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Validation of Microbial Recovery of Pharmaceutically Important Gram-negative

Bacteria from Peroxygen/Silver based Disinfectants and Evaluation of their
Degree of Corrosiveness

Article · January 2017

DOI: 10.5530/fpi.1.2.1

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 ISSN Online: 2456-4192

Validation of Microbial Recovery of Pharmaceutically

Important Gram-negative Bacteria from Peroxygen/Silver
based Disinfectants and Evaluation of their Degree of Corrosiveness

Mostafa Eissa*
Quality Compliance Section Department, HIKMA Pharma pharmaceutical
Company, P.O. Box 1913, Cairo 11511, Egypt
Received 06 February 2017; accepted in revised form 20 March 2017

Abstract: Evaluation of a neutralization method and the degree of corrosiveness of disinfectants is a

crucial initial step that procede biocidal agents efficacy study. One of the major factors affecting the choice of
the disinfectants is their aggressiveness towards materials of construction in the pharmaceutical facility. Two
dilutions of each of four commercially used peroxygen/Ag+-based disinfectants were examined against four
Gram-negative microorganisms using Fluid Thioglycolate Medium (FTM) broth for both chemical and physical
neutralization. The acceptance criteria of a test group was > 70 % of the control group for each replicate group
namely neutralizer efficacy and neutralizer toxicity. Statistical analysis using One-way analysis of variance
(ANOVA) was carried out to confirm success/failure of a suspect result. By this analysis; five suspect treatments
passed the neutralizer efficacy test. Modified FTM-thiosulfate (FTMT); FTM neutralizer, was tested under the
same conditions against the same index of microorganisms and compared to a previously used and tested FTM
neutralizer. Neutralization of the modified FTM neutralizing broth was more effective than the previously
reported FTM with peroxygens/Ag+ disinfectants especially 1:100 dilution. Using higher dilutions has minimized
the reaction time in the present study. In addition, a gravimetric corrosion test was performed to determine the
degree of corrosiveness of disinfectants. Bixco showed greatest corrosion rate when compared with other
commercially used peroxygens which were not significantly different from the control. Corrosion test is a useful
tool to determine the preference among a range of disinfectants especially if they belong to the same class.

Key words: Peroxygen/Ag+, Neutralizing broth, Gram-negative bacteria, Fluid Thioglycolate

Medium, Corrosion test.

Introduction tion and microbial recovery studies are designed

Diminishing of biocidal activity of chemical com- per compendial guidelines methods for validation
pounds is a crucial initial prerequisite for many of microbial recovery as detailed in the United
assessment studies that are focused on the evalu- States Pharmacopeia (USP) Chapter <1227>
ation of the potencies of the antimicrobial compo- “Validation of Microbial Recovery from Pharma-
nents and recovery of bioburden from controlled ceutical Articles” as described by Clontz 1.
environment. Neutralization procedure assess- The residual amount of the biocidal agent can
ment should be carried out for each organism/ be neutralized by chemical neutralizers; several
disinfectant combination, to demonstrate the abil- classes of biocides have well-established chemi-
ity of the culture medium to support growth of cal neutralizers 2 and examples of these are noted
any viable microorganism. In general, neutraliza- in (Table 1) as illustrated by Sutton, et.al.3. How-

*Corresponding author (Mostafa Eissa)

E-mail: < mostafaessameissa@yahoo.com > © 2017, Har Krishan Bhalla & Sons
 Table 1. Previously reported efficacy against biocides
and toxicity on microbial cells for neutralizers 3

Neutralizer* Antimicrobial compound to be neutralized Toxicity on microorganisms

Bisulphate Glutaraldehyde, Mercurials Non-spore forming bacteria

Dilution Phenolics, Alcohol, Glutaraldehyde None
Glycine Glutaraldehyde Growing Cells
Lecithin Parabens, Bis-Biguanides, Quaternary Bacteria
Ammonium Compounds (QACs)
Magnesium or EDTA None
Calcium ions
Polysorbate QACs, Iodine, Parabens None
Thioglycolate Mercurials Spores and Staphylococci
Thiosulphate Glutaraldehyde, Halogens, Mercurials Staphylococci

*All neutralization methods are based on chemical compounds except dilution technique
ever, a main challenge of neutralization of bio- bial activity, resistance to organic matter, stability,
cides using chemical compounds is the potential incompatibility, irritancy, toxicity or corrosivity. To
toxicity demonstrated by many classes of neu- overcome such limitations of an individual agent,
tralizers. Thus, the evaluation of a chemical neu- formulations consisting of combinations of agents
tralizer scheme should consider the examination are available. For example, some combinations
of the potential toxicity of the neutralizer as well are synergistic, e.g. hydrogen peroxide (interme-
as its efficacy. A common example of the toxicity diate antibacterial activity level) and peroxygen
of some neutralizer components is that of thiogly- compounds. The germicidal properties of hydro-
colate against Staphylococci; in addition to spores gen peroxide (H2O2) have been known for more
4-7
. Another example is thiosulfate toxicity against than a century, but the use of low concentrations
Staphylococci 5,8-10. of unstable solutions did little for its reputation.
Complete abolishing of disinfectants activity is However, stabilized solutions are now available
crucial for the accuracy of a biocidal efficiency and due to its unusual properties and antimicro-
determination, as microbicidal effect is usually bial activity, hydrogen peroxide has a valuable role
assessed as still-viable along definite time inter- for specific applications. Concentrations of 3-6
val and suppression of microbial growth by the % are effective for general disinfection purposes.
remaining non-neutralized amounts of biocide may Peracetic acid (CH3CO3H) is the peroxide of ace-
lead to over estimated measures of microbicidal tic acid and is a more potent biocide than hydro-
activity 11. A work done by Russell describes three gen peroxide, with excellent rapid biocidal activ-
principle criteria to judge effectiveness of the neu- ity against bacteria. Similarly, silver have long been
tralizer. First of which; the neutralizer must effec- known to have antibacterial properties and prepa-
tively stop the effect of the biocidal solution. Sec- rations of these metal were among the earliest
ondly, the neutralizer must not itself be unduly toxic used antiseptics, silver will inhibit enzymes in the
to the challenge organisms. Finally, both the neu- membrane, and for that matter in the cytoplasm,
tralizer and the active agent must not react to form which contain thiol, -SH, groups 13,14.
a toxic byproduct. On the same line, three meth- To obtain accurate data a from neutralization
ods have been published describing methods of study, considering the inoculums count of each
neutralizer evaluation 12. organism incorporated in the study is a must. The
Unfortunately, there is no ideal disinfectant, anti- most common ranges to be accepted for count-
septic or preservative. All chemical agents have able numbers of colonies on a plate are 30-300
their limitations either in terms of their antimicro- 15,16
. However, experimental studies have dem-
onstrated very poor accuracy in plate counts be- ard Analysis and Critical Control Points
low 25 CFU per plate at which Error is 1/5 of the (HACCP), which was developed in the 1970s by
mean. Theoretically, it can be argued that because the U.S. Department of Agriculture to address
the CFU follow the Poisson distribution, the error food safety, is a systematic, proactive, and pre-
of the estimate is the square root of the average ventative tool to identify, assess, and prevent or
17
. This leads to results as those illustrated in reduce the potential risks that can occur at spe-
(Table 2), Equations (1) and (2) were derived from cific steps in a process. Through the risk analysis
(Table 2) for count range from 1 to 30 CFU/plate. process, critical control points are identified and
Equations (3) and (4) are derived from (1) and monitored 19.
(2), respectively by applying the inverse of the Several new corrosive antimicrobial-based bio-
Logarithm: cidal agents such as chlorine and peroxygens that
are commercially available contain anticorrosive
Log E.P. = 2 - (Log N.C./2) (Eq.1)
substances. Corrosion Inhibitors (ex. 3-amino-5-
2 Log S.E. = Log N.C (Eq. 2)
mercapto-1,2,4-triazole (AMTA)) are widely used
E.P. = 10(2-((Log N.C.)/2)) (Eq. 3)
in the protection of metals such as copper against
S.E. = 10((Log N.C.)/2) (Eq. 4)
corrosion in different corrosive media 20. Simi-
Where, E.P. = is the error as percent of mean. larly, carbon steels are commercially available
S.E. = is the standard error. N.C. = is the number metals, which are used in the fabrication of reac-
of CFU/plate. tion vessels, storage tanks, etc. Corrosion behav-
Equation (3) demonstrates that increasing the ior of steels is widely investigated in inorganic acids,
number of CFU/plate reduces error percent of salts, non-oxidation organic acids, alkaline solu-
the mean. However, at the same time -from Equa- tions, and marine media, and many inhibitors were
tion (4) -standard error increases but in less mag- also suggested to retard the corrosion of steel in
nitude (1 to 5.48) than that of the error percent of the above-mentioned solutions. However, corro-
the mean (100 % to 18.3 %) when the number of sion behavior and corrosion inhibition of carbon
CFU increases from 1 to 30 per plate. steel in oxidation media, especially in strong oxi-
Regarding the process risk assessment; a vari- dizing disinfectant solutions were rather scant.
ety of tools has been used in pharmaceutical in- Strong oxidizing disinfectant solutions have wide
dustry to determine the critical points in the pro- range of biocidal activity; they can be used to kill
cess that are at high risk of being contaminated bacteria, algae, yeasts, moulds, fungi and viruses
with microorganisms 18. On the same line, Haz- 21
. Peroxygen disinfectants are very active, and
Table 2. Error as a percentage of mean for plate counts
covering range from 1 to 30 CFU/plate and standard error 17

