bztenmtional Journal of FocM Microbiology.

16 (1992) 25-39 25
© 1992 Elsevier Science Publishers B.V. All rights reserved 0168-16(}5/92/$05.1)0

FOOD tl0497

Review

Evaluation of the bacteriological quality of seafood

Lone Gram
Tecimological Laboratory. Danish MinixtO' of l'~.~heries. Lyngb), Denmark
(Received 16 March 1992; accepted 3 April 1992)

Bacteria largely determine the quality of fresh and lightly preserved fish products. This paper surveys
traditional and rapid methods for estimation of bacterial levels in scaffold. The u.~ of traditional agar
techniques is discussed with reference to development t)f substrates and procedures suited for fish and
fish products. This includes estimation of the bacteria specifically involved in the spoilage proce~,~.
During the last decade, several microbiological rapid methods or principles (DEFT. microcolonies,
Limulns lysate, ATP, conductance, microcalorimetry, reduction of trimethylamine oxide (TMAO)) have
been suggested for estimating the bacteriological qualily of seah~.ls. A brief survey of tbe~ methods
and the results obtained is given. Preliminary results on development of poly- and monoclonal
antibodies against Shewmu,lla putrefaciens are mentioned. Future research may involve the develop-
ment of DNA-probes against genes coding fl+rspecific spoilage reactions.

Key words: Bacteria: Seafood: Spoilage process: Microbiological methods

Introduction

T h e a m o u n t of bacteria in foods is interesting for several reasons. It serves as a

general indicator of hygiene; bacteria are important spoilers of m a n y products, and
many bacteria and their metabolites constitute a health risk for the consumer. For
these reasons n u m e r o u s methods have been developed for measuring levels of
bacteria in food products. T h e present p a p e r deals with such methods as applied
to seafood, and focuses on two bacteriological parameters: the bacterial population
as such and the specific spoilage bacteria.
Determination of bacterial numbers in food is widely used as indicator of
hygiene and most agar media and rapid methods focus on this determination. As
the spoilage of iced fish is caused by specific spoilage bacteria (Liston, 1980; H o b b s
and Hodgkiss, 1982), several methods have been developed for specific determina-
tion of such bacteria in fresh fish. T h e most important fish s~oilage bacteria are
characterized by the ability to produce H 2S and reduce T M A O , and these abilities
have been used in the development of indicative agar media and specific chemical
and physical assays (Levin, 1968; Gibson et al., 1984; Huss ¢t al., 1987; G r a m et al.,

Corresponden('e address: L. Gram, Technological Laboratory, Danish Ministry of Fisheries, Technical

University Bldg. 221, DK-2800, Lyngby, Denmark.
26

TABLE I
Methods fi)r determination of the content of bacteria in seafood

Method Temp. Incubation Sensitivity Reference

°C CFU/g
Plate count agar 20-25 3-5 days 10
Iron Agar 20-25 3-5 days 10 Chat ct al. 1968; Lcvin, 1968
"Rcdigcl' 211-25 3-5 days 10 Slabyj and Bolduc. 1987
'PetrifilmT M SM" 20-25 3-5 days 10 Slabyj and Bolduc, 1987:
Abgrall and Cleret, 19~)
Microcolony-DEFY 20-30 3-4 h 104-10s Rodriquesand Kroll, 1988
DEFT - 30 rain 104- l0 s Pettipher and Rodriques. 1982:
Abgrall and Bourgois, 1989
ATP - Ih 104-10s Sharpe et al.. 1971);
Ward et al. 1986
Limulus lysate test - 2-3 h 103-104 Sullivanet al.. 1983
Microcalorimetry 20-25 8-40 h I11 Gram and Scgaard. 1985
Dye reduction 37 i-4 h 10" Reddy et al., 1990
Conductance 211-25 4-36 h 10 Gibson el al., 1984:
Gram, 1985; Ogden, 1986:
Gibgm and Ogden, 1987
Capacitance 20-25 4-36 h 10 van Sprcekens and
Stckelenburg. 1986

1987). it has been reported that the expected shelf life of chilled cod fish can be
predicted by the number of specific spoilage bacteria (R~,;'tt-Jorgensen et al., 1988)
and the development of rapid methods for detection of these bacteria is therefore
of particular interest. For a thorough description of the principles behind the
methods used, the reader is referred to Adams and Hope (1989).

