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Research Article Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints

Authors:

Abstract

Introduction: Despite its long history as an antiseptic, the image of CS has been badly ‘tarnished’ by opportunistic promoters, lack of quality controls (QC), deliberate misinformation and reprehensible scare tactics. This article evaluates some commercial colloidal silver (CS) preparations for their efficacy as antiseptic agents. Aims: We examined the potential medicinal value of commercially available CS preparations testing them by a) various chemical and physical criteria and also b) in vitro assays for bio-efficacy and safety. Methods: Antibacterial activity of CS preparations was determined by disc diffusion growth inhibition assays against a panel of pathogenic bacteria and fungi. Toxicity (LC50) was assessed by the Artemia franciscanna nauplii bioassay. Results: Of the 12 CS preparations tested, 10 (83%) showed antimicrobial activity, albeit with varying specificity and efficacy. Argyrol and HLY displayed the broadest specificity, inhibiting the growth of all 14 bacteria tested (100%). These particular preparations also inhibited the growth of 3 (100%) and 2 (67%) of the fungal species tested respectively. The other preparations had varying degrees of efficacy and specificity. In general, only low concentrations of CS were required to achieve antibiotic activity, with MIC values ≤ 5μg/ml for some preparations against some microbial species. In contrast, 2 colloidal gold preparations were completely devoid of antimicrobial activity. All CS preparations were either nontoxic or displayed low toxicity in the Artemia franciscanna nauplii bioassay, further confirming their potential as antiseptics for medicinal use. Conclusions: The commercial CS preparations varied widely in their potential utility as complementary medicines. The establishment of quality controls for both antimicrobial efficacy and incipient toxicity to animal cells are badly needed. However, this study does demonstrate the effective antiseptic activity of certain CS preparations indicating that they should be seriously considered as medicinals for topical use e.g. treating burns, periodontitis, thrush etc.
(c) Copyright 2011 EManuscript Publishing Services, India 47
Research Article
Pharmacognosy Communications www.phcogcommn.org
Volume 2 | Issue 1 | Jan-Mar 2012
*Correspondence:
(Prof) M Whitehouse, PO Box 68, Stones Corner, Qld. 4120.
E-mail: whitehousemd@spin.net.au; Fax: +617 3349 3006
DOI: 10.5530/pc.2012.1.9
Colloidal Silver (CS) as an Antiseptic: Two opposing
viewpoints
Ian Cock1,2, Shimony Mohanty1,2, Alan White2, Michael Whitehouse3*
1Environmental Futures Centre, Griffith University, Nathan, Qld. 4111, Australia. 2Biomolecular and Physical Sciences, Griffith University,
Nathan, Qld. 4111, Australia. 3School of Medicine, Griffith University, Gold Coast, Qld. 4222, Australia.
INTRODUCTION
So often there is a singular lack of pharmacognosy literally
knowing your drug – when procuring, or even preparing,
an antiseptic ‘colloidal silver’ (CS) preparation. The history
of silver pharmacology is confused by lack of standardized
preparations other than topical silver nitrate used as a caustic
or soluble disinfectant, that rapidly stains surrounding tissues
black.[1]
CS preparations for topical and/or oral use were subsequently
developed to overcome the astringency of, and staining by,
simple silver salts[2] while retaining the long-known antiseptic
properties of metallic silver.[3] The older preparations, pre-1910,
usually contained high levels of oxidized silver (Ag+), not
precipitated by isotonic salt solutions, made from silver nitrate
and various carrier proteins or derived polypeptides. Argyrol®
is one such product still extant today and formerly much
used as an eye disinfectant to prevent neonatal blindness.
Finely dispersed silver metal (Ago) preparations were
originally made as pigments (green-yellow) for medieval
glass, manufactured by including a silver salt with a reducing
agent in the glass-making process. In the 19th Century
aqueous metallic/zerovalent silver colloids/hydrosols were
prepared chemically by reducing silver salts with organic
reductants e.g. sodium citrate (the so-called Carey Lea’s
preparation, 1889[4]), sodium tannate, etc. Their antiseptic
properties were generally of less interest than their physical
properties e.g. preparing mirrors, for electronic applications.
Since 1911, the availability of silver metal dispersions
prepared electrolytically from silver rods/plates provided bio-
accessible silver in the zerovalent state (Ago) and proved
ABSTRACT: Introduction: Despite its long history as an antiseptic, the image of CS has been badly ‘tarnished’ by
opportunistic promoters, lack of quality controls (QC), deliberate misinformation and reprehensible scare tactics. This
article evaluates some commercial colloidal silver (CS) preparations for their efcacy as antiseptic agents. Aims: We
examined the potential medicinal value of commercially available CS preparations testing them by a) various chemical
and physical criteria and also b) in vitro assays for bio-efcacy and safety. Methods: Antibacterial activity of CS
preparations was determined by disc diffusion growth inhibition assays against a panel of pathogenic bacteria and
fungi. Toxicity (LC50) was assessed by the Artemia franciscanna nauplii bioassay. Results: Of the 12 CS preparations
tested, 10 (83%) showed antimicrobial activity, albeit with varying specicity and efcacy. Argyrol and HLY displayed
the broadest specicity, inhibiting the growth of all 14 bacteria tested (100%). These particular preparations also
inhibited the growth of 3 (100%) and 2 (67%) of the fungal species tested respectively. The other preparations had
varying degrees of efcacy and specicity. In general, only low concentrations of CS were required to achieve antibiotic
activity, with MIC values ≤ 5µg/ml for some preparations against some microbial species. In contrast, 2 colloidal gold
preparations were completely devoid of antimicrobial activity. All CS preparations were either nontoxic or displayed
low toxicity in the Artemia franciscanna nauplii bioassay, further conrming their potential as antiseptics for medicinal
use. Conclusions: The commercial CS preparations varied widely in their potential utility as complementary medicines.