CFU Standard Error as CFU Standard Error as CFU Standard Error as

per Error % of per Error % of per Error % of
Plate Mean Plate Mean Plate Mean

30 5.48 18.3 20 4.47 22.4 10 3.16 31.6

29 5.39 18.6 19 4.36 22.9 9 3.00 33.3
28 5.29 18.9 18 4.24 23.6 8 2.83 35.4
27 5.20 19.2 17 4.12 24.3 7 2.65 37.8
26 5.10 19.6 16 4.00 25.0 6 2.45 40.8
25 5.00 20.0 15 3.87 25.8 5 2.24 44.7
24 4.90 20.4 14 3.74 26.7 4 2.00 50.0
23 4.80 20.9 13 3.61 27.7 3 1.73 57.7
22 4.69 21.3 12 3.46 28.9 2 1.41 70.7
21 4.58 21.8 11 3.32 30.2 1 1.00 100.0
are not affected by organic matter, but some prod- culture media were purchased from OXOID (Bas-
ucts may be corrosive to alloys, aluminum and ing-stoke, Hampshire) and chemicals from Sigma-
plain steel. Peracid solutions are a mixture of per- Alrich (St. Louis, MO 63103). (Table 3) shows
acetic acid, hydrogen peroxide and acetic acid; the list of microorganisms used in current study,
the two latter compounds possess a synergistic their sources, family and some of their biochemi-
effect with peracetic acid. They are extremely cal reactions.
effective biocides and have no toxic residuals 22, Standardized stable suspensions of test strains
but their acidic pH makes them corrosive to some were prepared and used as stated by their suppli-
materials when used at high concentrations. ers. Seed-lot culture maintenance techniques
Mechanistically, peracids are active against all (seed-lot systems) were used so that the viable
types of microorganisms, including spores, and microorganisms used for inoculation are not more
retain activity in the presence of organic mat- than 5 passages removed from the original mas-
ter 23 . ter seed-lot. All organisms were stored at -80°C
The current study was designed to optimize the in a validated -86°C Ultra low temperature freezer
use of combined chemical neutralization and dilu- (-86 Degree ULT Freezers, Qingdÿ Shandong,
tion for the recovery of Gram-negative microor- China) in validated cryogenic environment and
ganisms using commercial peroxygen-based bio- reactivated only prior to the study conduction us-
cidal agents. Herein, two studies were carried out, ing standard method illustrated by the supplier. All
firstly: disinfection validation program and the media were sterilized by autoclaving in a steam
degree of its corrosiveness. The second study in- sterilizer (FEDEGARI FOB3, Fedegari Autoclavi
volved the microbial recovery in the presence of SpA, SS 235 km 8, 27010 Albuzzano (PV), Italy).
residual disinfectant in environmental monitoring All pH measurements and weighing procedures
(EM) media. were done using Mettler-Toledo S20 Seven
Easy™ pH Meter and XPE Analytical Balance,
Materials and methods respectively (Mettler-Toledo, LLC 1900 Polaris
Preparation of microbial suspension Parkway Columbus, OH 43240).
Standard strains were purchased from the Suspensions were quantified by making serial
American Type of Culture Collection (ATCC, dilutions and performing duplicate plate counts us-
Manassas, Virginia) and handled as per a stan- ing conditions and media suitable for each micro-
dard procedure. As for the bacterial environmen- organism to choose suspensions of concentration
tal isolates, they were isolated and identified us- 3x102-103 CFU/50-100 μl as working suspensions.
ing the automated microbial biochemical identifi- Microbial test suspensions should be used as soon
cation system VITEK® 2 (bioMérieux, Inc., 100 as the results of serial dilutions could be enumer-
Rodol-phe Street, Durham, NC 27712). All the ated using a digital colony counter (Digital Colony
Table 3. List of Gram-negative rod microorganisms challenged in neutralizer validation
study with the source, family and some of the biochemical reactions of each

Challenged microorganisms Source Family Catalase/Oxidase/

Indole

Escherichia coli ATCC8739 Enterobacteriaceae +/-/+

Pseudomonas aeruginosa ATCC9027 Pseudomonadaceae +/+/-
Salmonella enterica subsp. ATCC14028 Enterobacteriaceae +/-/-
Enterica serovar Typhimurium
Sphingomonas picamobilis EM isolatea Sphingomonadaceae +/+/-

