44 PRESERVATIVES
Table 3: Comparison of Log reduction and % kill Initial (Ni)
5,000,000 5,000,000 5,000,000 5,000,000 5,000,000 5,000,000
Recovered (Nx) 500,000
50,000 5,000 500 50 10
Killed (Ni-Nx) 4,500,000
4,950,000 4,995,000 4,999,500 4,999,950 4,999,990
organisms. In contrast a population that has a narrow variation on MIC values will likely show slower initial action followed by a rapid and complete kill. The growth phase of the culture also
determines the tolerance to preservatives. The growth curve shown in Figure 2 shows the 4 phases of growth of any population. Test organisms are usually prepared in either log (exponential growth) or stationary phase and determined by the age of the culture. Standard Test methods usually stipulate the period of incubation of a test culture to ensure it is in the appropriate growth phase. Cultured bacterial cells supplied with excess nutrients grow quickly during exponential growth phase. Once there is a shortage of nutrients or excess of waste products, the culture enters stationary and then decline phases. Bacteria respond to the starvation stress with growth rate reduction and induction of defense mechanisms.3,4
As a
result, they may become more tolerant to preservatives. Cultures in exponential growth are
actively metabolising and are therefore more susceptible to the effect of preservatives due to increased free radical production from interference with normal metabolism. Bacteria usually become more resistant to environmental stress during slowing down of growth. This has been seen in both steady state and batch growth of cells and by comparing mid exponential with stationary phase growth, large differences in efficacy have been demonstrated. The most tolerant cells are those in decline phase. While only viable cells will be counted to determine the initial cell concentration, the population may contain up to ten times the number of dead cells than viable cells. The dead cells will undergo lysis and release cell debris and intact enzyme systems that may interfere with the efficacy of the preservatives.4 Test cultures may be prepared by
growing them in a liquid medium or on a solid agar. Cells grown in liquid media have been shown to be hardier and less susceptible to antimicrobial attack than those washed from solid agar surfaces. Almost all test methods include a
requirement for the number of cells to be PERSONAL CARE NORTH AMERICA (log10
Log reduction Ni-log10
1 2 3 4 5
5.6989 % kill Nx)
[(Ni-Nx)/Ni]*100 90 99
99.9 99.99 99.999 99.9998
included in the inoculum along with how they are prepared. Cells grown in liquid medium or washed from solid agar require diluting to the required concentration. The dilution of the cells may result in a carryover of nutrients into the inoculum, particularly from liquid cultures. This may be removed by centrifuging and resuspending the cells in a non-nutrient solution, such as isotonic saline. Other test methods require the cells to be diluted in nutrient broth5 nutrient broth.6
or even a 1:500 dilution of These variations result in
different supply of necessary nutrients to the cells to support growth and they may also inhibit the biocide under test. The age of the test organism suspension
and its storage can also affect the test results so many methods stipulate the temperature and time that the inoculum may be stored, such as 4°C for 24 hours for bacteria and yeast and 7 days for fungal spores. Cultures storage may affect the viability of the test organisms, or depending on the nutrient content, may allow the organisms to begin reproducing altering the number and growth phase of the inoculum. The number of cells included in the
inoculum is critical to the test result and will be further discussed later in this paper.
Step 2: Add test organisms to the sample under test The inoculum may vary in volume, placement and method of addition. The volume of inoculum added may affect the efficacy by diluting the test material if too much or repeated inoculations are conducted. The inoculum may be dripped in or onto a test samples, sprayed on and then mixed or left on the surface, all may affect the final outcome.
Step 3: Allow a desired contact period The contact time of the microorganisms with the test material must be realistic and sensible. Disinfectant tests typically allow 5-8 minutes contact while preservative efficacy tests allow days and weeks to see an effect. These times reflect actual in use periods. Tests for antibacterial hand wash are typically conducted using 30 seconds and 5 minutes contact. Results for 5
minutes contact are often reported and are entirely unrealistic if the hand wash is to be rinsed off. Current recommendations are for 20 seconds of washing, so any test contact times in excess of this may show results indicating better efficacy than will be seen in practice. However, if it is a leave on product, it may be a reasonable time. The standards for antibacterial surfaces and textiles utilizes a 24 hour contact period5,7
which may be totally
inappropriate, such as for medical staff uniforms or antimicrobial cutting boards while acceptable for antibacterial socks that just need to stop growth rather than kill any organisms.
Step 4: Neutralize the active All antimicrobial tests require that the effect of the antimicrobial agent must be neutralized or stopped following the required contact time so that surviving organisms can be recovered. If this does not occur, the killing effect on cells may be allowed to continue well past the required contact time and may even inhibit growth of surviving organisms resulting in incorrect test results. If available a neutralizer specific for the antimicrobial is used, such as sodium thiosulphate for chlorinated water. Otherwise general neutralizers, such as
Lecithin and Tweens are used in varying concentrations. A list of examples of neutralisers is included in Annex C of ISO 11930:2019.8
If it is not possible to
neutralize the biocide then dilution of the test solution may be used. It is usual to find the result where no organisms are recovered reported as <10cfu/mL due to at least a 1:10 dilution in neutralizer. If this cannot be validated, then a result of <100cfu/mL is often seen indicating further dilution to 1:100 was necessary. It is usually considered that if a 1:1000 dilution is required to validate the neutralizer system, then the test product is antimicrobial. All antimicrobial tests require that the
neutralizer is validated to ensure a correct result has been achieved.
Step 5: Determine survivors The enumeration method of surviving organisms may also affect the test result. The diluent and agar type chosen may influence recovery as surviving organisms may have been sub lethally injured. Agars with lower nutrient value, such as plate count agar (PCA) or synthetic agars like R2A, has been found to give better recovery than a high nutrient agar such as Tryptone Soya Agar (TSA) and the choice of agar used may affect the results obtained.11 Counts conducted using the spread plate technique, spreading the inoculum over the surface of pre-poured agar plates, have been claimed to give a higher recovery than
May 2020
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