burns 38 (2012) 529533

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The use of topical, un-buffered sodium hypochlorite in the

management of burn wound infection

E. Coetzee *, A. Whitelaw, D. Kahn, H. Rode

Department of Surgery and Microbiology, University of Cape Town, South Africa

article info abstract

Article history: Background: Burn wound infections are a major cause of morbidity and mortality. The
Accepted 20 October 2011 bactericidal action of sodium hypochlorite has been known for centuries and it has been
in clinical practice for over 70 years. Whereas a buffered sodium hypochlorite solution is not
Keywords: universally available, an un-buffered solution is cheap and easy to prepare.
Sodium hypochlorite Aim: The aim of this study was to determine the optimum concentration with regard to
Topical safety and efficacy, as well as shelf life of an un-buffered sodium hypochlorite solution for
Burns the topical management of burn wound infections.
Infection Methods: Human fibroblasts were exposed to serial dilutions of un-buffered sodium hypo-
chlorite solutions for 30 min and assessed for viability. Isolates of Pseudomonas aeruginosa,
Staphylococcus aureus and Streptococcus pyogenes were exposed to the same dilutions of un-
buffered sodium hypochlorite to establish the minimum bactericidal concentration. The pH,
osmolality and electrolyte concentrations were measured. These experiments were repeat-
ed with solution stored at room temperature for 6 consecutive days.
Results: 24% of fibroblasts were viable after exposure to a 0.025% solution and 98.9% with a
0.003% solution. The MBC for the P. aeruginosa isolates was 0.003%, for S. aureus was 0.006%
and for S. pyogenes was 0.0015%. This remained constant for 6 consecutive days. The un-
buffered 0.0025% solution has a pH of 10, an osmolality of 168 sodium concentration of
89 mmol/dl and chloride of 84 mmol/dl. This remained stable for 14 days.
Conclusions: An un-buffered solution of sodium hypochlorite with a concentration of 0.006%
would be suitable for the topical management of burn wound infections caused by common
pathogens. It has a shelf life of at least 6 days.
# 2011 Elsevier Ltd and ISBI. All rights reserved.

deaths in patients with burns exceeding 40% total burn surface

1. Introduction
The skin is an active immune organ and is the major epithelial
barrier between the body and a hostile environment [14].
Thermal injury causes instant coagulative necrosis which
rapidly becomes a suitable site for bacterial colonization and
proliferation due to its exclusion from the systemic circulation
and impaired local immune responses [1,2,5]. Despite signifi-
cant advances in burn care, infection is still a common cause
of death in burn patients and is responsible for 75% of all
area (TBSA) [1,2,6,7].
The introduction of topical antimicrobial agents has
dramatically reduced the number of deaths due to burn
wound infections [8]. However, resistance of pathogens, such
as Pseudomonas aeruginosa, to topical as well as systemic
antimicrobial agents is well described in burn units [9]. A
topical bactericidal agent which is effective, non-toxic to the
patient, does not lead to the development of resistance, and
which is cost effective, is vital in managing burn wound
infection.

* Corresponding author. Tel.: +27 825778437.