a = Slow-growing biofilm former organism isolated from early processing stages in water treatment station
and identified by VITEK 2 System version 5.02 (bioMerieux, France)
Counter Model: 361, Laxman Mahtre Rd. Nava- v) of the total volume in the tubes. Neutralizers
gaon, Dahisar West, Mumbai, India). used in the study were of normal strength for Fluid
Thioglycolate Medium Broth (FTM) and double
Neutralization validation study of peroxygen- strength for Fluid Thioglycolate Medium Thiosul-
based biocidal agents fate (FTMT).
The purpose of this study was to ensure that Inoculums of each of the used microorganisms
the assumed contact time is optimum. In other were added to each of the above described tubes
words, the time is valid for both the neutralizing so that the final count per plate of positive control
agent to efficiently stop the action of the tested was range 30 to 100 CFU per plate. Then about
sanitizer, and at the same time the neutralizing 20 ml of molten suitable medium at 45°C was
agent should not have any inhibitory or toxic ef- added; allowed to solidifying, then incubating at
fect on any of the microorganisms. It was sug- suitable temperature for 30-35°C for 3 days in
gested that two comparisons among three popu- BD 115 incubator (BINDER GmbH, ImMittleren
lations would be performed. The first study was Ösch 5 D-78532 Tuttlingen). After that; duplicate
concerned with the Neutralizer Efficacy (NE) plate counts were prepared for the 3 groups. The
which can be determined by evaluating the num- negative control study for each media with the
ber of survivors in the neutralizing broth in the same volume of diluents or neutralizers added was
presence and the absence of the biocide. Fur- carried out to ensure sterility of all used materi-
thermore, the ability of the neutralizing broth alone als. All the tests and control groups were per-
to allow the survival was a second important con- formed in triplicates for each microorganism, dis-
sideration in this analysis. The second compari- infectant and dilution. Residual peroxide was de-
son involved the Neutralizer Toxicity (NT). This termined using a semi-quantitative colorimetric
aspect of neutralization was determined by com- method by peroxide test strips (Peroxid-Test:
paring survivors in the neutralizing medium with- MQuant™, Merck KGaA, Frankfurter Str. 250,
out the biocide with the viability (growth) con- 64293 Darmstadt, Germany) at intervals in plain
trol 3,24. neutralizer with biocidal agents at two dilutions
The test solutions were freshly prepared and used in the test.
diluted in the same conditions that simulated ac-
tual usage environment of biocidal agents using Gravimetric corrosion test
the highest concentration which is 5 % (v/v) as Gravimetric corrosion measurement technique
recommended by the manufacturer. These com- was mainly based on the percentage weight-loss
mercial disinfectants were denoted Bixco (hydro- regime of the test specimens. In applying this
gen peroxide/Ag+), BafD (hydrogen peroxide/ method, the weights of test specimens (hollow
Ag+), Pur (hydrogen peroxide/peraceticacid/Ag+) cylindrical rings) were obtained before and after
and Mil (hydrogen peroxide/Ag+). Using a neu- a specified time interval of 96 hours (final weight,
tralizing broth as diluent 1:10 and 1:100 (v/v) dilu- mtfin.). The initial weights (initial weight, mtint.) of
tions of test solution- i.e. Disinfectant final con- the test specimens were recorded before immer-
centrations/10 ml of neutralizers were 5 % and sion in the test solution while the change in weights
0.5 % (v/v), respectively were made at working was taken after the test pieces were rinsed in
concentration, then 1 ml is transferred of each water and dried using a filter paper 25. The gravi-
dilution to each of duplicate petridishes this is test metric measurements were reproduced twice at
group. Neutralizer exposed group is prepared in room temperature range of 20 to 25°C. Control
parallel in the same manner as test group but us- coupons were exposed to the same conditions of
ing sterile saline or buffer instead of test solution. washing and time but not to the test solutions. The
While viability control group is prepared using initial and final weights of control coupons were
peptone water without test solutions or neutraliz- denoted by mcint.andmcfin., respectively 26. The
ing broth. Organisms were prepared so that the test was accelerated by using easily corroded steel
required inoculums did not exceed 0.5-1.0 % (v/ material of density approximately 7.6 g/cm3 and
challenged by the maximum working concentra- P = (KCorr/ρ) x 8.76
tion of disinfectant solutions of 5 % (v/v) and pu- Where,
rified water USP as control vehicle. P : the penetration index, mm.year-1.
At least three independent replicates of the ex- ρ : the density of metal, g/cm3
periment should be performed, and each should 8.76 : conversion factor, taken into account that
demonstrate that the average number of CFU one year has 8760 hours.
recovered from the challenge product is not less According to the penetration index stability of
than 70 % of that recovered from the inoculums the metal coupons against the liquid tested mate-
control (USP<1227>, 2013) and test for outlier rials used in this study and the degree of corro-
data within each set of groups was conducted sion could be determinedas detailed by some re-
using statistical package software to confirm the searchers 26. Finally, all statistical analysis was
results homogeneity and validity 27. If there were performed using GraphPad Prism version 5.00.288
suspect results within the groups, One-way for the corrosion test and version 6.01 for the
ANOVA was performed on Log 10 transformed microbial recovery test. Any interpretation or com-
results to confirm significance followed by plex calculation was performed using Microsoft
Dunnett’s Multiple Comparison Test which was Excel 2007.
used to confirm success or failure of the test and
Tukey’s Multiple Comparison Test to compare Results
between individual groups at α <0.001. The gravi- NT study revealed that both FTM and FTMT
metric corrosion test calculations were carried on did not possess any adverse or toxic effects on
according to the method described by Vasilescu tested Gram-negative microorganisms and passed
et al. 26. The weight loss (Δm, mg), the corrosion the acceptance criteria. The recovery ratio of the
rate (KCorr, mg.cm H2. h H1) were calculated as four microorganisms was e”1 except for 0.97 for
follow: Salmonella enterica subsp. entericaserovar
Δmc :mcint. - mcfin.Δmt = mtint. - mtfin.Δm = Δmt - Typhimurium with FTMT. All NT results did not
Δmc require statistical analysis by Dunnett’s multiple
KCorr = Δm/A.t comparisons test and they were not significantly
Where, different from each other using Tukey’s multiple
Δmc : weight loss of the control material, mg. comparisons test at α<0.001. The preliminary NT
Δmt : weight loss of the test material, mg. test results were presented in (Table 4) as geo-
Δm: the actual weight loss of the test material metric means and subjected to comparison with
due to exposure to specific corroding substance each other and against USP<1227>acceptance
to which control material is not exposed mg. criterion in (Fig. 1).
mcint. :the initial weight of control coupon mate- Table (4) shows the geometric means of NE
rial prior the initiation of the test, mg. ratio for the four microorganisms with four disin-
mcfin. :the final weight of control coupon mate- fectants at two dilution levels. Statistical analysis
rial after the completion of the test, mg. was conducted on the transformed microbial
mtint. :the initial weight of test coupon material counts of the four microorganisms’ groups includ-
prior the initiation of the test, mg. ing five suspect results then they were subjected
mtfin. :the final weight of test coupon material to Dunnett’s Multiple Comparison Test at
after the completion of the test, mg. α<0.001 which revealed non-significance of the
KCorr : the corrosion rate, mg.cm-2.h-1. difference between means. These suspect groups
t : the exposure time, hours. were from Pseudomonas aeruginosa and
A: the surface area of the coupon and is the Sphingomonas paucamobilis in FTM with Pur
exposure time in hours. and Bixco, respectively and from Salmonella
The penetration index (P, mm. year-1) in metal- enterica with Mil and Pur and Sphingomonas
lic mass was also determined using the following paucamobilis with Bixco in FTMT. These re-
expression: sults are demonstrated based on the USP <1227>
 Table 4. NT and NE ratios derived utilizing the geometric mean of the recovery in
the different populations of NT and NE for the tested Gram-negative bacilli
against Commercial peroxygen/silver biocidal agents at two
dilution levels: 1:10 and 1:100 (v/v)

