Dilution Series
One of the fundamental tools used in the laboratory is that of serial dilution. Serial dilutions are very important in disinfectant efficacy testing as all standards require that the disinfectant ‘kills’ a significant number of microbes either within a suspension or on a surface. Thus, disinfectant efficacy testing requires that high titres of microbes are used to enable the detection of large reductions in microbes as a result of the action of the disinfectant.
Without serial dilutions laboratory methods would not be able to determine the number of microbes in a high titre suspension of microbes. This is because if the suspension was directly analysed the number of microbes within the original suspension would be too numerous to be accurately resolved. For example if a high titre suspension of a bacteria was to be poured directly onto an agar plate then so many bacteria would grow that it would be impossible to accurately distinguish one bacterial colony from another to resolve the number present.
Serial dilutions are simply a standardised method to dilute a suspension of microbes, using a methodology that limits experimental mistakes and introduction of variability. The most frequently used serial dilution is a 1:10 dilution series, the process of performing a serial dilution is described in figure 1.
Laboratory mistakes are minimised as volumes are kept the same for each dilution step, and variability is reduced as the same laboratory equipment (e.g. pipettes) can be used for each step. The process is also self-validated, as the number of microbes recovered from each dilution suspension should reduce ten-fold with each dilution step. If results come back and the results from each successive dilution step do not show a roughly ten-fold reduction with each successive dilution step then there are probably issues with something within the experimental protocol e.g. pipette out of calibration, or problems with agar.
Once the number of microorganisms in a particular dilution have been determined then a mathematical back-calculation can then be used to determine the number of microbes in the original suspension.
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Log Reductions
In terms of disinfectant efficacy testing, ‘Lg Reductions’ or ‘Log Reductions’ convey how effective a product is at reducing pathogens. The greater the log reduction the more effective the product is at killing microbes.
‘Log’ is short for logarithm, a mathematical term for a power to which a number can be raised. For example, if using 10 as a given number, a Log 3 increase can be shown as 103 or 10 x 10 x 10 = 1,000.
A log reduction takes the power in the opposite direction. For example, a log reduction of 1 is equivalent to a 10-fold reduction or, to put it another way, moving down one decimal place or a 90% reduction. A 2 log reduction would therefore equate to a 99.0% reduction in the number of organisms.
All disinfectant testing standards state a minimum log reduction that needs to be achieved in order for a product to pass the standard and therefore have demonstrated that the product kills a sufficient number of microbes over a specific period of time.
During product efficacy testing, the microbiology laboratories count the number of microbes in the test suspension present at the start of the test. They then apply the disinfection product being tested, alongside a control product and wait the required test time before recounting the number of microbes present.
The result of the difference between the control and the test product is then expressed as a Log reduction. For example, if the number of microbes in the control was found to be 1,000,000 (or 106) and the end result using the product was only 1,000 (103), that would be a Log reduction of 3 or a reduction of 99.9%.
