Free Radical Biology and Medicine 159 (2020) 119–124

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Free Radical Biology and Medicine

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Short communication

Evaluating the bactericidal action of hypochlorous acid in culture media T

Louisa V. Ashby , Reuben Springer, Mark B. Hampton, Anthony J. Kettle,
∗

Christine C. Winterbourn
Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand

ARTICLE INFO ABSTRACT

Keywords: The bactericidal activity of the physiological oxidant hypochlorous acid (HOCl) is commonly studied in a variety
Hypochlorous acid of laboratory media. Reactive with numerous targets, HOCl will rapidly lose its toxicity via reduction or be
Reactive chlorine species converted to chloramines and other less toxic species. The objective of this study was to test the influence of
Oxidants various media, temperature and reaction time on the toxicity of HOCl. After incubating bacteria in media dosed
Bactericide
with reagent HOCl, the bactericidal outcome was measured by colony forming ability. In parallel, we determined
the HOCl and chloramine content after dosing media alone. Our results showed that more reagent HOCl was
required to kill bacteria in culture media than in aqueous buffer, and this corresponded to the lower con­
centration of reactive chlorine species achieved in the media. RPMI and MOPS minimal medium retained sig­
nificant oxidising equivalents after HOCl-dosing, but more nutrient-rich media such as MEM, DMEM, LB and
TSB, had higher scavenging capacity. Other factors that lowered the bactericidal strength of HOCl were longer
lag-times and raised temperature when pre-dosing media, and insufficient incubation time of cells with the
HOCl-treated media. In summary, we demonstrate that the choice of media as well as procedural details within
experiments crucially impact the cellular toxicity of HOCl. These factors influence the nature and concentration
of oxidants generated, and therefore are critical in affecting cellular responses.

1. Introduction
Hypochlorous acid (HOCl) can elicit a broad range of effects on
living cells [1,2]. Five decades on from the discovery that myeloper­
oxidase-generated HOCl can kill bacteria, there is still more to learn
about the interaction between bacteria and this oxidant [3,4]. Recent
insights into microbial responses to host-derived reactive chlorine
species have largely come from adding HOCl to clonal bacteria popu­
lations ([5–8] and reviewed in Refs. [9,10]). In most of these studies the
bactericidal effect of HOCl was assessed by finding the tipping point
between sublethal and lethal doses in order to compare genetically
modified strains, or inform on adaptive bacterial defence systems. In
these dosing experiments, the fate of reagent HOCl and whether it
reaches the cell surface will depend on the composition of the media.
HOCl is highly reactive with sulphur-containing molecules
[1,11,12]. With thiols, the initial product is the sulfenyl chloride
(R–SCl, reaction (i)) which can hydrolyse to the sulfenic acid (R–SOH,
reaction (ii)). Both intermediates readily condense with another thiol to
form a disulfide and can also yield higher oxidation products including
the sulfinic and sulfonic acids. The main product from methionine is the
sulfoxide. These reactions are likely contributors to the bactericidal
activity of HOCl, whereas when they occur in the medium they have the
effect of quenching its oxidising ability. HOCl also readily reacts with
amines (for example amino acids and ammonia) to form chloramines
(R–NHCl) and dichloramines (R–NCl2, reactions (iii and iv)) [13,14],
some of which are cytotoxic [13,15,16]. Chloramines are less reactive
than HOCl but they remain strong oxidants. They are more selective
towards thiols and preferentially form disulfides rather than more
oxidised species [12]. Ongoing chloramine exchange can further mod­
ulate their impact [17]. Previously, researchers have acknowledged
that complex media in bacterial experiments may impact the toxicity of
HOCl, raising the need to differentiate between primary and secondary
reactive species [9,18,19].
HOCl + R–SH → R–SCl + H2O (i)
R–SCl + H2O → R–SOH + HCl (ii)

Abbreviations: PBS, phosphate buffered saline; HBSS, Hanks balanced salt solution; RPMI, Roswell Park Memorial Institute 1640 medium; MOPS, 3-(N-morpholino)
propane sulfonic acid; MEM, Minimal essential medium alpha; DMEM, Dulbecco's Modified Eagle Medium; LB, Luria-Bertani broth; TSB, tryptone soya broth; RCS,
reactive chlorine species
∗
Corresponding author.
E-mail address: louisa.ashby@otago.ac.nz (L.V. Ashby).