The establishment of quality controls for both antimicrobial efcacy and incipient toxicity to animal cells are badly
needed. However, this study does demonstrate the effective antiseptic activity of certain CS preparations indicating that
they should be seriously considered as medicinals for topical use e.g. treating burns, periodontitis, thrush etc.
Key words: silver nanoparticles, colloidal silver, inorganic pharmacognosy, antimicrobial, Ago, Ag+
48
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
10 nm and 50 nm sized nanoparticles (Nano Composix,
San Deigo CA). The third reference CS was prepared
electrolytically with a mean particle diameter of 33 nm (kindly
donated by H. Laroo, Security Research, Ipswich Qld). For
antimicrobial testing, this was concentrated to 135 ug/ml.
Colloidal gold preparations
One gold colloid preparation was obtained commercially
from a local Brisbane health store. A second colloidal gold
preparation was prepared by reducing AuCl4 with sodium
citrate and contained particles with a mean diameter of
37 nm.[7]
Physical and chemical characterisation of colloidal
preparations
One CS sample (Argyrol branded as Argyrex) was obtained
as a semi-solid and readily dispersed into distilled water.
Aqueous samples were routinely examined for the following
properties:
Total silver content as ppm (mg/L) determined by atomic
absorption spectroscopy (AAS) or by Inductively Coupled
Plasma Mass Spectrometry (ICP-MS)
pH and electrical conductivity
colour and plasmon absorption over the range 410-
430 mm
light scattering at 532 nm, measured at right angles (90
degrees) in arbitary units/ppm using a 5 milliwatt green
laser and USB 2000 photometer (Ocean Optics,
Dunedin, FLA)
Free/contaminating silver ions (Ag+) by measuring the
amount of silver either a) removed by precipitation with
KSCN or after shaking with a suspension of the cation
exchanger Na+ Amberlite IR-120 (Rohm and Haas,
Philadelphia, Pa) or b) by cyclic voltammetry to measure
electro-reducible species.
Product stability: presence/absence of visible precipitate
after standing at room temperature in the dark for periods
up to three months.
Median particle size of the dominant population was
measured using a Nicomp 370 instrument (Particle Sizing
Systems, Santa Barbara CA) and are expressed as the
median value (by number weighting) of the dominant
species (≥ 99.5 %).
Some samples were further examined for contamination
by other metals or arsenic by ICP-MS. Samples which
showed zero silver content by AAS (due to matrix effects)
were specically re-analysed for silver by ICP-MS
Antimicrobial screening
Test microorganisms
All microbial strains were obtained from Michelle Mendell
and Tarita Morais, Biomolecular and Physical Sciences,
life-saving in World War I e.g. treating ‘trench fever’, a
rickettsial infection.[5]
Until the availability of sulfonamides (1930s) and antibiotics
(1940s), CS preparations were widely used whenever less
effective chemosterilants (alcohol, hypochlorite, iodine,
phenols) proved inadequate. Some questionable activities
then contrived to legally remove CS from the public domain
in the USA and ensure only patented anti-infective organic
pharmaceuticals could be sold to the public.
With the resurgent interest in do-it-yourself (DIY) medicine,
electrolytic CS generators have become widely available for
home use.[3, 6] Such generators produce variable mixtures of
soluble Ag+ and Ago particles, together with some less soluble
oxidised Ag products e.g. oxide, carbonate. These CS products
usually contain only low quantities of silver of the order
5-50 ppm (mg/L). Depending on the qualities of the silver
electrodes (<99.99 per cent pure) and of the water used as
electrolyte, signicant minor impurities may sometimes
contaminate these DIY preparations e.g. As, Cu, Mg, Pb.
Currently the US Food and Drug Administration (FDA)
and the Australian Therapeutics Goods Administration
(TGA) both proscribe the medicinal use of CS and prohibit
making any claims regarding its efcacy. These administrations
dogmatically proclaim that CS is both inefcacious and
toxic (surely an enigma). Nevertheless, the TGA does
recognise the value of CS for sterilising water, surely a
medicinal property but apparently not an (illegal) claim.
(Another enigma.) However, many CS preparations are
available in Australia, New Zealand, UK and USA from
pharmacies, health food stores and internet sales. Their
labels usually indicate only the total silver content. Some
also carry such disingenuous statements as, ‘We are not
allowed by law to tell you what this product is good for’.
It is important for the FDA, TGA and other drug regulatory
bodies to have available some practical criteria for sensibly
assessing CS products and that these criteria are available to
the general public. Otherwise, the present absurd restrictions
will be mindlessly perpetuated, despite the accepted use of
silver antibiosis by NASA, the US Army, various NGO’s
and other frontline agencies facing infections in the eld.
The current study examined the antiseptic properties of
some locally available commercial CS preparations against
a panel of microbial agents, to see whether their continuing
usage as antiseptic agents might be justied.
MATERIALS AND METHODS
Colloidal silver samples
CS samples were commercial products (mostly from Brisbane
health stores) with the exception of 3 reference samples.
Two of these were prepared chemically and supplied as
49
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
preparation. Linear regression was used to calculate the
MIC values.
Toxicity Screening
Reference Toxins for Toxicity Assay
Potassium dichromate (K2Cr2O7) (AR grade, Chem-Supply,
Australia) was prepared as a 1.6 mg/ml solution in distilled
water and serially diluted in articial seawater (see below)
for use in the Artemia franciscana nauplii bioassay. Mevinphos
(2-methoxycarbonyl-1-methylvinyl dimethyl phosphate),
obtained from Sigma-Aldrich as a mixture of cis (76.6%)
and trans (23.0%) isomers, was prepared as a 4 mg/ml
stock solution in distilled water. This was serially diluted
in articial seawater for use in the bioassay.