E-mail address: edt.coetzee@gmail.com (E. Coetzee).
0305-4179/$36.00 # 2011 Elsevier Ltd and ISBI. All rights reserved.
doi:10.1016/j.burns.2011.10.008
530 burns 38 (2012) 529533
The bactericidal action of sodium hypochlorite has been
known since the 1880s and it has been used in clinical practice
for more than seventy years [3]. Its bactericidal action is directly
related to its concentration and duration of exposure [3,10].
The toxic effects of sodium hypochlorite on wound healing
are confined to a restricted range of concentrations. However,
since the bactericidal activity of sodium hypochlorite is also
related to its concentration and time of exposure, it is
fundamental to use the optimum concentration which has
both adequate bactericidal capabilities as well as minimal
toxicity [3,9,10]. There have not been any reports of resistance
to sodium hypochlorite by bacteria or yeasts, which makes it a
valuable topical agent for use in burn wound infection [3,10].
A buffered sodium hypochlorite is not readily available in
developing countries. In contrast, an un-buffered solution of
sodium hypochlorite is cheap and easy to prepare. This study
was undertaken to determine the optimum concentration of
an un-buffered solution of sodium hypochlorite to be used as a
topical bactericidal agent in burn wound infections.
2. Methods
To establish the optimum concentration with regard to safety
and efficacy, and shelf life of an un-buffered sodium
hypochlorite solution for the use as topical agent in the
management of burn wound infections, serial dilutions of un-
buffered sodium hypochlorite were tested in vitro to deter-
mine:
 The effects of un-buffered sodium hypochlorite on fibroblast
proliferation.
 The minimum bactericidal concentration (MBC) of un-
buffered sodium hypochlorite against three common patho-
gens in burns patients.
 The pH and electrolyte concentrations of a 0.025% solution
at 24-, 48-, 72- and 96-h after preparation (to establish the
shelf life of this un-buffered solution).
2.1. Preparation of the un-buffered 0.025% sodium
hypochlorite solution
The solution was prepared in the sterile pharmacy. One liter of
sterile water for irrigation (SABAX, Adcock Ingram) was used
and 25 ml withdrawn from the bottle. Twenty five milliliters of
Milton1 (Cueta) was filtered through a 0.22 mm filter (Millex GS
0.22 mm, Millipore Ltd) and added to the sterile water to
produce a concentration of 0.025% of sodium hypochlorite.
2.2. Fibroblast proliferation assay
Human fibroblasts were cultured in DMEM containing 10%
foetal calf serum and penicillinstreptomycin at 37 8C in a 5%
carbon dioxide incubator. The fibroblasts were used when the
cultures reached 80% confluence.
The cells (3  107 per well) were seeded on a ninety six well
plate with 100 ml of DMEM, and allowed to grow for 24 h. The
cells were exposed to serial dilutions (0.0250.003%) of sodium
hypochlorite in sterile water for 30 min. The hypochlorite was
then removed by pipetting it out of the wells and the wells
rinsed with DMEM. The cells were then incubated in DMEM for
20 h before assaying for cell viability using the XTT cell
proliferation kit II (Roche).
2.3. Antimicrobial activity
Clinical isolates of P. aeruginosa (31), Staphylococcus aureus (12)
and Streptococcus pyogenes (5), from patients at the Burns Unit at
the Red Cross War Memorial Childrens Hospital, were collect-
ed. These isolates were chosen to provide a range of
susceptibility profiles, representative of those commonly seen
in the burns unit. The effectiveness of the un-buffered sodium
hypochlorite against P. aeruginosa, S. aureus and b-haemolytic
streptococcus (S. pyogenes) was tested after 24-, 48-, 72- and 96-h
of shelf life at room temperature, using the modified broth
dilution method.
2.4. The modified broth dilution method
0.5 McFarland standard of each organism (equivalent to
approximately 12  108 cfu/ml) was prepared by using an
overnight culture of organisms grown on blood agar. This was
diluted 1:150 in nutrient broth and added to serial dilutions of
sodium hypochlorite prepared to achieve final hypochlorite
concentrations of 0.025%, 0.0125%, 0.006%, 0.003%, 0.0015%,
0.0008% and 0.0004%.
Organisms were incubated (S. aureus and P. aeruginosa
aerobically; S. pyogenes in 5% CO2) at 35 8C for 1824 h.
After incubation, test tubes were read manually for
turbidity. The lowest concentration of hypochlorite that
produced no turbidity was taken as the MBC, and confirmed
by subculturing an aliquot of this suspension onto 2% blood
agar to prove bactericidal activity. An aliquot of the organism
suspension without sodium hypochlorite was sub-cultured
into blood agar, to check purity. The above assays were
repeated on a subset of isolates, using hypochlorite that had
been left at room temperature for 24-, 48-, 72- and 96 h.
2.5. Biochemical properties
Biochemical properties and pH of an un-buffered 0.025%
sodium hypochlorite solution were determined using standard
biochemistry methods, using freshly prepared sodium hypo-
chlorite. This was repeated daily for 14 consecutive days with
the un-buffered sodium hypochlorite stored at room tempera-
ture. The pH was measured using the ABL 520 Blood Gas
Analyzer (GMI Medical), the concentration of sodium and
chloride using the CX3 Chemical Analyzer (Beckmann) and the
osmolality measured using the OSMOMAT 030 osmolality
analyzer (Gonontec).
3. Results
3.1. Effects of sodium hypochlorite on fibroblast
proliferation
The effects of different dilutions of an un-buffered sodium
hypochlorite solution on fibroblast proliferation are shown in
 burns 38 (2012) 529533 531