https://doi.org/10.1016/j.freeradbiomed.2020.07.033
Received 8 June 2020; Received in revised form 23 July 2020; Accepted 27 July 2020
Available online 30 July 2020
0891-5849/ © 2020 Elsevier Inc. All rights reserved.
L.V. Ashby, et al.
HOCl + R–NH2 → R–NHCl + H2O (iii)
R–NHCl + HOCl → R–NCl2 + H2O (iv)
The aim of this study was to examine how media affect the delivery
of HOCl to bacteria. After adding HOCl to bacterial suspensions, we
measured bactericidal activity by colony forming capability. We ap­
plied a colorimetric assay that can distinguish between HOCl and
weaker chloramines to compare different cell media and to test the
influence of time and temperature when treating solutions. Our study
demonstrates that reactions within the media influence the amount and
nature of the reactive chlorine species that the bacteria are exposed to,
affecting reproducibility, outcomes and the conclusions that can be
drawn.
2. Methods
2.1. Reagents
Fresh HOCl stock solutions were prepared daily from sodium hy­
pochlorite (household bleach, Sara Lee Ltd, New Zealand) by measuring
A292 at pH 12 (ϵ292 350 M−1 cm−1). Taurine and sodium thiosulfate
were from Sigma, and 3,3′,5,5′-tetramethylbenzidine (TMB) was from
Fluka Chemicals. The buffers and cell media used in this study were:
Phosphate buffered saline (PBS), Dulbecco's formulation without cal­
cium or magnesium (Sigma D8537); Hanks balanced salt solution
(HBSS) (Sigma H8264); Roswell Park Memorial Institute (RPMI) 1640
medium (Gibco 11835030); Minimal essential medium alpha (MEM)
(Gibco 41061); Dulbecco's Modified Eagle Medium (DMEM) (Gibco
21063); Miller's Luria-Bertani broth (LB) and tryptone soya broth (TSB)
were made from powder (ThermoFisher Scientific). MOPS minimal
medium was made according to the original Neidhardt formulation
including micronutrients [20] with the modifications of low phosphate
(0.1 mM) and high glucose (4 g/L).
2.2. Assay for reactive chlorine species
A colorimetric platereader assay that employs the oxidation of
colourless TMB to a blue oxidised form, was used to measure reactive
chlorine species [21]. This chromophore can be produced directly by
HOCl [22], and in an iodide-catalysed reaction by chloramines [23]. A
standard curve of oxidised TMB absorbance at 645 nm was constructed
using taurine chloramine (as in reaction (iii)) [21], thereafter providing
a relative scale of oxidative equivalents termed Reactive Chlorine
Species (RCS). In brief, 150 μl standards or samples were mixed with
one quarter volume of freshly prepared developer solution (2 mM TMB
in 400 mM sodium acetate buffer at pH 5.4, with or without 100 μM
sodium iodide), and A645 was measured after 5 min. Where appropriate,
test samples were initially diluted in PBS (for example 50 μl sample plus
100 μl PBS) to yield absorbance values within the linear range
(0–100 μM RCS) of the standard curve, followed by adjustment for the
dilution.
2.3. Treatment of bacteria with hypochlorous acid
The wild-type reference strains of bacteria used were Staphylococcus
aureus USA300 [24] and Escherichia coli MG1655 [25]. For the S. aureus
experiments, overnight cultures in LB were washed twice in PBS and
resuspended in either PBS or RPMI at approximately 1 x 109 bacteria/
ml estimated by optical density. The actual concentration was de­
termined by colony counting the next day. Relevant doses of fresh HOCl
prepared in water were pipetted in small volumes (≤60 μl) into 400 μl
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Free Radical Biology and Medicine 159 (2020) 119–124
aliquots of bacteria in Eppendorf tubes while vortexing to achieve rapid
mixing and minimise inhomogeneous reactions. After incubation with
gentle end-over-end rotation (6 rpm) at 37 °C for 1 h, 50 μl aliquots
were quenched with 10 mM sodium thiosulfate (via reaction (v)) in ice-
cold PBS, then serially diluted for standard spread-plating on sheep
blood agar. The next day, colony counts were normalised against the
