Copper(II) sulphate is a desiccant. (Dries out bacterai)

Although copper is essential for most living organisms, copper(II) sulfate is poisonous.
Appl Environ Microbiol. 2001 November; 67(11): 5325-5327.
doi: 10.1128/AEM.67.11.5325-5327.2001.
Copyright © 2001, American Society for Microbiology

Concentrations of Copper Thought To Be Toxic to Escherichia coli Can Induce the Viable but Nonculturable
Brian Grey† and Todd R. Steck*

Department of Biology, The University of North Carolina at Charlotte, Charlotte, North Carolina 28223

*Corresponding author. Mailing address: Department of Biology, The University of North Carolina at Charlotte, Charlotte,
NC 28223. Phone: (704) 687-4393. Fax: (704) 687-3128. E-mail: [email protected].
†Present address:
National Health and Environmental Effects Research Laboratory, U.S. Environmental
Protection Agency,
Durham, NC 27711.

Received June 11, 2001; Accepted August 9, 2001.


We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of
Escherichia coli to enter the viable but nonculturable (VBNC) condition. Copper-induced VBNC cells could be
resuscitated for up to 2 weeks after entering the VBNC state.

Excess levels of copper are toxic to aerobic bacteria (14). It has been suggested that toxicity occurs due to membrane-
bound copper catalyzing the formation of hydroperoxide free radicals (16). Toxicity levels are determined by examining
bacterial growth in the presence of various concentrations of an agent on solid or liquid growth medium (5, 9, 13). A lack
of growth is considered to indicate that the agent has killed cells.
However, a lack of growth could also result from
the cells entering the viable but nonculturable (VBNC) condition.

The dormant-like VBNC condition occurs in response to a variety of environmental stresses and is considered to be a
long-term survival mechanism employed primarily by gram-negative bacteria (7). VBNC cells do not undergo visible
growth under conditions that would normally support growth. Because VBNC cells do not readily grow, growth-
independent viability assays are used to document the VBNC condition (11). That these assays are by necessity indirect
indicators of viability is what makes the VBNC condition controversial (2, 12). Regaining the ability to grow (i.e.,
resuscitation) is the most definitive proof of cells having been VBNC; however, resuscitation does not always occur by
reversing the initial VBNC-inducing conditions. There is a lack of molecular and genetic information about this condition
due in part to the existence of no known chemical inducer of the VBNC state. In Agrobacterium tumefaciens and
Rhizobium meliloti, copper was reported to be the first such chemical VBNC inducer (1). Resuscitation of these VBNC
cells was not examined, and no comparison of the VBNC-inducing concentrations of copper with those concentrations
considered to be toxic was made.

To determine if toxic concentrations of copper (9, 14) could induce the VBNC condition, mid- to late-exponential-stage
cells of Escherichia coli strain ED8739 (9) were grown with aeration at 37°C in Luria-Bertani (LB) liquid medium and then
added to LB plates containing various concentrations of copper sulfate. Copper toxicity has previously been examined in
this wild-type strain using these conditions (9). After 2 days of incubation at 37°C, cells were harvested from the plate as
follows. Five milliliters of 0.9% NaCl was repipetted over the plate surface; the released cells were then collected onto a
0.22-μm-pore-size polyvinylidene difluoride filter (Millipore Corp., Bedford, Mass.), washed with 0.9% NaCl, and then
removed from the filter by shaking in a culture tube in 1 ml of 0.9% NaCl. Cells were assayed for viability using the
LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Inc., Eugene, Oreg.) as described in the manufacturer's
instructions. This viability assay, which differentiates cells with an intact (i.e., viable) cell membrane from those with a
compromised (i.e., dead) cell membrane based on the differential ability of two fluorescent dyes to permeate them, has
previously been used to study the VBNC condition (4, 8, 15). Cells collected onto a 0.2-μm-pore-size black
polycarbonate filter (Poretics Corp., Livermore, Calif.) were scored using an Olympus BX-60 epifluorescence microscope
and a fluorescein isothiocyanate filter. At least 100 cells over at least four fields of vision were scored per tested sample.
A difference between the numbers of viable and culturable cells was used to quantitate the number of VBNC cells. The
average results of two trials, given in Table 1, indicate that growth was observed on agar plates of LB medium and of LB
medium containing 4 mM CuSO4 and that no growth was observed on LB medium containing either 6 mM or 25 mM
CuSO4. A previous study reported similar results and concluded that 6 mM CuSO4 was toxic to E. coli (9). Significant
concentrations of viable cells were recovered from the plates lacking observable growth (Table 1), indicating that VBNC
cells were present.

