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Crash Course in Microbial Identification

Welcome to this crash course in microbial identification methods! Here, you will get an overview of the traditional and modern methods available for the identification of bacteria, yeast or filamentous fungi to the species level.

Species level identification allows you to discriminate confidently between two species from the same genus, something that is often essential in the treatment of infectious diseases. For example, the bacterial genus Yersinia contains approximately 15 species, some of which form part of the normal human microflora, while others are serious pathogens and require medical treatment, e.g., Yersinia pestis (the causative agent of bubonic plague).

But before we get stuck in the methods, let’s look at a few of the main uses for accurate microbial identification:

  • Healthcare – accurate and fast identification of bacteria, fungi and parasites for correct and timely disease diagnosis, and appropriate treatment.
  • Epidemiology – for the tracking and tracing of disease spread and outbreaks, as well as the identification of new isolates, e.g., antibiotic resistant isolates.
  • Pharmaceutical industry – because microbes are a significant threat to sterility, accurate identification of environmental microbes is often a good manufacturing practice (GMP) requirement within the pharmaceutical industry.
  • Additional scenarios – include criminal investigation, microbial forensics (the investigation of bioterrorism threats), and environmental studies.

Traditional vs. Modern Methods

Traditional methods for microbial identification rely on phenotypic identification using staining, culturing and simple biochemical tests. Nowadays, newer and more powerful molecular, immunological, and biochemical analytical methods complement and sometimes replace traditional methods.

Traditional Methods – Macroscopic Features

Macroscopic features encompass the overall appearance of a microorganism, including its shape, size, color and smell. You should think of macroscopic features as those you can see with the naked eye. By examining the gross morphological/macroscopic features on an agar culture, you can often determine the type of microorganism.

A General Guide to Examining Agar Cultures

  • Filamentous fungi or molds appear as ‘hairy’ irregularly shaped colonies and often produce visible spores that may look powdery or dusty. Fungal colonies can contain more than one color, usually with a darker color in the center (often raised center) and a lighter color radiating from the center. Filamentous fungi grow radially from the center of an agar place, with the youngest on the outside, and the older darker material (rich with spores) on the inside. Filamentous fungi may also grow as a unicolored furry mat with no obvious sign of spores at all!
  • Bacteria often form distinct colonies, sometimes smaller than fungal colonies, which can be anything from slimy to very dry in texture. They range in color from white to bright red!
  • Bacteria often have a strong odor while filamentous fungi can be odorless or earthly smelling.
  • Yeast can be the trickiest to identify based on macroscopic features, because colonies can often look similar to bacterial colonies, depending on the species and type of agar used.
  • Note that the same species can appear differently depending on the culture media. Watch our for more details in a future article!

Traditional Methods – Microscopic Features

Ask yourself the following questions when looking at your unidentified species under the microscope. They can take you a long way in the identification process!

  • Are you looking at rods, cocci or spiral shaped bacteria?
  • Are you looking at bacterial cells with flagella?
  • Are you looking at budding yeast?
  • Are you looking at a filamentous fungus with branching hyphae?

Staining and Microscopy

Most often, researchers apply stains to microbial samples for easier visualization under a microscope. Covering all microbiological stains is beyond the scope of this article, but here is an overview of the most popular ones:

Gram Staining

Gram staining is often the first go-to test in bacterial identification. This purple stain, based on the crystal violet dye, is named after the Danish bacteriologist Hans Christian Gram, who developed it.

Typical Gram-positive bacteria include Bacillus, Staphylococcus, Streptococcus and Clostridium spp. while Escherichia, Helicobacter and Salmonella spp. are Gram-negative. Bear in mind that certain bacteria are Gram-variable and are, therefore, not amenable to Gram-staining.

Endospore Staining

This technique involves applying a stain to a bacterial sample to check for the presence of spores. Because not all bacteria produce spores, this information can be useful in identification. Several spore stains are available, and malachite green is probably the most popular one.

Ziehl-Neelsen Staining

This is an important tool for the staining of Mycobacterium tuberculosis (TB), which cannot be Gram-stained. The red stain carbol fuchsin is first used and followed by a counter stain, such as methylene blue. M. tuberculosis stain red while other bacteria stain blue.

Stains for Fungi and Yeast

Several fungal stains exist, although these are generally non-specific in nature.  They help visualize fungal elements for identification rather than discriminate between fungal species. Examples of fungal stains include:

  • Lactophenol cottonblue – blue stain which stains carbohydrates present in fungal cell walls.
  • Periodic-acid Schiff stain (PAS stain) – this stains carbohydrates and other moieties and leads to a magenta color in living fungi only. This is used in infection diagnosis.
  • Grocott’s methenamine silver stain colors fungal cell walls brown to black in color, but cannot distinguish between live or dead material.
  • Trypan blue, analine blue, and calcofluor white stain fungal and plant structures within the study of plant/fungal symbiosis and plant pathology.

Traditional Methods – Simple Biochemical Tests

Catalase Testing

  • If the unidentified bacterial species has catalase activity, bubbles of oxygen appear when hydrogen peroxide is added to a scraping of bacteria on a microscope slide.
  • Staphylococci, Micrococci, E. coli and the other Enterobacteriacaea as well as Salmonella spp. are amongst the most routinely encountered catalase-positive bacteria, while Streptococcus and Enterococcus bacteria are catalase-negative.

