Gram-Positive Bacteria Identification: Introduction, List of Common Bacteria, and Identification Keys

Introduction of Gram-Positive Bacteria Identification

Identification of Gram-positive bacteria is a little bit harder than Gram-negative bacteria since the most common bacterial etiological agents are Gram-negative bacteria and the vendors or suppliers are directly involved in supplying common tests reagents and test kit accessories. Due to both reagent unavailability and cumbersome technique for Gram-positive bacteria, isolation and identification are missed in the clinical laboratory. e.g. Corynebacterium species, Bacillus species, Staphylococcus species, Listeria species, etc.

List of Common Gram-Positive Bacteria

• Staphylococcus aureus
• Staphylococcus saprophyticus
• Staphylococcus epidermis
• Enterococcus species
• Streptococcus pneumoniae
• Streptococcus pyogenes
• Streptococcus agalactiae
• Corynebacterium diphtheriae
• Listeria monocytogenes
• Bacillus anthracis

Gram-Positive Bacteria Identification Keys

Staphylococcus aureus

Colony Morphology: Pinhead golden yellow or non-pigmented, beta-hemolytic or non-hemolytic colonies on blood agar

Beta-hemolytic colonies of Staphylococcus aureus

Catalase test: Positive

Gram stain: Gram-positive cocci in singles, pairs, and clusters

Coagulase test: Positive

Coagulase Test- Positive (Slide method)

S. aureus Growth on Mannitol salt agar- yellow colonies

Staphylococcus saprophyticus

Colony Morphology: Pinhead, non-pigmented, and non-hemolytic colonies on blood agar

Catalase test: Positive

Gram stain: Gram-positive cocci in singles, pairs, and clusters

Coagulase test: Negative

Growth on Mannitol salt agar: Pink colonies

Novobiocin Susceptibility Test: Intrinsic resistance to the novobiocin

Result Interpretation of Novobiocin Susceptibility Test

Sensitive – A zone of inhibition greater than 16 mm
Resistant – A zone of inhibition less than or equal to 16 mm

Note-The novobiocin disk is not helpful and can give misleading results if it is performed on isolates other than those from urinary samples. It is presumptive identification and thus biochemical, and immunological tests are further recommended for the confirmation of organisms.

Staphylococcus epidermis

Colony Morphology: Pinhead,non-pigmented, and non-hemolytic colonies on blood agar

Catalase test: Positive

Gram stain: Gram-positive cocci in singles, pairs, and clusters

Coagulase test: Negative

Growth on Mannitol salt agar: Pink colonies

Novobiocin Susceptibility Test: Sensitive to the novobiocin

Note-It is presumptive identification and thus biochemical, immunological tests are further recommended for the confirmation of organisms.

Enterococcus species

Fig. Colony Morphology: Pinpoint, non-pigmented, and non-hemolytic colonies on blood agar

Catalase test: Negative

Gram stain: Gram-positive cocci in singles, pairs, and short chains

Bile esculin Test: Positive

Note-It is presumptive identification and thus biochemical, immunological tests are further recommended for the confirmation of organisms.

Streptococcus pneumoniae

Colony Morphology: Pinpoint and alpha-hemolytic colonies on blood agar

Gram stain of specimen

Gram-positive elongated (lanceolate) diplococci may show evidence of capsule but no evidence of spore.

Gram-positive elongated (lanceolate) diplococci of Streptococcus pneumoniae in Gram-stained smear of culture

Note-

• Absence of capsules in Gram stain if organisms from cultures.
• It also forms short chains in Gram stain particularly the following culture but lacking such features in specimens.

Optochin susceptibility test: Positive

Note-It is presumptive identification and thus biochemical, immunological tests are further recommended for the confirmation of organisms.

Streptococcus pyogenes

Colony Morphology: Pinpoint, beta-hemolytic colonies on blood agar

Catalase test: Negative

Gram stain: Gram-positive cocci in singles, pairs, and chains

Bacitracin (0.04U) susceptibility test: Positive

Note-It is presumptive identification and thus biochemical, immunological tests are further recommended for the confirmation of organisms.

Streptococcus agalactiae

Colony Morphology: Pinpoint, beta-hemolytic colonies on blood agar

Catalase test: Negative

Gram stain: Gram-positive cocci in singles, pairs, and chains

Bacitracin (0.04U) susceptibility test: Negative

CAMP Test: Positive

Note-It is presumptive identification and thus biochemical, immunological tests are further recommended for the confirmation of organisms.

Corynebacterium diphtheriae

Culture: Black colony on tellurite blood agar

Albert’s stain: Metachromatic granules of Corynebacterium diphtheria stain purple-black against the light green counterstained cytoplasm
Biotype test
Molecular assay: PCR for diphtheria tox gene
Elek test (diphtheria toxin production): Positive

Listeria monocytogenes

• Specimens- Blood, CSF, amniotic fluid, pus, swabs from cervical and vaginal secretions, meconium, cord blood, etc.
• Direct Microscopy-In Gram stain smears of CSF sediments, the bacteria may be seen as Gram-positive coccobacilli.
• Culture- Blood agar shows small colonies surrounded by a narrow zone of β-hemolysis as shown above picture.
• Biochemical tests-Catalase test: Positive, Indole, oxidase, and urea hydrolyzation test: Negative. It ferments glucose and maltose with acid production and CAMP test: Positive.
• The motility test of L. monocytogenes shows tumbling motility.

Note-The characteristics motility and cultural characteristics of L. monocytogenes are usually sufficient to identify it without the need to use many biochemical tests.

Bacillus anthracis

Fig. Colony morphology: Colonies exhibit “tenacity” when teased up with a loop

Gram’s stain: Gram-positive bacilli with spores – look like railway carriages end-to-end
Polychrome Methylene Blue stain: Blue bacilli and spores surrounded by a thick red capsule (when taken from blood samples or Bicarbonate agar)
Motility: Non-motile
Growth on Blood Agar: No hemolysis in young cultures
Growth on Bicarbonate agar in CO2: Smooth mucoid colonies
Gamma phage lysis: Inhibition of growth

Note-

• The two medically important Bacillus species are Bacillus anthracis and Bacillus cereus.
• Bacillus anthracis is non-motile while most other Bacillus species are motile.

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