International Journal of Food Microbiology, 1 (1984) 127-134 127

Elsevier

JFM 00012

Estimation of the bacteriological quality of fish by

automated conductance measurements

D.M. Gibson, I.D. Ogden and G. Hobbs

Ministry of Agriculture, Fisheries and Food, Torrv Research Station, 135 Abbey Road, Aberdeen A B9 8 DG,
Scotland, U.K.
(Received 14 February 1984; accepted 1 April 1984)

The rapid automated conductance assay system has been used in experiments concerned with
estimating the bacteriological quality of fish in terms of total and selective counts. Results of the former
method were obtained in one-fifth of the time taken by conventional methods and the correlations
between the methods were good. Selective counts of Escherichia colt, coliforms, and vibrios were
completed in one-third of the normal time. The instruments were easy to use and the results easy to
interpret. The method holds out good promise for automating such routine fish bacteriology.

Key words: Fish bacteriology; Conductance assays; Rapid methods

Introduction

As bacteria grow in culture media metabolic p r o d u c t s accumulate. M a n y of these

are ionic a n d cause a change in the electrical c o n d u c t a n c e of the m e d i u m . Richards
et al. (1978) described i n s t r u m e n t s for m e a s u r i n g a n d displaying the changes in
c o n d u c t a n c e of bacterial cultures a n d showed that c o n d u c t a n c e changes were a good
i n d i c a t i o n a n d measure of bacterial growth. They showed that a plot of the change
of c o n d u c t a n c e (GB) with time had the same shape as a c o n v e n t i o n a l bacterial
growth curve a n d that G B was related to the change in bacterial n u m b e r s , T h u s
m e a s u r e m e n t of G B can be used to estimate growth rates. They also showed that the
time taken for a modest but detectable G B was inversely p r o p o r t i o n a l to the
logarithm of the n u m b e r of bacteria in the sample. This p e r m i t t e d total c o u n t s to be
d e t e r m i n e d by c o n d u c t a n c e m e a s u r e m e n t s .
The r o u t i n e bacteriological e x a m i n a t i o n of fish a n d fishery p r o d u c t s usually
includes an e s t i m a t i o n of the total count, the n u m b e r s of coliform bacteria a n d
E s c h e r i c h i a coli and, recently, a n e s t i m a t i o n of the n u m b e r of Vibrio p a r a h a e m o l y t i -
cus. This paper is c o n c e r n e d with the a d a p t a t i o n of some of these m e t h o d s for use
with an a u t o m a t e d c o n d u c t a n c e a p p a r a t u s a n d reports on the correlations with
results o b t a i n e d using c o n v e n t i o n a l microbiological techniques.

0168-1605/84/$03.00 1984 Elsevier Science Publishers B.V.

128

Materials and methods

Media

Nutrient broth (Oxoid CM 1), seawater broth (SWB) containing (g/l) beef extract
10, peptone 10, 750 ml sea water and 250 ml water, and the trimethylamine oxide
(TMAO) medium of Wood and Baird (1943) were used for total counts. MacConkey
broth (Oxoid) and modified Reasoner's medium (Silverman and Munoz. 1979)
containing (g/l) proteose peptone, 5; yeast extract, 3; lactose, 10; NaCI, 7.5: sodium
lauryl sulphate, 0.2; sodium deoxycholate, 0.1, pH 6.5; were used for counts of
coliform bacteria and E. cog respectively. Thiosulphate citrate bile salts broth
(TCBS, CM 333 recipe without agar, Oxoid Manual) was used for counts of vibrios.
When required the above media were solidified with agar (15 g/l).

Fish e x t r a c t s

Standard methods were used for the bacteriological examination of cod and
haddock obtained from commercial sources and stored in ice for various times.
Samples (usually 100 g) were homogenised in chilled 0.1% peptone-phosphate buffer
pH 7.0-7.2 (400 ml). Standard plate counts were determined on dilutions of the
homogenate using a modified Miles and Misra technique (Bousfield et al., 1973), the
plates were incubated at 2 0 ° C for 5 days. The selective counts were done similarly
on the appropriate medium with incubation at 3 7 ° C for 24 h.