Oxidase Testing

  • The oxidase test identifies bacteria with cytochrome c oxidase activity (CCO). This enzyme forms a part of the bacterial electron transport chain.
  • When present, CCO oxidizes the reagent (tetramethyl-p-phenylenediamine) to a purple-colored product.
  • When the enzyme is not present, the reagent remains in the reduced state and is colorless.
  • You should note that while oxidase-positive bacteria are aerobic, they are not necessarily strict aerobes and may be capable of anaerobic respiration.
  • Similarly, you will obtain a false-negative result if your bacterial species possesses an oxidase incapable of reacting with the test reagent.

Substrate Utilization Tests

  • A range of substrate utilization tests for microbial identification is available commercially.
  • These tests (usually for bacterial species) contain a panel of substrates, e.g., different carbon and nitrogen sources.
  • Bacterial species use these substrates differentially, and a record of the color changes in substrates after incubation with bacteria generates a key (or pattern) of substrate utilization.
  • This key is compared to the substrate utilization patterns of a computerized list of bacterial species.
  • These tests are probably more reliable in combination with catalase/oxidase testing and microscopic examination.

These tests overlap with the concept of selective growth medias. A wide range of selective medias exist for the isolation and identification of bacterial and fungal species and we hope to cover these in a future article.

Physiological Requirements for Growth

Addressing the following questions can help you narrow down the list of possible species.

  • Does the organism require oxygen?
  • In what temperature range does the organism grow best?
  • Can the organism tolerate acidic media?

Traditional Methods – Dichotomous Identification Keys

  • Dichotomous keys contain a series of steps, in which each step presents descriptions of two distinguishing features (e.g., Gram-positive or Gram-negative), with a direction to the next step in the key, until the identity is known.
  • The idea is that you use dichotomous identification keys alongside the traditional methods outlined above to help you identify your organism of interest.

Modern Methods for Microbial Identification

Although still widely used, traditional identification methods suffer from two major drawbacks. Firstly, they are only applicable to organisms that can be cultured in vitro. Secondly, some strains exhibit unique biochemical characteristics that do not fit the pattern of any known genus and species.

Fortunately, many modern methods are not dependent upon live cultures, and they can often reveal minute differences between organisms that escape detection by traditional means.

Modern Methods – PCR-Based Identification

  • PCR, including Real-Time PCR is probably the most widely used molecular technique for microbial identification. Using PCR, one can rapidly detect and identify microbial species directly from clinical samples, thus speeding up diagnostic procedures.
  • Several PCR-based methods exist. Most involve a universal set of PCR primers that identify bacterial/fungal samples by sequencing of the PCR amplicons.
  • The 16S rRNA gene is the gold standard sequence for bacterial identification by PCR, while the Internal Transcribed Spacer (ITS) region is the primary barcode marker for fungal species.

Modern Methods – Microarray-Based Identification

  • Microarray-based microbial identification relies on the hybridization of pre-amplified microbial DNA sequences to arrayed species-specific oligonucleotide probes. Each probe contains a distinct dye that fluoresces upon hybridization.
  • Microarrays are a versatile tool, which facilitate the detection and discrimination of different microbial samples on a single slide. Microarray is a fast technique, and speed is important within the clinic for diagnosis and timely initiation of proper antimicrobial therapy.
  • Microarray-based platforms typically use similar barcode regions as other PCR-based methods (above).

Modern Methods – Immunological

  • ELISA-based methods can be set up for microbial detection (usually within diagnostics) on a species-by-species basis.
  • These methods are highly sensitive and specific but rely on very specific antibodies and highly discriminating protein(s) within your organism of interest.

Modern Methods – Chemical Analytical

Fatty Acid Profiling

  • Fatty acids are essential within bacterial cell membranes, and different bacterial species produce different combinations of fatty acids. As such, the profile of fatty acids obtained from an unidentified bacterial species can be used to identify that species by matching against known fatty acid profiles.
  • Fatty acid profiling is typically performed using a combination of gas chromatography and mass spectrometry.

Metabolic Profiles /Chemo-Profiling

  • Besides primary metabolites (e.g., ATP, ADP) that are essential for growth, microbes produce a plethora of secondary metabolites that are not essential for growth but may be advantageous within certain environments, e.g., outcompeting other microorganisms for nutrients.
  • Secondary metabolites include antibiotics, immunosuppressive compounds, pigments, antioxidants, and others. These metabolites are a huge source of existing and newly emerging drugs.
  • Different species often produce unique secondary metabolite profiles, and these profiles allow us to identify known microbial species. Metabolic profiling is usually carried out using HPLC and mass spectrometry.

That brings us to the end of this crash course in microbial information. It was a lot of information, and I don’t claim to cover every technique out there! Do you have use any other methods to identify microbial species? Just drop us a line in the comments section and let us know!

Further Reading and Useful Resources:

  1. Printable dichotomous identification keys for bacteria.
  2. Houpikian P and Raoult D. (2002) Traditional and Molecular Techniques for the Study of Emerging Bacterial Diseases: One Laboratory’s PerspectiveEmerg Infect Dis. 8(2): 122–31. doi: 10.3201/eid0802.010141.
  3. Järvinen A-K, Laakso S, Piiparinen P, Aittakorpi A, Lindfors M, Huopaniemi L, et al. (2009) Rapid identification of bacterial pathogens using a PCR- and microarray-based assayBMC Microbiol. 9:161. doi: 10.1186/1471-2180-9-161.
  4. Khot Prasanna D, Fredricks DN. (2009) PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther. 7(10):1201–21. doi: 10.1586/eri.09.104.
Image credit: Iqbal Osman

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