Journal of Applied Microbiology 1998, 84, 399403

Development of a new culture medium for the rapid detection

of Salmonella by indirect conductance measurements
D. Blivet, G. Salvat, F. Humbert and P. Colin
Centre National dEtudes Veterinaires et Alimentaires, Ploufragan, France
5948/10/96: received 10 October 1996, revised 30 May 1997 and accepted 5 June 1997

The main difficulties in conductance

medium development are to allow Salmonella to grow and produce a conductance
signal while impeding growth of related species such as Escherichia coli and Citrobacter
freundii. Various selective agents were screened for these capacities and a new
medium was derived, named KIMAN (Whitley Impedance Broth basal medium
supplemented with three selective components: novobiocin, malachite green and potassium
iodide). This medium supported the growth of Salmonella serotypes and inhibited nonsalmonella strains in pure cultures.
D . B LI V ET , G . SA LV A T, F. H UM BE R T A ND P . C OL I N. 1998.

INTRODUCTION

In view of the widespread nature of Salmonella species, rapid

detection methods must be found in order to limit the release
of contaminated products and thus avoid food-borne illnesses.
Classic cultural techniques are labour intensive and take from
three to seven days to complete the confirmation of a positive
sample. Recent developments in rapid detection include various techniques such as hydrophobic grid membrane filter
techniques (Entis et al. 1982 ; Entis and Boleszczuk 1991),
genomic techniques (Aabo et al. 1995 ; Bandekar et al. 1995;
Lin and Tsen 1995), radiometric techniques (Lampi et al.
1974), immunoassays (Blore and Slavik 1992 ; Desmidt et al.
1994 ; Brigmon et al. 1995) or impedance technology (Bolton
1991 ; Silley and Forsythe 1996). Impedance technology is a
rapid, automated and qualitative technique which measures
in a medium the conductance change induced by bacterial
metabolism. The detection time is a function of both initial
bacterial concentration and growth kinetics of micro-organisms in the given medium (Eden and Eden 1984).
Different media have been proposed for the detection of
Salmonella by conductance measurements but they give false
detections due to: Citrobacter freundii (Easter and Gibson
1985 ; Bullock and Frodsham 1989 ; Di Falco et al. 1993) ;
Escherichia coli (Ogden and Cann 1987) ; Hafnia alvei (Ogden
1988 ; Di Falco et al. 1993) ; Serratia marcescens (Arnott et al.
1988) ; Enterobacter cloacae and Klebsiella oxytoca (Pless et al.
1994) ; Pseudomonas spp. (Saco 1993).
The indirect impedance method described by Owens et al.
(1989) allows the use of components inappropriate in the
direct method on account of high basal conductance. This
Correspondence to: Dr P. Colin, C.N.E.V.A., B.P.53, 22440 Ploufragan,
France.
1998 The Society for Applied Microbiology

method is based on the detection of CO2 released by microorganisms into the culture medium, and which is absorbed
in an alkaline solution in contact with the electrodes of the
tubes.
The present work was undertaken to develop a new culture
medium which would allow growth and detection of major
Salmonella serotypes isolated from poultry products and
inhibit non-salmonella organisms by the indirect impedance
technique.
MATERIALS AND METHODS
Pure cultures

Pure cultures identified as Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Braenderup, Salmonella St
Paul, Salmonella Agona, Salmonella Infantis, Salmonella London, Salmonella Heidelberg, Escherichia coli, Proteus vulgaris,
Klebsiella pneumoniae, Serratia odorifera, Citrobacter freundii,
Enterobacter cloacae and Providencia stuartii were used. This
collection was extended to 14 salmonellas with Salm.
Virchow, Salm. Newport, Salm. Heidelberg (a strain producing no H2S), Salm. Arizonae and Salm. Enteritidis (two
strains), and 12 non-salmonellas with Pr. mirabilis (three
strains), Ser. odorifera, Ent. sakazakii, to test the best conductance medium. No Gram-positive strains were tested in
view of their known sensitivity to most inhibitors (Arroyo
and Arroyo 1995).
All strains were sub-cultured on plate count agar (PCA;
AES, Combourg, France) and one colony forming unit (cfu)
was inoculated in brain heart infusion (BHI; Difco, Detroit,
MI, USA) broth and incubated at 37 C for 1824 h.
Dilutions in tryptone-salt (TS ; AES) were made to obtain

