Efficiency of Different Enrichment and Isolation Procedures For

7/27/2019 Efficiency of Different Enrichment and Isolation Procedures For

1/8

Journal

of

Appl ied

Bacteriology 1995,

79

360 367

Eff iciency of d i f ferent enr ich m ent and iso lat ion pro ced ures for

the detectio n of Salmonella seroty pes in edib le of fa l

G. A r r o yo and J.A. Arroyo

Departamento de Microbiologia 11 Facultad de Farmacia and 'Departamento de Microbiologia 111 Facultad de

Biologia Universidad Complutense Madrid Spain

5209/01/95: rece ived 24 January 1995, rev ised 5 Apr i l 1995 and accepted 10 Apr i l 1995

G . A R R O Y O

A N D

J . A . A R R O Y O . 1995. Rapid detection systems for Salmonella in foodstuffs are

currently being developed. However, existing standards still call for application of traditional

methods employing pre-enrichment followed by selective enrichment and isolation. The

efficacy of various methods was tested using

264

chicken and lamb organ meats.

Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in

Tetrathionate Brilliant Green Broth (TT B) at 37 C, Selenite Broth with Brilliant Green and

Sulphapyridine at 37C and 43 C, and Rappaport-Vassiliadis Broth (RV 10) at 42C. The

isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen

Enteric Agar (HEA) and Salmonella-Shigella Agar.

no.

6579

and enrichment in

TTB/37 C

followed by isolation in HEA, no longer

recommended

by

that standard, produced the best results. Low percentages of positive

samples and difficulties in detecting

Salmonella

are the result of interference by competing

organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment.

A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with

Sal m. enteritidis Sal m. kapemba

and

Salm . virchow

and the preceding experiment was

repeated. All the TSB and egg samples tested positive, but the percentage of positive samples

from the lamb and chicken liver was only 81-92%. Recovery of the salmonellas did not

depend upon the method employed or the serotype inoculated but instead on interference by

competing flora and the numbers of

Salmonella

present in the samples.

Enrichment in RV/42 C followed by isolation on BGA as recommended by I S 0 standard

I N T R O D U C T I O N

Salmonellas are responsible for most cases of food poison-

ing in

the developed world. Foods such as meat, eggs,

poultry and organ meats are common vehicles of salmonel-

losis. For that reason, rapid detection methods based pri-

marily on immunological and genetic characteristics are

under development. However, the standards presently in

force in many countries recommend traditional methods,

which are slower, requiring

a t

least

5

d, though they can be

quite reliable when applied by experienced laboratory tech-

nicians.

A variety of methods are in use, and their success rates

depend upon a number of different factors. They employ

pre-enrichment in buffered peptone water, followed by

Correspondence

t o

:

Dr G.

r royo Jul ian

Romea

9

28003-Madrid

Spain.

selective enrichment in more than one medium, usually

Muller-Kauffmann Tetrathionate Broth and Selenite

Cystine Broth incubated a t 37C or

a t

43 C, and subse-

quently selective isolation on various solid media (Anon.

1981). Van Schotthorst

et a l .

(1987) recommended enrich-

ment

in

Rappaport-Vassiliadis (RV) broth

a t

43 C, and

Beckers

e t

a l . (1987b) proposed replacing the Tetrathionate

method with the RV method

in

the I S 0 standard (Anon.

1990).

The selective effects of the different media are mainly

based on the addition of substances that inhibit the growth

of contaminating microflora and prevent the proliferation of

competing microflora, on the incubation temperature and

the inoculum size employed. Studies on some of these

inhibitors (Arroyo and Arroyo 1995) have often yielded dis-

appointing results. The numbers of competing Entcrobac-

teriaceae and Pseudomonadaceae

in

many natural foodstuffs

995 The

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2/8

M E T H O D S

FOR SALMONELLA D E T E C T I O N 301

must be assumed to be higher than the numbers of Salmon-

ella

and those competing organisms are also capable of

withstanding the same inhibitor concentrations as salmon-

ellas during pre-enrichment and isolation, thu s masking th e

presence of salmonellas and giving rise to false positives

(Rhodes and Quesnel 1986).

