Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Ughtfoot
pork and liquid egg (251). Growth has been
observed on beef, crab meat, and fish when these
have been stored at 8°C. Generation times on
meat at lOOC are long (8-26 h) compared to those
for spoilage organisms. Lower minimum growth
temperatures than these have sometimes been
reported when salmonellae have been cultured in
laboratory media.. Growth has been recorded in
broth at 5.9°C (257), and as low as 5.2°C (259) and
even 4°C (277) on agar media. In the last study,
one of 109 strains tested grew at 4°C, and 44
failed to grow at 7°C. There are difficulties in
determining the minimum temperature for
growth. Close control and monitoring of the
temperature over extended periods are needed.
Frequently, near the minimum, there may be a
period when there is an increase in cell mass (and
hence optical density) without cell division, and
growth is not sustained.
The QIJtimum tp.mpp.rature for growth is
35-37°('..., with growth of most strains occurnng
up to 45-47°C though at a :r;:educedrate. Growth
does not occur at 50°C. In Figure 8.1 the ~e
root of the aerobic growth rate of Salmonella
Typhimurium in mutton mince (352) is plotted
against temperature, and is compared with rates
predicted from a USDA ARS Microbial Food
Safety Research Unit model (52). Agreement
between the two sets of data is good, especially
considering one was obtained for one strain on
meat and the other from different strains growing
in laboratory medium.
Above the maximum temperature for growth,
salmonellae die. Salmonellae are sensitive to heat
and heat resista~ strains are uncommon. The
m~, decImal reduction tIme at b'/uC of
stationary phase cultures of 296 strains of
salmonellae heated in trypticase soy broth with
2% yeast extract (pH 6.8) was found to be about
1.3 minutes (285). One strain of S. Blockley had a
D-value of 5.8 minutes and S. Senftenberg strain
775W had a D-value of 31 minutes. All other
strains had D-values between 0.7 and 2.6
minutes. Similarly, another survey (27) found
that most of 221 strains had a D-value at 60°C of
0.4-0.6 minutes, and that only three strains (a
strain of S. Bedford, a strain of S. Senftenberg,
and S. Senftenberg 775W) were heat-resistant
strains (D-values at 60°C of 4-6 min). The z-value
(CO needed to reduce the decimal reduction time
by 10-fold) of normal heat-sensitive salmonellae is
4-5 Co.
Heat resistance depends on what conditions
the Sal;'!.onella cells are exposed to before
heatinglQells grown at slightly low pH (pH 5.8) \
(240), or at high pH (pH 8-9.75) (192) hav-eJ
increased heat resistance over cells grown at near
neutral pH. The higher the growth temperature,
228
the more resistant the cells (193, 285), and even
exposure for relatively short periods to sub-lethal
temperatures (37°-52°C) increases heat
resistance (53, 193). Storage of cells at 4-8°C
significantly decreased the heat resistance of
S. Enteritidis PT 4 (187) and, therefore,
S. Enteritidis in eggs should be more easily killed
by cooking following refrigerated storage than
following ambient storage. Stationary phase cells
are more resistant than log phase cells (194, 285).
Heat resistance of salmonellae in foods also
.jepe~s on the composition of the food, the pH
and the type of aCIdulant, and the ~. For
instance, S. Enteritidis is more readily destroyed
in egg albumen than in homogenised egg, and
survives heat better in egg yolk than in either of
the other two (194). Differences in pH account in
part for the greater heat resistance in egg yolk
than in egg white (282). If homogenised egg is
acidified with hydrochloric acid, heat resistance
increases to reach a maximum near pH 5.5; if
acetic or lactic acids are used, heat resistance
decreases with acidification (282).
Foods high in fat and low in moisture (e.g.
dried egg, dried animal feeds, chocolate, syrups)
may need severe heat treatments to kill
salmonellae: for instance, in milk chocolate with
less than 2% moisture the Dso'c value for
S. Typhimurium has been measured at 222
minutes (204). The addition of small amounts of
water can have a marked effect on reducing this
high heat tolerance: for instance, increasing the
moisture content to 3.7% decreased the D71'c
value from 20 to four hours for S. Anatum in milk
chocolate (33). Cells dried on membranes showed
little or no death when heated in an oven at lOOoe
for one hour, and one percent or more survived
even heating at 135°C for 20 minutes (221).
The solute responsible for the lowered awalso
1.75
:I:
u; 1.5
. 0
c 0
0 .
1.25
0
Cl>
c 1
Cl> .
Cl 0
'0 0.75
0 0 Mutton
0: 0.5
. USDA Model
<11
i
5- 0.25
rJ)
0
5 10 15 20 25 30 35
Temperature 'C
Figure 8.1. Comparison of growth rate for
Salmonella on mutton with rates predicted by
USDA model -

influences heat resistance. When sodium chloride
was used to reduce the aw in broth, there was a
small increase in resistance of salmonellae to a
maximum at near aw 0.95 (6.1% salt w/w) followed
by a decrease in resistance to aw 0.90 (13.8% salt
w/w) (27). When glycerol was used to lower the aw,
the heat resistance increased with concentration
of glycerol to a final aw of 0.85 (43.3% w/w). The
increase in D60°Cvalues was only 4-5 fold. When
sucrose was used to lower the aw, the heat
resistance also increased progressively with
concentration to an awof 0.90 (57.6% w/w), but, in
this case, the increase in D60°Cvalues was over a
lOO-fold.Furthermore, the z-value was considerably
higher in the presence of salt than in the presence
of either glycerol or sugar. Corry (68) found a
linear relationship between the log D65'c value
and the % concentration (w/w) of solute, but not
between aw and heat resistance, for sucrose,
glucose, fructose, sorbitol and polyethylene glycol.
There was not a similar linear relationship for
glycerol which behaved differently from the other
solutes and which also gave a much lower heat
resistance. At the same concentration, the heat
resistance of cells suspended in solutions of
different solutes increased in the order glycerol,
fructose, sorbitol, glucose, sucrose. The length of
habituation time of Salmonella at reduced water
activity also influences heat tolerance. Mattick et
al. (261) found optimal habituation time and the
extent of increase in heat tolerance depended on
the solute. Habituation in glucose-fructose to aw
0.95 for 12 h resulted in maximal heat tolerance,
with more than a fourfold increase in D54 values.
Habit,uation under the same conditions for 72 h,
however, resulted in heat sensitivity comparable
to non-habituated salmonellae. The combined
effects of food composition, acid and solute type,
pH and aw mean that it is not possible to predict
the heat resistance of salmonellae in foods of low
aw.
At chill temperatures below that permitting
growth, salmonellae can survive on foods for long
periods. They have been shown to survive for
more than 28 days at 2-4°C on a variety of
vegetables such as green beans, cabbage, lettuce,
beets, carrots, peppers, and tomatoes (207). On
inoculated pecan halves, little decrease in the
viable population of three strains of salmonellae
over 32 weeks storage at 5°C was found by
Beuchat and Heaton (38). Survival is dependent
predominantly on other factors like pH and aw,
and survival is longer at chilled than at ambient
temperatures: for instance, S. Enteritidis PT 4
inoculated into commercial mayonnaise is
destroyed more rapidly when the mayonnaise is
held at 20°C than when held at 4°C (249).
Salmonella
Freezing will reduce the numbers of
salmonellae by an amount that depends on the
freezing rate and the type of food. Rapid freezing
promotes survival. Log phase cells are more
sensitive than stationary phase cells. There is an
initial decrease in viable count as a result of the
freezing damage, followed by a slower rate of
decline during storage. Lower storage temperatures
and less fluctuations in temperature give greater
survival. Storage temperatures near the freezing
point result in most death or injury. In minced
chicken breast (pH 5.8), 60-83% of Salmonella
cells survived storage at -20°C for 126 days,
whereas at -2° and -5°C only 1.3% to 5.8% were
still viable after 5 days (140). Salmonellae can
survive for long periods in frozen foods and their
frequent isolation from frozen foods is ample
evidence of their ability to survive.
pH
In laboratory media, salmonellae can grow from
about pH 4.0 to near pH 9.J5!with the optimum
bemg in the range ~\The minimum pH
allowmg growth is influenced by the temperature
of incubation (66), and by the presence of salt and
nitrite (151). The minimum pH also depends on
the acidulant used. In broth acidified with
hydrochloric, gluconic, lactic, or acetic acids, the
minimum pH for growth has been reported to be
4.05, 4.20, 4.40, and 5.40 respectively (66).
Outside the range of pH allowing growth,
salmonellae die. At low pH values the nature of
the acidulant determines the rate of death.
Volatile fatty acids are more bactericidal than
acids such as lactic and citric acids. The
undissociated molecule is responsible for the
lethal action so that the effectiveness of acids
increases as the pH is lowered (157). For formic,
acetic, propionic and butyric acids, the
bactericidal effect decreases somewhat with
increasing chain length. The rate of death
decreases as the temperature is reduced, and, at a
given pH and temperature, is dependent on the
concentration of the acid. Organic acids are more
effectively bactericidal under anaerobic than
under aerobic conditions.
Tolerance of acidic conditions, apart from
being an advantage for survival in the
environment, is important for virulence because
ingested salmonellae have to pass through the
acid (pH less than 3) of the stomach. Tolerance of
acid conditions is influenced by how the
organisms are grown. Salmonellae have at least
three systems that enable cells to adapt to survive
potentially lethal acid exposure (234). One is a pH
independent general stress resistance produced
by stationary phase cells that is dependent on
RpoS and the production of the stationary-phase-
229
 Salmonella

Combinations temperature such as occur during cooling and

As is clear already, when salmonellae are in foods, during storage and distribution.
several factors act simultaneously to influence the Models for heat destruction depend on
growth rate or the extent of survival. It is the knowing the D- and z-value for the food. Models
combination of parameters like pH, aw and that can be used to predict survival of salmonellae
temperature, and the presence of inhibitors like in foods in other non-growth conditions are being
nitrite or short chain fatty acids that are developed. A preliminary one that is available
important in determining the response of examines the response of salmonellae to the
salmonellae. effects of temperature, percent salt (aw), and pH
A knowledge of the minimum and maximum with lactic acid as acidulant. The kinetics of
values for temperature, pH and aw that allow survival are not linear or simple, and as a result
growth under otherwise ideal conditions is useful. this model predicts the time for a 10 OOO-fold
Similarly, a knowledge of the combinations of reduction in numbers, or a 4-log decrease. Figure
these factors that will prevent growth is 8.3 has been derived from predictions given by
important in judging whether salmonellae may this model (USDA ARS Microbial Food Safety
grow in a food. More useful information is Research Unit Model) and shows how
obtained by taking advantage of 'user-friendly' combinations of temperature (5-30°C), aw (0.88
models that predict the growth of salmonellae in and 0.90) and pH (4.8-5.2; lactic acid) influence
foods once the temperature, pH, and aware the time taken for viable salmonellae to decrease
known. One is a USDA ARS Microbial Food by 4-log units.
Safety Research Unit Model (52) and a second is
Food MicroModelTM from the UK which was
developed from Ministry of Agriculture Fisheries Methodology
and Food funded research. The agreement
between predicted growth rates and rates
observed in many foods has been good. One Methods for the detection of salmonellae in foods
example is shown in Figure 8.1. In many have been intensely researched for many decades,
situations the major controlling factors are and this has led to a proliferation of analy.tical
temperature, pH and aw. Nitrite, high carbon procedures for their isolation and identification.
dioxide concentrations, and some natural Most of the procedures are lengthy to perform,
antimicrobial materials in foods may be and none are optimal for all food categories or for
important in some circumstances. In these cases all known serovars.
the prediction will be 'fail-safe' in that it will In the following discussion both conventional
predict faster growth than in fact occurs. The cultural techniques and rapid methods for the
models can also be used to predict the likely isolation or detection of salmonellae in foods are
extent of growth under changing conditions of reviewed. Despite the increased use of rapid
methods, it should be recognised that develop-
ments in cultural methods are still of considerable
importance, as they rely on cultural procedures to
6 provide enough cellular material for detection.
5 Cultural methods
The conventional examination of food products for
4
::I salmonellae requires the use of cultural methods
LL which are different from those used for clinical
0 3
Cl
0 specimens. This is mainly because salmonellae in
.J
2 foods are subjected to ecological conditions which
are unlike those of their preferred environment,
the gastrointestinal tract of man and animals.
During processing and storage of foods, for
0 example, they may be exposed to heat,
0 5 10 15 20 25 desiccation, preservatives, osmotic stress or
Days changes in pH. Furthermore, they usually have to
exist in association with a relatively large number
Figure 8.2. Effect of pH on the survival of of competing microorganisms which represent the
SalmonellaTyphimurium in pepperoni natural flora of the food. Therefore methods for
manufacture; fermentation at 35°C, maturation their isolation must be designed to enhance the
at 12°C,final Sw0.878 (350) survival and multiplication of salmonellae while