Determination of bacterial contents

Unspecific measurement of the content of bacteria is the most common method

for determination of the bacteriological quality of seafoods. Although this figure is
seldom a good indicator of the sensoric quality or expected shelf life of the product
(Huss et al., 1974), it is taken as a general indicator of the hygienic status of the
product. A summary of different methods used for fish and fish products is given
in Table 1.

Plate counts
Estimation of the number of culturable bacteria relies on the method first
described by Robert Koch: allowing each cell to multiply to form a visible colony.
Several studies have compared different types of agar, diluents, incubation temper-
atures etc. with the purpose of finding the procedure recovering the highest (most
true) number of microorganisms from the fish sample. Common plate count agars
(PCA) are still the substrates most widely used for determination of total counts.
However, examination at our laboratory of several types of seafood have shown
that a more rich agar (Iron Agar, Lyngby, Oxoid) gives significantly higher counts
than PCA (Gram, 1990). Whilst all studies agree that incubation temperature at
and above 30°C are inappropriate when examining seafood products held at chill
temperatures, results are contradictory on the use of surface and pour plating
(Fujii, 1985; Thampuran and lyer, 1985). At our laboratory we routinely use pour
plating and a 3-4 day incubation at 25°C.
As described at the previous conference held in Alaska in 1986, several
attempts have been made to ease the procedures for determination of the content
of bacteria (Fung et al., 1987). Redigel (RCR Scientific) is a substrate pre-treated
petri dish to which the sample and a gelling agent is added. PetrifilmTM SM (3M
Company) is a film precoated with dry media, a cold water soluble gel and the dye
triphenyl tetrazolium chloride which aids the counting. The film serves as a dish
when 1 ml of sample is added. These techniques have been compared to conven-
tional plate counting (Fung et al., 1987; Slabyj and Bolduc, 1987). Slabyj and
Bolduc (1987) concluded that counts on Redigel (20°C for 3-5 days) equalled PCA
counts (20°C for 3-4 days), whereas Petrifilm only recovered 55% of the PCA
count. Contrary to this, Petrifilm was found to yield significantly higher counts
than PCA in the examination of 130 samples of fish (Abgrall and Cleret, 1990).
This difference was attributed to the bacterium Photobacterium phosphoreum
which did not grow on the conventional media.
The total costs per analysis (including materials and labour cost) of Redigel,
Petrifilm and normal petri dish procedure have been calculated to $US 8.22, 8.22
and 13.62, respectively (Chain and Fung, 1991). The main advantage of Redigei
and Petrifilm compared to conventional plate counts in addition to the costs, is the
ease of handling. However, all agar-based methods share a common drawback in
the lengthy incubation required.

Direct counts (microscopy)

Microscopical examination of foods is a rapid way of estimating bacterial levels.
By phase contrast microscopy the level of bacteria in a sample can be determined
within one log-unit. One cell per field of vision equals approximately 5 x 105
C F U / m l at 1000 × magnification. Whilst microscopical methods are very rapid,
this low sensitivity must be considered their major drawback.
The staining of cells with acridine orange and detection by fluorescence mi-
croscopy has earned widespread acceptance as the direct epifluorescence filter
technique (DEFT). Pettipher and Rodriques (1982) found high correlation coeffi-
cients (approx. 0.9) when comparing DEFT counts and plate counts (104-10 u~
C F U / g ) of samples of fresh and frozen fish. Results of the DEFT counting were
obtained within 30 min. Acridine orange binds to DNA and RNA and produces an
orange-red or green fluorescence. Early work suggested that the dye could be used
to distinguish between viable and dead cells but the results on this point are
contradictory (Pettipher, 1989).
 Culturable cells can be counted using the DEFT procedure after a short period
of incubation on agar (Rodriques and Kroll, 1988). The sample is filtered on a
membrane which is then incubated for 3-4 h on agar. The membrane filter is
remounted, stained with acridine orange by the normal DEFT procedure and the
microcolonies counted. By this procedure only viable cells are counted. Normally a
rough differentiation of the microflora is possible via DEFT (e.g. distinguishing
between yeasts, rods and cocci). For the microcolony technique, the use of
selective media can allow for a distinction between different bacterial groups.
Recently the DEFT method was used to examine samples of raw fish, ham and
minced meat samples (Abgrall and Bourgois, 1989). Fish samples were more
difficult to examine than meat samples due to more food debris remaining as
interfering background fluorescence.

