Inactivated bacteria
BactoBox® is optimized for enumeration of bacteria in Active cultures. That said, BactoBox® can be customized to include enumeration of bacteria in other physiological states. Take enumeration of bacteria in bacterins and postbiotics products as an example.
Below we explain what happens to the electrical properties of bacteria when they are inactivated by alcohol and heat. In the subpages, we discuss if the default measurement program - BacTotal-10 - is suitable for enumeration of the inactivated states or if customization is needed.
The table below provides a quick overview of whether BacTotal-10 still classifies the detected objects as cells for a given inactivation method. Click the link for details.
It depends. Some heat-inactivated bacteria are enumerated as cells by BacTotal-10 while other heat-inactivated bacteria may fall outside the cell-definition.
Custom gating can include heat-inactivated cells in cell counts. Contact SBT to set it up.
No. Alcohol-treated cells usually fall outside the BacTotal-10 cell-definition.
Custom gating can include alcohol-inactivated cells in cell counts. Contact SBT to set it up.
No. An autoclave treatment causes bacteria to disintegrate to tiny fragments. Usually these fragments are too small to be detected by BactoBox®.
It is usually not possible to enumerate autoclaved cells. The objects are too small to be detected on BactoBox®.
It depends. Some aged cultures of bacteria are often still found within the BacTotal-10 cell-definition while other cultures — especially Gram-positive bacteria — often fall outside the cell-definition.
Custom gating can include aged cultures in cell counts. Contact SBT to set it up.
Advanced feature 🧩
Custom gating is an advanced feature. We only support advanced features through a custom solution. Contact support for details.
Products containing inactivated bacteria
Bacterins
A bacterin is a vaccine made from dead or inactive bacteria. Bacteria may be inactivated by chemicals such as formaldehyde/formalin, glutaraldehyde, β-propiolactone (BPL), phenol, and acetone. Physical methods like heat, gamma irradiation, and UV methods are also used.
Postbiotics products
Postbiotics are preparations of dead or inactive microorganisms and their cellular components. The dominant inactivation is heat — e.g. by pasteurization — but other methods like pressure, ultrasound, gamma-irradiation and supercritical CO2 are also used.
How inactivation influences electrical signatures
What matters most for BactoBox® measurements is the shape and size, membrane status, the thickness of the peptidoglycan, and the presence or absence of a conductive cytoplasm.
Inactivation of Gram-positive bacteria
Let's take a simple example first. S. epidermidis is coccoid (round) and has a simple cell envelope consisting of a thick layer of peptidoglycan and a relatively thin lipid membrane.
Before inactivation the untreated cell has the following electrical properties:
The membrane has a non-charged interior and therefore impedes electricity.
The cytoplasm is rich in ions and therefore good at conducting electricity.
The thick peptidoglycan carries charged amino acids and is therefore conductive.
As cells become smaller, the cytoplasmic volume is reduced, while the thickness of the membrane is constant. Because the cytoplasm is the conductive part, a small cell is typically less conductive than a large cell.
Alcohol
Alcohol mixtures like 70% ethanol and 70% isopropanol destabilize the lipid bilayer. They disorder the lipid structures, so the membrane no longer impedes electricity and small molecules from the cytoplasm may escape through the peptidoglycan. The alcohol-inactivated Gram-positive cell is a sack of conductive peptidoglycan.
Heat
Heat above ~75 °C, e.g. boiling for 15 min, collapses the membrane bilayer into a disordered state. Embedded proteins denature and leave gaps, where small molecules can leak out of the cytoplasm. In Gram-positive bacteria, the disordered membrane also stops impeding electricity. In contrast, the peptidoglycan cell wall is relatively thermostable. Most Gram-positive cells therefore become more conductive after heat treatment.
Some Gram-positive bacteria have membranes with higher heat-tolerances; they keep impeding electricity after heat-treatment and because some of the cytoplasmic content is lost they become overall less electrically conductive than the untreated cell.
Inactivation of Gram-negative bacteria
Gram-negative bacteria are more complex in their envelope structures.
Before inactivation the untreated cell has the following electrical properties:
Two membranes are present, and both impede electricity.
The cytoplasm is rich in ions and therefore good at conducting electricity.
The thin peptidoglycan is conductive but not as conductive as for the Gram-positive cell.
Alcohol
Alcohol affects both lipid membranes of Gram-negative bacteria: it destabilizes the structure and may strip lipids entirely. After alcohol-treatment the Gram-negative cell is left as a sack of thin, but still conductive peptidoglycan.
Heat
High temperature perturbs the structures of the inner and outer membranes in Gram-negative bacteria. But even with two membranes partially down, the pair of membranes still impede electricity to some degree. Molecules from the cytoplasm can diffuse through the pores in the leaky membrane. The conductive peptidoglycan is largely unchanged, but Gram-negative bacteria only have thin peptidoglycan and therefore it does not offer much propagation of electricity.
The biggest impact of heat is the leakage of conductive cytoplasm. After heat-inactivation the Gram-negative cells are overall less conductive.
In the next section Inactivation by heat we give examples on heat-mediated changes to electrical phenotypes.
Summary
Heat and alcohol cause damage to bacterial membranes. The regular structure is destabilized and the membrane may lose its ability to retain small molecules. Membrane damage is often easier to spot electrically for Gram-positive bacteria.
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