Evaluation of Toxicity
Toxicity was measured using the Artemia franciscana nauplii
lethality assay originally developed by Meyer et al[12] for
screening phytotoxins. The assay was modied as previously
described.[13, 14] Briey, Artemia franciscana Kellogg cysts were
obtained from North American Brine Shrimp, LLC, USA
(harvested from the Great Salt Lake, Utah). Synthetic seawater
was prepared using Reef Salt, AZOO Co., USA. Seawater
solutions at 34 g/l distilled water were prepared prior to use.
2 g of A. franciscana cysts were incubated in 1 L synthetic
seawater under articial light at 25oC, 2000 Lux with continuous
aeration. Hatching commenced within 16-18 h of incubation.
Newly hatched A. franciscana (nauplii) were used within 10 h
of hatching. Nauplii were separated from the shells and
remaining cysts and were concentrated to a suitable density
by placing an articial light at one end of their incubation
vessel and the nauplii rich water closest to the light was
removed for biological assays. Seawater (400 µl) containing
approximately 43 (mean 43.5, n = 248, SD 12.8) nauplii were
added to wells of a 48 well plate and immediately used for
bioassay. The CS preparations were diluted in seawater for
toxicity testing. 400 µl of the samples and the reference toxin
were transferred to the wells and incubated at 25 ± 1oC under
articial light (1000 Lux). A negative control (400 µl seawater)
was run in at least triplicate for each plate. All treatments
were performed in at least triplicate. The wells were checked
at regular intervals and the number of dead nauplii counted.
The nauplii were considered dead if no movement of the
appendages was observed within 10 seconds. After 72 h all
nauplii were sacriced by adding acetic acid and counted to
determine the total number per well. The LC50 with 95%
condence limits for each treatment was calculated using
probit analysis.[15]
RESULTS
Characterisation of commercial CS samples
Silver content ascertained by AAS varied from 64 %
(Courtenays Original CS) to 207 % (Burke-Hale) of the
Grifth University, Australia. Stock cultures of Aeromonas
hydrophila, Alcaligenes faecalis , Bacillus cereus, Citrobacter freundii,
Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas
uorescens, Salmonella newport, Serratia marcescens, Shigella sonnei,
Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus
pyogenes were subcultured and maintained in nutrient broth
at 4 oC. Stock cultures of Aspergillus niger, Candida albicans
and Saccharomyces cerevisiae were subcultured and maintained
in Sabouraud media at 4 oC.
Evaluation of antimicrobial activity
Antimicrobial activity was determined using a modied
disc diffusion method previously described.[8, 9] Briey, 100
µl of the test bacteria/fungi were grown in 10 ml of the
appropriate fresh broth until they reached a density of
approximately 108 cells/ml of bacteria or 105 cells/ml for
fungi (as determined by direct microscopic determination).
One hundred microliters of microbial suspension was spread
onto agar plates prepared with the broth in which they
were maintained.
CS were tested using 5 mm sterilised lter paper discs. Discs
were impregnated with 10 µl of the test sample, allowed to
dry, then placed on the microbially inoculated agar plates.
The plates were allowed to stand at 4 oC for 2 hours before
incubation with the test microbial agents. Plates inoculated
with Alcaligenes feacalis, Aeromonas hydrophilia, Bacillus cereus,
Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis,
Pseudomonas uorescens, Serratia marcescens, and Candida albicans
were incubated at 30 oC for 24 hours, then the diameters of
the inhibition zones were measured in millimetres. Plates
inoculated with Escherichia coli, Salmonella newport, Shigella sonnei,
Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus
pyogenes were incubated at 37 oC for 24 hours, then the
diameters of the inhibition zones were measured. Aspergillus
niger inoculated plates were incubated at 25 oC for 48 hours
before measuring the zones of inhibition. All measurements
were to the closest whole millimetre. Each antimicrobial
assay was performed in triplicate. Mean values are reported
in this study. Standard discs of ampicillin (2 µg),
chloramphenicol (10 µg) and nystatin (100 µg), obtained
from Oxoid Ltd. Australia served as positive controls for
antimicrobial activity. Filter discs impregnated with 10 µl of
distilled water were used as negative controls.
Determining Minimum inhibitory
concentration (MIC)
The minimum inhibitory concentration (MIC) of the
colloidal silver samples were determined by a modied
disc diffusion method[10, 11] across a range of doses. The
samples were serially diluted in deionised water. Discs were
impregnated with 10 µl of the test dilutions, allowed to
dry and placed onto inoculated plates. The assay was
performed as outlined above and graphs of the zone of
inhibition versus concentration were plotted for each CS
50
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
CS preparations displayed greater inhibitory activity towards
Gram-negative bacteria than to Gram-positive bacteria.
The Silver Magic sample inhibited the growth of 9 of the
10 Gram-negative bacteria (90 %) tested but only 2 of the
4 (50 %) of the Gram-positive bacteria tested. Similarly,
Suttons Bali Belly Buster inhibited the growth of 7 Gram-
negative bacteria (70 %) and only 2 (50 %) Gram-positive
bacteria respectively.
Other CS preparations showed varying degrees of
antimicrobial activity and specicity. Suttons Original CS
also had a relatively broad specicity for Gram-negative
bacteria, inhibiting the growth of 6 of the 10 Gram-negative
bacteria (60 %) tested but did not inhibit the Gram-positive
bacteria. Ionic silver (as AgNO3), Courtnays CS, Nano
Xact (10 nm), Burke’s CS and Holland and Barrett CS
inhibited 4 (40 %), 3 (30 %), 2 (20 %), 2 (20 %) and 0 (0
%) of the Gram-negative bacteria respectively. Neither the
silver nitrate nor Courtnays CS preparation inhibited the
growth of any of the Gram-positive bacteria tested, whilst
Nano Xact (10 nm), Burke’s CS and Holland and Barrett
CS each inhibited a single (25 %) Gram-positive bacterium.