231
..
 Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot

suppressing the growth of competitors. Because Standard reference methods

expert opinion is divided on many aspects of
Salmonella methodology, laboratories usually
choose techniques and procedures which have
been found to be best suited for their purpose.
Comprehensive reviews on the diverse, and often
contradictory reports on Salmonella methodology
have been presented by Litchfield (248),
Fagerberg and Averts (123), Flowers et al. (134)
and Fricker (141). There will always be disagree-
ment between microbiologists on the best choice of
media and incubation temperatures, but there
has been general approval of the following basic
procedure: pre-enrichment in non-selective broth,
selective enrichment in broth, isolation on
differential selective agar, biochemical confirmation
of suspected colonies, and serological identification.
Isolation of Salmonella Typhi
With the long incubation period of typhoid, it is
not always possible to isolate Salmonella Typhi
from the implicated food. In an outbreak situation
and as the ultimate. source of infection is the
human carrier, efforts must be concentrated on
screening all possible food handlers by culture of
both faeces and urine as well as by serological
methods for antibody detection, especially Vi
antibodies. It may be necessary to examine more
than one sample of faeces to detect the
intermittent excretor. Sewer swabs (273) can be
used to trace contamination up a sewer or
watercourse to its origin to detect the source of
infection of the water supply. This method has
been used successfully in many outbreaks. Swabs
are also useful in theinvestigation of outbreaks at
institutions. Development of a DNA probe could
prove useful in rapid diagnostic assays for S.
Typhi in mixed bacterial samples (338).
For the isolation of S. Typhi from food or
faeces, similar methods and media to those used
for other Salmonella serovars are employed.
However, S. Typhi will not grow at elevated
temperatures (e.g. 43°C) or at the concentrations
of brilliant green which are suitable for the
growth of other salmonellae. Selenite F and
The development of standardised reference
methods for the detection of salmonellae in foods
became necessary with the adoption of
microbiological specifications for this
microorganism in both state and national food
law. Standard reference methods have also proved
useful for arbitration purposes in the
international trade of foods, for assisting many
laboratories engaged in routine work to obtain
more uniform results, and for interlaboratory
comparison testing programs. I!js important to
realise that standard reference methods are
usually based on the best practical procedures
available and accordingly may not be the most
sensitive. Hence it is likely that some laboratories
experienced in Salmonella analysis will obtain
equivalent or better results using alterjj]ftive
techniques. For non-routine or research purposes,
such techniques may be more acceptable to some
laboratories.
In Australia, a committee of the Standards
Association of Au.s.tJ:alia (SAA) has promulgated a
iillIDdard rpfprpnpp method (AS 17~h ') ~ ~~
the isolatioILand identification of salmo.Dp]]aein..
f90ds (359). A flow diagram of this method is
presented in Figure 8.4. The analytical techniques
described in this method conform to the five
procedural steps listed previously. The discussion
to follow is centred on this published method.
,,-
Sample preparation
It is generally al:rppd th3t for adeqllatB rBcovery
of salmonellae from cont2minatBd food mMerials,
sample amounts of at least 25 g must be tested.
or some high risk food categories such as drIed
milk powder, sample sizes of hetween 50 to 100 g
qre frequently rppommended (19, 20, 134, 203).
While the probability of detecting salmonellae in a
contaminated product increases with sample size,
80
Q)

selenite cysteine broth are not very selective, '"

os -- pH 4.8 a. 0.88
E 60 -0- pH 4.8 a. 0.88
while Muller-Kauffman tetrathionate broth, c.>

Rappaport (RV) and Rappaport (RVS) broth are ~ -.- pH 5.2 a. 0.90
tn
inhibitory to S. Typhi (310). Iveson and Mackay- .3 40
~
Scollay (208) and Chau and Forrest (63) found ...
0
that strontium selenite was superior to selenite F ';;;
>-
20
os
for the isolation of S. Typhi. Tetrathionate broth will C
grow a wide range of serovars including S. Typhi. 0
S. Typhi grows well on desoxycholate citrate 0 10 20 30
agar (DCA) and on bismuth sulphide agar (BSA), Temperature .C
but xylose lysine desoxycholate agar (XLD) is not
\ very selective. Mannitol lysine crystal violet Figure 8.3. Interaction of temperature, awand
I brilliant green agar is not suitable for S. Typhi. pH on death of salmonellae

t ~32
 .
T
i
!
"

I' Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot
11

RESUSCITATION

g 25 g sample
+
225 mL Buffered peptone water
37"C/16-20h

~
0.1 mL 1 mL

+ +
SELECTIVE ENRICHMENT BROTHS

RV broth 8- - 8 Mannitol selenlte cystine broth

42°C/18-24h 31"C/18-24 h

!/ PI.!. o~
SELECTIVE SOLID MEDIA
~!
XLDagar IL-..J I IL-..JI bismuth sulphite agar
31"C/24-48 h

~
3 typical colonies/plate

~ ~
peptone water

37°C until turbid

~
BIOCHEMICAL TESTS

n lysine
IL-..JI CLEDmedium
Q nutrient agar t:j decarboxylase
broth g ONPG broth

37°C/18-24 h

~~ [] SEROLOGICAL TESTS

Figure 8.4. Flow diagram of method for detecting Salmonella

-234
 Salmonella

enrichment is given in the following sections. At shown to enhance Salmonella recovery from pork
this point, however, it is useful to consider sausages (144). The US FDA (19) recommends the
specifically sample preparation in relation to addition of 2.25 mL Tergitol 7 or Triton X100 to
enrichment procedures for detecting salmonellae 225 mL pre-enrichment medium used for recovery
in foods. of salmonellae from coconut, meats, meat
The recommended ratio of inoculum to substitutes, meat by-products, animal substances,
enrichment medium varies from 1:4 to 1:10, often glandular products and fish, meat and bone
according to the type of food and the size of the meals. When used, a minimum amount of
sample to be tested (19, 134, 171, 203, 359). For surfactant should be added. This is determined by
most purposes, a ratio of 1:10 is preferred. The titration and is simply achieved by adding just
higher dilution is particularly important where enough to initiate foaming. It is necessary to
food is added directly to a selective enrichment ensure that the combined effect of surfactant
broth. Silliker and Taylor (345) demonstrated concentration, enrichment composition and
that the reduction of selectivity of enrichment incubation temperature is not too inhibitory for
broths was directly proportional to the amount of the recovery of injured salmonellae. Morris and
water soluble components in a number of different Dunn (276), using Tergitol No. 7 at 6.0% in
foods including gelatin, albumen, egg yolk and tetrathionate brilliant green broth incubated at
dried beef. Since the addition of food samples may both 37°C and 43°C, demonstrated that this
adversely effect the performance of selective enrichment combination with pork sausage
media due to alteration in pH, salinity, nutrient samples may result in inhibition of salmonellae
composition, or other physical factors, it is when incubated at the higher temperature.
important to evaluate the influence of each It is also important for any laboratory using a
specific product on enrichment procedures. This surfactant, to test adequately each batch of
will be discussed further in later sections. surfactant to ensure that no toxicity towards
Mter the sample has been added to the salmonellae is apparent at the concentrations
appropriate volume of enrichment medium, it is used. Different manufactured batches of
important to ensure that salmonellae will be surfactant can vary significantly in regard to
released from the food into the diluent without toxicity, irrespective as to whether they are
loss of viability. If the sample is powdered, ground recommended for use in isolation of salmonellae.
or comminuted, it may be readily dispersed by D'Aoust et al. (94) studied the effects of nine
gentle swirling or stirring with a sterile glass rod. surfactants in pre-enrichment media. Tween 20,
Other products will require mechanical blending Teepo1610 and Brij 35 were eliminated because of
to obtain a homogeneous suspension. The risk of their toxicity to Salmonella. Tergitol 7, Tween 80,
destroying salmonellae by localised overheating Triton X100, Myrj 525 and Arlacel 80 and Tween 60
during this process must be minimised. Most food did not inhibit the growth of salmonellae but also
samples can be adequately homogenised using did not increase the isolation rate from 45 fatty
Stomacher machines which generate very little foods studied when compared to nutrient broth
heat during mixing. Some food materials exhibit a controls. It is therefore not clear whether
remarkable resistance to dispersion when added surfactants do enhance recovery of salmonellae to
to enrichment media. Casein and products any significant degree.
containing casein, for example, form large sticky
insoluble lumps which could prevent the recovery Non-selective pre-enrichment
of salmonellae trapped within the non-dispersed Various treatments related to food processing
casein. To obtain optimum dispersion of this such as heating, drying, freezing, irradiation,
material, the SAA (357) described a procedure for changes in pH, or addition of preservatives may
mixing the sample in chilled phosphate solution induce sublethal damage in bacterial cells (55).
(acid casein) or chilled buffered peptone water Since the presence of inhibitors or other selective
(other caseins and casein products) before the agents can reduce markedly the ability of media
addition of the pre-enrichment medium. to support the repair and growth of injured cells,
In the examination of gelatin, the US FDA most methods for isolating salmonellae from foods
recommends the inclusion of a gelatinase solution involve a pre-enrichment step using a non-
in the pre-enrichment medium to prevent selective medium.
solidification during incubation (19). Dilution of This phase of the isolation procedure,
gelatine 1:20 in pre-enrichment medium is also according to Flowers et al. (134), should provide:
successful in this regard. Similar dispersion a. nutrients for multiplication to favour the
problems can occur with fatty foods. Addition of ratio of Salmonella to non-Salmonella
. .
the emulsifying agents Tergitol No. 7 and Tween mIcroorgamsms;
80 to tetrathionate brilliant green broth has been b. repair of cell damage;
235
Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Ughtfoot

c. rehydration; and the inoculation ratio of 1:10 is almost

d. dilution of toxic or inhibitory substances. universal, these are not suitable for all food types.
Many different pre-enrichment media have Table 8.12 summarises the recommendations of
been recommended for various food materials. the US FDA and SAA.
One of the most widely used media is lactose There is general agreement that the
broth, originally developed by North (288) to incubation temperature for pre-enrichment
improve the recovery of salmonellae from dried should be around the optimum for growth of
egg products. It was claimed that in the presence salmonellae, i.e. 35°C to 37°C. The SAA
of a mixed flora, the fermentation of lactose recommends 37°C:!:: 1°C. The time of incubation
resulted in a lower pH which enabled salmonellae has been well studied and recommendations have
to survive and grow while other types of varied from 6-48 h. More recently, the time
microorganisms were inhibited. Silliker et al. recommended has shortened to 16-24 h.
(346), however, considered that where there was AS 1766.2.5-1991 states 16-20 h while the US
an unfavourable coliform to Salmonella ratio in a FDA states 24:!::2 h (19). D'Aoust and Maishment
food sample, this broth would favour the growth of (93) found that short incubation times of 6 h were
lactose fermenting competitors. Taylor et al. (377) associated with reduced recovery of salmonellae.
and Gerichter and Sechter (150) on the other On the other hand, prolonged incubation can also
hand, found that the carbohydrate used in pre- lead to reduced recovery due to overgrowth.
enrichment media did not influence their Fricker (141) reviews the issue at length and
effectiveness in the ultimate number of concludes that incubation of pre-enrichment
Salmonella isolations. These findings suggest cultures for 24 h before subculture probably
that other resuscitation media such as nutrient provides the best results.
broth and buffered peptone water are also Components of some food samples may
suitable for the pre-enrichment of most foods. In impair resuscitation and adversely affect the
addition, it is possible that highly nutritious foods recovery of salmonellae. Cocoa powder, for
such as dried eggs and milk may be simply example, is known to contain naturally occurring
reconstituted in either distilled water or substances which are bactericidal to some strains
physiological saline. Pre-enrichment in buffered of salmonellae (56). Zapatka et al. (421) showed
peptone water has increased in popularity that this bactericidal activity could be diminished
because it offers several important advantages. by the addition of casein to the pre-enrichment
The inclusion of peptone aids resuscitation and media (reconstituted non-fat dry milk or nutrient
the presence of phosphate buffer prevents broth containing 5% (w/v) casein). In the
excessive acidification. Baylis et al. (34) found examination of chocolate and products containing
that the recovery of heat injured cells of chocolate, the addition of skim milk to the
Salmonella can vary depending on the resuscitation medium has been recommended (19,
formulation of buffered peptone water. Moreover, 134, 359). D'Aoust and Sewell (92) concluded that
phosphates have been shown to assist in the rapid reconstituted skim milk powder with 0.002% w/v
repair of damaged cells (322, 323). Van Leusden brilliant green was marginally more successful in
et al. (390) recommended buffered peptone water detecting salmonellae compared with a method of
for routine use as does Fricker (141) and this is the International Office of Cocoa and Chocolate
the medium specified in the Australian Standard and International Sugar Confectionery
reference method AS 1766.2.5-1991 (359). Manufacturers' Association which used mannitol
Bailey and Cox (25) described a medium broth for pre-enrichment. The recovery of
called universal pre-enrichment (UP) broth for salmonellae from other foods containing natural
the simultaneous recovery of Salmonella and inhibitors or artificial preservatives could also be
Listeria. This medium was highly buffered and improved by the inclusion of non-toxic
low in carbohydrate. Good recovery of low neutralisers in pre-enrichment media. The US
numbers (10 cfu) of heat-injured Salmonella and FDA (19) for example, recommend the use of 0.5%
Listeria was demonstrated in both pure and K2S0a in the examination of dried onion and
mixed culture and several different foods. garlic, and dilution beyond their toxic levels of
Lactose broth is widely used in the USA and allspice, cinnamon and oregano. There is even
has been adopted as the official pre-enrichment now, limited information in the literature relating
medium for most foods by the AOAC and US FDA to neutralisers for use in resuscitation procedures
(19,20). The SAA specifies buffered peptone water for different foods.
for all foods except cocoa and cocoa products. The Repair of cell damage or resuscitation is an
ratio of food to medium is usually recommended important function of the pre-enrichment phase
as 1:10. However, although lactose broth and and has been extensively reviewed by Andrews
buffered peptone water are often interchangeable (7). Numerous studies on reconstitution of dried
 11
"