400 D . B LI V ET ET A L.

suspensions containing approximately 1 103 cfu ml1 salmonella and 1 107 cfu ml1 non-salmonella organisms.

growth change; decreased growth (DT is superior to DT

control by [] 24 h, [ ] 47 h, [ ] 710 h or [ ] more
than 10 h); [I] inhibition (no DT).

Conductance media

Conductance media were formulated by adding different

selective components to the Whitley Impedance Broth (WIB;
composed of tryptone, 115 g l1; lactalbumen hydrolysate,
10 g l1; meat peptone, 50 g l1; yeast extract, 30 g l1;
magnesium sulphate/calcium chloride 05 g l1), basal formula developed by Don Whitley Scientific Ltd (Shipley,
UK). The final concentrations of inhibitory components
were: sulfadiazine (80, 40, 80 mg l1; Sigma Aldrich, La
Verpillie`re, France), novobiocin sodium salt (20, 40,
80 mg l1; Sigma), brilliant green (50, 10, 30 mg l1; Merck
Cleveno, Chelles, France), crystal violet (50, 10, 20 mg l1;
Prolabo, Fontenay-sous-Bois, France), malachite green oxalate (20 mg l1; Merck), magnesium chloride hexahydrate
(36 g l1; Merck), sodium biselenite (10, 40 g l1; AES),
sodium thiosulphate (10, 10 g l1; Merck), sodium deoxycholate (10, 25, 50 g l1; Merck), trisodium citrate 2hydrate (10, 10 g l1; Merck) and potasssium iodide (10,
20, 40, 80 g l1; Merck). Aliquots of 100 ml of concentrated
solutions (39) sterilized by filtration were added to 38 ml
of WIB medium. After the effects of each inhibitory substance
were tested alone, combinations of two or three of them were
evaluated.
Indirect conductance assay

The apparatus used was a RABIT (Don Whitley Scientific

Ltd) and 900 ml of 06% (w/v) KOH solution (Dezenclos
et al. 1994) were added to each electrode tube. A 12 75 mm
glass tube in which the conductance medium was dispensed
was placed just above the electrodes. The electrode tubes
were tightly closed with a rubber bung and left in the dark at
4 C for at least 24 h to allow maximum absorption of
endogenous CO2. Diluted suspensions of the micro-organisms (100 ml) were dispensed in glass tubes so that approximately 1 102 cfu Salmonella and 1 106 cfu of each of the
other strains were used in the inoculum. Inoculated tubes
were placed in the RABIT apparatus and monitored for 24 h
at 37 C. Conductance changes were recorded every 6 min
and three consecutive changes of 15 microsiemens (mS)
determined the detection time (DT).
Interpretation

Growth of the 15 strains in each conductance medium was

compared to their growth in the WIB basal medium, considered as control. Growth changes were evaluated according
to the following scale: enhanced growth, [ ] (DT is
inferior by at least 2 h to DT control); [ ] little or no