Both the Nano Silver preparation and the Nano Xact (50
nm) failed to inhibit the growth of any bacteria.
These preparations varied widely in terms of total silver
content (ppm) (Table 1). It is therefore likely that some
preparations may appear to have low efcacy due to the
doses tested. For example, the Holland and Barrett CS
preparation inhibited the growth of only a single bacterium.
However, this particular preparation had a very low silver
content (2.5 ppm) compared to the other commercial CS
products which was below the MIC of many of the other
CS preparations. So it is possible that this preparation
(Holland and Barrett) might have displayed broader
specicity at a higher silver content. In general only low
concentrations of CS were required to achieve antibiotic
activity, with MIC values ≤ 5µg total silver/ml for some
preparations against some microbial species. In contrast,
2 nanoparticulate colloidal gold preparations (30-70 nm
diameter) were completely devoid of antimicrobial activity.
Neither of these colloidal gold preparations was effective
against any of the bacteria or fungi tested.
Toxicity Studies
All CS preparations were serially diluted in articial seawater
for toxicity testing in the Artemia franciscanna nauplii lethality
assay (Table 3). For comparison, the reference toxins
potassium dichromate and Mevinphos were also tested.
Both reference toxins were rapid in the induction of toxicity,
with mortality noted within 3 hours of exposure (unreported
results). Potassium dichromate was particularly toxic with
LC50 values at 24 h of 86.3 μg/mL. In contrast, none of
the CS preparations induced mortality signicantly above
that of the seawater control within the rst 24 h of exposure.
quantities displayed on the labels (Table 1). A few samples
(notably some originating from Malaysia) displayed zero
Ag content by AAS but had an Ag content plus or minus
10% the content indicated on the label when re-examined
by ICP-MS (unpublished results). This disparity may be
due to matrix effects inherent in the AAS assay. These
preparations were considered atypical and were not included
in the subsequent antimicrobial/toxicity studies.
pH was also quite variable, ranging from 8.1 to 10.7. Further
CS preparations tested prior to these studies displayed even
greater variability, with pH values as low as 4.7. These low
pH CS preparations were considered atypical and were not
included in the subsequent antimicrobial/toxicity studies.
Most samples were colourless, some opalescent and only
a few had distinctive yellow-brown tints. Semi-quantitative
determinations of scattering of green laser light gave values
ranging from 6 (Nanosized Silver) to 98 (HLY) arbitrary
units/ppm silver.
The content of free ionic silver was difcult to ascertain
as some CS samples appeared to have a very high Ag+
content (>80% by conventional analysis); but still caused
signicant light scattering due to the presence of colloidal
particles. This may be explained by the fact that even
reference CS samples (prepared by the chemical reduction
of Ag+) were variably removed by low speed centrifugation
techniques used in separating the Ag+.
Conductivities ranged from 18 (HLY) to 2540 μS (Argyrol);
those with high conductivity generally showing strong
absorption for Na+ in the AAS assay. Other impurities
detected included calcium (often high), arsenic, lead and
copper indicating use of impure water or silver metal for
preparing CS. Notably contaminated samples were excluded
from bioassays. Only one sample, contaminated with Pb,
signicantly exceeded current Australian standards for
permissible metallic constituents in drinking water and was
not included in subsequent antimicrobial/toxicity studies.
Antimicrobial activity
Antimicrobial activity of each CS preparation (10 µl) was
tested in the disc diffusion assay against a panel of 14
bacterial, 2 fungal and 1 yeast species (Table 2). Of the
12 CS preparations tested, 10 (83 %) were found to be
have inhibitory activity, albeit with varying specicity and
efcacy. Argyrol and HLY displayed the broadest specicity,
inhibiting the growth of all 14 bacteria tested (100%).
These preparations also inhibited the growth of 3 (100
%) and 2 (67 %) of the fungal species tested respectively.
Two other CS preparations (Silver Magic and Suttons Bali
Belly Buster) also displayed broad antibacterial activity,
inhibiting the growth of 11 (78 %) and 9 (64 %) of the
14 bacterial species tested respectively. Both these latter
51
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
Table 1: Characterisation of (a) some commercial CS products (shown in green), (b) some reference CS preparations (shown in grey) and (c) some
colloidal gold products (shown in yellow).
Physical Properties
F & B
Orig CS
BBB
SM
IS
NzS
H & B
Bion
Argyrol
NX 10
NX 50
HLY
Gold
Colloid
Gold (37nm)
Total Silver Content
(from company)
9-14 16-20 NS 25-30 10 10 3 3 NR 20 20 13.5 NR NR
Total Silver Content
(measured)
9.1 12.8 18.5 38.8 10.7 10 2.8 6.2 14500 20 20 13.5 NR NR
Conductivity
(μSiemens)
105 72 80 113 56 1096 60 97 2540 777 635 18 365 NR
pH 9.1 9.4 8.4 8.2 8.5 10.7 8.9 9 8.7 8.7 8.4 8.1 10.1 NR
Light Scattering Index 15 19 14 10 29 6 7 15 7 24 45 98 5 NR
Median Nanoparticle
Size (μm)
33±4.0 33±4.1 32.6±4.2 33±4.1 33 ± 4.1 73.5±9.7 33±4.1 33±4.1 33±4.0 32.5±4.2 33.1±4 33±4.1 286±58 32.6±4.2
F & B = First and Best, Courtenay, Montville Qld Australia; Orig CS = Original CS, Suttons, WA Australia; BBB = Bali Belly Buster, Suttons, WA Australia; SM = Silver Magic (extra strength), Lowood Qld Australia; IS = Ionic Silver,
Mineral Solutions, USA; NzS= Nanonized Silver, Trust Nature, Malaysia; H & B = Silver protein (dairy), Holland and Barrett, UK; Bion = Bionaid Ag.H2O, Burke Hale, USA; Argyrol = Mild Silver Protein SS 10% Argyrex, USA;
NX 10/50 = NanoXact, Nano Composix, San Deigo USA; HLY = electrolytic preparation, Hans Laroo, Ipswich Australia. Nanoparticle sizes are expressed as median values (± SD) of the dominant population (≥99.5%).