Indirect estimations
Bacterial numbers have been estimated in foods by measuring the amount of
bacterial adenosine triphosphate (ATP) (Sharpe et al., 1970). Bacterial ATP is
extracted and measured by the luciferin/luciferase bioluminescent reaction. The
method is very rapid and results are obtained within 1 h. However, the separation
of bacterial and somatic ATP can be difficult. Correlation coefficients of 0.97-0.99
were found when comparing bacterial ATP levels with plate counts for four fish
species. The ratio of bacterial ATP to colony count remained constant during
storage trials, and did not differ significantly between fish species (Ward et al.,
1986). As the level of ATP per cell varies depending on nutritional state, stress etc.
(Stannard, 1989) it must be recommended that a standard curve be produced for
each separate product.
Barak and Ulitzur (1980) used luminescence as indicator of the bacterial load of
fish samples. A sample was withdrawn and the development in luminescence
followed at 25°C. Good correlation between cell numbers and luminescence was
found when testing pure cultures of luminescent bacteria isolated from fish.
However, during chill storage of fish no correlation was found between change in
luminescence and traditional spoilage parameters, although luminescent bacteria
in one study have been linked with fish spoilage (van Spreekens, 1974).
The Limuh~s amoebocyte lysate (LAL) test relies on the reaction between
Gram-negative endotoxin (lipopolysaccharide) and the serum of the horseshoe
crab. Since the majority of bacteria on fish from temperate waters are Gram-nega-
tive, this test has been used for estimation of bacterial counts (Sullivan et al.,
1983). However, the increase in endotoxin during storage of flounder fillets only
followed the increase in bacterial numbers until a level of 107 C F U / g was reached.
Thereafter the level remained constant even though the bacterial count continued
to increase to 10'~ C F U / g . This study used the formation of a gel between the
sample and the LAL-rcagent, and this procedure is quite difficult to interpretc.
Measurements relying on rocket electrophoresis or chromogenic substrate reac-
tions have greatly standardized the LAL test (Jay, 1989), but neither have been
applied to seafoods (Sullivan et al., 1983). Enumeration of Gram-negatives has
been done by a variation of DEFT, where acridine orange is used as counterstain
in the normal Gram-stain. This procedure has been successfully applied to milk
samples (Rodriques and Kroll, 1985).
Several methods for rapid estimation of bacterial numbers are based on the
withdrawal of a sample, incubation at high temperature (20-25°C) and detection of
a given signal. The time taken to reach a significant change, the Detection Time, is
inversely related to the initial number of bacteria, i.e. early reaction indicate a high
bacterial count in the sample.
This has been used for several instruments relying on measurement of ab-
sorbance, e.g. Cobas (Roche Diagnostica Denmark) (Schultz e t a l . , 1988). The
homogenized sample is inoculated in a small well and the absorbance measured
continuously. A certain degree of selection can be obtained by inoculating the
sample in standard selective media. Basically, this turbidiometric procedure records
the time taken for the bacterial population to reach the detection limit of a
spectrophotomcter (106--10 ~ bacteria/ml). There is no reports in the literature
using this method on fish products, but the analysis has been successfully employed
to meat and meat products where 10-~-10'~ C F U / g were detected in 14-4 h at
21°C (Schultz et al., 1988).
The generation of heat by bacteria can be measured by a very sensitive
calorimeter. The instrument, e.g. LKB Bioactivity Monitor (LKB Produkter, Swe-
den), measures the heat transferred per second from a sample to the surrounding
metal chamber (Gram and S~)gaard, 1985). The detection limit is between i0 T and
10a C F U / m l . The sample is incubated and the time taken to reach a significant,
detectable heat production registered. Bacterial levels from 104 to l0 ~) C F U / g in
chilled cod fillets have been detected in 30-11 h at 21°C (Gram and Sogaard,
1985 ).