RESULTS
Effectiveness of potential inhibitory substances

The effects of potential inhibitors on the growth of salmonella

and non-salmonella strains by the indirect conductance technique are shown in Table 1. Some components did not affect
growth of the different strains: MgCl2.6H2O (36 g l1), sulfadiazine (8, 40, 80 mg l1), sodium thiosulphate (10,
10 g l1), sodium citrate (10, 10 g l1), sodium deoxycholate
(10 g l1) and potassium iodide (10, 20 g l1). On the other
hand, some components were too selective, inhibiting all or
some salmonella strains at the concentrations tested: brilliant
green (5, 10, 30 mg l1), crystal violet (10, 20 mg l1), sodium
biselenite (10, 4 g l1) and potassium iodide (80 g l1).
The strain Pr. vulgaris was the most easily repressed; it was
completely inhibited by malachite green (20 mg l1), sodium
deoxycholate (5 g l1) and crystal violet (5 mg l1). Novobiocin appreciably impeded the growth of Kl. pneumoniae
and Ent. cloacae when used at 20 mg l1. Malachite green
(20 mg l1) stopped the growth of both Pr. vulgaris and Ser.
odorifera, and slowed the growth substantially of E. coli, Ent.
cloacae and Prov. stuartii. Sodium deoxycholate (25 g l1)
inhibited the growth of Pr. vulgaris, E. coli and Ser. odorifera.
Proteus vulgaris and Prov. stuartii were inhibited, totally or
substantially, respectively, by crystal violet (5 mg l1). Potassium iodide (40 g l1) slowed the growth of E. coli and
completely inhibited the growth of Kl. pneumoniae, Ser. odorifera and Cit. freundii, while allowing good development of
all salmonellas. After consideration of these results, inhibitor
substances were combined so that prevention of growth of all
non-salmonella strains was achieved; Table 2 shows the
effects of each combination. The association between novobiocin (20 mg l1) and malachite green (10 mg l1) led to inhibition of three of the seven non-salmonella strains tested (Pr.
vulgaris, Ent. cloacae and Prov. stuartii) but also slowed the
growth of salmonella strains. The addition of potassium iodide (40 g l1) to that combination had an unexpected effect;
none of the seven non-salmonella strains was detected (very
low conductance change and no DT), and the salmonella
strains grew as well as in the WIB basal medium. This formulation was further called KIMAN (KI for potassium
iodide, MA for malachite green, N for novobiocin).
Effectiveness of the KIMAN medium

Twelve serotypes (14 strains) of Salmonella were successfully

tested for their capacity to grow and produce a conductance
curve in the new medium. The nine (12 strains) non-sal-

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 399403

R AP ID D ET EC T IO N O F SA LM O NE LL A 401

Table 1 Effect of selective substances on the 15 strains tested by indirect conductance

Inhibitor (concentration)

NVB
NVB
CV
MG
NDC
NDC
KI
Test strains
(20)
(40)
(5)
(20)
(25)
(5)
(40)

Salmonella
Salmonella Infantis
*

Salmonella Agona

Salmonella Heidelberg

Salmonella London

Salmonella Typhimurium

Salmonella Enteritidis

Salmonella St Paul

Salmonella Braenderup

Non-salmonella
Escherichia coli

Proteus vulgaris

I
I

Klebsiella pneumoniae

I
Serratia odorifera

I
Citrobacter freundii

I
Enterobacter cloacae

Providencia stuartii

*Enhanced growth; [] (DT by 2 h to DT control); [] little or no growth change; decreased growth DT to DT control by
[] 24 h; [ ] 47 h, [ ] 710 h or [ ] more than 10 h); [I] inhibition (no DT).
All concentrations in mg l1, except in g l1.
CV, crystal violet; KI, potassium iodide; MG, malachite green; NDC, sodium deoxycholate; NVB, novobiocin.

monella species were unable to grow in this conductance

medium (Figs 1 and 2).
DISCUSSION

In this study, the only criterion used for the selection of the
components of the new conductance medium for Salmonella
was the rapid detection by indirect conductance assays of
eight Salmonella strains (1 102 cfu) and the inhibition (no
detection) of seven other Enterobacteriaceae (1 106 cfu).
Some of the results obtained here are different from those
observed in previous studies. Brilliant green (BG) is widely
used in many Salmonella enrichment and isolation media
(Kristensen et al. 1925 ; Kauffmann 1935 ; van Schothorst
et al. 1987) and is not supposed to prevent Salmonella growth.
Despite these results and an awareness of the absence of bile
salts in WIB medium which may reduce toxicity of BG
(Fricker 1987), the strains tested in this study did not grow
at low levels of the dye (5 mg l1). Some differences between
the literature and the results of the present study were also
noticed with crystal violet (CV) (van Schothorst et al. 1987 ;
Arroyo and Arroyo 1995) and sodium biselenite; the sodium
biselenite used in this study was without cystine which is
supposed to decrease selenite toxicity (Leifson 1936 ; North
and Bartram 1953). On the other hand, components such as