The addition of stains, e.g. brilliant green, to the pre-

enrichment media combined with raising the incubation

temperature to 43C has produced varying results for

Tetrathionate Broth (Van Schothorst et al. 1977). Arroyo

(1990) reported that temperature affected the growth of

certain Salmonella serotypes, making detection more diffi-

cult.

T he use of antibiotics and chemical therap eutic agents in

the media has also been proposed. Osborne and Stokes

(1955) recommended adding sulphapyridine to enhance the

effectiveness of S elenite Br oth.

The success of the isolation media depends basically

upon the enrichment step employed and the number of

competing organisms that survive that step.

Besides the international standards, the recommen-

dations of the National Reference Center at the Carlos I11

Health Institute in Madrid are usually followed when ana-

lysing food and drink in Spain (Pascual Anderson 1993).

The object of the present study was therefore to evaluate

the efficacy of the recommended enrichment media and the

influence of incubation temperature using Selenite Broth

with added brilliant green and sulphapyridine. T h e efficacy

of four commonly used solid isolation media was also

tested.

T he foodstuffs employed in this study were typical natu-

rally contaminated sources of salmonellas, namely, lamb

organ meats and chicken liver, purchased at markets in

Madrid (Experiment 1). Tryptone Soy Broth (TSB), eggs

and lamb and chicken liver were also artificially contami-

nated with three Salmonella serotypes (Experiment 2).

MATERIAL S AND METHODS

Experiment 1: Naturally contaminated samples

A

total of 264 organ meats purchased retail at 10 markets in

Madrid were examined. The organs used were chicken

livers (78) and lamb organs (186), including liver (46), lung

(46), heart (46), oesophagus (46), and spleen

(2).

The organ

meats were transported to the laboratory in

a

portable

cooler at 4C. Examinations were performed on the day of

purchase.

Twenty-five grams of each sample were weighed out

using sterile instruments under aseptic conditions, added to

225 ml of Trypto ne Soy Broth (T SB), and homogenized in

sterile bags in a Stomacher model 400 Lab Blender for 2

min.

Microbiological examinations

The microbiological examinations employed differed some-

what from standard m ethods.

(a) Pre-enrichme nt was performed in T S B incubated a t

37C for 18 h (Arroyo 1990).

(b) Selective enrichment was performed by transferring

10

ml of the pre-enrichment culture to a flask containing

100 ml of Muller-Kauffmann Tetrath ionate Broth

(T TB ) containing

1

ml

of

a 0.1% (w/v) solution of bril-

liant green, followed by incubation at 37C for 24 h.

Two flasks, each containing 100 ml of Selenite Broth

(SC ) containing 1 ml of a 0.1 (w/v) solution of brilliant

green and sulphapyridine were likewise inoculated with

10

ml of the pre-enrichment hom ogena te; one of the

flasks was incubated at 37 C, the other at 43 C, for 24 h

(Arroyo 1990). Finally,

0.1

ml of pre-enrichment

medium was transferred to 10 ml Rappaport-Vassiliadis

Broth (RV 10) (Vassiliadis 1983) and incuba ted at 42 C

for 24 h.

(c) Selective isolation was performed by streaking the sur-

faces of plates containing Brilliant Green Agar (BGA),

Hektoen Enteric Agar (HEA), Salmonella-Shigella Agar

(SSA) and Deoxycholate Citrate Agar (DCA) with the

T T B and S C selective enrichment media. T he plates

were all incubated at 37C for 24-48 h. T h e RV selective

enrichment medium was inoculated only onto BGA

(Vassiliadis

et al.