G. meana E.coli S.enterica P.aeruginosa S.paucimobilis

FTM FTMT FTM FTMT FTM FTMT FTM FTMT

NT 1.07 1.14 1.07 0.97 1.20 1.10 1.11 1.08

Bixco 1:10 0.75 1.13 0.81 0.90 0.43 0.83 0.67b 0.58b
1:100 0.87 1.15 0.81 1.47 0.78 1.07 0.89 0.98
BafD 1:10 0.09 0.58 0.72 0.76 NA 0.26 NA NA
NE 1:100 0.56 0.90 0.80 1.10 0.49 0.86 0.96 1.00
Mil 1:10 0.22 0.59 0.62 0.67b NA 0.27 NA NA
1:100 0.55 0.88 0.81 0.83 0.36 0.82 1.01 1.05
Pur 1:10 0.22 0.43 0.23 0.73b NA 0.26 NA NA
1:100 0.54 0.75 0.54 1.02 0.58b 0.84 0.86 1.02

a = Geometric means
NA = Not applicable as the replicate contains one zero or more
b = Group set having result(s) below 70 % but passed statistical test for NE
Averages Log (CFU+1)

Neutralizer (FTM and FTMT)-Microorganism mixture

Fig. 1. Viability control bar of FTM and FTMT for Escherichia coli ATCC 8739, Salmonella
enterica subsp. enterica serovar Typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC
9027 and Sphingomonas picamobilis and NT of both chemical neutralizers on specified Gram-
negative rods. All results are expressed as averages of Log10 transformed (CFU+1) ± S.D. Dashed
line represents acceptance criteria for both neutralizers according to United States Pharmacopeia
Harmonized USP<1227>Validation of Microbial Recovery from Pharmacopoeial Articles at 70 %.
(Results were analyzed using GraphPad Prism for Windows version 6.01)
 acceptance criterion in (Figs. 2, 3, 4 and 5). while for FTMT, the success of total microbial
Statistical comparison between groups in NE recovery increased from less than 50 % to 100 %
study of the four microorganisms with the four thus, the impact of ten-times dilution of peroxygen-
biocidal agents revealed superiority of FTMT over based disinfectants on neutralization success of
FTM in terms of microbial recovery at both dilu- microbial recovery was significant and increased
tions with Bixco especially showing greatest re- the microbial recovery by more than a factor of
covery over the other three disinfectants. These two. In terms of total microbial recovery from
results were illustrated in (Figs. 2, 3, 4 and 5). By the four disinfectants after neutralization Salmo-
comparing FTMT and FTM neutralization profile nella enteric demonstrated highest recovery rate
of all groups, it was found that FTM total rate of from the chemical neutralization study (81 %) fol-
success and failure was 14 and 18 per 32 while lowed by Sphingomonas paucimobilus (63 %)
that of FTMT was 23 and 9 per 32 test groups, then Pseudomonas aeruginosa and Escherichia
respectively. The ease of neutralization of com- coli (44 %).
mercial peroxygen disinfectants was in the fol- `The results illustrated in (Table 5) indicated that
lowing decreasing order: Bixco (15/16)>BafD (8/ peroxide elimination reaction time in FTMT var-
16)>Mil=Pur (7/16). By increasing dilution ratio ied from nearly instantaneous with Bixco at both
from 1:10 to 1:100 more than half of the failed dilutions to delayed for 1.83 to 2 minute with BafD
results passed the test and the failed microbial at 1:100 (v/v). Mil and Pur 1:100 (v/v) were in
count plates decreased to about half for both FTM between both disinfectants i.e. 1.3 to 1.5 minute.
and FTMT together. However, for FTM alone BafD, Mil and Pur at 1:10 (v/v) showed continu-
there is 2.5 increase in the rate of success when ous presence of peroxides during test time in the
dilution ratio was increased from 1:10 to 1:100 neutralizer indicating inadequacy of neutralization.
Averages Log (CFU+1)

Disinfectant (5 %) Neutralizer (FTM and FTMT) mixture at 1:10 and 1:100 (v/v)
Fig. 2. NT control bar of FTM and FTMT for Escherichia coli ATCC 8739 and NE of both
neutralizers with Bixco, BafD, Mil and Pur at two dilutions ratios. All results are expressed as averages
of Log10 transformed (CFU+1) ± S.D. Dashed line represents acceptance criteria for both neutralizers
according to United States Pharmacopeia Harmonized USP<1227>Validation of Microbial Recovery
from Pharmacopoeial Articles at 70%. (Results were analyzed using GraphPad Prism for Windows
version 6.01)
Averages Log (CFU+1)

Disinfectant (5 %) Neutralizer (FTM and FTMT) mixture at 1:10 and 1:100 (v/v)
Fig. 3. NT control bar of FTM and FTMT for Pseudomonas aeruginosa ATCC 9027 and NE
of both neutralizers with Bixco, BafD, Mil and Pur at two dilutions ratios. All results are expressed as
averages of Log10 transformed (CFU+1) ± S.D. Dashed line represents acceptance criteria for both
neutralizers according to United States Pharmacopeia Harmonized USP <1227> Validation of Microbial
Recovery from Pharmacopoeial Articles at 70 %. (Results were analyzed using GraphPad Prism for
Windows version 6.01)
Averages Log (CFU+1)

Disinfectant (5 %) Neutralizer (FTM and FTMT) mixture at 1:10 and 1:100 (v/v)
Fig. 4. NT control bar of FTM and FTMT for Salmonella enterica subsp. enterica serovar
Typhimurium ATCC 14028 and NE of both neutralizers with Bixco, BafD, Mil and Pur at two dilutions
ratios. All results are expressed as averages of Log10 transformed (CFU+1) ± S.D. Dashed line
represents acceptance criteria for both neutralizers according to United States Pharmacopeia
Harmonized USP <1227> Validation of Microbial Recovery from Pharmacopoeial Articles at 70 %.
(Results were analyzed using GraphPad Prism for Windows version 6.01)
 Averages Log (CFU+1)

Disinfectant (5 %) Neutralizer (FTM and FTMT) mixture at 1:10 and 1:100 (v/v)
Fig. 5. NT control bar of FTM and FTMT for Sphingomonas picamobilis as water-born isolated
from biofilm in water treatment facility and NE of both neutralizers with Bixco, BafD, Mil and Pur at
two dilutions ratios. All results are expressed as averages of Log10 transformed (CFU+1) ± S.D.
Dashed line represents acceptance criteria for both neutralizers according to United States
Pharmacopeia Harmonized USP<1227>Validation of Microbial Recovery from Pharmacopoeial Articles
at 70%. (Results were analyzed using by GraphPad Prism for Windows version 6.01)