.1
Salmonella

I~

foodsin pre-enrichment media have resulted in enrichment media were those appropriate to the
acceptance of slow rehydration methods, which sample type as specified in the BAM 1984 6th ~
have been shown to increase Salmonella recovery edition, Chapter 7. This procedure did not claim
from some foods. Presumably, slow rehydration greater recovery of Salmonella from dry foods but I
reduces osmotic injury which occurs by rapid rather was designed to validate a method of
rehydration from a near dry state. Ray et al. (324) refrigeration of pre-enrichment cultures over a
found that for dried milk products, reconstitution weekend. 11

for 1 h in a high osmotic environment (solid:liquid There has been considerable conjecture
ratio 1:2.5) followed by normal pre-enrichment regarding the optimum equilibration time for
(solid:liquid ratio 1:10) gave higher recoveries of rehydration. According to van Schothorst et al. ill

salmonellae. This was later confirmed by van (394) the time of sample equilibration at the
Schothorst et al. (394) who found reconstitution of initial 1:2 sample/medium ratio is of little
dried milk powder in buffered peptone water (1:2) consequence. A further investigation by D'Aoust
for 30 minutes at ambient temperature followed and Sewell (89) showed no advantage between
by dilution to a final ratio of 1:9 substantially long (4 h) and short (15 min) equilibration at i!

increased recovery of salmonellae. In a variation ambient temperature. This study was carried out
of the reconstitution procedure, similar results with high sample to medium ratio m\xtures of :1
were obtained for dried instant non-fat milk feeds and feed ingredients followed by dilution to
powder using a slow rehydration soak method (9). normal enrichment culture levels. While it
In this method the sample was poured onto the appears that the control of aw during rehydration I

surface of the pre-enrichment medium and
allowed to dissolve slowly at ambient temperature.
This method has also been demonstrated
enhance recovery of Salmonella from dry whole
milk, lactic casein, non-instant non-fat milk and
rennet casein, but not from sodium casinate (308).
In a comparison between rapid hydration and the
soak method, Wilson et al. (416) found that the
soak method gave higher recoveries from soya
flour but not from brewers' yeast, dried active
yeast or onion powder.
A refrigerated pre-enrichment procedure for
dry foods was described by D'Aoust et al. (87) in
1993. Pre-enrichment cultures were incubated at
4°C for 72 h prior to selective enrichment. Pre-
of dry materials may be important for optimal
recovery of damaged salmonellae, slow rehydration
to methods have only been demonstrated to be
significant for certain dairy products and soya flour.
The significance of aerobic or anaerobic pre-
enrichment is not clear. Alford and Knight (1)
described a shortened pre-enrichment using
continuous shaking for 4 h at 37°C which was
followed by addition of selenite and further
shaking for 20 h. Improved recovery rates
obtained with this procedure were attributed to
actively multiplying cells being less sensitive to
exposure to toxic substances such as selenite in
subsequent pre-enrichment media. It was
conceded, however, that the value of aerated pre-

Table 8.12. Pre-enrichment media for the isolation of salmonellae from foods
Medium (volume, mL) Food (mass, g) Reference
Lactose broth (225) Eggs and egg products (25); prepared powdered mixes, e.g. cake AOAC(19)
cookie, doughnut,etc., cheese, dough and prepared salads (25) infant
formula (25), fruits and nutmeats (25); crusteans and fish (25); food dyes,
pH >6.0 (25); frog legs, food snails and shellfish (25)
Lactose broth (225) + Tergitol No 7 Coconut (25); heat processed and dried products (25), e.g. meats, AOAC (19)
(2.2) or Triton X100 (2.2) animal substances and meals
Lactose broth (225) + 5% aqueous Gelatin (25) AOAC (19)
gelatinase (5)
Sterile distilled water (250) + 1% Non-fat dry milk (25); dry whole milk (25) AOAC (19)
aqueous brilliant green (0.45)
Trypticase soy broth (225) Dried yeast (25); spices (25) except for allspice, cinnamon and oregano AOAC (19)
which are diluted 1 :100 and cloves 1: 1000

Trypticase soy broth with 1.25 g K2SO, Onion flakes, onion powder, garlic flakes and garlic powder (25) AOAC (19)
(225)
Nutrient broth (225) Sugar frosting and topping mixes (25) AOAC (19)
Sterile reconstituted non-fat dried milk Cocoa and cocoa products including chocolate (25) AOAC(19) and
(225) SAA (359)
Buffered peptone water (225) All foods unless otherwise specified SAA (359)

231

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 Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot

enrichment may be limited by the competition microorganisms such as coliforms, Proteus and
with other microorganisms, the composition of the Pseudomonas. Raw and unprocessed foods are
food, and the availability of shaker-incubation sometimes inoculated directly into selective
space. In a more recent study, pre-enrichment enrichment media because salmonellae in these
under anaerobic rather than aerobic conditions, products are unlikely to be sub-lethally injured.
Kafel (217) provided higher Salmonella isolation Moreover, pre-enrichment could be detrimental
rates from fish. It has been suggested that the with fresh foods where overgrowth by other
atmosphere of pre-enrichment may play an microorganisms can occur. According to Litchfield
important role in the repair of damaged cells. (248), this is particularly the case with lactose
Van Schothorst et al. (395) demonstrated that broth which is a good growth medium for the
pre-enrichment of foods may be particularly Enterobacteriaceae. Nevertheless, the ICMSF has
important where selective enrichment involves recommended that all food samples be pre- .
the use of tetrathionate brilliant green broth enriched in a non-selective medium prior to
incubated at the elevated temperature of 43°C. selective enrichment (203). Their recommendation
Their investigation was undertaken because of was based on the findings of Edel and
the conflicting evidence in the literature on the Kampelmacher (112) and Gabis and Silliker (143)
usefulness of the direct enrichment of foods in this that pre-enrichment gave higher isolation rates
medium at 43°C. From their experimental data, it than direct selective enrichment in the a~alysis of
was postulated that pre-enrichment provides a frozen meat, poultry and liquid eggs. The SAA has
large population of salmonellae where there are also specified a pre-enrichment step in its method
most probably cells present which are less for the detection of salmonellae in foods (359).
sensitive to tetrathionate, and these will multiply There appears to be insufficient comparative
especially when an equally large competitive flora data in the literature to justifiably exclude direct
is able to 'detoxify' the medium. selective enrichment for all raw and non-frozen
The compositing or pooling of samples prior to foods. In the examination of fresh or chilled
pre-enrichment has been discussed previously as poultry by the whole bird rinse technique, pre-
a practical and economical approach to enrichment is unnecessary and the rinse fluid
Salmonella testing. Wet compositing of pre- may be transferred directly to the selective
enrichment cultures is an alternative system enrichment broths. Cox et al. (72), for example,
which has been frequently employed by the food found that direct selective enrichment of the total
industry in the USA (342). This method involves volume of rinse fluid was effective in detecting low
the transfer of 1 mL aliquots of a number of levels of salmonellae on non-frozen broiler
individually pre-enriched sub-samples into an carcases. They used distilled water as the rinse
appropriate volume (1:10 v/v) of a single selective fluid and added a concentrated solution of
enrichment broth. Price et al. (311) and Silliker selective enrichment broth to give the resultant
and Gabis (342) showed that this method, as solution a single strength concentration. By
compared to individual sample analysis, could be comparison, Thomason and Dodd (378) examined
used for a variety of food products without loss of 208 naturally contaminated samples of raw meat
sensitivity. Furthermore, Price et al. (311) and poultry and concluded that while direct
demonstrated that as many as 25 pre-enrichment selective enrichment gave slightly higher
broth cultures could be pooled, and that the recoveries than did pre-enrichment followed by
enrichment ratio may be varied from 1/10 to 1/50 selective enrichment, both procedures should be
and 1/100, with no apparent reduction of used to obtain maximal recoveries of salmonellae.
efficiency in the recovery of salmonellae. The There are numerous modifications of many
relative advantages of wet compositing include: selective enrichment media reported in the
a. an increase in the capacity for Salmonella literature. Ideally, these media should support
testing of foods; the multiplication of salmonellae to detectable
b. retention of pre-enrichment broth cultures for levels, be sufficiently selective to prevent
subsequent individual analysis if positive over growth by competitors, maintain selectivity
tests are found; and after addition of the sample and allow recovery of
c. the avoidance ofthe hazards and inconvenience all serovars. The different types of selective
of handling large flasks of culture. enrichment media which have been used for the
isolation of salmonellae from foods are presented
Selective enrichment in Table 8.13.
Selective enrichment media are employed in the These media are based on inhibitors such as
examination of foods to encourage the tetrathionate, selenite, magnesium chloride,
multiplication of salmonellae whilst reducing or strontium chloride, the dyes brilliant green and
inhibiting the growth of competitive malachite green, and antibiotics including

