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An Innovative Challenge Test For Solid Cosmetics Using Freeze-Dried
An Innovative Challenge Test For Solid Cosmetics Using Freeze-Dried
a r t i c l e i n f o a b s t r a c t
Article history: Freeze-dried bacteria and fungi were used as inoculum in 28 days' PET. An electrical method was used in replace-
Received 30 May 2014 ment of the conventional plate count method. The use of freeze-dried microorganisms in association with the
Received in revised form 18 August 2014 electrical method can minimize the workload and the variability involved in PET for cosmetic powders.
Accepted 19 August 2014
© 2014 Elsevier B.V. All rights reserved.
Available online 28 August 2014
Keywords:
Preservative
Efficacy test
Solid cosmetic
Electrical methods
http://dx.doi.org/10.1016/j.mimet.2014.08.009
0167-7012/© 2014 Elsevier B.V. All rights reserved.
M.R.S. Ferreira et al. / Journal of Microbiological Methods 106 (2014) 104–109 105
the material, executing the test and counting plates. Several alternatives 2.2. Challenge test using electrical methods to determine the survivors
to traditional colony-count techniques have been developed.
Electrical methods have been proposed as promising alternatives. 2.2.1. Microorganisms
This method is based on the modifications of electrical properties in The test organisms consisted of strains of freeze-dried S. aureus ATCC
culture medium due to metabolism of the microorganism (Jeffrey 6538, P. aeruginosa ATCC 9027, A. niger ATCC 16404 and C. albicans ATCC
et al., 1989; Connolly et al., 1993, 1994; Zhou and King, 1995; Pinto 10231.
et al., 1999; Basa and Flores, 2000; Chorianopoulos et al., 2008; Yang.
and Bashir, 2008). Non-charged molecules are turned into charged 2.2.2. Electrical device
molecules for microorganism growth, so these electrical changes may A Bactometer® model-128 microbial monitoring system (Biolab
detect microbial growth by the automatic system using signals such as Merieux®) was used. It has eight plastic modules in one unit. Each mod-
conductance, capacitance and impedance (Pinto et al., 1999; Szita ule contains sixteen molded wells. Each well contains two stainless steel
et al., 2007). Among the advantages, this method requires less material, electrodes holding up to 2 mL of culture medium. A total of 128 samples
labor and time (Connolly et al., 1993; Connolly et al., 1994). Many can be monitored simultaneously in one unit. The instrument uses three
authors found promising results using this method for bacteria determi- detection modes: impedance, capacitance and conductance.
nation (Connolly et al., 1993; Chorianopoulos et al., 2008; Priego et al.,
2011).
2.2.3. Signal choice (impedance, capacitance and conductance)
In this context, the aim of this study is to evaluate the application of
Volumes of 1.5 mL of GPMplus Bactometer® medium were added to
freeze-dried microorganisms as inoculum in the preservative efficacy
the wells at least 24 h before use.
test for a solid cosmetic and to verify the applicability of the impedance
A suspension containing 10% of eye shadow was prepared using
method to determine survivor microorganisms instead of using the
Diluent 2 as described in the inactivation of the preservative system
plate count technique for bacteria.
for both bacteria. This mixture was held stationary for 30 min before
the test execution.
2. Materials and methods The freeze-dried microorganism was recovered in 10 mL of saline
solution 0.9% (w/v), and was diluted to give a bacterial concentration
2.1. Freeze-dried microorganisms in preservative efficacy test range from 102 to 107 CFU/mL. A total of 0.1 mL of each dilution was
withdrawn and transferred to a group of six wells containing the culture
2.1.1. Microorganisms media in order to obtain 10 to 106 CFU/well. The amount of 0.1 mL of
The test organisms consisted of strains of freeze-dried Staphylococcus the diluted sample described above was added to each well.
aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Candida albicans The detection time (DT) was determined using impedance, capaci-
ATCC 10231 and Aspergillus niger ATCC 16404 (Souza and Ohara, 2003). tance and conductance signals for each two wells.
The incubation temperature was 35 °C for bacteria for 24 h and
28 °C for the fungi for 100 h.