In addition, FTM showed similar pattern but at using a gravimetric method showed, according to
1:100 (v/v) dilution of BafD, Mil and Pur the time the results demonstrated in (Fig. 6), that mass loss
till reach undetectable level of peroxide (i.e. 0 was significant only for Bixco only while the re-
ppm) was between 3.5 to 4 minutes (i.e. about maining disinfectants were not significantly dif-
twice the time needed for FTMT to eliminate re- ferent from control at α<0.001. The relative cor-
sidual peroxides level). The pH of each of FTM rosion values were arranged in a descending or-
and FTMT, without additives, was 7.02 and 6.58 der as follows: Bixco (44.36), BafD (5.29), Mil
at normal laboratory temperature; respectively. (5.21) and Pur (2.64). Furthermore, (Fig. 7)
However, Bixco was an exception among the showed that the corrosion rates were in the fol-
tested disinfectants being rapidly and effectively lowing descending order: Bixco>Purified
neutralized with each of the two neutralizing water>Mil=BafD>Pur. However, according to the
broths. At 1:10 (v/v) dilution, the approximate av- penetration index of the metal coupons exposed
erage ranges of the tested residual peroxides of to each of the test solutions, the stability class/
BafD, Mil and Puralong an interval of 15 min- corrosion resistance degree towards each of
utes: 10-25, 5-10 and 10-25 ppm in FTM and 5- Bixco, purified water USP, Mil, BafD and Pur
10, 5-10 and 5-10 ppm in FTMT, respectively. was based on the results shown in (Table 6) as
When test was repeated but at 1:100 (v/v) dilu- follows II/4, II/3 and II/2 (for the last three), re-
tion till disappearance of detectable peroxides lev- spectively. It was noted that, the result of Pur was
els the results were: 10-25(3 minutes), 10-25(3 very close to perfectly stable I/1.
minutes) and 5-10 (2.5 minutes) ppm in FTM and
0.5 (2 minutes), 0.5 (1 minute) and 0.5-2 (1 minute) Discussion
ppm in FTMT, respectively. This study was carried on as a part of a major
On the other hand, the corrosion test performed project of sanitization validation including differ-
 Table 5. Semi-quantitative colorimetric Peroxide test performed on four disinfectants: Bixco, BafD,
Mil and Pur at two dilutions levels: 1:10 (v/v) and 1:100 (v/v) in both FTM and FTMT

Time Bixco (pH 4.74)a BafD (pH 4.03)a Mil (pH 3.91)a Pur (pH 3.25)a
(minutes) FTM FTMT FTM FTMT FTM FTMT FTM FTMT
1:10 1:100 1:10 1:100 1:10 1:100 1:10 1:100 1:10 1:100 1:10 1:100 1:10 1:100 1:10 1:100
(v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v)

0.3-0.5 - - - - + + + + + + + + + + + +
1.25-1 - - - - + + + + + + + ± + + + ±
1.3-1.5 - - - - + + + ± + + + - + + + -
1.83-2 - - - - + + + - + + + - + + + -
2.5-3 - - - - + ± + - + + + - + + + -
3.5-4 - - - - + - + - + - + - + - + -
4-4.5 - - - - + - + - + - + - + - + -
pHb 6.84 6.98 6.57 6.62 6.26 6.43 6.32 6.47 6.24 6.41 6.35 6.44 6.07 6.49 6.45 6.50

(-) = Zero mg/l (ppm)

(±) = 0-0.5 mg/l (ppm)
(+) = >0.5 mg/l (ppm)
a = pH determinations were done in range20-25°C for working concentrations of the disinfectant at 5% (v/v)
b = pH determinations were done in range 20-25°C for biocidal agent-neutralizer mixture
 Mass loss of steel after exposure to peroxygens

Weight loss (g)

Disinfectant solution 5 % (v/v)

Fig. 6. Mass loss (g) of steel after 96 hours exposure at room temperature to commercial peroxygen
biocidal agents and compared with control groups exposed to air only. Results are expressed as mean
± standard error of the mean (SEM). (Generated by GraphPad Prism version 5.00.288)
Gravimetric corrosion test of peroxygens on steel coupons
KCorr (mg/cm2/h)

Disinfectant solution 5 % (v/v)

Fig. 7. Gravimetric corrosion test of commercially used peroxygen disinfectants on metal coupon
material made of steel of identical geometrical shape after 96 hours exposure at room temperature.
Control group consisted of material exposed to purified water only. Results are expressed as mean ±
standard error of the mean (SEM). (Generated by GraphPad Prism version 5.00.288)
ent types of standard strains and environmental maceutical products as objectionable bugs that
isolates. Gram-negative organisms are generally should not be present in any pharmaceutical prepa-
found in aqueous environments (e.g. water sys- ration. In addition, these three organisms are
tems) and raw materials of natural origin. These members of the class of bile tolerant Gram-nega-
types of organisms are usually pathogenic and tive microorganisms which is also considered ob-
produce toxins such as endotoxins (lipopolysac- jectionable pharmacopeial class. EM isolate tested
charides in the cell wall of Gram-negative bacte- was added to the list as high risk organism that
ria). Bacterial endotoxins cause pyrogenic (fever) may contribute to a failure of water treatment sta-
reactions 1. Three out of 4 tested microorganisms tion system and hence, impacting the efficiency
are pharmacopeial and are to be tested in phar- of a classified area of sanitization, production ma-
 Table 6. The classification of corrosion behavior of metallic materials
according to stability class and corrosion resistance degree

P, [mm/year] Stability class Corrosion Liquids in which coupon were

resistance immersed
degree PuWa Bixco BafD Mil Pur

<0.001 I- Perfectly stable 1

0.001-0.005 II- Very stable 2 0.0027 0.0026 0.0010
0.005-0.01 3 0.0076
0.01-0.05 4 0.0272
0.05-0.1 III- Stable 5
0.1-0.5 IV- Relatively stable 6
0.5-1.0 7
1.0-5.0 V- Low stability 8