Dye reduction tests have, with equivocal results, been used to determine
contents of bacteria in several types of fish. Recently, Reddy et al. (1990) described
the use of rcsazurin strips for determination of total counts in different Mangalore
fish species. Strips were dipped in resazurin solution and dried. When examining
fish samples, the strips were dipped in fish homogenates and incubated at 37°C.
The time taken to complete reduction was compared to plate counts by regression
analysis and correlation coefficients varied from 0.85 to 0.94. Counts between 106
and I(P" C F U / g corresponded to reduction times of 4 - l h. This high temperature
will, however, not allow for growth of psychrotrophic bacteria and the reduction
time is therefore unlikely to relate to the relevant psychrotrophic microflora.
A rationale similar to the turbidiometric and microcalorimetric methods has
been used in the development of methods based on electrical measurements.
Gibson et al. (1984) first described the conductometric estimation of bacterial
counts in fish samples by the Malthus Growth Analyser '~ (Radiometer Denmark
A/S). Fish samples were inoculated in different broths and Detection Times (DT)
at 20°C ranged from 36 to 8 h with bacterial levels increasing from 10 to l0 s
C F U / g . Similar results have been obtained in other studies at the Terry Research
Station in Aberdeen, Scotland (Ogden, 1986, Gibson and Ogden, 1987) and in our
laboratory (Gram, 1985; Ravn-Je)rgensen et al., 1988).
The Bactometer ~" instrument (BioMeroux UK Ltd.), which can measure changes
in impedance, conductance and capacitance was used to estimate bacterial num-
bers in fresh cod fish (van Spreekens and Stekelenburg, 1986). Capacitance DT at
20°C decreased from 11 to 1 h as total counts increased from l0 s to 108 CFU/g.
The capacitance signal gave a better curve than the conductance signal, in
contrast, conductance curves when measured in the Malthus instrument are very
distinct and DTs easily determined (Gibson and Hobbs, 1987). The Malthus
instrument has been optimized for measuring conductance and for this reason use
a frequency of 10 kHz (Gibson and Hobbs, 1987). The Bactometer instrument was
originally designed for measuring impedance only and uses a lower frequency of
1.54 kHz (Firstenberg-Eden and Eden, 1984) which is less optimal for conducto-
metric studies (Gibson and Hobbs, 1987).
In experiments with fresh, chilled fish (cod, haddock, trout) comparisons of
plate counts and detection times by regression analyses have resulted in slightly
different regression lines, as discussed by Gibson and Ogden (1987). Slopes of the
regression lines have varied from - 0 . 2 to -0.45 (Gibson et al., 1984; Gram, 1985;
Ogden, 1986; van Spreekens and Stekelenburg, 1986; Gibson and Ogden, 1987;
Ravn-Jcrgensen et al., 1988). However, detection times have been determined at
two different temperatures (20 and 25°C), and taking this difference into account a
very good agreement is seen between different experiments. Any differences could
also be explained by different initial microflora on the fish varying as a result of
season or handling.
The generation time of the bacteria causing the electrical changes can be
calculated from the measurements. It appears that in average, the decrease in
instrument temperature from 25 to 20°C causes an increase in generation times of
the bacteria, and since a more rapid result is obtained at 25°C, this temperature
would be the obvious choice. However, it is not known whether the same bacterial
species are selected at the two temperatures.
It is assumed in all of the methods based on accelerated proliferation that the
DT registered in a non-selective medium is representing growth of the total
microflora. Few studies have investigated the composition of the bacterial ~ora
when the detection limit is reached. Only a fraction of the bacterial population is
selected by the chosen conditions of temperature and substrate. Therefore the
population with the shortest generation time at the instrument temperature will be
responsible for the change of the parameter measured (Bfilte and Reuter, 1984;
Gram, 1985). As the raw material and the composition of the microflora varies,
this could explain some of the differences observed.