Conductance measurements

The apparatus described by Richards et al. (1978) was used in the initial part of
this work. This consisted of modified glass test tubes bearing platinum electrodes
which were placed in a water bath controlled precisely to 20 °C for standard plate
counts or 3 7 ° C for selective counts and connected to the measuring and control
system. The data were presented in paper tape form to a computer, the results then
being obtainable in numerical or graphical form. In the main part of the study, a
commercial instrument ('Malthus') produced by Malthus Instruments Ltd., William
Clowes Street, Burslem, Stoke-on-Trent was used. It has been described by Baynes et
al. (1983) and Porter et al. (1983). Media and samples (up to 128 simultaneously)
were held in 12-ml glass ampoules, containing reusable platinum electrodes printed
on a ceramic base, which were placed in a water bath and connected to the control
box and a microcomputer. The data were available continuously in numerical form
and off line in graphical form. The microprocessor was programmed to display on a
video display unit when a given preset GB occurred with any particular sample. In
this case the computer display indicated when three consecutive changes of > 2/~S
had occurred. Readings were taken every 25 min, for 24 h.
 129

Results

A plot of Ga with time of a typical sample assayed in triplicate is shown in Fig. 1.

The initial G a occurs when the count reaches 106/g.
Richards et al. (1978) showed that the time taken for an obvious G B (8.14 #S) is
inversely proportional to the logarithm of the number of bacteria inoculated. The
conductance changes can be measured precisely (1 part in 105) (Richards et al.,
1978; Jason, 1983) but the accuracy of the calibration is also related to the accuracy
of the counts procedure used for calibration. However, this calibration enables total
counts to be determined in a relatively short time using conductance measurements.

T o t a l counts

The relationship between the detection times and the plate counts for fish extracts
tested in nutrient broth, seawater broth and T M A O broth is shown in Fig. 2 and the
correlation coefficients together with their standard deviations are shown in Table I.
The T M A O medium gives the best correlation with counts, the least scatter about
the line of correlation and the shortest detection time. The detection times were
much shorter than the conventional incubation time of 5 days.

Selective counts

It was known from experience that the numbers of coliforms and vibrios would
be low or zero in the commercial fish used. Accordingly, the fish samples were

120 -

80

GB

40

-20 I I I
10 20 3O
Time (h)
Fig. 1. Typical plots of conductance changes with time for a fish sample assayed in triplicate in nutrient
broth at 20 o C.
 130

8-

6--
o
o
4- °° o
0
0

2-

I I I I I
8 16 24 32 4O
Detection time (h)

7- b

4-
9
c~
0
3-- ~o Io
2--

1--

I I I I I I 1 °l°n~l"
4 12 20 28 36
Detection time (h)
7-
c

6-

5-
O
o 4-
O)
o
-- 3 -

2-

I i i , I I ?o%
4 12 20 2B 36
Detection time (In)

Fig. 2. Plots of detection times (h) and inoculum number ( N ) for assays in: (a) nutrient broth, (b)
seawater broth, (c) TMAO medium.
 131

TABLE 1

C o r r e l a t i o n coefficient ( r ) b e t w e e n c o n d u c t a n c e d e t e c t i o n times f o u n d in 3 m e d i a a n d c o n v e n t i o n a l c o u n t
with residual s t a n d a r d d e v i a t i o n (S.D.) of detection time. a n d regression e q u a t i o n by analysis of variance.

Medium Residual Regression

S.D. equation ~

TMAO - 0.89 0.45 .v = 5.6 - 0.16 x

Seawater broth - 0.85 0.55 y = 6.8 - 0.14 x
Nutrient broth - 0.85 0.65 v = 7.6 - 0.20 x

v is total viable count, x is d e t e c t i o n time.

spiked with mixtures of 10 recent isolates from fish grown in nutrient broth for 48 h
at 20°C, mixed, diluted, and 1 ml added to each of the three media. A typical
relationship between detection times and numbers for vibrios is shown in Fig. 3 and
the results are summarised in Table II. In the medium most appropriate for the
organisms, i.e. MacConkey's and modified Reasoner's for E. coil and coliforms and
TCBS broth for vibrios, there was a fast response and a good relationship between
numbers and detection times. Long detection times resulted from large numbers of
other organisms and were therefore not acceptable as a positive result. With the
tolerance levels as shown in the table such confusion did not occur. The relationships
appear to be independent of the total count of the sample (10°-105/g).

Discussion

The instruments for conductance measurements described by Richards et al.

(1978) were designed to measure growth rate, but because of the high correlation

I I 1 I I
2 4 6 8 10
Detection time (h)

Fig. 3. R e l a t i o n s h i p b e t w e e n d e t e c t i o n times a n d i n o c u l u m ( N o) for s a m p l e s spiked with vibrios.