238
 Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot
shown by North and Bartram (287) to greatly
improve the growth of salmonellae in the presence
of large amounts of organic material. They also
demonstrated that the quantity and nutritional
quality of peptones and phosphates used in
selenite media affected the recovery efficiency of
the broth. Occupational health and safety issues
for laboratory workers are beginning to impact on
the use of selenite in media. Although the risk is
small and the medium for Salmonella isolation
quite productive, it is possible that the use of
selenite containing media will be restricted or
cease altogether.
Other recommended modifications to selenite
media include the addition of brilliant green
and/or sulphapyridine to prevent the growth of
strains of Proteus, Escherichia, Enterobacter and
Pseudomonas which are not inhibited by high
concentrations of selenite. Stokes and Osborne
(362) showed that the incorporation of 0.0005%
brilliant green in a mannitol taurocholate selenite
medium prevented the development of
Escherichia and Proteus while supporting the
luxuriant growth of salmonellae from very small
inocula (one cell per mL of medium). In a
subsequent investigation, Osborne and Stokes
(299) found that egg products considerably
reduced the selective properties of the selenite
brilliant green medium. They also demonstrated
that the neutralising effect of egg products could
be eliminated by the addition to the medium of
sulphapyridine (0.05%). The latter modification
was considered suitable for the isolation of
salmonellae from a wide range of samples. In the
choice of enrichment media, it should be noted
that broths containing a number of different
selective agents may lead to the inhibition of
strains of salmonellae sensitive to their combined
activities.
Iveson and MacKay-Scollay (208,209) showed,
with their studies in Australia, that strontium
chloride broth gave equivalent or better results
than either Rappaport's or strontium selenite
media in recovery of salmonellae from human,
animal and environmental samples. The selective
property of this medium is based on the inhibition
by the strontium ion of non-pathogenic Gram
negative bacteria. In particular, it was claimed
that in strontium chloride broth Proteus was
suppressed to a greater extent than in selenite or
tetrathionate broths.
Rappaport medium, introduced in 1956, used
the selective agents magnesium chloride,
malachite green and low pH (321). This medium
was not used as widely as tetrathionate or
selenite media, but interest was renewed by
Vassiliadis et al. (402) who modified the medium
by reducing the malachite green (Rappaport R25
240
...
medium) and used it as a secondary selective
enrichment medium. Later the medium was again
modified by reducing the malachite green
concentration still further to 0.004% (401, 404).
This medium was known as Rappaport RIO broth,
later as Rappaport-Vassiliadis (RV) medium, with
a recommended incubation temperature of 43°C.
The R25 modification of Rappaport broth has been
shown to be superior to Muller-Kauffmann (MK)
tetrathionate and selenite F broths for isolation of
salmonellae from chicken giblets (173) and
superior to strontium chloride B for isolation of
salmonellae from polluted water (174). It was
suggested that this medium, incubated at 37°C,
should be used routinely as a single enrichment
medium. Van Schothorst and Renaud (392)
studied the growth of Salmonella in RV medium
and suggested that changing one of the
ingredients from tryptone to soya peptone would
enhance its performance. The RV modification
has gained favour over the R25 modification and
MK tetrathionate for analysis of foods, following
reports by Vassiliadis (398), Tong-pim et al. (386),
Northolt et al. (289) and Vassiliadis et al. (399).
RV medium was found to be superior to both
tetrathionate and selenite cystine broths by AlIen
and co-workers (2) in the analysis of frozen
shrimp. In the study conducted by Northholt and
co-workers, it was noted that MK tetrathionate
medium, when prepared in the laboratory from
base ingredients, gave equivalent results to RV
medium. However, two commercially available
dehydrated MK preparations compared poorly to
RV. This highlights another reported advantage
of RV medium, that is, it is easy to prepare
standardised and reproducible batches. In
addition, prepared RV medium is stable for at
least one month and this is considered an
advantage over other media (398).
Since various serovars may have different
sensitivities to inhibitory substances, it is
generally advised that two dissimilar selective
enrichments be employed for each test (203).
Litchfield (248) concluded in his review that
optimum recovery of salmonellae could be
obtained by using both selenite cystine and
tetrathionate broths in the examination of food
samples. Provision for the parallel use of these
media is specified in the official methods of many
food microbiology laboratories in the USA (19,
134). In Australia, the SAA altered its Salmonella
method (AS 1766.2.5) in 1989 and substituted RV
medium for tetrathionate broth. In this revised
method the RV medium is incubated at 42°C in
combination with mannitol selenite cystine broth
incubated at 37°C. Considering the sensitivity of
some strains of salmonellae to brilliant green dye,
it is considered unwise for laboratories to use only

1 !
Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot
and open-ended tube (Craigie tube). The method
involves the inoculation of mixed cultures into
Craigie tubes placed in larger tubes containing
the same medium. Motile microorganisms move
through twice the depth of medium to reach the
outer surface. Since salmonellae travel fastest
through the media, they are readily cultured from
the surface of the medium in the outer tube. This
technique has been recommended (171) as a
method of secondary enrichment for salmonellae
after culture in selenite F broth and on selective
agar media. In the examination of animal feeding-
stuffs the method was shown to more than double
the number of Salmonella isolations.
Harper and Shortridge (167) developed a
selective and differential motility medium in
Craigie tubes for the routine examination of
clinical specimens. The medium was a modified
Salmonella-Shigella broth in 0.2% agar, and the
method was shown to be more effective in
isolating salmonellae other than S. Typhi from
specimens heavily contaminated with
microorganisms of faecal origin. The spread of a
culture of salmonellae through the Craigie tubes
and surrounding medium is usually detected by
blackening caused by H2S production. Other
motile microorganisms are either inhibited by bile
salts in the medium or migrate more slowly
through the tube. In an earlier investigation,
Stuart and Pivnick (363) obtained similar results
using a modified U tube (test tube with a small
bore side-arm) containing a medium based on
Rappaport's broth in 0.6% agar. In the
examination of food samples, selective motility
media in either Craigie tubes or U tubes could
prove useful as a primary and/or secondary
selective enrichment procedure. The method may
not, however, be reliable in isolating strains
of salmonellae with low motility rates, e.g.
S. Choleraesuis var. Kunzendorf and S. Typhi.
Banwart (28) described a refined U tube
selective motility system for detecting
salmonellae consisting of a glass flask with a
central chamber and three side U tubes connected
to it. The central chamber contained lactose broth
and each side U tube contained different semi-
solid selective or differential agars overlaid with
brain heart infusion broth. In subsequent studies,
Banwart (29, 30), Banwart and Kreitzer (32), and
Banwart et aI. (31) modified the selective media
and demonstrated that Salmonella could be
reliably detected in a variety of foods such as egg
and poultry products, cake mixes and candies.
Moreover, salmonellae could be isolated and
identified in 48 h as opposed to 72 h with normal
cultural procedures. Fung and Kraft (142)
developed a less complex multi-layer motility agar
system using a glass flask with a single straight
242
....
r
\
j
side-arm. The side-arm contained successive
layers of solid agar composed of different selective 1
or biochemical media such as selenite cystine agar jI
and triple sugar iron agar. The body of the flask J
contained lactose broth into which the sample was I
inoculated. This system was shown to detect small I!
numbers of salmonellae in mixed cultures and I
poultry products in the presence of large numbers
of competitive microorganisms. The major
advantages claimed for both systems compared to
conventional procedures are that they are rapid,
sensitive, provide savings of time, labour and
material, and allow the examination of relatively
large sample amounts. The disadvantage is that
special bulky apparatus is required. In the routine
examination of large numbers of samples, the use
of such apparatus would be impractical and incur
additional incubation costs. A more recent method
by De Smedt et aI. (99) appears to alleviate these
short comings. Samples undergo a traditional pre-
enrichment procedure after which 0.1 mL aliquots
are inoculated onto the surface of a semi-solid,
modified RV medium (MSRV) in Petri dishes.
Motile bacteria which have migrated over the
surface of the medium are confirmed as
salmonellae by slide agglutination. This method is
considered to be a rapid alternative to standard
cultural detection of salmonellae and is reviewed
in more detail in the rapid method section of this
paper.
Differential selective agar media
Numerous selective plating media have been
recommended for the culture of salmonellae from
liquid enrichment broths. These media generally
consist of a basic nutritional medium with added
dyes, bile salts, antibiotics, and/or other chemicals
to inhibit the growth of competitors. In addition,
they usually contain an indicator system to
differentiate Salmonella from other
microorganisms. Indicator systems are often
based on H2S production and/or fermentation of a
particular carbohydrate such as lactose, sucrose
or xylose. Commonly employed agar media and
their modifications have been described and
reviewed by Litchfield (248), Fagerberg and
Averts (123) and Fricker (141). Selective plating
media can be differentiated into categories
according to their relative inhibitory action on
Gram negative microorganisms. Fagerberg and
Averts (123) listed in order of increasing
selectivity and differentiation eight agar media
commonly used (Table 8.14).
It is now almost universal practice to use
multiple selective agars to optimise the isolation
of salmonellae from selective enrichment culture.
This approach is used by the FDA, where bismuth
sulphite (BS), hektoen enteric (HE) and xylose

lysine desoxycholate (XLD) agars are used (20)
and by the SAA where XLD and BS agars are
used (359). As discussed previously it is a widely
held opinion in Australia that the selective agars
chosen should not both contain brilliant green
dye; e.g. it is considered inappropriate to use both
BS and brilliant green (BG) agars. An authori-
tative report by Andrews et al. (6) describes the
comparative efficiency of BG, BS, HE, XLD,
Salmonella-Shigella (SS) and desoxycholate (DC)
agars for the recovery of salmonellae from foods.
BG agar has been reported to give excellent
suppression of non-Salmonella enterics, and with
the addition of either sulphadiazine or
sulphapyridine (BGS agar), Proteus spp. and
pseudomonads are greatly inhibited (145, 299).
Read and Reyes (326) showed that the quality of
the brilliant green dye used in either laboratory
prepared or commercial dehydrated media had a
significant effect on the performance of BGS agar.
In an investigation to determine factors affecting
the selectivity of BG agar, Moats and Kinner (271)
concluded that more reproducible results could be
obtained when brilliant green dye is added after
sterilisation of a commercial base medium. Other
modifications of BG agar which have been
demonstrated to improve isolation of salmonellae
include the incorporation of an H2S indicator
(272), the addition of novobiocin (270), and the
combined antibiotic enrichment with sulphace-
tamide and mandelic acid (410). It has been also
suggested that increasing the incubation
temperature from 37-41.5°C or 43°C may further
enhance the selectivity of BG agars (326, 410).
The BS agar of Wilson and Blair is generally
considered to be the best medium to use in
combination with other media. In a Canadian
study published in 1994 (408), the authors
concluded that BS agar should be compulsory for
all Salmonella isolation methods. The advantage
of BS agar lies in its ability to detect rarer strains
of salmonellae which have atypical carbohydrate
reactions, for example, those that ferment lactose
or sucrose. BS agar does not rely on carbohydrate
fermentation to differentiate salmonellae from
Table 8.14. Selective agar plating media for salmonellae
Category
Slightly selective
Moderately selective and differential
Highly selective and differential
I
I
Salmonella
I1
,I I
other bacteria. Blackburn and Ellis (41), for
example, showed that BS agar was especially
useful in the isolation of lactose positive
salmonellae from dried milk and milk drying I
plants. Their investigation revealed that 86
(15.6%) of 552 cultures examined were positive in
lactose fermentation tests. Almost all salmonellae il
appear on this medium as black colonies with a
'11
characteristic jet black centre due to H2S
production. The inhibitory action to coliforms is
attributed to the combined effect of bismuth ~~
sulphite precipitate and sodium sulphite solution.
Although the medium can be used directly after
pouring for the isolation of S. Typhi, Cook (67)
found that freshly poured plates were inhibitory
to other serovars. This inhibition may be reduced ,:r
by 'ageing' the medium in the refrigerator for ~i
3-4 d after pouring. The 'ageing' of the medium is I
considered necessary for the production of the
characteristic Salmonella colony (387). 11:
One of the disadvantages of BSA is that it has
a very short shelf life and should be used shortly
after preparation. AlIen et al. (3) described a
stabilised BSA medium that was comparable with
freshly prepared and overnight aged BSA after
3-4 months of storage.
Other more recent media which aid the
identification of atypical biochemical strains are
novobiocin brilliant green glucose (NBG) agar
(102) and mannitol lysine crystal violet brilliant
green (MLCB) agar. MLCB was first described by
Inoue in 1968 at a meeting of microbiologists in
Japan and further studied and reported on by van
Schothorst et al. (393). These media will differen-
tiate lactose positive salmonellae although the
false positive rate'{or MLCB has been considered
unacceptably high ~ is claimeg . that NBG
agar provides a more con~iSten[ differentiation
reaction of all salmonellae than does BS agar.
Certainly, the reaction on BS agar may vary with
Salmonella strain, medium batch, age and
manufacturer.
In 1990, Rambach (320) described an agar
medium (RAM) which was formulated to provide
a marked colour differentiation of salmonellae
Medium
MacConkey agar
Salmonella-Shigella (SS) agar
Desoxycholate citrate(DS) agar
Hectoen enteric (HE) agar
Xylose lysine desoxycholate (XLD) agar
Bismuth sulphite (BS) agar
Brilliant green (BG) agar
Brilliant green sulphonamide(BGS) agar
243
. ..,
Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot
from other Gram negative bacteria. Salmonellae
were red in colour while E. coli were blue, Proteus
colourless and Citrobacter violet. S. Typhi was
also colourless. Differentiation of salmonellae was
achieved by their ability to produce acid from
propylene glycol and the incorporation in the
medium of the chromogenic substrate X-Gal
(5-bromo-4-chloro-3-indolyl-[3-D-galactopyranoside)
to allow different coloured colonies to be produced
by other members of the Enterobacteriaceae due
to their [3-D-galactosidase activity.
Due to the presence of [3-D-galactosidase
activity in some Salmonella strains, users of RAM
must be cautious in assuming all blue-green
colonies are non-salmonellae (227). Similarly,
Garrick and Smith (146) reported atypical reactions
with salmonellae on RAM and also Citrobacter
freundii as mimicking Salmonella on RAM.
Another approach to selective, differential
isolation was reported by Cox and Stallard (71)
and later expanded on by Cox in 1993 (70). The
agar medium described contained glycerol to
differentiate Salmonella from Citrobacter, mannitol,
lysine and a H2S detection system. The medium
was called lysine mannitol glycerol agar (LMG)
and was reported to be capable of the detection of
S. Typhi, distinguishing lactose and sucrose
positive salmonellae, although not capable of
detecting H2S or lysine decarboxylase negative
salmonellae. A number of chromogenic agars have
also been developed and commercially marketed.
Selective agar plates are usually incubated at
37°C for 24 h. Because BS agar is highly
inhibitory, incubation for 48 h is usually
necessary. It has been suggested that Salmonella
isolations can be increased by the reincubation,
for an additional 24 h, of BGS agar plates which
fail to show growth or have atypical colonies. It is
also relevant to note that Salmonella colonies
develop a wrinkled appearance on BG or BGS
agars when incubated plates are left at ambient
temperature for several days. This phenomenon is
extremely useful in that it often allows
salmonellae to be isolated from a plate which,
after initial incubation at 37°C, may be regarded
as negative. Recognition of typical colony
appearance can be particularly useful where
plates are . crowded with lactose fermenting
.
I mIcroorganIsms.
I It is often recommended that two to three
typical colonies should be selected from each plate
I for biochemical and serological confirmation (19,
1 20, 134, 203, 359). When examining samples for
multiple serovars, at least 20 colonies per plate
should be examined. Maximum yields of
salmonellae may also be obtained by increasing
I
the number of different selective enrichments and
plating media. Harvey and Price (171) claimed
r, L
....
244
that the isolation rate of salmonellae could be
dramatically increased by subculturing swabs
taken from apparently negative plates into
modified Craigie motility tubes containing semi-
solid nutrient agar. The improved recovery rates
were attributed to secondary enrichment in media
independent of selective toxic chemicals.
Biochemical confirmation
There is an extensive number of biochemical tests
which can be used to characterise presumptive
Salmonella isolates. The reactions which are of
diagnostic importance in their identification have
been listed previously in Table 8.1. A detailed
description of these tests is provided by Edwards
and Ewing (122) and Cowan and Steel (69). In the
routine examination of foods, identification of
cultures can usually be accomplished ~ith a
limited number of biochemical tests followed by
agglutination tests with polyvalent Salmonella
antisera. Some of the more commonly employed
confirmatory tests are shown in Table 8.15.
Many non-selective differential agar media
have been developed as biochemical screening
procedures to eliminate false positive isolates.
The advantage of multiple test media is that they
reduce the number of inoculations and the
equipment needed to identify salmonellae rapidly
and with reasonable accuracy. The more frequently
used media have been reviewed by Litchfield (248)
and details are presented in Table 8.16.
According to the methods prescribed by the
SAA (359), suspect colonies are sub-cultured to
peptone water and incubated at 37°C until turbid.
This culture is used to inoculate two nutrient agar
slopes which are used for serological confirmation
and the two biochemical tests recommended,
ONPG and lysine decarboxylase (LD). Generally
both tests are carried out separately in broth
cultures. Proudford (315) developed a simple
procedure for performing these tests in a single
test tube containing a differential agar medium
composed of lysine decarboxylase medium in the
butt and ONPG medium in the slant (OL
medium). This medium subsequently has been
demonstrated to provide a useful method for
screening cultures of suspected salmonellae
isolated from a wide range offoods (314) and from
poultry (64, 96). Workers using the SAA method
(359) must recognise that atypical Salmonella may
give a positive ONPG or a negative LD reaction.
All microorganisms showing a negative
ONPG reaction irrespective of the LD reaction or
a positive LD reaction irrespective of the ONPG
reaction, should be investigated further before
concluding they are not salmonellae. Most
laboratories use additional biochemical tests as a
matter of course. For laboratories using the SAA
 Salmonella