a= Purified water USP

chine cleaning with the consequence of high pos- By contrast, sulfur-containing compounds such as
sibility of compromising drug quality. sodium bisulfite, and sodium thiosulfate were all
The test for neutralizer validity was performed unable to neutralize Ag+ activity. These and other
originally between FTMT and FTM with the 3 findings imply that interaction of Ag+ with thiol
tested standard strains to compare between the 2 groups in enzymes and proteins plays an essential
tested neutralizers in their efficiency and capac- role in bacterial inactivation29.It was observed that
ity. Later, Sphingomonas paucimobilus was NaCl present in both neutralizing broths can aid
tested among other isolated microbial species to in Ag+ neutralization by precipitation as hazy white
confirm the validity of FTMT for the recovery of colloidal AgCl turbidity. Thioglycolate in the pres-
environmental isolates. Effective neutralization of ence of hydrogen peroxide is oxidized to dithio-
a biocidal agent is crucially important to the accu- glycolate. It is well known that sodium thiosulfate
racy of information obtained from any disinfec- is used to neutralize hydrogen peroxide solution
tant efficacy study 28. The determination of NT used in disinfection of contact lensesand it is su-
and of NE should be a comparison between a perior over other neutralizers such as catalase in
test and a control population. NT was determined terms of stability 30. However, the possible reac-
as the ratio of recovery between a viability popu- tion products of thiosulfate with peroxygens are
lation, and a population exposed to the neutral- the oxidation product of thiosulfate by peroxygen
izer. This comparison directly examined the tox- such as tetrathionate and other polythionates
icity of the individual neutralizing media for the which have no effect on microbial lethality. The
different microorganisms. The efficacy of a neu- selectivity of tetrathionate broth depends on the
tralizer wasdefined as the ratio of recovery be- ability of the thiosulfate and tetrathionate in com-
tween the neutralizerand the biocide, and the neu- bination to suppress commensal coliform organ-
tralizer exposed populations. Therefore, only the isms 31. Tetrathionate reacts with free sulfhydryl
effect of the biocide in the system was measured. groups of enzymes and to cause their inactivation
These ratios allowed for a threshold value (> 0.70) 32
. Thiosulfate can also react with sulfhydryl
as the first test. The second test was a statistical groups. Thus, it is suggested that tetrathionate
one to confirm success or failures of the suspect broth interferes with the synthesis, the activity, or
results by Dunnett’s Multiple Comparison Test. both, of sulfur-containing enzymes or cell wall and
As for the sodiumthioglycolate, being a thiol membrane components 33.
group containing compound, it could neutralize the The lethality of tetrathionate broth is directly
activity of silver nitrate against P. aeruginosa. related to the concentrations of thiosulfate and
tetrathionate in the medium. Decreasing the con- tralization of antimicrobial effect of peroxygen
centration of either salt reduces the lethal action compounds much more than silver salts. How-
of the mixture. The concentrations of 0.0736 M ever, based on the results of (Table 6), as the ini-
and 0.0236 M of thiosulfate and tetrathionate, re- tial concentration of the neutralized disinfectants
spectively (3:1 ratio), appear to be optimal 34. The is getting low the time needed for neutralization
ability of a microorganism to reduce tetrathionate becomes shorter in this study and hence increases
may play a role in decreasing the inhibition for the chance for microorganism to be recovered
certain species. When part of the tetrathionate is from the recovery medium. For FTMT neutral-
reduced, the ratio would be altered. It can be con- izer, the inhibitory effect of thiosulfate-tetra-
cluded that the outcome of the neutralization pro- thionate combination could be altered by: first, di-
cess may be toxic for the tested microorganisms. lution and the second factor is modifying three-to
This part needs more investigation to be performed one ratio by microorganism itselfand its intrinsic
on the mechanism of chemical neutralization and resistance (ex. by production of tetrathionate re-
the effect of reaction products accumulation on ductase enzyme) and/or variation in disinfectant
microbial viability. The above findings were, in part, composition and/or concentration.
in agreement with ours which demonstrated that Cystine (SCH2CH(NH2)CO2H)2 is the amino
Salmonella enterica was recovered at >70 % acid formed by the oxidation of two cysteine mol-
from all FTMT-peroxygen exposed groups which ecules that covalently link via a disulfide bonds
gave the highest recovery rate if compared with that cleave more rapidly at higher temperatures
Escherichia coli, Sphingomonas paucimobilus (ex. temperature of media preparation and ster-
and Pseudomonas aeruginosa. Salmonella ilization) 36. Cystine is present in FTM in half
enterica highest recovery rate from FTM is most amount of that in FTMT. Cysteine amino acid has
probably attributed to its intrinsic tolerance to re- reducing properties like thioglycolate with
sidual hydrogen peroxide and the support of this peroxygens. However, auto oxidation is expected
assumption the considerably high microbial recov- on standing from atmospheric oxygen in the head
ery from FTM and FTMT at 1:10 (v/v) where space of the reservoir tube thus neutralizing broth
there persistent relatively high level of the perox- must be either prepared fresh, heated once di-
ides based on the results shown in (Table 6). FTM rectly before use and/or incorporation of thicken-
possesses much less chemical neutralization ca- ing agent in the neutralizing broth (ex. agar in small
pacity (the reducing compounds components are amount to render media thick but not solid) which
half that of in-house made neutralizer and there is retard atmospheric oxygen diffusion. This prop-
no thiosulfate) than FTMT for peroxygens be- erty is found to greater extent in FTMT than FTM.
cause no difference in the ratio of pass/fail tests Redox indicator such as resazurin is a good indi-
was observed between 1:10 and 1:100 groups. If cator for such situation to judge visually the pres-
this property is coupled with well-known low con- ence and the degree of oxygen diffusion into the
centration exponents (ç) of hydrogen peroxide media for either reheat or discard. A further in-
(peroxygen) if compared to silver nitrate (ç=0.5 vestigation is needed to measure the possible ef-
and 0.9 to 1 respectively) then the intrinsic micro- fect of auto oxidation upon storage of the neutral-
bial resistance will play its major rule in surviving izer on its capacity of neutralization of peroxygen-
Gram-negative bacteria cells in the presence of based biocidal agents.
residual amount of peroxygen disinfectants. Ac- Considering the NT study performed for both
cording to USP<1072> Disinfectants and Anti- FTM and FTMT, used herein, its results revealed
septics (2014): Biocidal activity reduction factor= that they were non-toxic and could be used in the
(Dilution folds) ç. Thus, the reduction of activity validation program. The other important subse-
of hydrogen peroxide is about 3.2 to 10 reduction quent aspect is NE. The scheme followed was
in activity only for 1:10 and 1:100, respectively based on FTM as primary neutralizer that has very
while for AgNO3 it is 7.9 and 10 to 63.1and 100 close composition to NIH Thioglycolate (supported
35
. This means that dilution has little effect of neu- by previous work). FTM Thioglycolate was found
to be non-toxic or of low toxicity against microor- centration that meet the predefined acceptance
ganisms. In-house made neutralizer FTMT is in criterion. For example, critical concentration for
between DEB and NIH Thioglycolate in compo- Bixco was about 3.5-4 % (v/v) for Sphingomonas
