How do we know if a microbe is dead?
By Prof. Maria Marco PhD, University of California, Davis
“Kills 99.9% of bacteria and viruses.” This percentage and others like it are frequently found on disinfectant labels.
Ideally, the microbicidal effect of the product is sufficient to kill more than the numbers of the target pathogen or pathogens expected to be in the environment where the disinfectant is used. However, if more of the target pathogen is present, 99.9% can be misleading or even result in illness. For example, for a surface with one million pathogenic E. coli cells, a 99.9% reduction would mean that 1000 living and infectious E. coli cells remain. That amount of E. coli could be sufficient to cause life threatening illness.
Declaring a microbe dead
The issue of quantity is only one of the conundrums when relaying information about microbial death. Another issue is how do we even know if a microbe is really dead? For viruses, they are not cells and so the terms “live” and “dead” do not apply. Viral inactivation is inferred by a loss in the ability to infect and multiply in host cells. For bacteria, the answer is not so simple. Bacteria can form dormant states, whereby their metabolic activity is minimal, but then, when conditions are right, they grow and divide again. Endospores formed by some bacteria are a great example of dormancy. But even bacteria that do not form endospores can be viable (alive) but dormant for long periods of time and may be inferred to be “dead”. Sometimes these dormant states are desirable, such as when drying bacterial strains for use as probiotics and retaining their capacity to reactivate when the conditions are right.
Measuring dead microbes
Another problem with microbial death is how we measure it. Colony forming unit (CFU) enumeration on laboratory culture media is the gold-standard for quantifying viable bacterial cell numbers. However, it is now well known that only a very small fraction of all bacteria on Earth have been grown or “cultured” in the laboratory. Many microbes are “unculturable” mainly because the nutrients and environmental conditions needed for their enrichment in the laboratory have yet to be found. So, it is not possible to quantify viability of “unculturable” microorganisms using the gold standard approach.
Even for microbes that grow well in the laboratory (for example E. coli), they too may not always be culturable. After exposure to certain conditions, such as nutrient limitations, some bacteria can form Viable But Non Culturable (VBNC) states. Those VBNC microbes will not grow using routine culture methods, but they are viable and may return to a metabolically active, reproductive state later, such as when nutrients or environmental conditions change. Testing via CFU therefore may underestimate levels of viable bacteria – with important implications for measuring and monitoring both beneficial and pathogenic organisms.
Dead bacterial cells can best be described as having different states. Completely lysed bacterial cells, wherein the cell membrane is destroyed, and intracellular contents are released, are obviously dead. Yet, this state may constitute only a minor fraction of dead bacteria in a population. Bacteria can also be dead, but still have an intact cell membrane. Several methods have been developed to assess viability of cells in that state, including measurements of cellular enzymatic activity (MTT conversion assay) and uptake of fluorescent dyes impenetrable to intact cell membranes (SYTO9-propidium iodide staining). While this question of evaluating cell viability is far from resolved, an intriguing recent recommendation was to use multiple tests, assessing both metabolic activity and reproductive capacity (1).
Implications for biotics
How does all this relate to biotics? Probiotics should be alive (viable) at the time of administration. Postbiotics are preparations of dead (inanimate) microbes. Both must deliver a health benefit. Decisions on how viable and dead microbes are enumerated in biotic preparations should address the fact that there are different bacterial viability states. The use of a single method such as CFU enumeration can lead to underestimating numbers of viable cells and will not be helpful for quantifying dead cells. Although we may never be able to say that a microbial population is either absolutely 100% alive or dead, such viability states may affect how well either a probiotic or a postbiotic performs for its intended purpose of conferring a health benefit.