NR = not recorded.
52
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
Table 2: Relative antimicrobial potencies of (a) 9 commercial (shown in green) and 3 reference (shown in grey) CS samples, (b) 2 gold hydrosols (shown in
yellow) and (c) 4 standard antibiotic discs (shown in blue).
MIC (μg/ml) Inhibition by Antibiotics
Courtnays
Suttons
Suttons Bali Belly Buster
Silver Magic
Ionic Silver
Nano Silver
Holland and Barrett
Burke’s
Argyrol
Nano Xact (10nm)
Nano Xact (50nm)
HLY
Gold Colloid
Gold (37nm)
Ampicillin (2 μg)
Chloramphenicol (10 μ g)
Ciprafloxicin (2.5 μg)
Nystatin (100 μg)
Gram negative rods
Aeromonas hydrophilia 6.3 7.4 0.1 17.4 10.7 7.1 2.5 + + + NT
Alcaligenes faecalis 4.1 0.3 10.7 6.2 4.5 20 6.6 + + + NT
Citrobacter fruendii 13 0.6 7.7 6.2 8.4 1 + + + NT
Escherichia coli 12.2 4.9 9.1 + + + NT
Klebsellia pneumoniae 7.6 0.8 0.3 10.2 1 4.1 + + + NT
Proteus mirabilis 0.3 0.7 2.5 + + + NT
Pseudomonas fluorescens 9.1 12.8 15.4 0.6 10.7 16 0.2 + + + NT
Salmomella newport 12.8 0.5 11.8 8.4 20 1.6 + + + NT
Seratia marescens 0.8 6.7 18.5 5.2 + + NT
Shigella sonnei 0.8 0.6 7.1 6.1 + + + NT
Gram positive rods
Bacillus cereus 12.9 5.5 4.6 13.7 + + + NT
Gram positive cocci
Staphylococcus aureus 2.5 6.2 1.5 20 10.2 + + + NT
Staphylococcus epidermidis 16.1 2.7 + + + NT
Staphyloccocus pyogenes 12.9 0.6 2.2 11.3 + + + NT
Fungi
Aspergillus niger 1207 +
Candida albicans − − − − − − − 278.8 135 + +
Yeast
Saccharomyces cerevisiae 129.9 135 + +
Numbers indicate the mean MIC values of triplicate determinations; − indicates no inhibition; + indicates that the antibiotic standard disc inhibited microbial growth and an MIC was not obtained (as only a single dose disc was tested);
NT indicates that the antibiotic disc was not tested against an individual bacterium.
53
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
disinfectant to purify and store drinking water.[16] More
recently, silver has been used for similar purposes on the
Apollo spacecraft,[17] NASA space shuttles[18] and the MIR
space station.[19] In the 19th century, silver preparations
were used as antiseptics post surgery, in dentistry, as well
as for the prevention of ophthalmia neonatorum in
newborn children.[16] CS proved life-saving in World War
I and gained widespread acceptance as an antiseptic agent.
[5] In the 1920’s the US Food and Drug Administration
(FDA) approved CS for wound treatment. Later, the
discovery of sulphonamide, penicillin, and macrolide
antibiotics led to a decline in the use of CS antibiotics.
However, the development of super-resistant bacterial
strains has rekindled some interest in colloidal silver as
a medicinal agent. Despite the long history of effective
usage of silver preparations as antiseptic agents as well
as their acceptance by the FDA in the 1920’s, the US
FDA and the Australian Therapeutics Goods
Administration (TGA) have recently reversed this
approval. They now prohibit individuals making any claims
regarding the efcacy of commercial CS preparations,[20, 21]
offering next to no scientic evidence to justify this
position.
The present study has veried the antimicrobial properties
of a selection of commercially available CS preparations
tested in vitro against a panel of pathogenic bacteria and
fungi. Indeed, 83% of the CS preparations tested were
able to inhibit the growth of 1 or more bacterial species.
Numerous recent publications have also demonstrated
the antimicrobial activity of laboratory synthesised colloidal
silver preparations. A poly-N-vinyl-2-pyrrolidone (PVP)
Indeed, only the HLY preparation induced high enough
mortality for the determination of an LC50 (46.4 μg/mL)
within the rst 48 h of exposure. Whilst an increased
induction of mortality above that of the seawater control
was also evident for the Courtneys, Suttons, Suttons Bali
Belly Buster and Ionic Silver preparations at 48 h, the
mortality was below 50%. Therefore it was not possible
to determine an LC50 for these CS preparations and they
were considered of low toxicity at 48 h. Mortality induction
was above 50% for the Courtneys, Suttons, Suttons Bali
Belly Buster and Ionic Silver preparations at 72 h enabling
the determination of LC50 values. However, Artemia nauplii
toxicity studies usually only reports LC50’s at 24 h and/or
48 h. Therefore the reporting of a 72 h LC50 may be
considered excessive. All CS preparations tested in this
study can therefore be considered either nontoxic (Silver
Magic, Nano Silver, Holland and Barrett, Burkes, Agyrol
and the two Nano Xact CS preparations) or of low toxicity
(Courtneys, Suttons, Suttons Bali Belly Buster, Ionic Silver
and HLY CS preparations).
