PRESERVATIVES 43
that is, that they would need to be controlled by the material under test. Cosmetic preservatives are tested with a selection of organisms to cover the range of Gram positive and Gram negative bacteria as well as a yeast and a mold. The species selected have either been isolated from infected cosmetics or are expected to be placed into cosmetics during production or by the consumer. Likewise, the bacteria selected to test antibacterial hand wash, typically S. aureus and E. coli or a Klebsiella sp. are selected on the basis that they are either skin or gut commensals and would expect to be encountered on hands. All microorganisms exhibit an intrinsic
tolerance to biocides and it is determined by the naturally chromosomal controlled property or adaption of an organism. This may be expressed by a number of factors, including morphology, biofilm formation, nutritional starvation or growth rate control. Bacteria are divided into two groups:
Gram positive and Gram negative, based on the ability of a dye to bind to the cells. Gram negative bacteria possess an additional outer membrane, which stops the dye binding to the cell and often makes them tolerant to higher concentrations of biocide than Gram positive bacteria. The outer membrane acts as a further permeability barrier to entry of biocides, many of which must penetrate the cell membranes to reach their targets within the cytoplasm of the cell. Pseudomonads are commonly more
tolerant to antimicrobial actives than most other microorganisms. They have been found to have differences in their outer membrane permeability of up to 400 times that of other Gram negative organisms, making it more difficult for biocides to enter.1,2 The strain chosen is not as important,
provided the same strain is used by all laboratories conducting the test as variability in susceptibility occurs across any population. The maintenance of cultures is paramount in keeping the culture homogeneous in this respect. Continuous subculturing with inherent mutation/variation can result in a culture that behaves quite differently to the parent stock and care must be taken to minimize passage numbers or generations away from the original culture held in a reference collection.
Table 2: Calculation of log reduction and % kill Method
Calculation
Log reduction (R) R = log10 (Ni ÷ Nx) = log10 Ni – log10 Nx
% kill
% = (Ni– Nx)/Ni * 100 = (number killed/number added) * 100
Figure 2: Bacterial growth curve. Without preservation insufficient preservation
Stationary phase Decline phase
Log phase
Lag phase Time
Cell count
Microbiostasis
sufficient preservation Disinfection
Time Figure 3: Organism kill rates. Natural variation that occurs across
generations results in offspring with varying tolerance to antimicrobial actives. Obviously, the more generations the greater the potential for variations to occur. The USP has set a limit of 5 passages from the original ATCC culture to minimize this potential variation. If factory isolates known to express tolerance to the preservatives/biocide are a concern then
Expressed as
Number, e.g. 2
%, e.g. 99.9%
Ni: Number of microorganisms at time 0 Nx: Number of recovered microorganisms at time x in the product
May 2020
they should be used in addition to or in place of the usual culture collection organism. Special care must be taken to ensure the tolerance is not lost by the cultured organisms as this can rapidly occur when passaging with no selective pressure to maintain the tolerance is allowed. It must also be considered that the use of these special organisms in conducting tests will generally require higher levels of preservatives to pass the test requirements than that required by usual test organisms. The effect of this variation in the
population may affect test results. A population with a wide variation in the Minimal Inhibitory Concentration (MIC) (Fig 1) against a preservative will likely show a result with a fast initial kill due to the higher number of cells showing a low MIC value followed by a slower and less complete kill due to the number of more tolerant
PERSONAL CARE NORTH AMERICA
Log10 Cell Count
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