Microscopy for K

Counting of bacteria is typically done using a specialized counting chamber like the improved Neubauer. A small volume of the culture sample is transferred to the counting chamber and the cells in the small squares are manually counted in a microscope. Often, this is a manual procedure, but automated cell counters are also available.

Bacteria can be counted using phase contrast microscopy without labels. Fluorescence microscopy is another common method, where bacteria are stained with fluorescent dyes before counting.

Counting of bacteria using a microscope. Illustration of improved Neubauer counting chamber.

Advantages of microscopy for K

Microscopy is a direct method for cell counting, unlike optical and dry weight methods, which require calibration. With microscopy, cells are observed and counted directly, providing accurate species identification, rather than inferring concentration from turbidity or biomass.

Direct enumeration by microscopy offers a key advantage over the plate count method: it maintains a constant total cell concentration once the stationary phase begins.

In addition, microscopy can reveal is the sample contains of single cells or aggregates. While challenging, this method is fundamentally the only way to count cells in clumps and chains.

Disadvantages of microscopy for K

On the flip side, microscopy is time-consuming and low throughput. The sample-workup is involved and cell-counting is slow. Because only a fraction of the sample size is inspected, sampling error can occur if cells are unevenly distributed in the counting chamber.

Precision is relatively low because relatively few objects are enumerated. The method is also known to be somewhat operator-dependent and requires extensive training for reliable results.

Practical recommendations

The low throughput and low precision makes microscopy is unsuited as a real-time method for multiple time points. It is, however, a great qualitative technique to inspect cell morphology and check for the presence of clumps and contaminants. With a microscope it is easy to evaluate if the sample needs further workup for a single-cell suspension.