To determine if this response to copper was strain specific, the same experiment was conducted with E. coli strain ES80
(a spontaneous rifampin-resistant derivative of the uropathogenic E. coli strain 536 [10]) with similar results (data not
shown). Because the efficiency of recovery of bacteria in different physiological states from agar plates is unknown, the
percentage of cells in each plate that were VBNC could not be determined. Therefore, cells in liquid cultures were

Liquid cultures of strain ES80 were established as follows. Cells grown in LB medium were harvested by centrifugation
when at an optical density at 600 nm of 0.8 to 1.0, washed three times in 0.9% NaCl, added to a final concentration of 1
× 108 to 3 × 108 cells per ml in 20 ml of 0.9% NaCl with or without 500 μM CuSO4 in a 125-ml Erlenmeyer flask, and
incubated at 25°C. This concentration of
copper sulfate was chosen because it would be considered toxic in liquid
medium; previous experiments had shown that this concentration as well as lower concentrations of copper sulfate (100
μM) resulted in the removal of all culturable cells from a liquid culture within a few days (unpublished results). At various
intervals over a 4-week period, samples were removed and subjected to culturability and viability assays. Viability was
determined as described above. Culturability was determined as follows. Cells collected onto a 0.2-μm-pore-size
cellulose nitrate filter (Whatman, Inc., Clifton, N.J.) were washed and suspended in 0.9% NaCl, diluted as necessary in
saline, and plated in triplicate onto LB plates. Colonies were counted after the plates were incubated at 37°C for at least
3 days. Occasionally, plates were recounted after 10 days; when this was done, no difference in colony counts was
observed. The results of one representative experiment are given in Fig. 1. For all three trials, no culturable cells were
observed by day 10. When viability was measured, all cultures contained at least 107 viable cells per ml throughout the
experiment. Therefore, although CuSO4 killed over 90% of the cells, those cells that escaped killing were in the VBNC
state. Similar results were obtained with a starting cell concentration ranging from 107 to 109 cells per ml (data not

To confirm that the lack of observed growth was due to cells being VBNC and not dead, resuscitation of copper-induced
VBNC ES80 cells was examined. At various times after VBNC induction, samples were removed and cells were collected
onto a cellulose nitrate filter, washed with 3 to 5 volumes of 500 μM EDTA, suspended in the original volume of 0.9%
NaCl, and incubated at 25°C. Samples of cells were diluted 25- and 1,000-fold in 0.9% NaCl. At various times, samples
of cells were removed, diluted if necessary in 0.9% NaCl, and plated onto LB medium to monitor culturability; any
resulting colonies were transferred to LB medium containing rifampin (100 μg per ml). It was found that, if this treatment
was performed within 2 weeks of loss of culturability, the washed cells regained culturability to a level proportional to
their dilution and to a level expected if resuscitation, and not growth, was occurring (Fig. 2). That all colonies were
rifampin resistant indicated that the colonies did not arise from contamination. Cultures that contained no detectable
culturable cells for 4 weeks did not undergo resuscitation under these conditions.

The reappearance of culturable cells could be due to VBNC cells being resuscitated or to the growth of a few
undetected culturable cells (12). That no nutrients were known to be present in the liquid cultures argues against the
latter possibility. If growth of a few culturable cells was occurring due to the presence of some unknown nutrients, the
concentrations of culturable cells present in the EDTA-washed cultures would be approximately the same regardless of
the dilution and would not have been proportional to the fold dilution. Furthermore, that the cells were washed makes it
unlikely that lysed dead cells could have provided nutrients, especially in an amount sufficient to support growth to the
observed levels.

An alternate explanation for the reappearance of culturable cells is that the viable cells present prior to washing with
EDTA were nonculturable, not because they were VBNC but because they were in a hydrogen peroxide-sensitive state.
The observation of a hydrogen peroxide-sensitive culturable cell population (3) was used to argue against an earlier
report of resuscitation of nonculturable Vibrio vulnificus cells (18). Hydrogen peroxide-sensitive cells grow on rich
medium only when plates are supplemented with sodium pyruvate or catalase (3). Hence, V. vulnificus cells that did not
give rise to colonies on unsupplemented rich medium may not have all been VBNC, and the subsequent appearance of
culturable cells following warming (18) may not have been the result of resuscitation but may have resulted instead from
the growth of residual hydrogen peroxide-sensitive culturable cells. We wanted to determine if copper was inducing a
similar hydrogen peroxide-sensitive culturable cell population. The culturability of copper-treated cells was assayed as
described above; in addition, samples were plated on 25-ml agar plates supplemented with 80 mg of sodium pyruvate
(3). In both of two trials, there was no difference in the decrease in the number of culturable cells on plates containing or
lacking sodium pyruvate (data not shown). Therefore, the appearance of culturable cells shown in Fig. 2 is due to
resuscitation and not the growth of a hydrogen peroxide-sensitive culturable cell population.

