pneumoniae (, optochin susceptible)

Srep Viridans (, optochin resistant)

Next test is hemolytic growth

Hemolytic Patterns
Which Streptococcus?

Gamma
No hemolysis
See organisms but no hemolysis around the
organism (typical of enterococcos or strep bovis)

Beta
Zone of hemolysis
Can see clear zones around
small colonies

Alpha
Green (vs. translucence of beta)

Hemolytic patterns on blood

agar
(Streptococcus: catalase -)

Alpha
S. pneumoniae
or Strep
Viridans
Beta
S. pyogenes or
S. agalacticae
Gamma
E. faecalis or
S. bovis

Bacitacin Test
Catalase -, hemolytic

Determine if Strep. Pyogenes (bacitracin

sensitive)
Plate organism and put bacitracin disc in center
If see zone of inhibition around disc then
indicates strep pyogenes (bacitracin senstive)

If grows up to disc then it is beta hemolytic but

not strep pyogenes (could be S. agalactiae)

Optochin Test
Catalase - (no bubbling with peroxide), alpha hemolytic (green)

If there is a zone of inhibition around

optichin disc then probably Strep.
Pneumonia
Growth up to the disc shows
resistance to optichin so most likely
Virdians Strep

Staph or Strep

Gram Positive

- Coagulase test (add to hydrogen peroxide)

- Bubbles (coagulase +) then Staph
- No bubbles (coagulase -) then Strep

Which Staph?
- Catalase test (check for coagulation with Ab or serum)
- Coagulation (catalase +) then Staph aureus
- No coagulation (catalase -) then Staph epidermidis or saprophyticus

Which Strep?
- Hemolytic test
- Alpha (green)
- Beta (zone of hemolysis)
- Gamma (no hemolysis) E. faecalis or S. bovis

Which beta hemolytic Strep?

- Bacitracin sensitive (zone of inhibition) then Strep pyogenes

Which alpha hemolytic Strep?

- Otpochin sensitive (zone of inhibition) then S. pneumoniae
- Optochin resistant (grow up to disc) then Viridans (S. mutans or intermedius)