Gross Colony Characteristics Used to Differentiate and Identify Presumptively Microorganisms
Microbiologist can easily identify or guess an isolate by observing the colony characteristics of the organisms that have been isolated. The descriptions that follow are commonly used to investigate colony characteristics. Many of the colony characteristics listed below may differ between species and strains of the same genus.
Hemolysis
On sheep blood agar , hemolysis observed in the medium immediately surrounding or underneath the colony is caused by bacterial enzymatic or toxin activity. Hemolysis (e.g. α, β, or no hemolysis with other colony characteristics) on sheep blood agar aids in presumptive identification, particularly of streptococci and enterococci. It is critical to decide whether true hemolysis is present or whether the discoloration of the medium is due to the organism’s growth on the plate. To visualize the hemolytic pattern, the colony is often removed with a loop. To decide if the organism is hemolytic, proper technique necessitates the passage of bright light through the bottom of the plate (transillumination) as seen below. Many species have no lytic effect on RBCs in sheep blood agar and are referred to as nonhemolytic.
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α-Hemolysis is the partial lysis of RBCs in an sheep blood agar plate around and underneath the colony, which causes the medium to turn green. Streptococcus pneumoniae and some viridans streptococci are examples of species that cause α-hemolysis.
β -Hemolysis
β-Hemolysis is the complete removal of erythrocytes in sheep blood agar around or underneath colonies as a result of RBC lysis. Many species, such as Streptococcus pyogenes, produce a broad, deep, clear zone of β-hemolysis, while others, such as Streptococcus agalactiae and Listeria monocytogenes (a gram-positive rod), produce a small, diffuse zone of β-hemolysis close to the colony. These characteristics may aid in the identification of specific bacteria species. Since the RBCs in the medium is lysed during media preparation, CHOC agar does not display hemolysis. On CHOC agar, organisms that are α -hemolytic or β -hemolytic on sheep blood agar typically have a green coloration around the colony. This coloration, however, should not be confused with a hemolytic characteristic.
Size
Colonies are classified as large, medium, small, or pinpoint. A microbiologist, on the other hand, often takes a ruler and measures a colony. In most cases, size is a visual contrast between genera or species. Gram-positive bacteria, for example, tend to grow smaller colonies than gram-negative bacteria. Staphylococcus species are typically larger than Streptococcus species.
Form or Margin
The colonies’ edges should be examined, and the shape, or margin, should be defined as smooth, filamentous, rough or rhizoid, or irregular. Bacillus anthracis colonies are visually characterized as “Medusa heads” due to their filamentous appearance. Certain Proteus genera, especially Proteus mirabilis, can swarm on nonselective agar such as blood or CHOC agar. Swarming is a hazy blanket of development that stretches well beyond the streak lines on the soil. Diphtheroids produce colonies, with the exception of group B (S. agalactiae), which is defined as translucent. S. agalactiae forms semiopaque colonies of species clustered in the middle, which is often referred to as a bull’s-eye colony. The majority of staphylococci and other gram-positive bacteria are opaque. The majority of gram-negative rods form opaque colonies. On blood-containing media, Bordetella pertussis is defined as shiny, resembling a half-pearl.
Illustration of form or margin to describe colony
morphology
Swarming colonies of Proteus spp. The organism was
inoculated in the middle of the sheep blood agar plate (arrow)
Elevation
Elevation can be measured by tilting the culture plate and looking at the colony’s edge. Elevation may be raised, convex, flat, umbilicate (concave base, depressed center), or umbonate (raised or bulging center, convex). Unless the colonies are mucoid due to the presence of a polysaccharide capsule, S. pneumoniae usually produces umbilicate colonies. S. aureus colonies are usually convex. β-hemolytic streptococci, on the other hand, typically form flat colonies.
Density
The colony’s density may be clear, translucent, or opaque. It is useful to look at the colony by using transillumination to see the variations in colony density. Translucent colonies let some light through, while opaque colonies do not. Except for group B (S. agalactiae), β-hemolytic streptococci are classified as translucent. S. agalactiae forms semiopaque colonies of species clustered in the middle, which is often referred to as a bull’s-eye colony. The majority of staphylococci and other gram-positive bacteria are opaque.
Color, as opposed to pigmentation, is a term used to describe a specific genus in general. Colonies may be white, gray, yellow, or buff in color. Staphylococci that lack coagulase are white, while Enterococcus spp. can be gray. Nonpathogenic Micrococcus spp. and Neisseria spp. are yellow or off-white. Diphtheroids have a buff appearance. On sheep blood agar, the majority of gram-negative rods are gray.
staphylococci on blood agar plate.
Consistency
Touching the colony with a sterile loop determines consistency. Colony consistency can range from brittle (splinters) to creamy (butyrous), dry, or waxy; on rare occasions, the entire colony adheres (sticks) to the loop. Certain Neisseria spp. are sticky, while S. aureus is creamy. Colonies of Nocardia spp. are brittle, crumbly, and wrinkled, resembling bread crumbs on a plate. Diphtheroid colonies are usually waxy and dry. Except for mucoid forms, most β -hemolytic streptococci are dry, and when moved by a loop, the entire colony remains intact.
Pigment
Pigment formation is an intrinsic feature of a particular organism that is usually confined to the colony, though some pigments will diffuse through the culture medium. Growing bacteria at room temperature improves pigment production in general. The following are some examples of species that contain pigment:
- P. aeruginosa—green, sometimes a metallic sheen
- Serratia rubidaea—brick red especially at room temperature
- Kluyvera spp.—blue
- Chromobacterium violaceum—purple
- Prevotella melaninogenica—brown-black (anaerobic) Pigment production for these organisms is variable.
Odor
When the lid of the culture plate is removed and the odor dissipates into the surrounding atmosphere, the odor should be determined. Never inhale directly from the plate as a microbiologist. The following are some examples of microorganisms that emit distinct odors:
- S. aureus—old sock (stocking that has been worn continuously for a few days without washing); this odor is evident when bacteria are growing on mannitol salt agar
- P. aeruginosa—fruity or grapelike
- P. mirabilis—putrid
- Haemophilus spp.—musty basement, “mousy” or “mouse nest”smell
- Nocardia spp.—freshly plowed field
Colonies with Multiple Characteristics
Other bacteria, in addition to the species already described, fit into several descriptive categories of colony morphology. On SBA, Bacillus cereus forms large, rough, greenish, hemolytic colonies. On SBA or CHOC agar, Eikenella corrodens forms a small, “fuzzy-edged” colony with an umbonate center. In the agar, roughly half of the strains can corrode or form pits.
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