Bacterial testing is the foundational process of microbiology, designed to identify, quantify, and characterize microorganisms present in a sample. This analysis is required across many fields, from diagnosing infections to monitoring environmental quality and ensuring food safety. Determining what bacteria are present allows scientists to make informed decisions about public health interventions, medical treatments, or process control. The methodology has evolved from simple visual observation to highly complex molecular techniques.
Culturing and Initial Identification
The first step in characterizing a bacterium often involves growing it in a laboratory setting, a process called culturing. Samples are spread onto solid agar plates or introduced into liquid broths containing the specific nutrients, pH, and temperature needed for the bacteria to multiply. On solid media, a single bacterium replicates into a visible mass known as a colony, allowing for the isolation of a pure culture for further testing.
Once a colony is isolated, initial classification is performed using microscopy and Gram staining. This differential stain separates bacteria into two large groups based on their cell wall structure. Gram-positive bacteria, which have a thick layer of peptidoglycan, retain the crystal violet dye and appear purple. Gram-negative bacteria, possessing a thinner peptidoglycan layer, take up a pink or red counterstain after the alcohol wash. Microscopy also reveals the organism’s morphology, classifying it by shape as cocci (spherical), bacilli (rod-shaped), or spirilla (spiral).
Biochemical and Metabolic Analysis
Following the initial Gram stain and shape determination, identification is refined by analyzing the bacterium’s metabolic capabilities. These biochemical tests look for the presence or absence of specific enzymes and the ability to break down certain compounds. For instance, the catalase test detects the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen gas, seen as visible bubbling. This reaction helps differentiate genera, such as the catalase-positive Staphylococcus from the catalase-negative Streptococcus.
Another marker is the oxidase test, which determines if a bacterium possesses the enzyme cytochrome c oxidase. Tests for sugar fermentation are also common, where bacteria are grown in media containing carbohydrates and a pH indicator; a color change signals acid production. Modern laboratories frequently use commercial multi-test systems containing a dozen or more miniaturized biochemical tests in a single strip or plate. These systems generate a unique numerical profile that is entered into a database for final species-level identification.
DNA and RNA Based Identification
Molecular methods provide the most specific and fastest means of bacterial identification, focusing on the organism’s genetic material. Polymerase Chain Reaction (PCR) rapidly amplifies specific segments of bacterial DNA, allowing for the detection of tiny quantities of genetic material directly from a sample. This speed is advantageous for diagnosing acute infections where timely results are important for patient care. PCR is particularly useful for bacteria that are difficult or impossible to grow in a culture dish, often called non-culturable organisms.
For definitive species identification, scientists often sequence the 16S ribosomal RNA (rRNA) gene. This gene is found in all bacteria and contains regions that are highly conserved across all species, alongside regions that vary significantly between species. By sequencing the 16S rRNA gene, a bacterium’s genetic signature can be compared against public databases for accurate identification, even of novel species. This method is considered a gold standard in bacterial taxonomy because it offers accurate classification independent of the organism’s growth characteristics.
Determining Effective Treatments
Once a bacterium is identified, the next step, especially in clinical settings, is determining which antibiotics will be effective against it. This process is called Antibiotic Susceptibility Testing (AST). Identification alone is insufficient because bacteria within the same species can exhibit different levels of drug resistance.
One common AST method is the Kirby-Bauer disk diffusion test. Antibiotic-impregnated paper disks are placed on an agar plate inoculated with the bacterial isolate. After incubation, the antibiotic diffuses into the agar, and a clear area around the disk, known as the zone of inhibition, indicates that the antibiotic prevented bacterial growth. The size of this zone determines if the bacterium is susceptible, intermediate, or resistant to the drug.
Minimum Inhibitory Concentration (MIC)
A more quantitative approach is the Minimum Inhibitory Concentration (MIC) test, which uses serial dilutions of an antibiotic in broth culture. The MIC value is the lowest concentration of the drug that prevents visible growth of the bacterium, providing a precise measure of potency that guides the selection and dosage of treatment.