Diagnostic microbiology

Diagnostic microbiology is the scientific study of microbial identification. This field focuses on harvesting and observing specific organisms to understand their functions. By identifying these microbes, physicians and scientists can provide more accurate diagnoses for patients. This process relies on comparing unknown organisms to a reference of previous studies. Scientists use various methods to find differences between species. These differences allow them to determine exactly what an organism is.

One primary method involves culturing microbes in a controlled environment. Microbes can be aerobic or anaerobic. Anaerobic organisms require an environment that is completely free of oxygen. To grow these, scientists often flush broths with nitrogen gas to remove oxygen. They may also use a specialized chamber to prevent oxygen from entering. Sodium resazurin can be added to the medium to indicate the redox potential. These anaerobic cultures are typically incubated at 35 °C for 48 hours before growth is examined.

Microbial growth follows a specific pattern known as a growth curve. This curve consists of four distinct stages: the lag, log, stationary, and death phases. During the lag phase, microbes adjust to their environment by synthesizing specific proteins. Next is the log phase, where the culture experiences rapid, logarithmic growth. In the stationary phase, the concentration of cells is at its highest, but reproduction stops. Finally, the death phase occurs when nutrients are depleted and toxic metabolites become abundant. Because of these stages, incubation times vary significantly. For example, Escherichia coli requires less than 24 hours of culture time. In contrast, Mycobacterium tuberculosis may require 6 to 8 weeks to show results.

Modern technology has introduced much faster ways to identify microbes. DNA-based PCR diagnostics provide faster results than traditional overnight biochemical tests. These DNA tests match the specificity of biochemical tests in about 90% of cases. Another advanced tool is MALDI-TOF, which stands for matrix-assisted laser desorption/ionization-time of flight. This is a specific type of mass spectrometry used for rapid identification. A scientist places a pure culture on a target and covers it with a matrix. A laser then ionizes the protein complexes, which travel through a vacuum. A detector measures them based on their mass and charge. The resulting protein spectra are compared to a known database. For best results, the culture must be less than 72 hours old. This ensures the microbes are in the log phase with many ribosomal proteins available.

Optical and immunological methods provide other ways to see microbes. Immunofluorescence involves using anti-antibodies that have a fluorescent molecule attached. These molecules are chemiluminescent, meaning they glow when they are hit with ultraviolet light. Scientists can also use light scattering to detect and classify bacteria. This involves analyzing how light bounces off the cells at different angles. Spectroscopic methods, such as elastic light scattering, can even identify bacteria directly from whole blood. This provides a very fast way to analyze a patient sample.

Biochemical tests are also used to look at the metabolic pathways of microbes. These tests often use indicators that change color when a specific reaction occurs. For example, the catalase test checks for an enzyme that breaks down hydrogen peroxide. If bubbles form on a glass slide, the test is positive. The 6.5% salt broth test determines if bacteria can tolerate high salt levels. This helps identify species like Staphylococci or Enterococci. The acetate utilization test helps distinguish Escherichia coli from Shigella. In this test, an indicator changes color when the pH increases due to acetate use.

Scientists also use antibiotic disks to test for drug susceptibility. This method is commonly used with Mueller–Hinton agar. A scientist seeds bacteria across a petri dish and places an antibiotic-treated disk on top. If the antibiotic works, a clear ring called a zone of inhibition forms. This ring shows where the bacteria could not grow. This helps doctors choose the most effective medicine for an infection.

Other specialized tests include the CAMP test, which identifies Streptococcus agalactiae. This test looks for a specific substance that causes a characteristic arrowhead shape on blood agar.

By combining these many different tools, diagnostic microbiology provides a complete picture of the microbial world.