AAT Bioquest

How are microbes identified?

Posted September 18, 2023


There are several methods that can be used to identify microbes including: 

  • Morphological Characteristics: Microbes can be initially identified by their size, shape, and cellular structures when observed either with the naked eye or under a microscope. The type of microbe can be identified by observing features such as cell shape, size, and arrangement, and presence of flagella, capsules, or spores.

  • Staining and Microscopy: There are various types of stains that enable easier visualization and identification of microbes under a microscope. Some of the more popular microbiological stains include: 

    • Gram staining, a purple stain that helps to differentiate between Gram-positive and Gram-negative bacteria

    • Endospore staining checks for the presence of spores, which helps to separate bacteria that produce spores from those that don’t produce spores. 

    • Ziehl-Neelsen staining uses red stain carbol fuchsin first, followed by a counterstain such as methylene blue to identify Mycobacterium tuberculosis (TB) which cannot be Graim-stained. 

  • Polymerase Chain Reaction (PCR): PCR amplifies specific DNA regions, facilitating faster detection and identification of microbial species directly from clinical samples. This helps to speed up diagnostic procedures. PCR is among the most widely used molecular methods for identifying microbes. 
  • Simple Biochemical Tests: Microbes possess distinct biochemical properties due to their metabolic processes. Testing for the presence or absence of specific enzymes, metabolic pathways, or substrate utilization patterns can aid in identification. These tests involve adding specific substrates to cultures and observing color changes, gas production, catalase activity, tolerance to acidic media, optimum growth temperature, or other reactions.
  • Microarray-Based Identification: This technique involves binding pre-amplified microbial DNA to specific probes on an array, where each probe emits unique fluorescence upon binding. Microarrays are versatile, enabling simultaneous detection and differentiation of various microbial samples on a single slide. This swift technique is crucial in clinical scenarios for prompt diagnosis and appropriate antimicrobial treatment. 
  • Immunological Identification: This method involves the use of species-specific ELISA techniques primarily used in diagnostic applications. While these techniques are highly sensitive, their success depends on highly discriminating proteins within the target organism.  
  • Dichotomous Identification Keys: Dichotomous keys involve a series of steps with each step presenting paired characteristics such as Gram-positive or Gram-negative. Making a selection at each step leads to the next pair of choices, which helps to narrow down the possibilities, ultimately arriving at the correct identification. 
  • Fatty Acid Profiling: Performed using a combination of mass spectrometry and gas chromatography, this technique is based on the principle that different bacterial species produce different combinations of fatty acids, which are located within bacterial cell membranes. Fatty acid profiling helps to identify unknown bacterial species by comparing against known profiles. 
Additional resources

Medical Microbiology. 4th edition. Chapter 3 Classification

Polymerase Chain Reaction (PCR)

Helixyte™ Green *20X Aqueous PCR Solution*