magnesium chloride hexahydrate, sulfadiazine, sodium thiosulphate, sodium deoxycholate or sodium citrate, commonly
used in Salmonella media (Hawa et al. 1984 ; De Smedt and
Bolderdijk 1987 ; Fricker 1987 ; Miller and Tate 1991), did
not show any significant inhibitory activity.
Novobiocin (NVB) is used in many selective media, at low
concentrations (545 mg l1), even though salmonella growth
occurs up to 80 mg l1 (Restaino et al. 1977). The effectiveness of that antibiotic was confirmed in this study, i.e.
good development of all Salmonella test strains with inhibition
of Kl. pneumoniae, Ent. cloacae, Ser. odorifera, Prov. stuartii
and Cit. freundii at 40 mg l1. Potassium iodide (KI) is used
in tetrathionate broth and concentrations vary from 5 to
1375 g l1 (DAoust 1981). In the present study, while
20 g l1 of KI had no effect on all 15 test strains and 80 g l1
of KI totally inhibited all strains, the concentration of 40 g l1
was successful in allowing salmonella growth and inhibiting
Kl. pneumoniae, Ser. odorifera and Cit. freundii.
The differences in effectiveness of inhibitors observed
between studies could be explained by the use of different
media, strains and conditions. Nevertheless, and according
to previous results, a combination of three selective agents
was added to the WIB basal medium and tested by indirect
conductimetry on the 15 test strains. The KIMAN formulation, WIB supplemented with novobiocin (20 mg l1), mala-

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 399403

402 D . B LI V ET ET A L.

Table 2 Effect of associations of selective substances on the 15

4500

strains tested by indirect conductance

Inhibitor (concentration)

NVB MG NVB MG NVB MG KI

Test strains
(20 10)
(20 20)
(20 10 40)

Salmonella
Salmonella Infantis
*

Salmonella Agona

Salmonella Heidelberg

Salmonella London

Salmonella Typhimurium

Salmonella Enteritidis

Salmonella St Paul

Salmonella Braenderup

4000

Conductance (S)

3000
2500
2000
1500

1000

22:30

18:30

14:30

20:30

d
12:30

10:30

08:30

06:30

04:30

c
be f g
02:30

00:30

16:30

500

Time (h)

Fig. 2 Conductance curves of pure cultures of (a) Salmonella

Heidelberg, (b) E. coli, (c) Pr. vulgaris, (d) Kl. pneumoniae, (e) Ser.
odorifera, (f) Cit. freundii, (g) Ent. cloacae and (h) Prov. stuartii
in KIMAN medium determined by the indirect impedance
technique at 37 C for 24 h

chite green (10 mg l1) and potassium iodide (40 g l1), was
successful in rapidly detecting (around 12 h) approximately
1 102 cfu salmonella organisms and completely inhibiting
(no detection within 24 h) approximately 1 106 cfu nonsalmonella organisms.
Conductance media described previously yield Citrobacter
and Hafnia as false positives (Ogden 1988) and are recommended to be used in conjunction to limit both false
positives and negatives. The conductance medium defined in
this paper could be a challenger to these media, particularly
as formulations have been tested under hard conditions (only
1 102 cfu salmonella against 1 106 cfu non-salmonella
organisms). Further work is necessary on naturally contaminated foods to verify if (i) Salmonella recovery rate is
optimum and (ii) no or few false positives occur.
If confirmed, this new conductance medium would be of
great interest for the rapid detection of Salmonella spp. in
food; the whole method takes less than 48 h, which shows once
more that impedance technology is a promising technique for
pathogen detection.

c
22:30

a
20:30

f
18:30

16:30

14:30

b e g
12:30

10:30

08:30

06:30

04:30

h d
02:30

5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0

00:30

Conductance (S)

Non-salmonella
Escherichia coli

I
Proteus vulgaris
I
I
I
Klebsiella pneumoniae

I
Serratia odorifera

I
Citrobacter freundii

I
Enterobacter cloacae
I

I
Providentia stuartii
I
I
I

*Enhanced growth; [] (DT by 2 h to DT control); []

little or no growth change; decreased growth (DT to DT
control by [] 24 h; [ ] 47 h; [ ] 710 h or [ ] more
than 10 h); [I] inhibition (no DT).
All concentrations in mg l1, except in g l1.
KI, potassium iodide; MG, malachite green; NVB, novobiocin.

