Article Figures & Data
Figures
- Fig. 1.
Groups of cells within a microbial population may exhibit heterogeneous uptake of fluorescent stains and thus be classified into more subpopulations than “live” and “dead.” The route from “live” to “dead” contains many steps (not all of which will occur or be observed to occur in all cases), and while the extremes are relatively clear-cut, the reversibility of these steps and indeed the moment of death are far from easy to define. Metabolic activity can be demonstrated by cleavage of fluorescein diacetate or uptake of rhodamine 123, RNA content can be measured using pyronin Y, and membrane damage can be measured by entry of stains that are normally excluded, such as propidium iodide.
- Fig. 2.
Flowchart indicating the steps in adjusting a published protocol for a new flow cytometer, microorganism, or experimental condition.
Tables
- Table 1.
Interpretation of the results of plate counting
Observed result Usual interpretation Alternative interpretations A colony is formed A viable cell gave rise to the colony At least one viable cell gave rise to the colony—but it may have been two or more cells coinciding at the same place on the plate or a clump of cells that contained at least one viable individual No colony is formed There were no viable cells in the sample (i) The growth medium and/or incubation conditions were incorrect (ii) The cells were damaged/stressed and therefore unable to grow on solid medium (iii) The population density was low and therefore cell-cell communication could not take place, resulting in no observable growth (iv) Insufficient time was allowed for visible colony development in slowly growing cells - Table 2.
Comparison of methods for determining viability of microorganisms
Method Speed No. of cells analyzed Ease of use Typical costs (excluding labor) Plate counting Preparation of dilutions and plating take minutes. Hundreds of plates can be prepared per day. Incubation of plates for 1 to 7 days typical before results are obtained. Viable counts are typically based on plates with 30 to 300 cells. Minimal training required in aseptic technique and safe handling of microbes. Plastic consumables and media components. Incubation at growth temp. Microscopy Dilution (if necessary) and staining take minutes. Some stains may require incubation of, e.g., 10 to 30 min. Manual microscopic analysis may take several minutes per sample. A hundred samples could conceivably be processed in a day. Results are obtained immediately. Typically 100 to 500 cells per sample are scored as viable or dead. Image analysis can be used to automate the process of identifying and scoring viable/dead cells. Minimal training in safe handling of microbes and stains (some of which are carcinogenic). Microscope slides and coverslips. Stains. Cost of purchasing and maintaining microscope or fluorescence microscope. Flow cytometry Dilution (if necessary) and staining take minutes. Some stains may require incubation of, e.g., 10 to 30 min. Manual sample presentation may take several minutes per sample. Automated samplers can be loaded with, e.g., a 96-well plate of samples. Hundreds of samples can be processed in a day. Results are obtained immediately, although postacquisition analysis of data is common. Typically 10,000 to 100,000 cells per sample are analyzed. As stain uptake is quantified, intermediate results between live and dead are possible. In addition to the above, training is needed in operation and quality control of flow cytometer. Experience required for protocol development and data analysis. Sample tubes and stains. Costs of purchasing and maintaining flow cytometer.