Table 1 Percentage fibroblasts surviving 30-min ex- Table 2 Percentage fibroblasts surviving 30-min ex-
posure to dilutions of un-buffered sodium hypochlorite posure to dilutions of un-buffered sodium hypochlorite
solutions in sterile water. solutions in 0.9% saline.
Day 0.025% 0.0125% 0.006% 0.003% Day 0.025% 0.0125% 0.006% 0.003%
1 24.0 86.2 88.0 98.9 1 23.38 42 85 93.1
2 16.7 81.7 100 100 6 21.6 29.6 71.9 100
3 27 74 82.7 87.2
Average 22.5% 35.8% 82.3% 96.6%
4 27.4 63.4 83.5 100
5 15 39.4 64.6 64.4
6 30.1 95.4 100 100

Average 25.0% 80.1% 90.8% 97.2%

Table 1. Following exposure of the human fibroblast to a
0.025% un-buffered sodium hypochlorite for 30 min, only 24%
of fibroblasts were viable. After 30 min exposure of 0.0125%
sodium-hypochlorite, 86.2% of fibroblasts were viable. Thirty
minutes exposure to a 0.006% solution left 88% fibroblasts
viable and 98.9% of fibroblasts were viable after being exposed
to a 0.003% solution of un-buffered sodium hypochlorite for
30 min.
The effects of the different dilutions on fibroblast prolifer-
ation remained constant for six consecutive days. As the
results of day five were not consistent with all the other results
of day one to six, it was disregarded as a technical error most
likely caused the inconsistency.
The same experiment was repeated with sodium hypo-
chlorite solutions diluted in 0.9% saline, instead of sterile
water. This yielded similar results, as shown in Table 2.
3.2. The effectiveness of sodium hypochlorite against
bacterial isolates
The minimum bactericidal concentration (MBC) of the un-
buffered sodium hypochlorite solution is shown in Table 3. A
concentration of 0.006% of sodium hypochlorite was bacteri-
cidal to all the bacterial isolates tested.
The MBC for the P. aeruginosa isolates was 0.003%. The MBC
for the S. aureus isolates was 0.006% and for the S. pyogenes
isolates was 0.0015%. The MBC remained unchanged for all the
isolates that were tested with un-buffered sodium hypochlo-
rite stored for up to 96 h at room temperature (Table 4).
3.3. Biochemical properties and pH of the un-buffered
0.025% sodium hypochlorite solution
The un-buffered 0.025% sodium-hypochlorite solution had a
pH of 10 and an osmolality of 168. The sodium concentration
was 89 mmol/dl and the concentration of chloride was
84 mmol/dl. These results remained stable with the 0.025%
stored at room temperature over 14 days (Table 5).
4. Discussion
Burn wound infection is a major cause of morbidity and
mortality in burns units. However, the introduction of
topical, antimicrobial agents has resulted in a significant
reduction in the number of deaths due to burn wound
infections.
The bactericidal properties of topical sodium hypochlorite
are well known and it has been in clinical use for many years.
The sodium hypochlorite solution currently used in the
treatment of burn wounds has been buffered by adding
0.3 N sodium dihydrogen phosphate-disodium monohydro-
gen phosphate (NaH2PO4Na2HPO4). Unfortunately this buff-
ered sodium hypochlorite solution is not readily available and
costly. An un-buffered solution is cheap and easy to prepare.
This study was therefore undertaken to determine the
optimum concentration of an un-buffered sodium hypochlo-
rite solution to be used as topical agent in burn wound
infections.
The ideal topical bactericidal agent should be effective,
non-toxic to the patient, not lead to the development of
resistance, and be cost effective. In this study an un-
buffered sodium hypochlorite solution was bactericidal to
all burn wound pathogens tested at a concentration of
0.006%.
The advantage of using the MBC assay in this study was
that it allowed us to determine a concentration at which the
compound was bactericidal. While agar well diffusion meth-
odology has been shown in some studies to correlate well with
in vitro efficacy [11], others have not shown this [12]. Agar well
diffusion does not allow one to determine whether the
compound is cidal or inhibitory, and does not allow for
quantification of the concentration at which cidal activity
occurred. In addition, agar well diffusion assays have been
reported to be labour intensive and difficult to standardize [13].
However, it is important to note that regardless of the method
of testing in vitro activity, clinical studies showing efficacy
using the these concentrations determined need to be
conducted.

