Bacterial identification begins with the agar plate, a foundational tool in microbiology. These plates hold a nutrient-rich medium solidified with agar, providing a supportive environment for microorganisms to grow and form visible colonies. Accurate bacterial identification provides information crucial for public health, medical diagnostics, and food safety. The systematic identification of an unknown bacterium moves through distinct phases, starting with visual assessment on the plate, progressing to microscopic confirmation, and concluding with functional biochemical tests. This multi-step approach allows scientists to narrow down the organism’s identity to a single species.
Macroscopic Clues: Analyzing Colony Morphology
The first step in identification involves a detailed visual examination of the bacterial colonies directly on the agar surface, known as morphology analysis. The overall shape of a colony is a defining characteristic, often described with terms like circular, irregular, filamentous (thread-like), or punctiform (pinpoint in size). The edge of the colony, or the margin, is also observed and can be categorized as entire (smooth), undulate (wavy), or lobate (lobed).
Observing the colony from the side provides information about its elevation above the agar, which might be flat, raised, convex (dome-shaped), or umbonate (having a raised center). The surface texture is another clue, with descriptions ranging from shiny and smooth to dull, wrinkled (rugose), or mucoid if the colony is slimy. Color and opacity are similarly noted, where pigmentation may be white, opaque, or a specific color, and the colony’s transparency can be opaque, translucent, or transparent.
A specialized observation involves the use of blood agar, a medium enriched with blood, to test for hemolysis, the bacterium’s ability to break down red blood cells. Complete lysis, or \(\beta\)-hemolysis, results in a clear zone surrounding the colony where all red blood cells have been destroyed. Partial breakdown, known as \(\alpha\)-hemolysis, is indicated by a greenish or brownish discoloration around the colonies. If no visible change occurs in the agar surrounding the colony, the reaction is termed \(\gamma\)-hemolysis, signifying a lack of the hemolytic enzymes.
Microscopic Insights: Using Staining Techniques
Following the macroscopic assessment, a small sample of the colony is used for microscopic examination, with the Gram stain being the most informative initial test. This differential staining technique separates bacteria into two large groups based on the structural properties of their cell walls. The procedure involves four steps:
- Applying the primary stain crystal violet.
- Adding a mordant (iodine) to form a large crystal violet-iodine complex.
- Using an alcohol-based decolorizer.
- Applying the counterstain safranin.
The distinction lies in the thickness of the bacteria’s peptidoglycan layer. Gram-positive bacteria possess a thick peptidoglycan layer that shrinks upon exposure to the alcohol decolorizer, effectively trapping the crystal violet-iodine complex within the cell. As a result, Gram-positive cells retain the primary stain and appear purple or violet under the microscope.
In contrast, Gram-negative bacteria have a much thinner peptidoglycan layer sandwiched between the cytoplasmic membrane and an outer membrane. The alcohol decolorizer dissolves the outer membrane and causes the crystal violet-iodine complex to wash out. These decolorized cells then take up the counterstain, safranin, causing them to appear red or pink.
The microscopic view also reveals the cell’s morphology, which is the shape and arrangement of individual cells. The three basic shapes are coccus (spherical), bacillus (rod-shaped), and spirillum (spiral or curved). These shapes organize in characteristic patterns: cocci in pairs are diplococci, in chains are streptococci, and in grape-like clusters are staphylococci. Identifying both the Gram reaction and the cellular morphology provides a strong preliminary classification, allowing the scientist to move toward more specific functional tests.
Biochemical Confirmation: Functional Testing
After narrowing the possibilities with morphology and Gram staining, the final stage of identification relies on a suite of biochemical tests to determine the organism’s unique metabolic capabilities. These tests look for the presence of specific enzymes or the ability to utilize certain nutrients. The Catalase test detects the enzyme catalase, which protects the bacterium from the toxic byproducts of aerobic metabolism.
To perform the Catalase test, a small sample of the bacterium is mixed with hydrogen peroxide. A positive result is indicated by the rapid, visible formation of oxygen bubbles, demonstrating the enzymatic breakdown of the peroxide. This test is useful for distinguishing the catalase-positive Staphylococcus species from the catalase-negative Streptococcus species within the group of Gram-positive cocci.
Another standard test is the Oxidase test, which identifies bacteria possessing the enzyme cytochrome c oxidase, a component of the electron transport chain. The Oxidase test involves introducing the bacterium to a reagent that changes color when oxidized; a positive result is a quick change to a deep blue or purple color. This helps distinguish oxidase-positive organisms like Pseudomonas from oxidase-negative organisms such as those in the Enterobacteriaceae family.
Fermentation tests are also employed, using media containing a specific carbohydrate, like lactose, and a pH indicator. If the bacterium ferments the sugar, it produces acid end-products, which lower the medium’s pH and cause the indicator to turn from red to yellow.
For complex identification, many laboratories use miniaturized systems like the Analytical Profile Index (API) strips. These strips contain multiple dehydrated substrates for up to twenty different biochemical tests in a single strip. The results from these tests—a pattern of positive and negative reactions—are combined to generate a numerical profile that is then matched against a reference database, leading to the definitive species identification.