132

T A B L E II

D e t e c t i o n times ( 1 0 5 - 1 0 ° / a s s a y ) a n d relationships with n u m b e r s (a, b or c) for s a m p l e s spiked with

m i x t u r e s of recent isolates of E. coil, n o n E. coil coliforms a n d vibrios in selective m e d i a

D e t e c t i o n time (h)

Modified TCBS MacConkey

Reasoner

E. colt 2-6.5 8.5-20 2-6.5

a c b

Coliforms 5-10 7-14 6-12

b c c

Vibrios 6-10 2-7 6-10

b a b

a, r > 0.95; b, 0.85 < r < 0.9; c, r < 0.85.

between GB and change in bacterial numbers they can be used to measure initial
numbers in a sample. The detection routine in effect measures the time taken for the
population of bacteria to adapt to the medium and to grow. Such results are
available long before those of conventional plate count methods in which the colony
forming unit (cfu) has to replicate until the colony is visible. Experience in our own
and other laboratories indicates that the correlation coefficient between detection
times and counts rarely exceeds 0.9. One reason for this is that each cell in the
inoculum whether present in the free state or as part of a clump contributes to the
overall change, but both single cells and clumps gi+e rise to single cfus in the plate
count. Also, injured cells recover better in liquid than on solid media. The conduc-
tance measurements are made with a precision of 1 part in 105 but it is well known
that the error in routine bacteriological counts is high (see Hall, 1982; Postgate,
1969). It is difficult, as Richards et al. (1978) pointed out, to calibrate a precise
method against an inaccurate method however well established. Thus, higher correla-
tion coefficients would not necessarily be expected.
It is assumed that conductance assays depend upon the increased number of
charged ions resulting from the metabolism of the medium by the microbes in the
inoculum. Accordingly, the choice of culture media even for total counts can
influence the results. It has been shown recently (Easter et al., 1983) that some fish
spoilage bacteria can use T M A O as an electron acceptor during microaerophilic
growth. As T M A O is a neutral uncharged molecule, and the product of its reduction
trimethylamine is basic and highly charged, it is an excellent choice of substrate in a
medium for fish spoilage as it is crucial to the metabolism of the culture and yields a
good conductance change. The microaerobic growth conditions in a conductance
assay system may be more relevant to the changes in quality of fish as they are more
akin to the redox conditions existing during spoilage. The results may therefore
correlate more highly with other spoilage parameters such as taste panel score and
chemical indices than the routine conventional bacteriological count. It is clear,
however, from the data presented that the results for total counts are available far
 133

sooner, in less t h a n 1 day c o m p a r e d with 5 days for the r o u t i n e bacteriological

analysis. The results from high count, ie u n a c c e p t a b l e quality samples are available
in a few hours. With c o n v e n t i o n a l microbiological analysis there is no difference in
i n c u b a t i o n time needed for samples with a very high or a very low count.
The results presented for the selective c o u n t s are extremely promising, consider-
ing that no work had been d o n e on devising the most a p p r o p r i a t e selective media for
the various organisms considered. All i n c u b a t i o n s were d o n e at 37 ° C because at the
time no a d d i t i o n a l i n c u b a t o r s were available, e.g. at 44.5 ° C for E. coll. W h e n the
media are used in plates it is possible to use the a p p e a r a n c e of the colonies as an aid
to their identification. With liquid culture this is not possible: however, the detection
times o b t a i n e d (Table II) a n d change of colour of m e d i u m do allow for a partial
identification of the types growing. In general the o r g a n i s m s for which the m e d i u m
is considered to be most favourable gave the shortest detection time even for very
low i n o c u l a t i o n n u m b e r s a n d the quality of the relationship between detection times
a n d n u m b e r s was good. Long detection times, ie more than 8 - 1 0 h, indicated the
growth of other organisms since the media used were only selective a n d not
differential or diagnostic.
Both sets of a p p a r a t u s were reliable a n d easy to use. With the original a p p a r a t u s
of Richards et al., it was necessary to wait until the end of the e x p e r i m e n t before
r u n n i n g the tape through the c o m p u t e r to find the detection time. The ' M a l t h u s '
i n s t r u m e n t has the a d v a n t a g e that the status of each sample is displayed c o n t i n u -
ously on the V D U of the c o m p u t e r a n d u p d a t e d every 25 rain. The electrode
assemblies were easy to handle, robust a n d were used repeatedly without any change
in their characteristics.
In conclusion, the a u t o m a t e d c o n d u c t a n c e e q u i p m e n t has been used successfully
to predict the bacteriological quality of fish in less t h a n one-fifth the time taken by
c o n v e n t i o n a l m e t h o d s so that a p p r o p r i a t e action can be taken before the p r o d u c t s
are offered for sale. N o r m a l l y the fish would have been sold a n d c o n s u m e d before
the bacteriological data are available. The i n s t r u m e n t s were easy to use a n d there
was less l a b o u r needed for sample h a n d l i n g as n o serial d i l u t i o n of the h o m o g e n a t e s
was necessary.

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