untreated bacterial count, and plotted against the HOCl doses given per
109 bacteria, also adjusted for the actual starting bacteria concentra­
tion.
HOCl + 2S2O32− → S4O62− + Cl− + OH− (v)
For the E. coli experiments, 1 ml of an overnight culture grown in LB
was washed in PBS and resuspended in 15 ml MOPS minimal medium
in a conical flask, then returned to a shaking incubator for 3 h at 37 °C,
200 rpm, to establish growth phase and an optical density of A600 ≈
0.6. Bacteria were harvested by centrifugation such that each pellet
would give A600 = 0.35 when suspended in 2.5 ml of dosed media.
HOCl doses were added by pipette to MOPS minimal medium while
vortexing, followed by a timed incubation before exposing to cells. The
2.5 ml suspensions were in round-bottomed loose-cap 14 ml tubes and
incubated at 37 °C, 200 rpm. To harvest bacteria after treatment, 500 μl
aliquots were mixed with 500 μl MOPS minimal medium containing
20 mM sodium thiosulfate, then pelleted and resuspended in 500 μl
saline solution (0.9% w/v NaCl). These undiluted 500 μl suspensions
are hereby referred to as “neat”. Note that for a sample without bac­
terial growth or lysis, this suspension would be equivalent to the
starting concentration (A600 = 0.35). From each neat suspension, a 10-
fold dilution series was prepared in saline, and 4 μl spots were dis­
pensed onto agar plates. The first spot of each row was from the 1/10
dilution of the neat suspension. Plates were either LB agar or sheep
blood agar, with razor-drawn grids for spot placement. Photographs
were taken after overnight incubation at 37 °C.
3. Results and discussion
3.1. The dose of reagent HOCl experienced by cells depends on the
suspension medium
The bactericidal effect of HOCl on S. aureus was determined for
bacteria suspended in either PBS or RPMI (Fig. 1). Single bolus doses of
increasing concentrations of reagent HOCl were given to bacterial
suspensions at ambient temperature, followed by incubation for an
hour at 37 °C before processing for colony growth. A dose of 100 nmol
HOCl per 109 bacteria was completely toxic for S. aureus in PBS,
whereas doses in excess of 500 nmol were required to kill all the bac­
teria in RPMI (Fig. 1A). RPMI is nutrient-rich including an amino acid
content at approximately 6.8 mM [26]. To determine the fate of HOCl
added to RPMI, we used the TMB oxidation assay. This assay distin­
guishes between HOCl and chloramines such as taurine chloramine,
because the latter require iodide as a catalyst to facilitate the oxidation
of TMB (Fig. 1B). The signal from reagent HOCl in PBS was linear and
independent of iodide, whereas no signal was detected from HOCl-
dosed RPMI unless iodide was present in the developer. This result
indicated that no HOCl remained in the dosed RPMI solution (Fig. 1C).
These results explain why higher doses of HOCl were required for
bactericidal activity in RPMI than in PBS – they were needed to achieve
oxidising equivalence of the reactive chlorine species (RCS) generated
by HOCl's reaction with constituents.
Reactions of HOCl and its secondary oxidants with a range of targets
have been shown to contribute to its bactericidal activity. Enzyme in­
activation, protein unfolding, ATP loss, metabolic shut down and
L.V. Ashby, et al.
inhibition of replication have been observed [4,13,19,27,28] and the
contribution of each is likely to vary depending on the oxidant species
involved. Bacterial resilience against reactive chlorine also depends on
the complexity of the culture media [29]. In the hour following the
addition of HOCl (Fig. 1A), the bacteria will be responding to the toxic
assault with defence processes known to be rapidly initiated by sub­
lethal doses [18,28,30–32]. Nutrient-rich media will be better than
buffer at supporting responsive survival mechanisms involving upre­
gulation of metabolism and protein synthesis. Thus, improved bacterial
recovery could contribute to the bacteria in RPMI having a higher ca­