2.1.2. Cosmetic sample Plate counting was performed in parallel using 1.0 mL of the same
The samples used in this study consisted of a powdered eye-shadow microorganism suspension used above diluted to present around
containing 1.0% (p/p) of Glydant plus® (DMDM hydantoin and 102 CFU/mL. Tryptic Soy Agar was used as media and the incubation
iodopropynyl butylcarbamate). time was 24–48 h at 32 ± 2.5 °C.
The choice of the better signal was based on the data obtained in this
test and also a calibration was conducted to correlate plate count with
2.1.3. Inactivation of the preservative system the 3 signals used.
The preservative system was inactivated by using the decimal dilu-
tion of three different diluents: 1) Peptone 1.0%, sodium thiosulfate 2.2.4. Challenge test and determination of the survivors by impedance
0.6%, sodium bisulfite 0.25%, soy lecithin 0.7%, and polysorbate 80 0.5% method
in distilled water to the bacteria (Diluent 1). 2) Peptone 2.0%, sodium The test was conducted as described in the challenge test using
thiosulfate 0.6%, sodium bisulfite 0.1%, soy lecithin 1.0%, and polysorbate freeze-dried microorganisms in the preservative efficacy test. Samples
80 3.0% in distilled water was added to yeast (Diluent 2). 3) Casein soy were aseptically removed after 0 h, 2 h, 4 h, 8 h, 24 h, 48 h, 7 days,
broth 3.0%, soy lecithin 0.5% and polysorbate 20 4.0% in distilled water 14 days, 21 days and 28 days to determine the detection time (DT).
(Diluent 3) to the mold. All of the media were previously sterilized. Two tests were performed in duplicate for S. aureus, P. aeruginosa and
A. niger.
The wells were incubated at 35 °C for 24 h to obtain the detection
2.1.4. Challenge test
time (DT). The number of survivors was established using the calibra-
One-vial containing 107 CFU of the freeze-dried microorganisms
tion curve.
(Souza and Ohara, 2003) was mixed gradually with 15 g of the sample.
The inoculated samples were maintained in glass bottles at room tem-
perature and samples were aseptically removed after 0 h, 2 h, 4 h, 8 h, 2.2.5. Statistical analysis
24 h, 48 h, 7 days, 14 days, 21 days and 28 days for viable counting. PET results obtained from electrical and pour plate methods were
The sample was diluted as described in the inactivation of the preserva- compared using a linear least square regression analysis. We assumed
tive system, and after 30 min of contact, 10-fold serial dilutions were that both impedance and pour plate methods were equivalent if the
made. confidential intervals for slope and intercept include the values 1.0
The pour plate technique was performed on a 1-mL aliquot taken and 0.0, respectively.
from the appropriate dilution using Tryptic Soy Agar for the bacteria
and Sabouraud Dextrose Agar for the fungi. The incubation time was 3. Results
48 h at 32 ± 2.5 °C for bacteria and yeast; for the mold it was 72 h at
22.5 ± 2.5 °C. 3.1. Use of freeze-dried microorganisms in the challenge test
At least six tests were performed for each microorganism and the re-
sults were compared with the specifications of the official compendia Table 1 shows the average number of survivors of S. aureus,
and CTFA. P. aeruginosa, C. albicans and A. niger. On the 7th day, the number of
106 M.R.S. Ferreira et al. / Journal of Microbiological Methods 106 (2014) 104–109
survivors was lower than 10 (ten) Log CFU/g for the bacteria. C. albicans Table 2
presented a faster die-off with no recovery in 24 h. Correlation coefficient using S. aureus ATCC 6538, P. aeruginosa ATCC 9027, C. albicans
ATCC 10231 and A. niger ATCC 16404 for impedance, conductance e capacitance.
A. niger, presented the opposite behavior with a recovery rate of
4.7 CFU/g in 28 days. While the C. albicans was the least resistant micro- Microorganisms Correlation coefficient
organism, the A. niger was the most resistant. Impedance Conductance Capacitance
Table 4
Detection Time (DT) and number of survivors (Log CFU/g) calculated using the calibration curve for S. aureus (impedance), P. aeruginosa and A. niger (capacitance).
T0, T2, T4, T8, T24 and T48: enumeration immediately after contamination, 2, 4, 8, 24 and 48 h, respectively.
T7, T14, T21 and T28: enumeration 7, 14, 21 and 28 days, respectively.
Faster reduction was observed when C. albicans was used as a chal- It is also important to consider that this inoculum could be used in
lenge organism (Table 1) attending all specifications used in this study any sort of sample including liquid or semi-solid.
