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How easy is it to enumerate your biological indicator?

To enumerate a biological indicator is to ascertain the number of spores or microbes present and can be a process as simple as 1...2...3... Enumerations are important for determining the population size which is in turn important for use in a sterilisation process with predetermined kill target. Too low a population and the process could be falsely assumed to have passed as there is less to kill. Too high and the process could be falsely assumed to have failed, with too many to kill.

There are numerous enumeration methods, most of which incorporation some form of series dilutions. Serial dilutions is a process of reducing the substance (or population when in terms of biological indicators) through a number of calculable steps. When looking at counting microbes for example a solution of a suspected population of 1X106/ 1 million, counting to 1 million on an agar plate may be difficult but using a series of steps it can be reduced to 100. A lot more manageable.

A density based process can be more complex and involve even more equipment and steps. For example, neutral red assay works on the basis that the cells in question when submerged in a neutral red dye solution can absorb the dye into their cell structure. Once absorbed the solution is washed of the excess solution leaving behind essentially red stained cells. These cells are then transferred into a destructive alcohol solution causing them to rupture and release their contents including red dye. The solution can be placed into a spectrophotometer where a light passes through and the observed light can be associated with a density reading. The more viable cells present, the greater the concentration of absorbed dye and thus the greater the concentration released when destroyed. The advantage of certain techniques based around concentration is the lacking need to count individual cells.
Many techniques are available, which one you choose can be dependent on your counting skills and patience.
If you need assistance with your enumeration process please contact Bioquell.

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