These results indicate that copper can induce E. coli to enter the VBNC condition. The concentrations of copper used in
this study include those considered toxic (9, 13) and are higher than those used to examine copper-induced cell injury
(6, 17). That high concentrations of copper do not kill all cells suggests that current growth-based microbiological
methods for assaying toxicity result in an undercount of the number of viable cells through incorrect scoring of VBNC
cells as dead. Additional non-growth-based assays should be used to obtain accurate toxicity data. Although cells could
be resuscitated only from VBNC cultures less than 1 month old, it is possible that other conditions could induce
resuscitation in older cultures. Current studies are under way to determine if similar results can be achieved with other
heavy metals.


We are grateful to Joerg Hacker (University of Wurzburg) and Henry Wu (Uniformed Services University, Bethesda, Md.;
deceased) for bacterial strains and to Jordan Gottlieb for assistance with collecting preliminary data.

This work was supported by the University of North Carolina at Charlotte.

1.Alexander E, Pham D, Steck T R. The viable-but-nonculturable condition is induced by copper in Agrobacterium
tumefaciens and Rhizobium leguminosarum. Appl Environ Microbiol. 1999;65:3754-3756. [PubMed]
2.Barer M R, Harwood C R. Bacterial viability and culturability. Adv Microb Physiol. 1999;41:93-97. [PubMed]
3.Bogosian G, Aardema N D, Bourneuf E V, Morris P J L, O'Neil J P. Recovery of hydrogen peroxide-sensitive culturable
cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state. J Bacteriol. 2000;
182:5070-5075. [PubMed]
4.Cole S P, Cirillo-Daniela D, Kagnoff M F, Guiney D G, Eckmann L. Coccoid and spiral Helicobacter pylori differ in their
abilities to adhere to gastric epithelial cells and induce interleukin-8 secretion. Infect Immun. 1997;65:843-846. [PubMed]
5.Diaz-Baez M C, Roldan F. Evaluation of the agar plate method for rapid toxicity assessment with some heavy metals
and environmental samples. Environ Toxicol Water Qual. 1996;11:259-263.
6.Domek M J, Robbins J E, Anderson M E, McFeters G A. Metabolism of Escherichia coli injured by copper. Can J
Microbiol. 1987;33:57-62. [PubMed]
7.Gauthier, M J. Environmental parameters associated with the viable but nonculturable state. In: Colwell R R, Grimes D
J. , editors; Colwell R R, Grimes D J. , editors. Nonculturable microorganisms in the environment. Washington, D.C.:
American Society for Microbiology; 2000. pp. 87-112.
8.Ghezzi J I, Steck T R. Induction of the viable but non-culturable condition in Xanthomonas campestris pv. campestris in
liquid microcosms and sterile soil. FEMS Microbiol Ecol. 1999;30:203-208. [PubMed]
9.Gupta S D, Lee B T O, Camakaris J, Wu H C. Identification of cutC and cutF (nlpE) genes involved in copper tolerance
in Escherichia coli. J Bacteriol. 1995;177:4207-4215. [PubMed]
10.Hacker J, Ott M, Blum G, Marre R, Heeseman J, Tschape H, Goebel W. Genetics of Escherichia coli uropathogenicity:
analysis of the O6:K15:H31 isolate 536. Zentbl Bakteriol. 1992;276:165-175. [PubMed]
11.Huq, A., I. N. G. Rivera, and R. R. Colwell. Epidemiological significance of viable but nonculturable microorganisms, p.
301-323. In R. R. Colwell and D. J. Grimes (ed.), Nonculturable microorganisms in the environment. American Society
for Microbiology, Washington, D.C.
12.Kell D, Kaprelyants A, Weichart D, Harwood C, Barer M. Viability and activity in readily culturable bacteria: a review
and discussion of the practical issues. Antonie Leeuwenhoek. 1998;73:169-187. [PubMed]
13.Liu D, Kwasniewska K. An improved agar plate method for rapid assessment of chemical inhibition to microbial
populations. Bull Environ Contam Toxicol. 1981;27:289-294. [PubMed]
14.Nies D H. Microbial heavy-metal resistance. Appl Microbiol Biotechnol. 1999;51:730-750. [PubMed]
15.Rigsbee W, Simpson L M, Oliver J D. Detection of the viable but nonculturable state in Escherichia coli O157:H7. J
Food Saf. 1997;16:255-262.
16.Rodriguez-Montelongo L, de la Cruz-Rodriguez L C, Farias R N, Massa E M. Membrane-associated redox cycling of
copper mediates hydroperoxide toxicity in Escherichia coli. Biochim Biophys Acta. 1993;1144:77-84. [PubMed]
17.Singh A, Yeager R, McFeters G A. Assessment of in vivo revival, growth, and pathogenicity of Escherichia coli strains
after copper- and chlorine-induced injury. Appl Environ Microbiol. 1986;52:832-837. [PubMed]
18.Whitesides M D, Oliver J D. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. Appl Environ
Microbiol. 1997;63:1002-