pacity to survive reactive chlorine species than those in PBS.
A range of cell culture media were tested for their capacity to
quench HOCl (Fig. 2). The glucose in HBSS did not change the HOCl
signal compared to that in PBS, as expected from its low reactivity with
HOCl [33]. Stronger signals were detected after adding HOCl to MOPS
minimal medium compared to those from RPMI, with some activity in
the absence of iodide. MOPS minimal medium is devoid of amino acids
but MOPS is a reactive tertiary amine and the medium also has other
amine constituents. It contains 40 mM MOPS and 4 mM N-tris(hydro­
xymethyl)-methyl glycine (tricine) for their buffering capacity, and
9.5 mM ammonia chloride as an essential nitrogen source. We found
that adding HOCl to 9.5 mM ammonia chloride alone, or a solution of
40 mM MOPS with 4 mM tricine, also gave significant oxidation in the
assay with and without iodide (not shown). Since all of the HOCl would
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Free Radical Biology and Medicine 159 (2020) 119–124
Fig. 1. Bactericidal potency of HOCl added to
cells in PBS buffer or RPMI medium. (A)
Stationary phase S. aureus, at 109/ml in PBS or
RPMI, were dosed with increasing concentrations
of reagent HOCl with rapid mixing on a vortex.
After 1 h at 37 °C, 10 mM sodium thiosulfate was
added, then aliquots of bacteria were spread on
agar. Bacterial survival was measured by colony
counting and normalised against untreated bac­
teria. Data (means of 4 replicate counts ± SD) are
from multiple separate experiments; PBS (n = 9),
RPMI (n = 3). Parallel analysis of oxidising spe­
cies in HOCl-treated solutions without bacteria
was by the colorimetric TMB assay (B & C). (B)
The taurine chloramine standard curve (solid line)
was constructed by adding the indicated con­
centrations of HOCl to PBS containing 5 mM
taurine, then the iodide-catalysed oxidation of
TMB was detected by the increase in absorbance at
645 nm. This standard curve was used to convert
A645 of test samples to relative Reactive Chlorine
Species (μM). (C) Room temperature PBS and
RPMI were dosed with HOCl by rapid mixing on a
vortex, then assayed 15 min later. Developer so­
lution (400 μM TMB in 400 mM sodium acetate
buffer pH 5.4) was either without iodide (to detect
HOCl oxidation of TMB; hollow symbols and
dotted lines) or contained 20 μM iodide (to detect
HOCl and weaker chloramine oxidation of TMB;
solid symbols, solid lines).
be rapidly converted to chloramines under these conditions [21,34] this
suggests some of the resulting chloramines must be sufficiently oxi­
dising to not require iodide catalysis in the assay. Tertiary amines such
as MOPS are known to catalyse chlorination reactions [35], as do other
species including imidazoles [36].
In contrast to the MOPS minimal medium and RPMI results, 200 μM
HOCl was completely quenched when added to MEM, DMEM, LB or TSB
media. In a recent study of the response of Pseudomonas aeruginosa to
HOCl, LB and an artificial sputum medium were chosen to imitate the
inflammatory environment in patients with cystic fibrosis [37]. Typical
of studies using bacteria suspended in high-nutrient media, HOCl was
added in the supra-millimolar range. The exact fate of such HOCl doses
will depend on the medium used and its capacity to quench its oxidising
ability via the generation of non-reactive products. The specific mole­
cules causing cellular effects may also vary due to which targets in the
medium are oxidised. Our results show that cellular exposure to oxi­
dants in complex media is likely to be via secondary products of the
added HOCl that are formed after a substantial degree of quenching by
the media.
3.2. Time dependence of HOCl addition
Some cell culture protocols require prior addition of oxidants to
media before adding the treated media to cells. We compared adding
L.V. Ashby, et al. Free Radical Biology and Medicine 159 (2020) 119–124