Table 3 MBC of sodium hypochlorite against different isolates.

Organism Number isolates MBC 0.006% MBC 0.003% MBC 0.0015% MBC 0.008% MBC Overall MBC
tested <0.004%
P. aeruginosa 31 20 11 0.003%
S. aureus 12 4 4 1 3 0.006%
S. pyogenes 5 2 1 2 0.0015%

Total 48 0.006%
532 burns 38 (2012) 529533

Table 4 Stability testing of the MBC over time with un-buffered sodium hypochlorite solution stores at room
temperature.
Organism ID MBC 24 h 48 h 72 h 96 h
P. aeruginosa 0.003 0.003 0.003 0.0015 0.003
P. aeruginosa 0.003 0.003 0.003 0.0015 0.003
P. aeruginosa 0.003 0.003 0.003 0.003 0.003
P. aeruginosa 0.003 0.003 0.003 0.003 0.003
S. pyogenes <0.0004 <0.0004 <0.0004 <0.0004 <0.0004
S. pyogenes <0.0004 <0.0004 <0.0004 <0.0004 <0.0004
S. aureus 0.006 0.006 0.006 0.003 0.003
S. aureus <0.0004 <0.0004 <0.0004 <0.0004 <0.0004
S. aureus 0.003 0.003 0.003 0.003 0.003

In addition, 88% of fibroblasts were viable after 30 min

exposure to a 0.006% solution and 99% of fibroblast were viable
after 30 min exposure to a 0.003% un-buffered solution.
The optimum concentration of the un-buffered sodium
hypochlorite solution with regard to efficacy and safety was
the 0.006% solution. This was bactericidal to all the organisms
tested, and almost 90% of the fibroblasts remained viable after
30 min of exposure.
Previous studies have found that the optimum concen-
tration of the buffered sodium hypochlorite solution was
0.025% [10]. This concentration was found to be bactericidal
within 30 min and had no influence on fibroblast cell
architecture and viability. The 0.025% sodium hypochlorite
solution was buffered by adding 0.3 N sodium dihydrogen
phosphate-disodium monohydrogen phosphate (NaH2PO4-
Na2HPO4), which resulted in a solution with an osmolality of
354 mosm/l and pH of 7.5. In the current study, the un-
buffered 0.025% sodium hypochlorite had an osmolality of
168 mosm/l and pH of 10.
In an audit from the Red Cross War Memorial Childrens
Hospital Burns Unit, in Cape Town, South Africa, a 0.0025% un-
buffered sodium hypochlorite solution was successfully used
in the management of children with P. aeruginosa burn wound
infection. Soaking the wounds for 30 min with this solution
with a pH of 10 and osmolality of 168 did not result in more
pain or any other side effects.
The bactericidal effects as well as the effects on fibroblast
proliferation of the un-buffered sodium hypochlorite solu-
tion remained stable for a minimum of six days. Furthermore,
the biochemical properties remained unchanged for 14 days.
It is therefore safe to use the un-buffered sodium hypochlo-
rite solution for at least 5 days when stored at room
temperature.
5. Conclusion
The toxic effects of sodium hypochlorite on wound healing
have been confined to a restricted range of concentrations.
Similarly, the bactericidal concentration of sodium hypo-
chlorite is also related to the concentration and time of
exposure. Thus it is of fundamental importance to use the
optimum concentration that has both adequate bactericidal
properties as well as minimal toxicity. In this study the
optimum concentration for un-buffered sodium hypochlorite
was found to be 0.006%. Clinical studies would be required in
order to assess the in vivo effect of this concentration both on
bacterial clearance as well as potential toxic effects on wound
healing.
Ethics
This study was approved by the University of Cape Town,
Heath Sciences Faculty Research and Ethics Committee.
Conflicts of interest
None to declare.
Acknowledgements
The authors would like to thank Fay Manuel and Toni Wiggins
for their tireless work and enthusiasm in helping to conduct
the experiments as well as Lester Davids for writing the
fibroblast proliferation experiment protocol.

Table 5 Biochemical properties of 0.025% sodium

hypochlorite solution stored at room temperature. references

Time Sodium Chloride pH Osmol

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