Neither of the gold hydrosols tested in this study induced
mortality above that of the seawater control at any
concentration/time point tested. It was therefore not
possible to determine LC50 values for the gold hydrosols
and they are therefore also considered nontoxic.
DISCUSSION
CS preparations have been used as medicinal agents for
centuries. In ancient times, metallic silver was used as a
Table 3: LC50 (95% confidence interval) for brine shrimp nauplii exposed to (a) 9 commercial (shown in green) and
3 reference (shown in grey) CS samples, (b) 2 gold hydrosols (shown in yellow) and (c) positive (potassium
dichromate and mevinphos) and negative (seawater) controls.
LC50 (μg/mL) Control (μg/mL)
Courtnays
Suttons
Suttons Bali Belly Buster
Silver Magic
Ionic Silver
Nano Silver
Holland and Barrett
Burke’s
Argyrol
Nano Xact (10nm)
Nano Xact (50nm)
HLY
Gold Colloid
Gold (37nm)
Potassium Dichromate
Mevinphos
Seawater
24 hour LC50 − − − − 86.3 1346
48 hour LC50 − − − − 46.4 − 80.4 505
72 hour LC50 3.8 5 1.5 4.5 − − − 3.1 77.9 103.9
Numbers indicate the mean LC50 values of triplicate determinations; −denotes values that were not obtained as ≥ 50 % mortality was not obtained at this time point for
any concentration tested.
54
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
suspensions of metallic silver and the different products
may have very different physical properties as well as bio-
efcacies. The quality of CS preparations will vary between
products and may also vary from batch to batch of the
same product. So CS preparations may vary widely in terms
of efcacy and indeed, probable safety. Currently, there
are no industry standards for manufacturers to adhere to
and no guidelines and government regulation of the industry
(aside from prohibiting claims of efcacy).
Historically the original types of CS were the silver-protein/
polypeptide products prepared by reacting silver salts (Ag+)
with a polypeptide ‘carrier’ (class 1 above). The Argyrol
and the Holland and Barrett products examined in this
study are 2 examples of silver-protein preparations still
available today. Argyrol had the broadest antimicrobial
specicity of any CS preparation tested in this study. While
Argyrol inhibited the growth of all bacterial and fungal
species tested, it was not particularly potent: the recorded
MIC’s against some of microbial species is relatively high.
For example the MIC of Argyrol for S. marcescens (18.5
ppm) is quite high compared to other CS preparations
tested. This is even more apparent for the antifungal activity
of Argyrol. Whilst Argyrol was the only preparation which
inhibited all 3 fungal species tested, in all cases the MIC
was quite high (≥ 100 ppm). It is possible that similar
activity of the other CS preparations was not evident
either because their silver content was either too low or
they contained the wrong size of particles for antifungal
activities.
The most commonly available class of CS products seem
to be the electro-CS preparations (class 2a above). These
are usually prepared by low voltage electrolysis using silver
electrodes in deionised water yielding dispersed metallic
silver preparations, ranging from 2-150 ppm total Ag. Ideally,
this procedure would produce pure silver hydrosols
suspended in water without contaminants. The clusters of
silver atoms constituting the metallic nanoparticle in these
preparations generally carry a positive electrical charge.[28]
Most of the CS preparations examined here were electro-
CS preparations. The most effective reference CS preparation
with antimicrobial activity (HLY) was produced by
electrolysis.
The third type of CS preparations (class 2b) are produced
chemically by adding reagents (e.g. reducing agents) to
soluble silver salts (Ag+) to produce a hydrosol (water
dispersible) form of zerovalent metallic silver (Ag0). As
with the other types of CS, the concentration of these CS
preparations can vary widely, depending on the preparative
procedures employed. The resultant silver particles generally
carry a negative electrical charge,[28] the particles being readily
precipitated by charged cations (e.g. Al3+, La3+). However,
even these CS preparations will invariably contain non-
stabilised silver nanoparticle preparation displayed potent
antibacterial activity against S.aureus and E. coli.[22] Silver
nanoparticles synthesised by the inert gas condensation
method were effective at inhibiting the growth of E. coli,
albeit at a higher MIC (60 µg/ml) than seen in our study
or by Cho et al.[22] In an interesting recent report, the
bacteria Klebsiella pneumonia was used to reduce aqueous
Ag+ to produce a biogenic CS preparation which was an
effective inhibitor of S.aureus and E. coli. [23] This same
report also documented the ability of these biogenic silver
nanoparticles to increase the efcacy of various other
antibiotics, highlighting the potential of CS/antibiotic
co-treatments.
By contrast, other studies have questioned the efcacy of
CS preparations as antiseptic agents.[24, 25] The CS preparations
tested in one of these studies[24] were produced from silver
salts by chemical reduction. Studies in our laboratory have
also shown that chemically produced CS preparations may
have lower antibacterial activities than CS produced by
other methods.[26] The other study[25] used CS preparations
with low silver content (5 ppm). Our results conrm the
low efcacy of CS preparations with similarly low
concentrations (e.g. the Holland and Barrett CS preparation
with only 2.5 ppm silver).
Bacteria may also develop resistance to ionic silver
preparations.[27] Several studies have tested CS preparations
against antibiotic multi-resistant bacterial strains. It is
possible that the low efcacies reported from some of
these studies are related to the bacterial strains tested.