Determinations of spoilage bacteria

The total number of bacteria on fish rarely indicates sensoric quality or

expected storage characteristics (Huss et al., 1974). However, it is well recognized
that certain Gram-negative bacteria arc the main cause of sp' "'-ge, i.e. their
metabolism generates the unpleasant off odours and off flavour,~ sociated with
spoilage (Liston, 1980; Hobbs and Hodgkiss, 1982). Many studies have shown that
Shewanella (formerly Alteromonas formerly Pseudomonas) putrefaciens is the most
important fish spoilage bacteria of marine fish stored at 0°C ( C h a i e t al., 1968;
Hobbs and Hodgkiss, 1982; Gram et al., 1987). Thus a model has been constructed
correlating remaining shelf life of chilled cod with ',he number of Shewanella
putrefaciens (Ravn-Jorgensen et al., 1988). Several attempts have been made to
enumerate spoilage organisms (in particular Shewanella putrefaciens) specifically.
Most methods are based on the ability of this bacterium to produce H2S and
reduce trimethylamine oxide (TMAO) (Levin, 1968; Gibson et al., 1984; Gram et
al., 1987). In future, it may also be possible to detect the organism directly using
specific antibodies.

Plate counts
Studies of the biochemical changes occurring during chill storage of fish have
shown that volatile sulfides are major components of spoilage off-odours (Herbert
et al., 1975). Levin (1968) showed that the ability to produce H2S was a prominent
characteristic of Pseudomonas putrefaciens isolated as spoilage organism from
chilled white fish. This has recently been confirmed by an exhaustive study on the
characteristics of this organism now named Shewanella putrefaciens (Stenstrem and
Molin, 1990).
The Peptone Iron Agar (Difco) is a peptone rich substrate containing ferric
citrate. The substrate is used for detection of HeS-producing bacteria such as
Shewanella putrefaciens, which can be seen as black colonies due to precipitation of
FeS (Levin, 1968). Later both Ogden (1986) and Gram et al. (1987) detected
H2S-producing bacteria on ferric citrate containing agars by FeS-precipitation. In
both studies thiosulfate and L-cysteine were added to the media to facilitate
H2S-production. These compounds have been incorporated in Iron Agar (Gram et
ai., 1987) which is now available as a pre-mixed substrate from Oxoid (Iron Agar,
Lyngby). Several studies have shown that during iced storage of fish, the bacteria
forming black colonies on Iron Agar are all characterized as specific spoilage
bacteria since all reduce trimethylamine oxide (TMAO) and all produce spoilage
off-odours in a sterile fish broth, while on the contrary the bacteria isolated from
white colonies do not contribute to the spoilage (Gram et al., 1987; Ravn-JOrgen-
sen and Huss, 1989).
While ShewaneUa putrefaciens is the most important bacterium during spoilage
of iced marine fish, other spoilage bacteria belonging to I/ibrionaceae are impor-
tant in spoilage of fish stored at high temperatures (Gorzcyka et al., 1985; Gram et
al., 1987, 1991). Spoilage potential of the isolated bacteria has in all these studies
been determined by testing for production of off-odours. In an Australian study,
the ability of bacteria to produce spoilage off-odours did not coincide with ability
to produce H2S (Gorzcyka et al., 1985) whereas the specific spoilage bacteria of
cod and Nile perch spoiled at high temperatures all produced H2S and all reduced
TMAO (Gram et ai., 1987, 1991). To detect these organisms, it is essential that the
substrate contains organic sulfur (e.g. L-cysteine) since several strains do not
produce H2S from inorganic sulfur (Gram et al., 1987).
 Other factors than the sulfur source are important for the formation of a black
precipitate. Already Levin (1968) noted the importance of redox-potential, thus
seeing a more pronounced formation of FeS if a cover layer was added to the
substrate or pure cultures were tested in tubes with Peptone Iron Agar. This may
be due to oxidation of FeS to yellow Fe(OH).~ under aerobic conditions (Gram et
al., 1987). Also Shewanella putrefaciens can use ferric ions as electron acceptors in
an anaerobic respiration (Semple and Westlake, 1~87) in which amino acids (like
cysteine) serve as substrates (Ringo et al., 1984). The latter reactions will facilitate
the formation of FeS under an-oxic conditions where Fe 3+ is reduced to Fe 2+ as
H2S is produced.
Incubation temperature, length of incubation and pH of the substrate will also
influence the formation and stability of black precipitate. If plates are incubated at
37°C a n d / o r incubated for more than 7 days, FeS disappears rapidly from some
colonies (Gram, 1990). As indicated above, this can be due to oxidation of FeS. As
FeS is soluble in acid, pH of the medium influences the strength of precipitate.
When comparing pH of 6.8 and 7.4 a more pronounced precipitate was noted at
the higher pH (Gram et ai., 1987).
The ability of Shewanella putrefaciens to form a r e d / b r o w n pigment has been
used in the gelatine-iron medium of Long and Hammer (1941) and in a modified
version (van Spreekens, 1974). All bacteria isolated from r e d / b r o w n pigmented
colonies were identified as P~eudomonas or Alteromonas putrefaciens, s. She-
wanella putrefaciens.
Several studies have related fish spoilage to proteolysis and have therefore
developed agars for specific counting of proteolytic bacteria. Chai et al. (1968)
used a soft gelatin agar whereas others have used a medium to which a heat
denatured fish protein was added (Chandrasekaran et al., 1985). Proteolytic
colonies appeared with a clearing zone (Chandrasekaran et al., 1985). However,
bacteria may attack a denatured protein more easily than a native fish protein. The
authors did not find an unambiguous correlation between formation of clearing
zones and production of off-odours by the bacteria (Chandrasekaran et al., 1985).
Furthermore, some studies have concluded that proteolysis is of minor importance
in fish spoilage as compared to breakdown of easily digestible amino acids and
peptides (Lerke et al., 1967). Table 11 gives a summary of agars used for estimation
of spoilage bacteria.