sition. The combination of microorganism, neu- paucimobilus and >5 % (v/v) for the remaining
tralizer and disinfectant is unique and thus the suc- microorganisms in FTMT.
cess of one combination with one microorganism Data transformation to Log 10 was done to ap-
does not mean that same combination with other proximate normal distribution following approach
microorganisms will do accordingly 3. However, outlined by 39. In Log 10 transformation, in the
some researchers allowed for predetermined con- event where the mean count is zero, this requires
tact time between disinfectants and neutralizers the addition of “1” to each value of zero. So, that
before addition of microorganisms. This sequence this does not distort other results, a value of “1” is
is different from practical situations in which dis- added to each item of data 40.
infectant carrying microorganism is neutralized by The gravimetric corrosion measurements indi-
neutralizing media either in disinfectant validation cated that Mil, BafD and Pur disinfectants con-
study or EM sampling. Our results showed that tain effective anticorrosive substances while
some disinfectants may be neutralized slower than Bixco does not. Normal test takes long time that
others for the same chemical neutralizer taking may reach in some cases 90 days but using easily
some time till elimination of residual biocide to corroded steel material accelerated the test such
undetectable level. This time cannot be neither that it took few days and this is important in pre-
controlled nor calculated which may lead to ex- liminary rapid judgment for selection of the most
aggerated estimation of biocidal potency. suitable biocidal agents 41. Long term inspection
Bacteria are known to degrade H2O2 using two study of prolonged contact time could be done
classes of enzymes: catalases and peroxidases after that for the selected disinfectants on corro-
37
. Salmonella typhimurium encodes three cata- sion resistant materials used in the pharmaceuti-
lases (KatE, KatN, KatG) and three peroxidases cal facility. peracetic acid (PA) is a wide spec-
(AhpC, TsaA, Tpx) 37,38. This fact agreed with trum, rapid disinfectant that is more expensive
that of the current study in which Salmonella than hypochlorite, but without its corrosivity and
typhimurium showed greatest tolerance to the degree of inactivation by organic matter. PA is
residual peroxides levels in the investigated neu- currently used as a disinfectant for flexible fiber
tralizing broth. Thus, the critical factor in the mi- optic endoscopes, where corrosion is highly un-
crobial recovery in this test is the intrinsic bacte- desirable. It is believed that PA could be used in
rial cell resistance to the low level of peroxides in laboratories handling mycobacteria for disinfec-
the neutralizing broths which was persistent at 1:10 tion in areas where corrosion must be avoided
(v/v) till more than 15 minutes but progressively 42,43
. This is in agreement with the finding in the
decaying at 1:100 (v/v) in neutralizing broths at current study where Pur gave the lowest corro-
rate in FTMT higher than FTM. It is apparently sion rate if compared with the other disinfectants
that this tolerability to peroxygens traces was at especially Mil and BafD (where anticorrosive sub-
minimum with Escherichia coli and Pseudomo- stance is present in their formulae) as PA syner-
nas aeruginosa, maximum with Salmonella gistic combination with hydrogen peroxide made
enterica subsp. Entericaserovar typhimurium the concentration of the later lower in the biocidal
with Sphingomonas paucimobilus in between. agent formula.
This is supported by (Figs. 1, 2, 3 and 4) where Finally, the results in (Table 6) are indication for
critical disinfectants concentrations (concentra- that the reaction rate at ordinary room tempera-
tion of disinfectant in neutralizing broth that meet ture played significant role for the process of
> 70 %) could be determined roughly. Critical con- chemical neutralization in addition to neutralizer
centration could be determined for each disinfec- capacity in this study since both FTM and FTMT
tant by performing serial dilutions of biocidal agent at 1:100 (v/v) dilution were capable to neutralize
in diluting broth and determining the highest con- tested disinfectants with the former reaction time
was longer (about twice the time of the later). necessary in the process of chemical neutraliza-
The differences in the approximate average range tion to achieve rapid diminishing of residual bio-
of peroxide level were also significant especially cidal agents otherwise variable outcomes of mi-
at 1:100 (v/v) between both neutralizers - till com- crobial recovery will be obtained according to in-
plete neutralization-indicating that not only the time trinsic microbial resistance to the disinfectant be-
of exposure of microorganisms to residual disin- ing neutralized. Thus, in the study of chemical neu-
fectants affected the viability but also the level of tralization process microorganism suspension must
biocidal agents in neutralizing broths. This be added directly after mixing of disinfectant-neu-
uncalculated time in neutralization process may tralizing broth directly as the delay may mask the
give false over estimation of cleaning efficacy true neutralization process capability and hence
monitoring and/or sanitizer efficacy if microorgan- probably leads to the skewness in the actual po-
isms were added later to the neutralizer-disinfec- tency of the biocidal agent and/or misjudgment of
tant broth after certain time. During this time, in- the sensitivity of procedure of microbial recovery
nate microbial resistance played significant role from residual antimicrobial substances.
for its survival till complete neutralization and gave
variable observed outcomes of chemical neutral- Conclusion
ization of peroxygen-based disinfectants. Again, An ideal chemical neutralizer must be non-toxic
this could be evident from critical value. This value to microorganisms and neutralizes chemical bio-
is between 1.5 and 2 % (v/v) for Sphingomonas cidal agents immediately after mixing without toxic
paucimobilus, Escherichia coli and Pseudomo- byproducts formation to microbial cells. If such
nas aeruginosa with BafD and Mil in FTMT purpose could not be accomplished at low dilution
while for Salmonella enterica is > 5 and 4 % (v/ ration increasing dilution level may be the first aid
v) respectively. In FTMT, Pur critical concentra- step in achieving this goal to reduce or diminish
tion for organisms was in the following descend- exposure time and magnitude of toxicity of anti-
ing order: Salmonella enterica > Sphingomonas microbials and/or toxic neutralization reaction
paucimobilus>Pseudomonas aeruginosa > Es- products. FTMT neutralizer was effective with
cherichia coli. For FTM, critical value of Pur index commercial peroxygen-based disinfectants
for the three standard strains was less than 0.1 % (Bixco, BafD, Mil and Pur) tested at 1:100 (v/v)
(v/v). For Mil and BafD, the recovery of Escheri- dilution ratio. Chemical neutralizers based on re-
chia coli and Pseudomonas aeruginosa was dox neutralization reactions must be ensured to
less than 0.2 % (v/v) while Salmonella enterica be in its full reduced form to obtain maximum
showed greater resistance with critical value of > neutralization capacity and hence efficacy for dis-
4.5 % (v/v) approximately. Further mechanistic infectants based on oxidizers. Gravimetric corro-
study is needed to provide evidence that peroxygen sion test could be used as fast, simple and useful
neutralization process takes place faster with thio- tool to judge the selection of disinfectants which
sulfate than thioglycolate and cystiene in men- are based on corrosive active biocidal compounds
tioned neutralizers. where they can affect life span of many materi-
It is clear from the previous findings that it is als in pharmaceutical facility.