Presumably, it is these opposing reports on the efcacy of
CS as antimicrobial agents that led to both the FDA and
the TGA prohibiting general claims regarding the efcacy
of all CS preparations.[20, 21] Furthermore, these
administrations proclaim CS is also toxic without considering
whether the toxicities are due to contaminant Ag+, rather
than colloidal Ag0.
It is likely that the opposing viewpoints of the efcacy of
CS as antimicrobial agents may be due to the nature of the
CS preparation itself. The term “colloidal silver” is a blanket
designation used to refer to at least 4 different types of
product:
1. Silver impregnated proteins prepared from silver salts
and stable in NaCl.
2. Particulate silver metallic dispersions prepared by:
a. electrolytic dissolution of metallic silver rods.
b. chemical reduction of silver salts with excess
reductant ensuring virtual absence of free Ag+.
c. (dry) sintering of metallic silver.
It is very important to understand that whilst all of these
products may be marketed as CS, not all are colloidal
55
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
ingestion may interfere with the absorption and thus
bioavailability of some drugs, thus reducing their
effectiveness. Further studies are needed to examine the
consequences arising from using CS orally. Interestingly,
we were unable to nd any reports of argyria directly
attributed to the usage of CS preparations produced by
electrolysis, which may contain a lesser content of impurities.
The antibacterial activity[32] and toxicity[33] of silver
nanoparticles have previously been related to the size of
the CS particle rather than to the concentration of silver
alone. The correlation of toxicity with particle size may be
fortuitous as previous studies indicate that antimicrobial
efcacy may have an inverse relationship with toxicity. Whilst
previous studies indicate that CS toxicity decreases with
decreasing particle size, efcacy appears to increase with
decreasing size.[31] Thus it is likely that a CS preparation
with small particle size would be likely to have greater
antibacterial activity, yet lower toxicity, than preparations
containing larger metallic particles. Furthermore, production
of these small particles by protocols which diminish the
chances of contamination (e.g. electrolysis) may produce
somewhat safer preparations.
Our studies conrm the dependency of efcacy on particle
size. Two preparations of different particle size at the same
concentration from the same supplier (Nano Xact) were
tested in these studies. Only the CS preparation with the
smaller particles (10 nm) inhibited the growth of any
bacteria, whilst the 50 nm CS preparation was completely
ineffective. Furthermore, antimicrobial efcacy can also
depend upon the shape of the CS nanoparticles. In some
instances, triangular nanoparticles may be more effective
antimicrobial agents than spherical nanoparticles, which
in turn are more effective than rod shaped particles.[34]
Further studies are required to examine the relationship
between colloid size and shape with antimicrobial efcacy
and safety.
As efcacy and safety may be related to the various factors
described above (concentration, particle size, the charge
on the nanoparticulate silver, possible contaminations),
quality control of CS preparations is required to ensure
reproducible medicinal properties. Further studies are
needed to determine the optimum conditions to produce
non-toxic, higher efcacy CS preparations. Rather than
placing a blanket ban on statements concerning the
antibacterial activity of CS, regulatory bodies such as the
FDA and the TGA may be better servants of thier
communities by establishing Quality Control guidelines
and allowing (instead of forbidding) further research on
silver pharmacognosy; so helping biomedical scientists to
learn more about the safe and effective usage of CS, a
product that has long been used and will no doubt continue
to be used as a topical antiseptic.
silver contaminants (e.g. buffers, reductants), which may
affect their stability, efcacy and toxicity.
The fourth class of CS preparation (class 2c above),
sometimes referred to as “powdered silver”, is obtained
when a silver wire is disintegrated by a high voltage electrical
discharge analogous to an old photographic ash bulb.
The resultant silver ‘dust’ is either dispersed into aqueous
milieu, or added directly to creams and salves for topical
usage. No “powdered” CS preparations were tested in this
study. Therefore we can not comment on their antimicrobial
potential or toxicity.
Most of the commercial CS preparations tested (7 of the
12 tested) were completely nontoxic at the concentrations
supplied. Of the few preparations that were toxic (5 of
the 12 tested), toxicity was low and then only after extended
exposure. This nding opposes the opinion of the FDA
and TGA. In a 2007 report, the TGA describes 4 cases of
silver toxicity resulting from the ingestion of “homemade”
CS preparations.[21] However, this report signicantly failed
to dene the nature of these CS preparations (i.e. purity
and how they were made), nor the levels of CS ingested.
This same report also states that the TGA has received no
reports of toxicity associated with “legitimate therapeutic
goods containing presentations of silver that remain
appropriate”. Whilst not citing examples of toxicity linked
to CS treatment, the FDA issued a ruling in 1999 stating
that CS preparations are not recognised as safe or effective.
[20] As with the TGA report, no discrimination was made
in this ruling between the different classes of CS products,
the physiochemical properties of the colloids (size, ionic
state etc), nor the dosage and method of administration.
A further report by a US government agency (US Dept of
Energy) is also sometimes cited as proof of the toxicity
of CS preparations.[29] This study reported that exposure
to high doses of silver (specically silver nitrate and silver
oxide) may result in a range of symptoms including irritation
of the skin, eyes, gastrointestinal and respiratory tracts,
and mucous membranes as well as more serious
complications, and even death at very high doses. However,
whilst this report is cited as proof of the toxicity of CS,
the author makes it very clear that she is reporting on the
toxicity of silver in general, not CS specically.
The only reports of CS toxicity in humans relate to the
consumption of CS (not its topical use) and these describe
relatively mild responses. The worst adverse reaction
documented from the medicinal usage of CS is argyria, a
condition characterised by a bluish discoloration of the
skin.[2, 30, 31] Argyria is primarily a cosmetic condition which
causes no discomfort and has no other known side effects.
