Viability and Quantification Stains

Created by Monica Z. Bruckner, Montana State University, Bozeman

What Are Viability and Quantification Stains?

Many reasearchers are interested in quantifying total numbers of cells as well as quantifying live vs. dead cells in an environment to determine bacterial biomass. The following stains offer a glimpse of common staining techniques used for cell enumeration and viability; this list is not exhaustive.

Fluorescent Staining Using DAPI

Fluorescence image of Giardia lamblia cysts, purified from Mongolian gerbil fecal material and stained with DAPI. DAPI interacts with nucleic acids and stains the nuclei within the cyst. There should be 4 nuclei in each cyst. Cysts that appear to have fewer than 4 stained nuclei, may have 4 nuclei with the others not visible in this plane of focus. Cysts with no nuclei visible, may be dead, may be resistant to DAPI staining, or may be organisms other than G. lamblia. Photo courtesy of H.D.A Lindquist, US EPA.
DAPI (4', 6-diamido- 2-phenylindole) is a commonly used dye for bacterial enumeration, or cell counting. DAPI is a fluorescent dye that stains nucleic acids and is relatively unreactive with inert, non-biological matter, thus making it useful in differentiating between biotic and abiotic components in a sample. DAPI-stained cells appear blue under an epifluorescent microscope. This stain is non-specificin that it stains all cells, not differentiating between live and dead cells or specific species or organisms.

Viability Staining

Giardia lamblia (intestinalis) cyst as imaged at different instrument settings by confocal microscopy. (A) is the cyst imaged by transmission (differential interference contrast), only. (B) is the cyst wall selectively imaged through use of fluorescent-labelled (TRITC) antibody that is cyst wall specific. (C) is the cyst imaged through use of carboxy fluorescein diacetate, a viability stain. (D) is a composite image of (B) and (C). (E) is a composite image of (A), (B), and (C). Bar = 10 microns; sample from gerbil feces. Image courtesy of US EPA

Viability stains differentiate between live and dead bacterial cells in a sample. These fluorescent stains base differentiation on whether or not the cytoplasmic membrane of the cell is intact. A red and green dye are added to a sample; the green dye penetrates all cells (live and dead), whereas the red dye, which contains propidium iodide, only penetrates cells whose cell membranes are no longer intact (and are therefore dead). Hence, cells that retain the green stain are live, while cells that take the red stain are dead.

Fluorescent Antibodies

An antibody is a biologic entity with a high specificity toward other another biological entity. Fluorescent antibodies can be used to identify or track a particular organism in a complex and/or diverse microbial community. Specific antibodies are created in the laboratory to bind with particular organisms; their creation is often time consuming and expensive.

Green Fluorescent Protein (GFP)

This type of staining involves the insertion of a gene encoding protein, a green fluorescent protein (GFP), into the genome of a bacterium via genetic engineering. When viewed with ultraviolet microscopy, GFP-tagged cells fluoresce green. GFP cannot be used in studying natural populations, as the GFP must be inserted into the genome. However, GFP-tagged cells can be introduced to an environment and monitored over time since the GFP is passed to offspring.

Limitations to Viability and Quantification Staining

While these stains are useful in the laboratory to gain insight into total numbers of bacterial cells in an environment, they also have several limitations. Some key limitations are listed below.

  • Small cells may be difficult to visualize, even with a microscope. These cells may be overlooked or hidden behind abiotic particulate matter in a sample.
  • Large numbers of cells may be difficult to quantify, necessitating a dilution of the sample.
  • Cell aggregates or cells attached to substrates may be difficult to quantify, as individual cells cannot be differentiated in the mass.
  • It may be difficult to differentiate between dead cells and non-living matter in natural samples stained for viability.
  • These stains cannot diffentiate among species, and thus cannot measure species diversity.

While these limitations exist, viability and quantification stains such as those dicussed above are useful and commonly used in characterizing the biotic component of an environment. These methods may be enhanced when they are used in conjunction with other molecular methods, such as those that use genetics to differentiate between species.

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