References
1. Clontz, L. (2009). Microbial Limit And Bioburden Tests: Validation Approaches and Global
Requirements. 2nd ed. Boca Raton: CRC Press.
2. MacKinnon, I.H. (1974). The use of inactivators in the evaluation of disinfectants. Journal of
Hygiene. 73(02): 189-195.
3. Sutton, S.V., Proud, D.W., Rachui, S., Brannan, D.K. (2002). Validation of microbial recovery
from disinfectants. PDA Journal of Pharmaceutical Science and Technology. 56(5): 255-266.
4. Croshaw, B., Groves, M.J. and Lessel, B. (1964). Some Properties of Bronopol, A New
Antimicrobial Agent Active Against Pseudomonas Aeruginosa. Journal of Pharmacy and Pharma-
cology. 16 (S1), 127T-130T.
5. Kayser, A. and Van der Ploeg, G. (1965). Growth inhibition of staphylococci by sodium thio-
sulphate. Journal of Applied Bacteriology. 28(2): 286-293.
6. Mossel, D.A.A. and Beerens, H. (1968). Studies on the inhibitory properties of sodium thio-
glycolate on the germination of wet spores of clostridia. Journal of Hygiene. 66(02): 269-272.
7. Hibbert, H.R. and Spencer, R. (1970). An Investigation Of The Inhibitory Properties of
Sodium Thioglycollate In Media For The Recovery Of Clostridial Spores. Journal Of Hygiene.
68(01): 131-135.
8. Weber, G.R. and Levine, M. (1944). Factors Affecting Germicidal Efficiency of Chlorine and
Chloramine. American Journal of Public Health and the Nations Health. 34(7): 719-728.
9. Hugo, W.B. and Newton, J.M. (1964). The adsorption of iodine from solution by microorganisms
and by serum. Journal of Pharmacy and Pharmacology. 16(1): 49-55.
10. Cox, Claire B., Feeley, J.C. and Margaret Pittman. (1973). Sterility Testing: Detection of
Fungi and Yeasts in the Presence of Preservatives. Journal Of Biological Standardization. 1(1):
11-19.
11. Block, S.S. (1983). Disinfection, Sterilization and Preservation. 1st ed. Philadelphia, PA: Lea
& Febiger.
12. Russell, A.D. (1981). Neutralization procedures in the evaluation of bactericidal activity. Dis-
infectants: their use and evaluation of effectiveness. Academic Press, London. p 45-59.
13. Russell, A.D. (1990). Bacterial spores and chemical sporicidal agents. Clinical microbiology
reviews. 3(2): 99-119.
14. Hugo, W.B. and Russell, A.D. (1998). Pharmaceutical microbiology. 6th ed. London, UK:
Blackwell Science Ltd.
15. Maturin, L. and Peeler, J. (1998). Chapter III: Aerobic Plate Count, Bacteriological Analysis
Manual. Standard Practice for Determining Microbial Colony Counts from Water Analyzed by
Plating Methods. Retrieved 10 August 2016, from http://www.fda.gov/Food/Sd.enceResearchf
LaboratoryMethodsfBacteriologicaiAnalyticaiManualBAMfucrn063346.htm.
16. Maturin, L. and Peeler, J. (2011). Aerobic Plate Count. Bacteriological Analysis Manual.
Retrieved 10 August 2016, from http://www.fda.gov/Food/Sd.enceResearchLaboratoryMethods
BacteriologicalAnalyticalManual.htm.
17. US Pharmacopoeia. (2014). Validation of Microbial Recovery from Pharmacopoeial Articles,
In: US Pharmacopoeia 37/National Formulary 32. Washington, DC: United States Pharmacopoeial
Convention.
18. World Health Organization (WHO). (2003). Application of Hazard Analysis and Critical
Control Point Methodology to Pharmaceuticals, In WHO Expert Committee on Specifications for
Pharmaceutical Preparations, Thirty-Seventh report, Annex 7 (WHO Technical Report Series,
No. 908), World Health Organization, Geneva.
19. Van Schothorst, M. (1997). A simple guide to understanding and applying the hazard analysis
critical control point concept. ILSI Europe. 3rd edition. 1-30.
20. Es-Saheb, M., Elzatahry, A.A., Sherif, E.M., Alkaraki, A.S. and Kenawy, E.R. (2012). A
novel electrospinning application for polyvinyl chloride nanofiber coating deposition as a Corrosion
Inhibitor for Aluminum, Steel, and Brass in Chloride Solutions. Int. J. Electrochem. Sci. 7: 5962-
5976.
21. Qu, Q., Yuan, R., Li, L., He, Y., Luo, J., Wang, L., Xu, H., Gao, Y., Song, Y. and Ding, Z.
(2013). Corrosion Behaviour of Cold Rolled Steel in 84 Disinfectant Solutions. Int. J. Electrochem.
Sci. 8: 11625-11640.
22. Block, S.S. (2001). Peroxygen compounds. In: Disinfection, Sterilisation and Preservation, 5th
edition, Block SS (ed), Lippincott Williams & Wilkins, London.
23. Jeffrey, D.J. (1995). Chemicals used as disinfectants: active ingredients and enhancing additives.
Revue scientifique et technique (International Office of Epizootics) 14(1): 57-74.
 24. Eissa, M.E., Ashour, M.S. and Mansy, M.S. (2012). Neutralizer evaluation study of some
microbial isolates against two strong disinfectants with and without the presence of synthetic
detergent. World Applied Sciences Journal, 20(6): 823-831.
25. Adeosun, S.O., Sekunowo, O.I., Balogun, S.A. andObiekea, V.D. (2012). Corrosion
Behaviour of Heat-Treated Aluminum-Magnesium Alloy in Chloride and EXCO Environments.
International Journal of Corrosion. Article ID 927380, 9 pages.
26. Vasilescu, M. and Vasilescu, I. (2009). Contributions Regarding the Corrosion Behaviour of
Some Al-Cu-Mg Superalloys. Revista de Chimie. 60(1): 23-28.
27. US Pharmacopoeia. (2013). Validation of Microbial Recovery from Pharmacopoeial Articles,
In: US Pharmacopoeia 36/National Formulary 31, Washington, DC: United States Pharma-
copoeial Convention.
28. Langsrud, S. and Sundheim, G. (1998). Factors influencing a suspension test method for anti-
microbial activity of disinfectants. Journal of Applied Microbiology. 85(6): 1006-1012.
29. Liau, S.Y., Read, D.C., Pugh, W.J., Furr, J.R. and Russell, A.D. (1997). Interaction of
silver nitrate with readily identifiable groups: relationship to the antibacterial action of silver ions.
Letters in Applied Microbiology. 25(4): 279-283.
30. Ogunbiyi, L. (1986). The use of sodium thiosulfate for inactivating residual hydrogen peroxide
on contact lenses after disinfection. Clinical and Experimental Optometry. 69(1): 16-21.
31. Knox, R., Gell, P.G.H. and Pollock, M.R. (1943). The selective action of tetrathionate in
bacteriological media: A report to the medical research council. Journal of Hygiene. 43(03): 147-
158.
32. Parker, D.J. and Allison, W.S. (1969). The mechanism of inactivation of glyceraldehyde 3-
phosphate dehydrogenase by tetrathionate, o-iodosobenzoate, and iodine monochloride. Journal
of Biological Chemistry. 244(1): 180-189.
33. Postgate, J.R. (1963). The examination of sulphur auxotrophs: a warning. Microbiology. 30(3):
481-484.
34. Palumbo, S.A. and Alford, J.A. (1970). Inhibitory action of tetrathionate enrichment broth.
Applied microbiology. 20(6): 970-976.
35. Aslaksen, M.A., Romarheim, O.H., Storebakken, T., Skrede, A. (2006). Evaluation of
content and digestibility of disulfide bonds and free thiols in unextruded and extruded diets containing
fish meal and soybean protein sources. Animal Feed Science and Technology. 128(3): 320-330.
36. US Pharmacopoeia. (2014). Disinfectants and Antiseptics, In: US Pharmacopoeia 37/National
Formulary 32. Washington, DC: United States Pharmacopoeial Convention.
36. Imlay, J.A. (2008). Cellular defenses against superoxide and hydrogen peroxide. Annual Review
of Biochemistry. 77: 755.
37. Sokal, R. and Rohlf, F. (1995). Biometry. The Principles and Practice of Statistics in Biological
Research. W. H. Freeman and Company, New York. p 411-422.
38. Fang, F.C. (2011). Antimicrobial Actions of Reactive Oxygen Species. Mbio. 2(5): e00141-11-
e00141-11.
39. Sandle, T. (2004). An approach for the reporting of microbiological results from water systems.
PDA Journal of Pharmaceutical Science and Technology. 58(4): 231-237.
40. Standard Test Procedures For the Evaluation of Wildland Fire Chemical Products. (2000).
Test Method 5 - Corrosion Tests. [online] Available at: http://www.fs.fed.us/rm/fire/wfcs/tests/
documents/stp_cont.pdf [Accessed 8 Aug. 2016].
41. Holton, J., Nye, P. and McDonald, V. (1994). Efficacy of selected disinfectants against Myco-
bacteria and Cryptosporidia. Journal of Hospital Infection. 27(2): 105-115.
42. Griffiths, P.A., Babb, J.R. and Fraise, A.P. (1998). Mycobacterium Terrae: A Potential
Surrogate For Mycobacterium tuberculosis In A Standard Disinfectant Test. Journal Of Hospital
Infection 38(3): 183-192.

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