Colony morphology is a visual key and diagnostic tool for identifying bacterial species based on their growth characteristics on solid media. In pure culture, derived from single-cell or clonal populations, bacterial colonies typically exhibit uniform morphological characteristics that are visible macroscopically. Although these visual features provide valuable initial information for identification, additional confirmatory testing is required for definitive species determination due to the potential phenotypic variation, genetic diversity, and overlapping morphological characteristics among different bacterial species.
This guide covers detailed observation techniques, variations in colony appearance, and their clinical significance.
Proper culture techniques are essential for identifying morphological characteristics of pure bacterial cultures. Media selection and preparation are important steps in this process. While general purpose media such as nutrient agar and tryptic soy agar (TSA) support the growth of most microorganisms, selective and differential media are required for specific identifications. Examples include:
- McConkey agar (for Gram-negative bacteria),
- Blood agar (for hemolysis studies),
- Mannitol salt agar (for Staphylococcus),
- SS agar (for Salmonella and Shigella).
Streaking techniques are used to obtain isolated colonies, and the method chosen depends on the bacterial species and research needs. Common streaking methods include:
- Quadrant streaking (four-quadrant method) – The most widely used technique, in which the agar plate is divided into four sections. The inoculum is streaked continuously, decreasing the bacterial density in each quadrant to obtain isolated colonies.
- T-streaking – Similar to quadrant streaking but divides the plate into three sections in a “T” shape.
- Continuous streaking – The inoculum is spread continuously from one edge to the center without lifting the ring.
- Radial streaking – The inoculum is streaked vertically and then diagonally for even distribution.
Optimal incubation conditions must be maintained for accurate results:
- Temperature: 37°C (for most pathogens) or 25–30°C (for environmental bacteria).
- Duration: 18–24 hours (extended to 48–72 hours for slow-growing organisms such as Mycobacterium).
- Atmosphere: Aerobic, anaerobic, or CO₂-enriched (5–10% for fastidious bacteria).
2. Macroscopic Colony Characteristics
Examine colonies under good lighting and use a stereomicroscope for finer details.
A. Size (Colony Diameter)
| Category | Size Range | Examples |
|---|---|---|
| Pinpoint | <0.5 mm | Streptococcus pneumoniae |
| Small | 0.5–1 mm | Haemophilus influenzae |
| Medium | 1–3 mm | E. coli, Staphylococcus |
| Large | >3 mm | Bacillus, Proteus |
B. Shape & Form
| Type | Description | Example Bacteria |
|---|---|---|
| Circular | Perfectly round | Staphylococcus aureus |
| Irregular | Uneven edges | Bacillus anthracis |
| Filamentous | Thread-like extensions | Nocardia |
| Rhizoid | Root-like spreading | Bacillus mycoides |
| Spindle-shaped | Elongated, tapered ends | Fusobacterium |
C. Margin (Edge) Characteristics
| Margin Type | Appearance | Example Bacteria |
|---|---|---|
| Entire | Smooth, even border | E. coli |
| Undulate | Wavy edge | Proteus mirabilis |
| Lobate | Lobed, flower-like | Mycobacterium |
| Filamentous | Thin, spreading strands | Actinomyces |
| Erose | Irregular, jagged | Corynebacterium |
D. Elevation (Side View)
| Elevation Type | Description | Example Bacteria |
|---|---|---|
| Flat | No elevation | Shigella |
| Raised | Slightly elevated | Klebsiella |
| Convex | Dome-shaped | Staphylococcus |
| Umbonate | Raised center, flat edges | Salmonella |
| Crateriform | Depressed center | Yersinia pestis |
E. Surface Texture
| Texture | Description | Example Bacteria |
|---|---|---|
| Smooth | Glossy, moist | E. coli |
| Rough | Dry, granular | Mycobacterium tuberculosis |
| Mucoid | Slimy (due to capsule) | Klebsiella pneumoniae |
| Wrinkled | Folded (like cauliflower) | Bacillus cereus |
| Dull | Matte, non-reflective | Streptococcus pyogenes |
F. Color & Pigmentation
| Color | Pigment Type | Example Bacteria |
|---|---|---|
| White | Non-pigmented | Staphylococcus epidermidis |
| Yellow | Staphyloxanthin | Staphylococcus aureus |
| Golden | Carotenoid pigments | Micrococcus luteus |
| Green | Pyocyanin (fluorescent) | Pseudomonas aeruginosa |
| Red/Pink | Prodigiosin | Serratia marcescens |
| Black | H₂S production (FeS) | Salmonella Typhi (on XLD) |
G. Opacity
| Type | Description | Example Bacteria |
|---|---|---|
| Opaque | Light does not pass through | Staphylococcus |
| Translucent | Partial light transmission | Proteus |
| Transparent | Almost clear | Haemophilus |
H. Hemolysis (Blood Agar)
| Hemolysis Type | Appearance | Example Bacteria |
|---|---|---|
| Alpha (α) | Greenish partial lysis | Streptococcus pneumoniae |
| Beta (β) | Clear complete lysis | Streptococcus pyogenes |
| Gamma (γ) | No hemolysis | Enterococcus faecalis |
3. Special Colony Morphologies
A. Swarming Motility
Proteus mirabilis: Forms concentric waves on moist agar.
Clostridium tetani: Thin, spreading growth.
B. “Fried Egg” Colonies
Mycoplasma: No cell wall, embedded in agar.
C. “Medusa Head” Colonies
Bacillus anthracis: Curled, hair-like projections.
D. “Ground Glass” Colonies
Mycobacterium tuberculosis: Rough, granular appearance.
4. Clinical & Diagnostic Significance
Staphylococcus aureus (golden yellow, β-hemolytic) vs. Staph. epidermidis (white, non-hemolytic).
E. coli (pink on MacConkey, lactose fermenter) vs. Salmonella (colorless, non-lactose fermenter).
Pseudomonas aeruginosa (green, grape-like odor) vs. Klebsiella (mucoid, lactose fermenter).