With argyria the skin discolouration may be misdiagnosed
as cyanosis, methaemoglobinaemia or haemochromatosis
leading to inappropriate treatment. It is possible that CS
56
Cock, et. al.: Colloidal Silver (CS) as an Antiseptic: Two opposing viewpoints
13. Cock IE. High performance liquid chromatographic separation and
identification of a toxic fraction from Aloe barbadensis Miller leaf gel using
the Artemia naulpii bioassay. The Internet Journal of Toxicology 2008; 4 (2).
14. Ruebhar t DR, Wickramasinghe W, Cock IE. Protective effect of the
antioxidants vitamin E and Trolox against Microcystis aeruginosa and
microcystin-LR in Artemia franciscana nauplii. Journal of Toxicology and
Environmental Health, Part A 2009; 72 (24): 1567-1575.
15. Finney, D.J. 1971. Probit Analysis, 3rd ed., Cambridge University Press,
Cambridge.
16. White RJ, An historical overview on the use of silver in modern wound
management. British Journal of Nursing, 2002; 15(10): 3-8.
17. Albright CF, Nachum R, Lechtman MD, Electrolytic silver ion generator for
water sterilisation in Apollo spacecraft water systems. Apollo applications
program, NASA Contract Rep, 1967; Report Number NASA-CR-65738.
18. http://www.sti.nasa.gov/tto/Spinoff2004/er_1.html, Water treatment systems
make a big splash. 2004, Accessed 30 September, 2011.
19. Conrand AH, Tramp CR, Long CJ, Wells DC, Paulsen AQ, Conrand GW,
Ag+ alters cell growth, neurite extension, cardiomyocyte beating, and
fertilised egg constriction. Aviation, Space and Environmental Medicine,
1999; 70(11): 1096-1105.
20. Federal Register, Over-the-counter drug products containing colloidal silver
ingredients or silver salts. Department of Health and Human Services
(HHS), Public Health Service (PHS), Food and Drug Administration (FDA).
Final rule. Federal Register 1999; 64 (158): 44653-44658.
21. Therapeutic Goods Association, 2007, Australian adverse drug reactions
bulletin, 26 (5), http://www.tga.gov.au/pdf/aadrb-0710.pdf. Retrieved 4 Oct
2011.
22. Cho K, Park JE, Osaka T, Park SG, The study of antimicrobial activity and
preservative effects of nanosilver ingredient. Electrachimica Acta, 2005;
51: 956-960.
23. Shahverdi AR, Fakhimi A, Shahverdi HR, Mianaian S, Synthesis and effect
of silver nanoparticles on the antibacterial activity of different antibiotics
against Staphylococcus aureus and Escherichia coli. Nanomedicine:
Nanotechnology, Biology and Medicine, 2007; 3: 168-171.
24. van Hasselt P, Gashe BA, Ahmad J, Colloidal silver as an antimicrobial
agent: fact or fiction. Journal of Wound Care 2004; 13 (4): 154-155.
25. Spratt DA, Pratten J Wilson M, Gulabivala K, An in vitro evaluation of the
antimicrobial efficacy of irrigants on biofilms of root canal isolates.
International Endodontic Journal 2001; 34 (4): 300-307.
26. White A, Cock IE, Mohanty S, Laroo H, Whitehouse M. Unpublished
results 2011.
27. Silver S, Bacterial silver resistance: molecular biology and uses and misuses
of silver compounds. FEMS microbiology Reviews 2003; 27: 341-353.
28. Key FS, Maass G. Ions, atoms and charged particles. Silver Colloids 2001:
http://www.silver-colloids.com/Papers/IonsAtoms&ChargedParticles,
accessed 27 October 2011.
29. Faust RA. Toxicity Summar y for Silver. Oak Ridge Reservation
Environmental Restoration Program, Oak Ridge 1992.
30. Bouts BA. Argyria. New England Journal of Medicine 1999; 340: 1554.
31. Legat FJ, Goessler W, Schlagenhaufen C, Soyer HP. Argyria after short-
contact acupuncture. Lancet 1998; 352: 241.
32. Morones JR, Elechiguer ra JL, Camacho A, Holt K, Kouri JB, Ramirez JT,
Yacaman MJ. The bactericidal effect of silver nanopar ticles.
Nanotechnology 2005; 16 (10): 2346-2353.
33. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of
nanoparticles in BRL 3A rat liver cells. Toxicology in Vitro 2005; 19: 975-983.
34. Pal S, Tak YK, Song JM. Does the antibacterial activity of silver
nanoparticles depend on the shape of the nanoparticle? A study of the
gram-negative bacterium Escherichia coli. Applied Environmental
Microbiology 2007; 27 (6): 1712-1720.
CONCLUSIONS
The commercial CS preparations tested in this study varied
widely in their potential utility as complementary medicines.
Quality controls for their content, antimicrobial efcacy
and incipient toxicity to animal cells are certainly needed.
This study clearly demonstrated the effective antiseptic
activity of some CS preparations, indicating that they should
still be seriously considered as medicinals for topical
treatments e.g. burns, periodontitis, thrush. Concerns about
safety (previously raised by the US FDA and the Australian
TGA) need to be debated on scientic grounds. Claims
about inefcacy are meaningless without attempts to dene
composition, concentration and bio-evaluation.
ACKNOWLEDGEMENTS
We are indebted to Prof. J. Ng, National Centre for
Environmental Toxicology, Coopers Plains, Australia for
ICP-MS analyses.
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... The sample, which contained nanoparticles with a mean diameter of ~33 μm ( Figure 1b; Table 1) was prepared electrolytically as previously described. 11 The physical parameters of the colloidal silver preparations were determined in the earlier study and are reproduced in Table 1. ...
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