Bacterial spores

There are many spore-forming bacterial strains and they are very different from each other. Therefore, there is no one-size-fits-all protocol. Instead, we highlight the best practices for how to measure bacterial spores with BactoBox®.

Shake flask sporulation study

Grow bacteria in a shake flask to study sporulation across the bacterial life cycle.

After the initial lag phase, bacteria enters the exponential growth phase. In the exponential growth phase BactoBox® consistently counts and classifies them as cells.

At the onset of nutrient loss in the stationary phase, the mother cells and spore populations appear.

In the later stages, the vegetative cells are almost gone. All that is left are:

• Free spores

• Spores caught inside mother cells

• Dead cells (ghosts)

Spores do not form at all in some nutrient-rich media like TSB. Do a literature review to find a medium that increases the yield of spores.

Use phase-contrast and fluorescence microscopy

Under a phase-contrast microscope, vegetative cells appear as dark bodies while endospores appear highly refractive and bright.

Spores are generally difficult to stain with nucleotide dyes. Nonetheless, SYBR green works well as a total stain for vegetative cells. It also provides some degree of spore visualization.

Membrane-impermeable dyes like TOTO®-3 or propidium iodide provides information on dead cells and the presence of spore-containing mother cells with an impaired lipid membrane.

Use heat and/or enzymatic treatment to release endospores

Mother cells may trap or encage endospores. Release the spores with heat treatment in a water bath (85 °C, 15 min).

Other spore-purification approaches typically combine a peptidoglycan-degrading enzyme with surfactants like SDS.

ASTM E2111-00 describes how to use ethanol extractions.

Use plate counts to evaluate the spore/vegetative status

Plate counts are the typical gold standard to enumerate spores. Compare your BactoBox® results with the plate counts to confirm your findings.

Plate counts before heat treatment gives information on both vegetative cells and spores. Plate counts after heat treatment only enumerates the heat-tolerant spores.

The spores germinate at different time points. Tnitial colonies often grow mucoid and dominate later colonies. This leads to underestimation and difficulties with the enumeration. To avoid colony domination, incubate plates at, e.g., room temperature and count colonies early. Inspect plates daily for occurrence of new colonies.