method (359) it is easy to extend the range of such as the API system (Biomerieux), Microbact
biochemical tests by: biochemical test strips (Oxoid), Minitek systems
(a) performing an indole test on the peptone (Becton, Dickinson and Company), and the Micro
water culture used to inoculate the ONPG ID system (General Diagnostics) offer a practical
and lysine decarboxylase tests after and economical method for the rapid
continuing its incubation up to 18 to 24 h, identification of suspected Salmonella isolates.
(b) performing an oxidase test on the nutrient ,
agar slope. Serological confirmation
Substituting tryptone water for peptone Once an isolate has been identified as a
water will enhance the sensitivity of the indole Salmonella sp. by biochemical and morphological I
test. These two additional tests will assist in methods, laboratories must confirm this
screening out some microorganisms which mimic identification serologically. In most cases
Salmonella. Other tests which are useful to commercially available polyvalent 0 and
include are the Voges-Proskauer and urease tests. polyvalent H antisera are used. If positive results
Where serovars with atypical biochemical are obtained, the cultures are forwarded to the
characteristics are suspected, a wider range of reference centre for complete identification. Some
confirmation tests should be performed as a laboratories may wish to proceed further with
matter of routine. identification and the range of commercial
There have been a number of combination antisera available makes possible the
reaction systems developed commercially to identification of many commonly occurring
simplify the biochemical identification of serovars. Laboratories using commercially
Enterobacteriaceae, particularly for laboratories prepared antisera should precisely follow the
with limited facilities for media preparation. manufacturers' instructions. Cultures should,
Many of these systems are miniaturised and allow however, always be forwarded to a Salmonella
up to 24 biochemical tests to be performed Reference Laboratory for verification and
simultaneously. For some laboratories, test kits inclusion in the surveillance reports prepared by
that laboratory.
With the common serovars which are found
Table 8.15. Biochemical tests used for
from a wide variety of sources, e.g. S. Typhimurium,
confirmation of salmonellae there is a need to further characterise strains
Test Typical reactions within the particular serovar. With phage typing
of salmonellae using standard sets of typing bacteriophages it is
KCN tolerance possible to subdivide strains of the serovar into
Urease production phage types which allow for more detailed
+ epidemiological tracing.
Lysine decarboxylase production

p-D-Galactosidase (ONPG) production Enumeration

Citrate utilisation + The numbers of salmonellae in foods may be
H,S production + estimated by direct plating onto selective agar or
Sucrose fermentation by the most probable number (MPN) technique.
Lactose fermentation Direct plating procedures are considered
+
impractical because the development of typical
Dulcitol fermentation
colonies may be obscured by the presence of food
Indole formation

Table 8.16. Non-selective differential agar media for the biochemical characterisation of salmonellae
Medium
Triple sugar iron agar H,S production; fermentation (acid + gas) of dextrose, lactose and sucrose.

Triple sugar iron urea agar As for TSI agar + urease activity.

Lysine iron agar H,S production; decarboxylation of lysine; fermentation of dextrose.

Kligler's iron agar H,S production; fermentation of dextrose and lactose.

Gillies' two tube media i) fermentation of glucose and mannitol; urease activity
ii) fermentation of salicin and sucrose; H,S production; motility.
Kohn's two tube media i) fermentation of dextrose and mannitol; urease activity
ii) fermentation of sucrose and salicin; H,S production; indole formation; motility

Dulcitol sucrose salicin iron urea agar fermentation of dulcitol, sucrose and salicin; urease activity; H,S production

245
.,
Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Ughtfoot
material and/or the growth of large numbers of
the other organisms usually present. By
comparison, the MPN procedure is useful in
enumerating salmonellae in food provided that
consideration is given to the nature of the
product, proper preparation of the sample to
eliminate clumps and to ensure dispersion of
salmonellae, and proper selection of pre-
enrichment and selective enrichment broths and
selective agar media (248). The ability to use an
MPN method is limited in most routine
laboratories, however, because of increased costs,
time and demand on incubation space.
Rapid methods
Due to the time involved (4-5 days) for clearance
of foods for Salmonella by conventional cultural
methods, the development of more rapid detection
techniques generated considerable interest and
development of such methods has and will
continue into the foreseeable future. According to
Swaminathan et al. (367) the possible advantages
of rapid detection of salmonellae are:
(a) reduction of warehousing costs for the food
industry,
(b) ability to respond quickly to contamination
problems,
(c) increased testing of food products for the
presence of Salmonella, thus increasing food
safety, and
(d) an opportunity to buy raw meats and poultry
and perishable food ingredients that have
been tested and found free of Salmonella.
Rapid methods for Salmonella detection from
food have included fluorescent antibody (FA)
stains, enzyme immunoassay (EIA), enrichment
serology, immuno-sensors, fluorogenic staining,
bacteriophage methods, hydrophobic grid
membrane filtration, electrical measurements
metabolic by-products, shortened liquid
enrichment, geneprobes and polymerase chain
reaction (PCR). These techniques have been
reviewed by Ibrahim and Fleet (197), Feng (126),
Blackburn (42) and in Chapter 5 of this book.
Enrichment serology. Semi-solid media have
been used for many decades for isolation of
Salmonella from mixed cultures based on their
ability to migrate faster than most competitive
bacteria. Semi-solid media and serological detec-
tion (enrichment serology) were at first shown to
have unacceptable levels of false negatives.
Attempts to rectify this led to extended cultural
enrichment, giving the technique little advantage
over traditional cultural methods (47). During the
late 1980s however, significant advances in
enrichment serology techniques occurred.
Modified semi-solid Rappaport- Vassiliadis
medium. A selective motility technique using
246
"'---
.
serological detection was reported in 1987 by
De Smedt et al. (99), allowing presumptive
detection of Salmonella within 48 h. Pre-
enrichment was for 20 h, followed by inoculation
of the surface of a MSRV agar plate and
incubation for 24 h to allow for selective migration
of motile salmonellae. Presumptive detection was
performed by serological tests on migrating
cultures. Significantly, this method required no
additional equipment or expense above that of
traditional cultural procedures. In a subsequent
study, De Smedt and Bolderdijk (97) reported that
as few as 60 salmonellae per mL of pre-
enrichment culture, even in the presence of 107
competitive bacteria, were needed for detection of
Salmonella using MSRV. A collaborative study by
15 laboratories where an MSRV method, was
compared to a traditional cultural procedure for
analysis of cocoa, chocolate and sugar
confectionery was published in 1990 (98). The
MSRV procedure was pre-enrichment for 20 h at
37°C followed by inoculation of three drops of the
pre-enrichment culture onto the surface of a
MRSV agar plate which was incubated at 42°C for
24 h. In addition, after 8 h of incubation, three
drops from each of the two selective broths used in
the traditional procedure were inoculated onto
MSRV. These MSRV plates were incubated at
42°C for 16 h. Any migrated cultures were
confirmed as Salmonella by biochemical and
serological means. In this study it was discovered
that some strains of S. Typhimurium did not fully
develop flagella and therefore did not migrate.
Each participating laboratory was requested to
check all MSRV cultures, whether migrating or
non-migrating. No significant difference between
the traditional cultural procedure and the MSRV
procedure was noted after all Salmonella positive
of results (migratory and non-migratory) from the
MSRV technique were taken into account. In
another collaborative study (101), similar
methods were compared and a variety of foods
used. Again no statistical difference between
MSRV and a traditional cultural procedure was
noted. These two collaborative studies tested all
MSRV cultures, whether migratory or non-
migratory for the presence of Salmonella. This
negated the advantage of the original MSRV
procedure, that is, all samples without migratory
zones are considered negative.
Work by O'Donoghue and Winn (294)
published in 1993, showed that a MSRV method
was equivalent to an in-house conventional
culture procedure for the detection of Salmonella
in low and high moisture foods. In this study only
MSRV agar plates exhibiting migratory growth
zones were tested for the presence of Salmonella.
The AOAC has adopted a MSRV procedure for
~