(Magee et al., 1997; European Pharmacopeia, 2007; British Pharmaco-
peia, 2010; Unites States Pharmacopeia, 2000.
A. niger (Table 1) was the most resistant microorganisms evaluated 4.2. Challenge test using the electrical methods to determine survivors
in the preservative system. Nonetheless this mold was within the guide-
lines for optic products stated by the United States pharmacopeia prod- The traditional method using the plate count technique has been
ucts having no increase from the initial calculation for the 28 day test conventionally used in the preservative efficacy test to verify the micro-
considering that it is necessary to evaluate the burden on the 7th, bial reduction (Curry et al., 1993; British Pharmacopeia, 2010; Unites
14th and 28th day. On the other hand, the test was not consistent States Pharmacopeia, 2012). However methods considered more rapid
with other guidelines (Magee et al., 1997; European Pharmacopeia, have been recommended to determine the number of microorganisms,
2007; British Pharmacopeia, 2010). including among them, electrical methods (DePasquale et al., 1985;
Once more, the use of freeze-dried inoculum presented good repeat- Jeffrey et al., 1989; Connolly et al., 1993; Muscatiello, 1993; Connolly
ability among the tests performed for fungi. et al., 1994; Pinto et al., 1999).
It is possible to conclude from the results that a freeze-dried inocu- These methods are based on the detection time (DT), which is the
lum could be used in the preservative efficacy test to challenge solid time that the microorganism takes to reach the threshold level neces-
samples. The main goal of this paper was to provide an innovative chal- sary for its detection (approximately 107 CFU/mL). Nevertheless this
lenge test using freeze-dried microorganisms and an electrical method time depends on the generation time of each microorganism presenting
to evaluate microbial growth. It was not our purpose to optimize the different curves (Jeffrey et al., 1989).
preservative system, considering different types of preservatives and On the other hand, if this method is used to determine the number of
their concentration in the formulation. Further studies using other pre- survivors in the preservative efficacy test that consists of using pure cul-
servative systems are needed to provide a better understanding of using tures of microorganism, the interference of the generation time would
this method in PET, since only one preservative was evaluated in this be eliminated and calibration curves could be constructed. Connolly
study. et al. (1993) concluded that the impedance method is the only one
that presented satisfactory results to be used in the challenge test
compared to the direct epifluorescence technique (DEFT) and ATP
bioluminescence (ATP –B).
Despite that the major advantage of this method consists of reducing
analysis time, this is not a determinant factor, since in the preservative
efficacy, the last evaluation of the sample burden is performed 28 days
after its contamination. The major advantage claimed for these methods
is that they are more economical in the use of materials, labor and the
results are obtained within a shorter time period than the 18–60 h typ-
ical for colony count methods (Connolly et al., 1993, 1994). Its use also
spares the need for multiple dilutions and exhaustive colony counting
in plates using the conventional method (DePasquale et al., 1985).
Comparing the results obtained using impedance for determining
the burden and the traditional method, S. aureus presented similar be-
havior as it was possible to detect this microorganism in a 48-hour
Fig. 1. Linear least square regression analysis for electrical and pour plate results using test and no recovery for the remaining 28 days. When these findings
S. aureus, P. aeruginosa and A. niger. are compared with the specifications adopted in this study (Magee
108 M.R.S. Ferreira et al. / Journal of Microbiological Methods 106 (2014) 104–109
Tan, A.S.B., Tuysuz, M., Otuk, G., 2013. Investigation of preservative efficacy and microbiolog- Yang, L., Bashir, R., 2008. Electrical/electrochemical impedance for rapid detection of
ical content of some cosmetics found on the market. Pakistan J. Pharm. Sci. 26, 153–157. foodborne pathogenic bacteria. Biotechnol. Adv. 26, 135–150. http://dx.doi.org/10.
Unites States Pharmacopeia, 2000. USP 24. Unites States Pharmacopoeial Convention, 1016/j.biotechadv.2007.10.003.
Rockville. Zhou, X., King, V.M., 1995. An impedimetric method for rapid screening of cosmetic
Unites States Pharmacopeia, 2012. USP 35. Unites States Pharmacopoeial Convention, preservatives. J. Ind. Microbiol. 15, 103–107.
Rockville.