Direct counting of spoilage bacteria

Several specific organisms (Salmonella, Listeria) and many microbial toxins are
today detected in foods by immunological methods (Notermans and Wernars,
1991). As the spoilage of chilled, lean fish is related to a specific organism, the
possibility of detection by immunochemistry is evident. Many immune-based meth-
ods have relied on detection of the organism by fluorescence microscopy. However,
the detection limit of the microscope will be the restricting factor. For use in e.g.
predictive models, a detection of 10-103 C F U / g is necessary (Ravn-Jorgensen et
ai., 1988).
TABLE II
Agars for detection of spoilage bacteria on fish

Principle Medium Temp. Incubation Reference

°C
Pigment Long& tlammer 21 2 days Longand Hammer. 1941
Pigment+ H2S lamg & Hammer 15 7 days vanSpreekens, 1974
Proteolysis Soft-agar-gelatin 211 3 days Levin, 1968
Fish-flesh-agar 28 I-2 d a y s Chandrasekaranet al.,
1985
H2S-prt~uction Peptoneiron Agar 21) 3 days Levin, 1968
H,S-medium 211 4 days Ogden, 1986
Iron Agar. Lyngby 211-25 3-5 days Gram el al., 1987

it can be concluded, in accordance with other studies, that the concentration of

bacteria has been a neglected step in the development of rapid methods (Gut-
teridge and Arnott, 19891. This problem may be attacked by the latest develop-
ment in immunosorbent techniques where, as an example, the use of immunomag-
netic separation appears to allow for a selective recovery and concentration of the
antigen: small iron-containing particles are coated with antibodies against the
bacterium, to be detected (Dynal, 1989). The antibody-coated particles are added
to the sample (e.g. 1 ml of a 10- t dilution of a fish sample) and placed in a tube or
in a microwell. Following a short period of incubation, a strong magnet is held at
the side of the tube or the well. When the small iron-containing particles (includ-
ing the immunosorbed antigen) have assembled at the magnet, the remaining
sample is discarded. The particles are resuspended in buffer and it is thereby
possible to concentrate specific bacteria from a large volume into a smaller one.
Such magnetic beads pre-coated with antibody are commercially available (Dynal
A / S , Norway).
Detection and quantification of the captured bacteria may be done by further
antibody-reactions like sandwich enzyme-linked immunosorbent assay (ELISA) or
in the case of live bacteria - - by measuring enzymatic activity. The immunosor-
bent technique has been used to detect enterotoxic E. coli (Lund et al., 1988),
Pseudomonas putida in lake water (Morgan ct al., 1991) and Clostridium perfringens
toxin in food samples (Cudjo¢ et ai., 19911.
At our laboratory, we have recently started the development of poly- and
monoclonal antibodies against Shewanella putrefaciens. The project is carried out
in collaboration with the Department of Biochemistry and Nutrition at the Techni-
cal University of Denmark and the Department of Microbiology and Plantphysiol-
ogy at Bergen University. The antibodies are raised in mouse against the type
strain (ATCC 80711 and against wild-types. Antigenic epitopes are characterized
through SDS-PAGE and immunoblotting. The type strain contains all the bands
found in wild-types, however, these are more diverse than the type strain. The
antibodies tested so far cross react with other Gram-negative bacteria with the
largest cross-reactivity seen towards Vibrionaceae. Preliminary experiments with
immunosorption on magnetic beads of Alteromonas dinitrificans have shown that
as little as 100 C F U / m l can be detected. Presently it is estimated that examination
of a sample will last 3-4 h; all steps included.
The two main obstacles are at present (1) the development of antibodies specific
for Shewanella putrefaciens and (2) the design of a detection procedure following
the immunocapture. While these problems can be solved, the practical application