analysis of cocoa and chocolate as first action
status in 1994 (20, 100). The method adopted
." involves pre-enrichment for 20 :!: 2 h at 35°C
followedby inoculation of this culture onto MSRV
1 and into tetrathionate broth. After 24 h of
incubation at 35°C the MSRV agar plate is
checked for a migratory zone which, if present, is
tested for Salmonella. The tetrathionate broth is
inoculated onto MSRV after 8 h of incubation at
35°C.Any migratory zone is likewise checked for
the presence of Salmonella after 16 h incubation
at 42°C. It should be noted that this procedure
does not include testing of non-migratory MSRV
cultures. Presumptive results are obtained within
48 h. A brief Australian report published in 1996
by Dziedziczak and Kabilafkas indicated excellent
results when analysing milk powder samples
using a method based on MSRV. Negative results
were obtained after pre-enrichment and 20 h of
incubation of MSRV (105). Results from Perales
and Erkiaga (306) suggest that semi-solid
Rappaport broth is superior to MSRV when used
at 35°C. Test methods based on the use of MRSV
have AOAC final action status (20).
1-2 TeseM. A product from BioControl
Systems Inc. USA to detect salmonellae in foods is
called the 1-2 TestTM. The test apparatus consists
of a two chambered plastic vial. One chamber
(enrichment chamber) is used for inoculation and
can be supplied containing tetrathionate-brilliant
green broth. The other chamber (motility
chamber) contains a semi-solid motility agar. The
original method employed pre-enrichment of
samples for 18 h at 35°C after which 0.1 mL of
pre-enrichment culture was added to the
enrichment chamber. Polyclonal flagellar antiserum
was added to the top of the motility chamber and
the test incubated at 35°C to 37°C. Salmonella
detection is apparent from a white line of immo-
bilised salmonellae in the motility chamber. The
test was checked after 8 and 24 h of incubation.
A study by D'Aoust and Sewell (91) in which
186 foods were analysed by a standard cultural
method and the 1-2 TestTM revealed that the
1-2 TestTM detected 25 Salmonella positive
samples while the culture method detected 43.
However, two raw poultry samples found to be
Salmonella positive by the 1-2 TestTM were found
to be negative by the cultural method. It is
interesting to note that although the 1-2 TestTM
detected salmonellae in only 25 samples,
salmonellae were isolated from 38 samples
following subculture from the 1-2 TestTM
enrichment chamber. D'Aoust and Sewell made
some suggestions for improving the performance
of the 1-2 TestTM and recommended further study.
Although the 1-2 TestTM received AOAC first
action status for foods in 1989 (133), research into
Salmonella
enrichment protocols which would improve its
performance continued (186, 281, 296). These
efforts lead to a modified procedure for raw flesh
and highly contaminated foods which used a
selective enrichment step performed outside the
1-2 TestTMinoculation chamber. In this procedure,
after pre-enrichment, selective enrichment is
performed in tetrathionate-brilliant green broth
incubated at 42°C for 6-8 h. The inoculation
chamber of the 1-2 TestTM is emptied of its
supplied broth (or the 1-2 TestTM is supplied with
an empty chamber from BioControl) and 1.5 mL of
selective enrichment culture is inoculated into the
chamber. The test is then incubated at 35°C for
14-30 h, although absence of an immobilised band
of cells at 14 h is considered negative. This
modification has received AOAC first action
approval in 1994 (20, 409). The procedure for
foods other than ra w flesh and highly
contaminated foods remained unchanged and the
selective enrichment stage of the test is performed
with the tetrathionate brilliant green broth
supplied in the 1-2 TestTM inoculation chamber.
Oxoid Salmonella Rapid Test. Oxoid Ltd,
UK have also developed a rapid selective motility
enrichment serology method in kit form called the
Oxoid Salmonella Rapid Test (OSRT). The kit
detects motile salmonellae in raw ingredients,
finished products and in factory environmental
samples. The test is performed in a culture vessel
consisting of a plastic culture jar containing two
tubes. Each tube consists of two parts separated
by a porous partition. For each tube the section
below the partition contains a different selective
medium and the section above the partition
contains a different indicator medium. Samples
are pre-enriched in the usual manner after which
a 1 mL aliquot of the pre-enrichmentculture is
used to inoculate a special Salmonella selective
medium contained in a culture vessel. The culture
vessel is then incubated at 41°C for 24 h. If
salmonellae are present in the sample they
migrate from the culture vessel into the tubes,
'I
first through the selective media and then into the j
11
indicator media. A positive reaction which is
indicated by a colour change, must be further
confirmed using a latex agglutination test (also .
supplied by Oxoid). Presumptive positive results I
can only be reported after completion of the
agglutination test. The kit, like most traditional
cultural methods, uses two dissimilar selective
media (RV medium and a modified lysine iron
desoxycholate medium) and differential indicator
media (a modified lysine iron cystine neutral red
medium and a modified Brilliant green medium).
Presumptive results are provided in 48 hand
confirmation is easily done by direct sub-culture
of the indicator media. Performance results
247
 Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot
published by Holbrook et al. (178, 179) indicate
that this technique has good selectivity and
sensitivity when compared to traditional
techniques and other rapid methods. In a pre-
collaborative study the OSRT yielded an
unacceptable false negative rate (8).
Imrnunoassays
Radioimmunoassay. Radioimmunoassay
methods have been developed which are both
selective and sensitive (199). One such method by
Stew art et al. (361) employed the specific
inhibition of dulcitol fermentation by Salmonella
poly H agglutinating serum. Carbon 14 labelled
dulcitol was used and salmonellae were detected
by the absence of production of 14C02 from
cu1tures with added agglutinating serum
compared to control cultures. However, such
assays employ radioisotopes which present
difficulties with safety and disposal. In addition,
the equipment required is expensive.
Enzyme immunoassay (EIA). The EIA is a
well established technique for assaying antigens
or antibodies. It has the advantage over
radioimmunoassays in that it does not rely on
radioactivity as the detection system. Antibodies
labelled with an enzyme detect antigen by
enzymatic conversion of a substrate, usually
resulting in a colour change which can be read
visually or spectrophotometrically. Heterogeneous
EIAs are used to detect bacterial antigens. In this
type of assay, when the enzyme labelled antibody
(ELA) binds in the test system, the enzyme label
remains unchanged. The unbound ELA must be
removed from the test system, leaving only bound
ELA prior to the addition of the substrate. This is
usually achieved by having one of the reactants
fixed to a solid matrix, such as the surface of a
micro titre plate well, a plastic bead or other solid
matrix like titanous hydroxide (198), enabling
unbound ELA to be simply washed from the
system with a buffered detergent solution.
Krysinski and Heimsch (226) first reported
the use of an EIA for detection of salmonellae in
foods in 1977. Since then there has been
considerable development (262, 269, 348, 366)
resulting in commercial EIA kits and excellent
reviews of the subject have been published by
Swaminathan et al. (367) and Ibrahim (196). For
most laboratories, commercial kits offer a conve-
nient way of introducing EIAs for routine use.
The TECRA@ Salmonella visual immunoassay
from TECRA@ Diagnostics Australia (TECRA@
International Pty Ltd) detects flagella antigens
using the sandwich principle of immunoassay.
Salmonellae are detected after pre-enrichment,
selective enrichment and culture in M broth to
ensure flagella development. Cultural procedures
and the EIA can be completed in 48 h. The
248
~---=
TECRA@ EIA is accepted by the Australian
Department of Primary Industry and Energy for
testing foods for compliance with export
standards, and has AOAC Official First Action
status (137). Studies of the TECRA@ EIA by
Flowers et al. (137), Hughes et al. (184) and Jay
and Comar (212) have shown it to be a sensitive
and reliable method. Specificity is high, with false
positive rates between 2% and 7% reported.
Hughes et al. (184) and Jay and Comar (212)
reported no false negatives while Flowers et al.
(137) reported no significant difference between
the TECRA@ EIA and the FDA cultural procedure
(19). A positive result is a definite blue-green
colour in the microtitre well. The assay can be
read visually with no difficulty in interpretation.
This feature of the TECRA@ EIA is &n advantage
over assays which require spectrophotometric
equipment. The TECRA@ EIA is supplied with a
modification of the standard 96 well microtitre
plate produced by Dynatech Laboratories Inc.,
USA. The micro titre plate consists of a base plate
with the wells as separate strips of 12 units.
Wells, used as complete or part strips, are
inserted into the base plate to perform the EIA,
thus making the EIA more flexible than with the
standard microtitre plate format. TECRA@ have
also released TECRA@ ULTlMATM which is a
visual EIA that provides a result in less than 36 h,
reduces selective enrichment to a single broth and
eliminates the need to use M broth.
Organon Teknika Corporation, USA, released
the first commercially available Salmonella EIA
in Australia, the Salmonella Bio-EnzaBeadTM
Screening Kit. The assay detected flagella
antigens, also by the sandwich principle and
utilised magnetic force to transfer the solid matrix
through each reagent. The solid matrix was a
polycarbonate coated metal bead to which
monoclonal antibodies were bound.
The EIA was extensively studied (88, 90, 11O,
115, 131, 132, 135, 385) and received AOAC
official first action approval (13, 132) but has
since been replaced by the Salmonella- TePM
ELISA test system (bioMerieux SA.). The
Salmonella-TekTM detects flagella antigens
monoclonal antibodies coated onto the inside of
microtitre wells instead of onto magnetic beads.
The Salmonella- Tek TM assay has been shown to
consistently detect lower levels of Salmonella in
mixed culture than the Bio-EnzaBeadTM and a
99.1 % agreement between the Salmonella- TePM
method and the standard AOAC culture method
was obtained (81).
A study by van Poucke (391) showed
Salmonella detection as low as 1 to 5 cfu per 25 g
food sample by the Salmonella- TeFM kit method.
When used with raw chicken samples, the

Salmonella-TekTMgave a high incidence of false
positive reactions (356). Modifications to the ElA
method were introduced and included elevated
temperature incubation (42°C) of the
tetrathionate broths to inhibit growth
competing bacteria; addition of novobiocin into
the M broth to reduce growth of Proteus species
and the elimination of the microtitre plate
, agitation and centrifuging of the post enrichment
M broths to simplify the assay. The modified
Salmonella-TekTM was granted first action
approval by the AOAC in 1994 (20, 111).
BioMerieux S.A. have developed a Salmonella
EIA to be used with the Vitek lmmuno Diagnostic
Assay System (VIDA8") to detect Salmonella in
food and environmental samples. The ElA is
performed in the fully automated VIDAS@
instrument. Monoclonal capture antibodies coat
the internal surface of a disposable pipette-tip-
like device. This device acts as the solid phase and
the pipette for the assay. Test sample from the
reagent strip is drawn into the antibody coated
device followed by washing and subsequent
injections of ElA reagents. A fluorescent substrate
is exposed to the enzyme conjugate remaining in
the device and a relative fluorescence value is
calculated automatically by the VIDAS@
computer. Assay results are obtained in 45 min,
following a 42 h cultural procedure. The VIDAS@
Salmonella Assay was compared in a study
published in 1994 with a conventional cultural
method for Salmonella detection in naturally and
artificially contaminated foods. A 92.9% overall
agreement between the two methods and a
sensitivity of 106 to 108 Salmonella per mL of
M broth was obtained (43). A later study in 1997
compared the VIDA8" Salmonella Assay with a
conventional cultural method for Salmonella
detection in naturally and artificially contaminated
foods with 99% overall agreement between the
two methods (82). The VIDAS@ Salmonella has
gained AOAC first action status (20, 82) and it is
now a widely accepted method.
Elisa Systems (Queensland Australia)
produce an EIA for analysis of Salmonella in food
and related products.
Immuno-captureII mmuno-concentration.
The ElAs so far discussed rely on the standard
cultural procedures of pre-enrichment and
selective enrichment to provide enough
Salmonella cells which can then be detected. An
ElA technology which would enable detection at
the resuscitation culture stage, would provide
even more rapid results (184). Two assays have
been commercialised that take advantage of this
approach.
A dipstick-based assay (TECRA@ UniqueTM)
has been developed by TECRA@ Diagnostics in
Salmonella
Australia to detect Salmonella in foods that
utilises an antibody-coated dipstick which is
initially used to capture Salmonella from a pre-
enrichment sample. After washing, the dipstick is
of transferred to M broth for 4 h to allow replication
of the captured Salmonella. The dipstick is then
transferred through ElA reagents and presence of
Salmonella detected via colour development on
the dipstick. All reagents are supplied in a 6 tube
test module and the assay is completed in 4-5 h
after a 16 h pre-enrichment protocol. Total test
time is 22 h. Evaluations of the TECRA@ UniqueTM
in artificially contaminated foods have given a
sensitivity of 1.5 x 104to 6.5 X 104 cfu per mL of
enrichment culture (214) and a 97.5% agreement
between the assay and conventional cultural
methods (17). The TECRA@ UniqueTM has AOAC
first action status (183).
TECRA@ have also developed an automated
system known as the Unique PlusTM which uses
an automated immunoenrichment to eliminate
further selective enrichment steps.
The VIDAS@ lmmuno-Concentration
Salmonella (lCS) has been developed by
bioMerieux S.A. It is an immunological
enrichment technique that relies on immuno-
concentration and a result can be obtained within
24 h of sample enrichment. The sample is
enriched overnight and then 0.8 mL is transferred
to the lCS strip that performs the immuno-
concentration. lmmuno-concentration takes 40
min and the concentrated salmonellae are then
transferred by pipette into lCS broth (bioMerieux)
and incubated for 5-6 h. One mL of the broth is
then heat treated and assayed in the VIDAS@
using the VIDA8" Salmonella strip. The method
has been recommended for AOAC first action
status (238).
Alternatively, the immuno-concentrated
salmonellae can be transferred via a swab from
the lCS strip onto specialised agar plates, the
agar plates incubated and typical Salmonella
colonies confirmed by the usual means. Two
methods using this approach have been
recommended for AOAC first action status (237,
239).
DNA-DNA hybridisation - gene probes. DNA
probes are single strands of DNA which are used
to detect complementary DNA in a target. This
technology has the potential to be used to detect
any microorganism, although discovering
appropriate DNA sequences for use is often a
complex task. A paper by Fitts (128) describes the
typical DNA hybridisation assay:
a. The bacteria or other organisms are applied
to a solid support such as a nitrocellulose
membrane filter. This can be done by simply
spotting a bacterial culture onto the filter, by
249
\
'I Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot

touching the filter to colonies on agar plates, nation of DNA sequences, as in the Gene-TrakTM
or by actual filtration through the membrane. system, provides great assurance that even
b. The bacteria are lysed to release their DNA, mutated strains of Salmonella will not be missed.
and the bacterial DNA is denatured into The use of radioactivity in DNA probes was a
separate strands. disadvantage, especially in the food industry. The
c. The separated DNA strands are then fixed to expense of the equipment needed to quantify
the solid support so that they will not wash radioactivity is also a disadvantage. To this end
away later. Gene- TrakTM has replaced the isotopic kit with a
d. The filters are soaked in a hybridisation DNA hybridisation assay that utilises an
cocktail that contains probe DNA. The probe enzymatically labelled probe and a colorimetric
DNA is most often a double-stranded DNA end point. The colorimetric Gene- TrakTMSalmonella
segment that is labelled by nick-translation. assay consists of a polydeoxyadenosine-tailed
During nick-translation, a DNA strand is capture probe and a fluoresceinated detector
first nicked by a DNAase, then DNA probe which target regions of ribosonal RNA
synthesis is allowed to occur at the site of the (rRNA) that are unique to Salmonella. If rRNA is
nick. The nicked strand serves as a primer for present in the sample, hybridisation takes place.
DNA synthesis, while the opposite strand in The probe target complexes are captured with a
the duplex molecule serves as the template. polydeocythynidine-coated dipstick and the entire
Nucleotides must be added for synthesis to complex is detected by an anti-fluorescein-
occur. These nucleotides can be labelled with horseradish peroxidase conjugate and a
a radioisotope, or with a nonisotopic reporter colorimetric enzyme substrate. For food analysis,
molecule. The double-stranded probe molecule the assay can be completed in 2.5 h, following a
must be denatured before hybridisation. 44 h cultural period (59).
e. When a probe is mixed with test DNA on the Comparative studies have demonstrated that
nitrocellulose filter, the probe sequence finds the colorimetric hybridisation method was
its complementary sequence in the test DNA equivalent to standard cultural methods (59, 80,
and forms hybrid molecules. These hybrids 337, 418) and close correlation to various rapid
contain one radio-labelled strand that can methods (28, 317, 356). The assay has been
then be detected by autoradiography. reported to be non-reactive with sub-genus v.
The first use of a DNA probe to detect Salmonellae (80) and was modified to include a
salmonellae in foods was by Fitts et al. (129). A probe to detect this sub-genus. The modified
DNA probe of 10 unique sequences isolated from colorimetric hybridisation assay was granted
S. Typhimurium reacted with 23 Salmonella official first action method by the AOAC in 1992
species and not with five other members of the (80). D'Aoust et al. (86) reported in 1995 good
Enterobacteriaceae. Further extensive testing, results with the assay and suggested a
using hundreds of Salmonella reference strains modification that would, according to the authors,
from the Centers for Disease Control (Atlanta, enhance the performance of the assay "to a level of
GA, USA) collection demonstrated that two of the unfailing sensitivity and specificity" (86). DNA or
sequences reacted with all Salmonella, three with RNA probes provide rapid detection of Salmonella
over 95% and five with under 50%. A probe in foods, however, specificity is limited and a pre-
comprising all sequences, has been developed enrichment step is still needed to improve
for routine use as the Gene- Trak TM Salmonella sensitivity.
Assay manufactured by Gene-Trak Systems Polymerase chain reaction (PCR).
(Framingham, MA, USA). Polymerase chain reaction (PCR) is a method for
The kit used radioactive labelled DNA which creating multiple copies of a target DNA
detected Salmonella at 1 x 105 organisms per mL sequence. The PCR reaction relies on three
of enrichment broth (106). Results were obtained significant steps; denaturation of the DNA
in 46-53 h and the assay could analyse 96 strands, annealing of the primer to the template
samples simultaneously. Data showed the and the synthesis of new strands via DNA
technique to be specific and sensitive (97, 130, polymerase. The PCR reaction occurs in a series of
136) and the Gene-TrakTM Salmonella assay cycles, resulting in duplication of each piece of
received AOAC official first action approval in DNA with each repetition of the cycle.
1988 followed by final action status in 1996 (20). PCR technology has advanced since the
A major advantage of a DNA probe is that a development of a heat stable DNA polymerase
microorganism always carries its unique DNA. and an automated machine called a thermal
DNA probes are not dependent on the expression cycler. An enzyme isolated from Thermus
of antigens or enzymes which may be subject to aquaticus, a thermophilic bacterium, led to the
environmental influences. The use of a combi- development of Taq DNA polymerase. This

250

polymerase can withstand the high temperatures
involved in the PCR process, therefore only has to
be added to the sample once and not at every
repetition of the PCR cycle.
PCR denaturing, annealing and strand
synthesis occurs at different temperatures, thus
the process is carried out in a thermal cycler. The
thermal cycler allows the denaturation of the two
DNA strands to occur at 90-95°C, annealing of
the primers to the DNA strands at 55°C and strand
synthesis via Taq polymerase at around 75°C.
As the polymerase chain reaction (PCR)
targets the bacterial DNA directly, the degree of
specificity, sensitivity and speed of detection of an
organism within a sample can be increased.
Recently, PCR primers that target specific regions
of the Salmonella gene have been developed and
investigated. A multiplex PCR technique for the
detection of Salmonella on chicken skin was
compared with standard cultural methods. The
PCR technique was found to be more sensitive,
15% positives to 7.4% positives and quicker, 24 h
compared to 3-4 d than the conventional method
(253). Primer sets such as the oligonucleutide S18
and S19 from the omp gene have successfully
detected 40 Salmonella serovars in vitro (229) and
detection of Salmonella via these primers in
biological samples is under investigation.
Detection of Salmonella species by PCR in
contaminated oyster samples has been
investigated. Mter a pre-enrichment step of 3 h,
1-10 cells of Salmonella species were detected by
the PCR (35). Unique PCR primers to detect
Salmonella typhi have been developed based on
the 5S-23S spacer region of a cloned DNA
fragment. The PCR method showed a sensitivity
of approximately 40 Salmonella Typhi cells in a
spiked chicken rice sample (422).
PCR will also target DNA from dead cells
within a sample, however in practice there needs
to be around 103 cfu of target bacteria per mL of
pre-enrichment culture for detection to occur and
this will almost certainly mean that live
Salmonella cells exist in the sample tested. PCR
also requires strict hygiene to minimise the
possibility of contamination of the assay with
genetic material from extraneous sources. The
thermocycling step is usually performed in a
separate room to other parts of the Salmonella
assay procedure. Some laboratories use a type of
cleanroom for this procedure. It has been
demonstrated by Stefanovicova et al (360) that
PCR can be utilised for the confirmation of
presumptive Salmonella colonies, thus reducing
the time required for confirmation to a maximum
of six hours.
A commercial PCR kit for the detection of
Salmonella has been developed by QualiconTM a
Salmonella
subsidiary of E. 1. Du Pont de Nemours and
Company, Wilmington, Delaware, USA. This is
the BA.)('"Salmonella system. A result is available
28 h after commencing analysis (36). A
comparative study of the BA.)('" gel-based assay
and a traditional culture test method has been
completed by Campden and Chorleywood Food
Research Institute (37) and found that the BA.)('"
assay was highly specific and sensitive with the
BA.)('" system generating more positive detections
than the cultural test method. Similar results
were obtained by a study undertaken by Bailey in
1998 (24). DuPont Qualicon have developed an
automated system for performing the assay that
provides for many improvements. The automated
instrument integrates the amplification and
detection steps and a closed-tube system eliminates
the possibility of cross contamination. Custom
software, that is continually being upgraded to
increase sensitivity, analyses the results of the
PCR. The AOAC Research Institute awarded the
gel-based assay performance tested method
status in 1998 (275) and the automated system is
currently undergoing evaluation. The agent for
the BA.)('"in Australia is Oxoid Australia Pty Ltd.
Electrical measurements - conductance. This
technique relies on the change of conductance
which occurs in a medium as microorganisms
grow. Microbial growth in nutrient media results
in an accumulation of small, highly charged
molecules such as amino acids, fatty acids and
organic acids. The electrical conductance of
microbiological media alters with these changes
in media constitution. Usually conductance and
capacitance increase and impedance decreases.
These changes can be detected by appropriate
electronic detection systems. The three
commercially available systems are the
Bactometer (bioMerieux, Australia), Malthus-AT
(Radiometer Ltd., Manor Royal, Crawley, West
Sussex) and RABIT impediometer (Don Whitley
Scientific, Australia). All are computer driven
automated systems.
Conductance detection of salmonellae was
first reported by Easter et a1. in 1982 (108). The
reduction of trimethylamine-N-oxide (TMAO) to
trimethylamine (TMA) by salmonellae was used
to produce a large change in medium
conductance. Later, Easter and Gibson (107)
proposed a method for Salmonella detection in
food, using a pre-enrichment broth of buffered
peptone water with 0.1 % TMAO and 0.5% dulcitol
at pH 7.2 (BPW/T/D) and selective enrichment in
selenite cystine broth with 0.5% of sodium
biselenite, 0.5% dulcitol instead of lactose and
0.5% TMAO (SC/T/D).
Since most Salmonella ferment dulcitol but
not lactose, dulcitol was used in place of lactose in
251
 Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot

the medium in order to enhance the conductance A comparison between conventional culture
change. Conductance was measured by a method and a conductance method using SC/T/D
prototype device, a Malthus instrument or a and a lysine medium demonstrated that the
Bactometer. For Salmonella detection, the methods were equivalent for analysis of animal
magnitude of conductance and the time for this to feeds (353). A conductance method using SC/T/D
occur are important. Normal conductance and MLD has been given official first action
responses for salmonellae in SC/T/D are status by the AOAC (153) in 1992.
approximately 500 pS and a maximum rate of Salmonellae can be recovered from the
conductance change of 15-25 3rS/l0 min (295). Bactometer, Malthus and RABIT systems by
The conductance method was in complete direct plating onto selective agar for confirmation
agreement with conventional cultural methods for and further identification. The conductance
the naturally and artificially contaminated foods methods developed to date can screen for
tested. However, some Citrobacter strains were salmonellae within 48 h, 18-24 h for resuscitation
found to produce positive conductance responses. and up to 24 h for detection of the conductance
The SC/T/D medium was modified by Gibson change in the detection medium. At present, the
(152) and Ogden and Cann (295) chiefly by electronic equipment is expensive and this is
replacing dulcitol with mannitol (CS/T/M). In likely to restrict its use. Despite a great initial
addition, the selenite concentration was reduced interest and much published work, this metho-
from 0.5% to 0.4%. The use of mannitol enabled dology has not gained widespread acceptance.
dulcitol negative salmonellae to be detected. Hydrophobicgrid membrane filter method.
Gibson (152) recommended resuscitation in The hydrophobic grid membrane filter (HGMF)
buffered peptone water with added mannitol and allows the enumeration of a higher number of
dimethylsulphoxide (DMSO). DMSO is less colonies than on a conventional filter. The
expensive than TMAO and successfully induces hydrophobic grid restricts the lateral spread of
TMAO reductase. Some strains of Salmonella most microbial colonies and this property makes
tested were not detected using SC/T/M and possible the detection of specific microorganisms
moreover some Citrobacter and E. coli strains even in the presence of quite high levels of
gave positive responses (295). Accordingly it was background microflora. The HGMF also provides
recommended that both SC/T/D and SC/T/M a suitable subject for computer scanning since the
should be used in conjunction with one another. colonies are of regular size and appear only in
Arnott et al. (21) in 1988 described the use of defined areas of the filter (between the grid lines).
a modified lysine decarboxylase medium (MLD) Detection of salmonellae in foods by HGMF
used in combination with SC/T/M for the detection was reported by Entis et al. (119). This method
of salmonellae in confectionery products. This employed a pre-enrichment step of 18-24 h, 6 h
medium contained yeast extract, glucose, selective enrichment in broth followed by
L-Iysine, ferrous ammonium sulphate, sodium filtration through a HGMF. The HGMF was
thiosulphate and 0.08% sodium biselenite at placed onto selective agar and incubated for 24 h.
pH 6.1. The two media detected all salmonellae Replicate plating was then performed using fresh
tested except Salmonella Pullorum. Microorga- HGMF transferred to a range of selective agars
nisms which gave false positive reactions with the with incubation for a further 24 h. Suspect
system were strains of C. freundii, E. coli, colonies were then sub-cultured and confirmed
Klebsiella pneumoniae and Serratia marscescens. biochemically and serologically. This method
Pugh et al. (316) claim to have increased the achieved results in 2-3 d after sample receipt and
confidence of conductance detection of was able to be automated. A manual version of
salmonellae. Their approach was to use, after pre- this method was adopted as Official First Action
enrichment, SC/T/D and MLD as previously by the AOAC in 1984 (116).
described and as an addition include The HGMF method was further improved by
bacteriophages specific for Salmonella. The assay using immunological detection (58). An enzyme
then comprised SC/T/D and MLD with and labelled antibody (ELA) stain was used to directly
without bacteriophages. The bacteriophages used detect Salmonella colonies on the HGMF. This
were Felix 0-1 and G47 of Gudel and Fey (163). eliminated the need for replicate plating to a
Two conductance responses are produced if range of selective agars. The ELA comprised
salmonellae are present; the normal response for pooled Spicer-Edwards H antisera and conjugate
Salmonella and an altered response for (HRP-protein A). Mter the HGMF is stained it
bacteriophage effected Salmonella. The can be submitted to computer counting. Using
combination of the two response curves provides a ELA detection, results are available in 48 h. False
more sensitive detection criterion. positive results can occur due to strains of C.

252
l

freundii being found to cross-react with the ELA.
This method was updated in 1990 and given
official interim first action status for all foods
(117). This update utilised EF-18 agar as a
replacement for selective lysine agar (SLA). Entis
and Boleszczuk (118) reported the equivalence of
[ the HGMF method using EF-18 agar to the
AOAC/BAM Salmonella reference method. The
HGMF procedure using EF-18 agar currently has
first status from the AOAC (20).
Magnetic separation. Magnetic separation
involves the separation and concentration of
specific target bacterial cells from a mixed
suspension. Immunomagnetic separation (IMS)
utilises antibody-coated magnetisable beads to
trap target cells and a high magnetic field to
separate the target cells from a mixed suspension.
IMS is a rapid selective enrichment technique.
Subsequent selective plating, or rapid detection
techniques, are required for the detection of
Salmonella. Parmar et al. (305) used IMS and
conductance measurements to detect Salmonella
in milk powder. Salmonella Enteritidis was
detected in 7.5 h, after a 6 h incubation period,
from an initial inoculum size of 20 cells per mL of
pre-enrichment culture.
DynabeadsTM, from Dynal Biotech, Oslo,
Norway are superparamagnetic, polystyrene
beads coated with anti-salmonella serum. These
can be used to detect Salmonella from 6 h pre-
enrichments of minced meat (405). DynabeadsTM
Anti-Salmonella (Dynal, Oslo, Norway) is a
commercially available immunomagnetic separation
procedure: 20 fll of the DynabeadsTM are added to
1 mL of an overnight pre-enrichment broth and
immunocapture occurs for 10 minutes. A to perform and relatively inexpensive. Automation
magnetic field is applied resulting in capture and
isolation of the bead target bacteria complex, the
beads are washed and plated onto selective agar.
In a study to detect Salmonella from 180 poultry
samples, the IMS technique proved superior (135
positives) to the MSRV method (33 positives) and
the conventional Salmonella method (98
positives) (78). IMS used to detect Salmonella in
45 spiked skim milk powders showed no false
negative results, compared with seven for selenite
cysteine, two for RV and one for Muller-Kauffman
tetrathionate enrichment broths (105). Further
performance studies conducted by Cudjoe et al.
(79) and Shaw et al. (340) demonstrated that
Dynabeads showed superior recoveries to the
standard cultural techniques.
Rapid methodology overview
Regardless of the methods and techniques
employed, isolation and culturing of salmonellae
from samples remains important for final
Salmonella
confirmation and complete identification. Identifi-
cation of isolates is of particular importance for
epidemiological data collection. Accordingly, it
remains desirable that all samples positive by
rapid methods should be confirmed by sub-culture
of an appropriate portion of the test. This requires
that selective enrichment and/or enrichment
media are kept until completion of the assay.
While the culture of salmonellae remains the
accepted procedure for confirming the presence of
Salmonella, some rapid techniques will always
yield unconfirmed positive results. Indeed many
investigators of rapid methods conclude that
unconfirmed positive results, which cannot be
satisfactorily explained as false positives, may in
fact be true Salmonella detections, which are
unable to be confirmed culturally. This is not
surprising, as it is well understood that for a
variety of reasons traditional cultural procedures
will not always detect small numbers of
salmonellae in certain samples. The factors that
can influence recovery rates include the sensitivity
of the methods, the susceptibility of the strains of
salmonellae to inhibitors in the food or added to
the media, over growth by competitors during
incubation, etc. Despite these deficiencies,
cultural confirmation will remain the criterion of
Salmonella detection until another method or
methods becomes widely accepted as equivalent
or an improvement.
There is no doubt about the benefits of rapid
Salmonella detection (at a minimum it allows a
faster response time to contamination). However,
in order to gain acceptance, rapid methods must
be at least as sensitive as cultural methods, easy
is not essential but is an advantage. For a rapid
method to be considered, a significant advance
over traditional cultural methods, the detection
time must be within 48 h and preferably within
24 h. Most rapid methods now meet this criterion.
Introducing new techniques of any type into a
laboratory should not be done without consider-
ation of the costs and benefits. Traditional
methods of Salmonella analysis are often claimed
to be laborious, complicated and in some cases
beyond the capabilities of small laboratories.
However, for many food microbiology laboratories
the cultural detection of salmonellae is a very
mundane, routine procedure, which is accomplished
economically and with minimal labour input per
sample. This is not to suggest that some alter-
native techniques do not offer potential savings in
labour, but such claims should be well studied. In
the main, the major advantage of rapid metho-
dology centres on shortening the duration of the
test rather than reducing the labour per test.
253
\
 Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot
Control
To reduce the incidence of salmonellosis, a multi-
faceted approach is needed. Epidemiological
surveillance can define the incidence
salmonellosis, identify vehicles and factors that
are important in the spread of Salmonella, and
evaluate the effectiveness of preventative
measures.
Education and training are important
because much foodborne disease results from
ignorance. People handling foods should have the
knowledge needed to control the hazard of
Salmonella infection. This extends from the farm,
where good animal husbandry and horticultural
practices have an influence on the extent of
contamination, to the consumer, where
epidemiological data indicate that considerable
mishandling occurs. Food service and home
preparation are the major places where
mishandling has caused foodborne illness (206).
In food processing and food service establish-
ments' foods undergo a number of processes
which have significant impacts on the incidence
and on the numbers of salmonellae. The risk of
foodborne illness is increased when there is a lack
of awareness of the microbiological hazards
associated with failure to control these processes
or treatments.
Sampling foods for salmonellae as a means of
assessing the safety of the food is neither practical
nor cost-effective (206). Sampling and testing
cannot detect low levels of contamination with a
high degree of confidence. Failure to detect
salmonellae implies that the extent
contamination is below a level set by the sampling
plan. If 30 samples from one batch of food were
tested and no salmonellae were found, we would
have 95% confidence that the extent of contami-
nation was below 11.6%. This would be true
provided that the organisms were homogeneously
distributed and that the methodology used was
able to detect the salmonellae if they were
present. To have the same confidence that
salmonellae would be detected in a batch with an
incidence of 1%, 300 samples would need to be
tested (205). For ready to eat foods,
manufacturers would aim to have contamination
rates several orders of magnitude below 1%, and
so it is clear that end product sampling has severe
limitations in assuring food safety.
The HACCP approach should be applied to
control salmonellae in foods (206, 347). The
establishment of an effective HACCP program
requires microbiological expertise (a) to analyse
the Salmonella hazards associated with different
foods; (b) to identify the control points critical in
254
keeping growth, death and survival of
salmonellae, and contamination with
salmonellae, under control; (c) to establish
monitoring procedures, acceptable limits at these
critical control points (CCP) and the action to be
of
taken when the limits are exceeded; and (d) to
determine the nature and frequency of
verification procedures to ensure that HACCP is
working correctly. Though it is not possible to
outline control steps for all the variety of foods in
which salmonellae should be controlled, some
have been outlined for a number of different foods
(206, 347).
For unprocessed agricultural commodities
such as vegetables, fruit and spices, contamination
may be from irrigation water and other water
inputs, manures, some fertilisers, or from workers
or equipment used in harvesting. The application
of HACCP approaches can reduce the risk of
Salmonella. Chemical treatment of minimally
processed fruits and vegetables, seeds and their
sprouts, has been extensively investigated to
reduce contamination levels of pathogens
including Salmonella. Whilst some treatments
are effective in reducing Salmonella numbers on
sprouted seeds, elimination of the pathogen is
difficult (39, 413, 414). High concentrations of
chlorine are required to achieve reductions in
Salmonella of 1 to 2.5 loglO cfu/g. Chemical
treatment on its own cannot be relied upon to
render the products safe. Irradiation of alfalfa
sprouts may eliminate Salmonella (319).
Salmonella outbreaks in unpasteurised orange
juice and apple ciders have elicited investigations
for alternative controls. Combinations of
of intervention treatments using heat and freeze-
thaw cycles can result in 5 10glOreductions in S.
Typhimurium DT104 numbers (388).
For intensively reared animals, a number of
steps can be taken to reduce the entry and spread
of salmonellae. A partial list includes: the use of
Salmonella free breeding stock and feed including
meat and bone meal, regular cleaning of premises
and particularly between herds or flocks, cleaning
of watering devices and feed troughs, replacement
and disinfection of used litter, restricting access of
insects, rodents, wild birds and people to the
animals. Some countries appear to be able to
produce animals with low contamination rates,
but in general salmonellae are often present in
intensively raised poultry and frequently found in
pigs. At the farm level, the problem of excluding
salmonellae is complex and a number of factors
have to be simultaneously controlled.
For free range or pasture raised animals, it is
more difficult to exclude salmonellae since they
may be present in the soil and on pasture from
previous herds, in natural surface waters,
 Salmonella

incoming stock, and wild animals. Good animal
husbandry practices can minimise contamination
ofareas of the farm and of the animals.
Salmonellae, at present, cannot be eliminated
from animals on farms, so that animals will be
slaughtered that are carrying these organisms on
their external surfaces and in their intestinal
applicable for preventing typhoid fever. As
S. Typhi in endemic areas is transmitted by food
and water, it is important to use cleanliness in
food preparation and handling, provide suitable
hand washing facilities for personal hygiene and
to boil milk and water before consumption.
Latrines have to be organised at a site distant I

tracts. Skinning, dehairing, or defeathering, from a source of drinking water. Chlorination of

evisceration and chilling are critical control points suspected public and private water supplies is
in the abattoir and control at these steps can recommended. Travellers in countries with risk of
reduce the extent of carcass contamination with typhoid should avoid consumption of raw
salmonellae. There is no system of slaughter used vegetables, shellfish, or peeled fruits. They should 'I
that will entirely prevent carcase contamination, only drink capsulated or closed bottled beverages.
and it should be assumed that raw meats will, Vaccination is highly recommended (138).
from time to time, contain salmonellae. However
the use of carcase surface treatment with organic
acid solutions is a well accepted practice to assist References
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255