Fig. 2. Recovery of RCS from various HOCl-dosed buffers or nutrient-rich

media. A range of cell culture media were tested in the colorimetric assay that
measures TMB oxidation (see Fig. 1): PBS, phosphate buffered saline; HBSS,
Hanks balanced salt solution; MOPS minimal medium; RPMI, Roswell Park
Memorial Institute 1640 medium; MEM, Minimal essential medium alpha;
DMEM, Dulbecco's Modified Eagle Medium; LB, Miller's Luria-Bertani broth;
TSB, tryptone soya broth. Samples of each solution were dosed with 200 μM
HOCl while mixing on a vortex. After 15 min at room temperature, aliquots
were assayed for Reactive Chlorine Species with the presence (black) or absence
(grey) of iodide in the developer. No signal was observed using either developer
for MEM, DMEM, LB and TSB media. Data are means ± SEM of 3 independent
experiments.

HOCl to MOPS minimal medium either 10 or 45 min before re­

suspending E. coli for a 30 min incubation (Fig. 3). Lower doses of HOCl
were bactericidal if the cells were exposed to the media within 10 min
of dosing (lethal at 220 μM) compared those required when added after
45 min (lethal only at 1.5 mM, Fig. 3A). The RCS detected in media
45 min after treatment was largely diminished compared to after
10 min (Fig. 3B). We saw a similar drop over time in RCS from HOCl-
dosed RPMI (not shown). The toxicity of HOCl-treated MOPS media
measured by colony formation corresponded with the level of the RCS
detected. That is, the loss in the reactive chlorine content that occurred
with an extended lag-time meant that a higher HOCl-dose was required
to achieve the same bactericidal effect. This has implications for the
importance of timing in HOCl-dosing of media.

3.3. Considerations of RCS lability, temperature and duration of exposure

To further test the instability of RCS in media, we looked at the

effect of temperature on their toxicity. Adding HOCl to MOPS minimal
medium at room temperature rather than pre-warming it to 37 °C, had a
dramatic effect on its toxicity when it was subsequently incubated with
bacteria (Fig. 4A). E. coli did not survive incubation in the media that
had been dosed with 2 mM HOCl at room temperature. The toxicity of
the warm HOCl-dosed media was lower, and decreased further with
time, indicating ongoing breakdown of reactive oxidants. The duration
of incubating bacteria in the dosed media was also an important de­
terminant of bactericidal activity (Fig. 4B). If E. coli were exposed for a
short time to the dosed media, then no bactericidal effect was evident.
However, with longer exposure, the weaker dose of 0.5 mM HOCl was
sufficient to kill them.
Fig. 3. Temporal changes in the toxicity of HOCl-treated MOPS media.
Increasing doses of reagent HOCl were added to room temperature MOPS
minimal medium either 10 (black) or 45 min (red) prior to incubating with
E.coli for 30 min at 37 °C. (A) After 30 min, bacterial suspensions were quen­
ched with 10 mM sodium thiosulfate prior to serial 10-fold dilutions being
spotted onto agar. (B) Just prior to the addition to bacteria, aliquots of the
dosed media were tested in the taurine chloramine assay with iodide in the TMB
developer. Increasing toxicity (X) is indicated, through to the fully lethal dose
(☠) determined in (A). (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)

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L.V. Ashby, et al.
4. Conclusions
[7]
A popular experimental approach for studying the cellular effects of
HOCl is its bolus addition into suspensions of cells. We have demon­
strated that the bactericidal potency of reagent HOCl is dependent on
the nature of the media components, and variations in reaction time, [8]
exposure time and temperature. The fate of HOCl upon addition to
complex media is either to be immediately scavenged, leading to no
cellular effect, or converted to a different reactive species such as a
[9]
chloramine, which in turn may be scavenged or react with the bacteria.
The relative reactivity of chlorinating reagents and the scavenging ef­ [10]
fect of full media have also been discussed in relation to mammalian
[11]
cell culture studies [17,38–40]. The striking differences between media
treatments, observed in our colony forming assays using single strains
of bacteria, raises the need for caution, for example when comparing
[12]
genetic mutants for altered survival. Subtle changes in media and
handling between experiments could potentially obscure true differ­
ences in bacterial sensitivity and responses to HOCl and its products. [13]
Acknowledgements
[14]
This work was supported by the Health Research Council of New
Zealand (grant number 15/479). [15]
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