3500

Time (h)

Fig. 1 Conductance curves of pure cultures of (a) Salmonella

Heidelberg, (b) E. coli, (c) Pr. vulgaris, (d) Kl. pneumoniae,

(e) Ser. odorifera, (f) Cit. freundii, (g) Ent. cloacae and (h) Prov.
stuartii in WIB medium determined by the indirect impedance
technique at 37 C for 24 h

ACKNOWLEDGEMENTS

The authors thank A.E.S. Laboratoire for financial assistance,

and especially Mr Butin for scientific advice and reading of
the manuscript.
REFERENCES
Aabo, S., Andersen, J.K. and Olsen, J.E. (1995) Research note:

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 399403

R AP ID D ET EC T IO N O F SA LM O NE LL A 403

Detection of Salmonella in minced meat by the polymerase chain

reaction method. Letters in Applied Microbiology 21, 180182.
Arnott, M.L., Gutteridge, C.S., Pugh, S.J. and Griffiths, J.L. (1988)
Detection of salmonellas in confectionery products by conductance. Journal of Applied Bacteriology 64, 409420.
Arroyo, G. and Arroyo, J.A. (1995) Selective action of inhibitors
used in different culture media on the competitive microflora of
Salmonella. Journal of Applied Bacteriology 78, 281289.
Bandekar, J.R., Ussuf, K.K. and Nair, P.M. (1995) Detection of
Salmonellae in chicken and fish samples using DNA probe. Journal of Food Safety 15, 1119.
Blore, P.J. and Slavik, M.F. (1992) A new rapid technique for
Salmonella antigen detection using a particle concentration fluorescence immunoassay (PCFIA). Journal of Rapid Methods Automation in Microbiology 1, 5965.
Bolton, F.J. (1991) Conductance and impedance methods for detecting pathogens. In International Congress on Rapid Methods and
Automation in Microbiology and Immunology, 1990, Helsinki,
Finland, ed. Vaheri, A., Tilton, R.C. and Barlows, A. pp. 176
181. Springer-Verlag.
Brigmon, R.L., Zam, S.G. and Wilson, H.R. (1995) Detection of
Salmonella enteritidis in eggs and chicken with enzyme-linked
immunosorbent assay. Poultry Science 74, 12321236.
Bullock, R.D. and Frodsham, D. (1989) Rapid impedance detection
of salmonellas in confectionery using LICNR broth. Journal of
Applied Bacteriology 66, 385391.
DAoust, J.-Y. (1981) Update on pre-enrichment and selective
enrichment conditions for detection of Salmonella in foods. Journal of Food Protection 44 , 369374.
De Smedt, J.M. and Bolderdijk, R.F. (1987) Dynamics of Salmonella
isolation with Modified Semi-Solid Rappaport-Vassiliadis
medium. Journal of Food Protection 50 , 658661.
Desmidt, M., Haesebrouck, F. and Ducatelle, R. (1994) Comparison
of the Salmonella-Tek ELISA to culture methods for detection
of Salmonella enteritidis in litter and cloacal swabs of poultry.
Journal of Veterinary Medicine Series B: Infectious diseases and
Veterinary Public Health 41, 523528.
Dezenclos, T., Ascon-Cabrera, M., Ascon, D., Lebeault, J.-M. and
Pauss, A. (1994) Optimisation of the indirect impedancemetry
technique; a handy technique for microbial growth measurement.
Applied Microbiology Biotechnology 42, 232238.
Di Falco, G., Giaccone, V., Amerio, G.P. and Parisi, E. (1993) A
modified impedance method to detect Salmonella spp. in fresh
meat. Food Microbiology 10, 421427.
Easter, M.C. and Gibson, D.M. (1985) Rapid and automated detection of salmonella by electrical measurements. Journal of Hygiene
Cambridge 94, 245262.
Eden, R. and Eden, G. (1984) Impedance Microbiology. Letchworth,
UK: Research Studies Press.
Entis, P. and Boleszczuk, P. (1991) Rapid detection of Salmonella
in foods using EF-18 agar in conjunction with the Hydrophobic
Grid Membrane Filter. Journal of Food Protection 54, 930934.
Entis, P., Brodsky, M.H., Sharpe, A.N. and Jarvis, G.A. (1982)
Rapid detection of Salmonella spp. in food by use of the ISOGRID Hydrophobic Grid Membrane Filter. Applied and Environmental Microbiology 43, 261268.