of the method in the fish industry will probably be the most difficult step. The
method will allow for estimation of levels of spoilage bacteria within 3-4 h. The
full appreciation of this can only be found in industries which are able to diversify
their production immediately depending on the quality of raw material.

Measurement of spoilage reactions

Several spoilage reactions can be used for evaluation of the bacteriological

status of fish products. As described above, agars on which H2S-producing
organisms are counted have been developed. During spoilage of white lean fish,
one of the major spoilage reactions is the bacteriological reduction of trimethyl-
amine oxide to trimethylamine (Liston, 1980; Hobbs and Hodgkiss, 1982). The
bacteria producing H~S are also potent reducers of TMAO and a number of
studies have evaluated the bacteriological quality, especially the spoilage potential
of the bacteria in a fish sample, by measuring the ability of the microflora to
reduce TMAO (Easter et al., 1982; Gibson et al., 1984; Gram, 1985; Ogden, 1986;
Gibson and Ogden, 1987; Huss et al., 1987; Ravn-Jcrgensen et al., 1988).
Three changes can be measured in a substrate when TMAO is reduced to
TMA: the redox-potential decreases, the pH increases and the electrical conduc-
tance increases. These changes are caused by the metabolic activity of the bacterial
flora and may thus be a more true estimation of the actual spoilage potential of the
bacterial population in a given sample than a colony count.
Trimethylamine is a sligthly basic compund (pK a = 10.4) and as TMAO is
reduced to TMA, pH of an unbuffered solution will increase. The ability of the
mixed microflora on fish to reduce TMAO was measured by mixing 2-3 ml of fish
homogenate with a standard solution of TMAO. The tube was covered with
mineral oil and pH measured at hourly intervals (Malle et al., 1986). Three fish
samples characterized by different degree of spoilage (81, 37 and 17 mg TVN-
N/100g) were examined. After 8 b of incubation at 25°C, pH had increased 2.5, 1.4
and 0.2 units. The pH changes were not immediate and could not be attributed to
the addition of bases from the fish samples. To obtain a rapid method, the authors
have set a fixed time of 8 h incubation (Maile et al., 1986). However, fish with low
counts of bacteria will not be detected within this time limit and the set-up of the
method therefore only allows for evaluation of fish which is close to spoilage.
Huss et al. (1987) described a method in which reduction of TMAO to TMA
was measured by change of colour of a redox indicator, resazurin. One ml of fish
sample was mixed with 2 ml of a sterile TMAO broth and the time taken for
TMAO to be reduced to TMA measured by the colour change of resazurin was
read (RT -- the reduction time). In storage trials, good correlation (r -- 0.99) was
found between RT and number of hydrogen sulfide producing bacteria. From 100
to 108 H2S-producing bacteria per gram were detected in 20-6 h.
Several of the studies using conductance measurements for estimating bacterial
counts have used a TMAO broth. When TMAO is reduced to TMA an ionized
molecule is produced, and Easter et al. (1982) showed that the conductance
change in a TMAO-medium was caused by reduction of TMAO to TMA. Simi-
larly, increase in conductance coincides with TMAO-reduction in a fish slurry
(Huss et al., 1987). As the reduction of TMAO in chilled fish is caused by the
specific spoilage bacteria that are also H,S producers (Gram et ai., 1987; Ravn-
Jergensen and Huss, 1989), the conductance detection times have been correlated
to the number of H2S-producing organisms (Gram, 1985; Ogden, 1986; Ravn-
J~rgensen ct al., 1988). It is therefore not surprising that a better correlation
(higher correlation coefficients and less scatter) was found when comparing detec-
tion times in TMAO broth to H 2S-producing organisms than when comparing to a
standard plate count.