Fricker, C.R. (1987) A review: The isolation of salmonellas and

campylobacters. Journal of Applied Bacteriology 63, 99116.
Hawa, S.G., Morisson, G.J. and Fleet, G.H. (1984) Method to
rapidly enumerate Salmonella on chicken carcasses. Journal of
Food Protection 47, 932936.
Kauffmann, F. (1935) Weitere Erfahrungen mit dem kombinierten
Anreicherungsverfahren fur Salmonella bazillen. Zeitschrift fur
Hygiene und Infektionskrankheit 117, 2632.
Kristensen, M., Lester, V. and Jurgens, A. (1925) Use of trypsinised
casein, brom-thymol blue, brom-cresol purple, and brilliant green
for bacteriological nutrient media. British Journal of Experimental
Pathology 6, 291299.
Lampi, R.A., Mikelson, D.A., Rowley, D.B., Previte, J.J. and Wells,
R.E. (1974) Radiometry and microcalorimetry. Food Technology
28, 5258.
Leifson, E. (1936) New selenite enrichment media for the isolation
of typhoid and paratyphoid (Salmonella) bacilli. American Journal
of Hygiene 24, 423432.
Lin, C.K. and Tsen, H.Y. (1995) Development and evaluation of
two novel oligonucleotide probes based on 16S rRNA sequence
for the identification of Salmonella in foods. Journal of Applied
Bacteriology 78, 507520.
Miller, R.G. and Tate, C.R. (1991) Xylose-Lysine-Tergitol 4: An
improved selective agar medium for the isolation of salmonella.
Poultry Science 70, 24292432.
North, W.R. and Bartram, M.T. (1953) The efficiency of selenite
broth of different compositions in the isolation of Salmonella.
Applied Microbiology 1, 130134.
Ogden, I.D. (1988) A conductance medium to distinguish between
Salmonella and Citrobacter spp. International Journal of Food
Microbiology 7, 287297.
Ogden, I.D. and Cann, D.C. (1987) A modified conductance medium for the detection of Salmonella spp. Journal of Applied Bacteriology 63, 459464.
Owens, J.D., Thomas, D.S., Thompson, P.S. and Timmerman,
J.W. (1989) Indirect conductimetry: a novel approach to the
conductimetric enumeration of microbial populations. Letters in
Applied Microbiology 9, 245249.
Pless, P., Futschik, K. and Schopf, E. (1994) Rapid detection of
Salmonellae by means of a new impedance-splitting method.
Journal of Food Protection 57, 369376.
Restaino, L., Grauman, G.S., McCall, W.A. and Hill, W.M. (1977)
Effects of varying concentrations of novobiocin incorporated into
two Salmonella plating media on the recovery of four Enterobacteriaceae. Applied and Environmental Microbiology 33, 585
589.
Saco, M.M. (1993) Impedimetric detection of Salmonella in poultry.
Prevention and control of potentially pathogenic micro-organisms
in poultry and poultry meat processing. 13. Consequences of the
zoonosis order: monitoring, methodology and data registration.
FLAIR NO 6/COST NO 906, 3337.
Silley, P. and Forsythe, S. (1996) Impedance microbiology-a rapid
change for microbiologists. Journal of Applied Bacteriology 80,
233243.
van Schothorst, M., Renaud, A. and van Beek, C. (1987) Salmonella
isolation using RVS broth and MLCB agar. Food Microbiology 4,
1118.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 399403