Summary and conclusions

Several methods exist for estimation of the number of bacteria in fish. All agar
assays depend on the formation of a visible colony and thus require long incuba-
tion periods. Redigel and Petrifilm eases the handling of samples but certainly
have all other drawbacks of the conventional method (e.g. length of incubation and
lack of correlation with sensoric quality or expected shelf life). Using a rich agar
and incubating plates at 5-25°C is preferable.
Several methods (Limulus, ATP, DEFT) have been used for rapid estimation of
the content of microorganisms in seafood. All methods require preparation of the
sample (filtering, enzyme-treatment etc.) which impede the normal laboratory
routine. The DEFT method is certainly useful if a rapid estimation of the bacterial
level is needed. It is said that some differentiation of the microflora is possible
based on differences in microbial morphology (Pettipher and Rodriques, 1982) but
this is of little use in many seafoods where the majority of the microflora are
Gram-negative rods. Methods based on microscopy will not indicate the activity or
viability of the cells and are hampered by a very low sensitivity. One disadvantage
of the method, the tiresome microscopy, may be eliminated by usage of automatic
counting by image analysis (Abgrall and Bourgeois, 1989).
Automated equipment (calorimetric and impediometric) measuring physical
changes occurring as a result of bacterial growth has been used for detection of
bacteria in seafood. The only really successful principle is based on electrical
measurements. Little work has been done on fish using the Bactometer instru-
ment, but the Malthus Growth Analyser is capable of estimating the number of
bacteria present and specifically the number of spoilage bacteria within 30 h,
depending on number of bacteria in the sample. The instrument is optimal for
assessment of spoilage potential of the bacterial flora in a fish sample since one of
the major spoilage reaction, the reduction of TMAO, is especially well suited for
being monitored by this type of measurement.
All methods rely on incubation at 20-25°C and since only a small proportion of
the microflora will be responsible for the changes measured the scatter around
regression lines, e.g. comparing colony counts to detection times, as well as
different regression lines can be explained by variation in initial microflora.
Many studies have bccn devoted to the specific enumeration of spoilage
bacteria, and agars detecting H2S-producing organisms like Shewanella putrefa-
ciens are available commercially and they are found very useful. The length of
incubation is one of the major drawbacks of these agars. Since reduction of T M A O
to T M A is a major cause of quality deterioration in white, lean fish, several
methods for monitoring spoilage bacteria are based on this reaction. The time
taken for a fish sample to reduce a known amount of TIVlAO is inversely
correlated to the number of spoilage bacteria. Maximum length of the analysis is
approximately 25-30 h, depending on the incuabtion temperature and number of
spoilage bacteria.
Many of the methods for detection of specific spoilage bacteria can only be used
in fish and fish products characterized by a T M A and H2S spoilage, it is not
possible by the same methods to enumerate specific spoilage bacteria on fish
spoiling due to action of pseudomonads which do not produce HzS or reduce
T M A O (Gram et al., 1991).
An interesting and obvious direction for future research is the use of specific
antibodies and gone-probes for the estimation of bacteria and spoilage reactions
(Adams and Hope, 1989; Gutteridge and Arnott, 1989). In products where
TMAO-reduction is of major importance, a probe directed against the genc(s)
coding for this specific enzyme system could be used in a rapid technique for
quantifying spoilage bacteria.

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