1978) and incubated a t 37C for 24-48

h. Colonies suspected of belonging to the genus Salmon-

ella were isolated in test tubes containing Tryptone

Soy

Agar (TS A) incubated at 37C for 24-48 h.

(d) Identification of salmonellas was carried out using mor-

phological, biochemical (H olt 1993) and serological tests.

Strains identified as Salmonella were serotyped at the

National Reference Center at the Carlos I11 Health Insti-

tute in Madrid.

Experiment

2:

Arti fic ially contaminated samples

Pure cultures.

Pure cultures of

Salm . enteritidis Salm .

kapemba

and

Salm. virchow

isolated from lamb and chicken

organ meats were grown on TSA slants for 24 h and then

washed from the slant with sterile physiological saline

solu-

tion. T he suspension was diluted with sterile saline solution

to an optical density of 0.2 at 540 nm measured with a

Bausch and Lomb model Spectronic 20 spectrophotometer.

Serial dilutions were prepared from this stock suspension to

995 The Society for Appl ied Bacter io logy, Journal of Applied Bacteriology 79, 360 367


3/8

362

G . A R R O Y O A N D

J . A .

A R R O Y O

yield the appropriate number of cells. That number was

determined by plating 0.1 ml of the diluted suspension on

TSA plates and incubating at 37C for 24 h.

Table 2 Efticiency of the four enrichment procedures in terms of

the number of isolated serotypes

Enrichment Isolationt

Y

Serotype

No.

I n o c u l a t i o n . Inoculation took place as follows :

(a) Three sets of 44 samples of 225 ml of TSB were each

inoculated with cultures containing 1000 cells of S a l m .

enteritidis Salm.

kapemba

and S a l m .

virchow

respec-

tively. These

132

samples were incubated at

37C

for

18

h. Inoculation of the enrichm ent and isolation media and

detection of salmonellas were then as already described

in Experiment

1

(b)

A

total

of 132

pasteurized eggs purchased at

10

markets

in Madrid were washed in sterile water under aseptic

conditions, cracked and the contents of each egg homog-

enized in 225 ml of TSB. Thre e groups

of

44

of

these

samples were then inoculated with cultures containing

1000 cells of each of the three aforementioned serotypes,

respectively, and incubated at 37C for 18 h. Inoculation

of the enrichment and isolation media and detection of

salmonellas were then as already described in Experi-

ment

1 .

(c) A total of 132 samples of each lamb liver and chicken

liver, purchased at 10 markets in Madrid, were homoge-

nized in a Stomacher model 400 Lab Blender (each

sample consisting of 25 g of organ meat in 225 ml of

TSB). Three groups

of 44

samples

of

each

of

these two

organ meats were inoculated with cultures containing

1000

cells of each

of

the aforementioned serotypes,

respectively, and incubated at

37C for 18

h. Inoculation

Table

1

Number and percentage of the different Salmonella

serotypes detected in the 260 processed and confirmed strains

TTB/37 C*

SC137 C

SC/43 C

RV142 C

Total

47 18.07 Salm. enteritidis

t yphimurium

virchow

worthington

kapemba

give

n antis

cubana

Salm. autoagglut.

96 36.92 enteritidis

typhimurium

virchow

worthington

kapemba

give

havana

arizona


virchow

worthington

kapemba

Salm.

autoagglut.


typhimurium

virchow

morthington

kapemba

infantis

brandenburg

anatum

agona

260 99.98

10

7

14

4

3

3

3

1

2

4

7

40

25

5

4

4

7

6

37

12

1

2

5

8

19

4

3

12

3

2

3

260

Serotype No.

Salm . enteritidis

typhimurium

virchow

worthington

infantis

kapemba

give

brandenburg

havana

anatum

arizona

agona

cubana

Salm.

autoagglutinable

14 serotypes

25

22

110

45

15

12

7

3

4

2

7

3

1

4

260

9.61

8.46

42.30

17.30

5.76

4.61

2.69

1.15

1.53

0.76

2.69

1.15

0.38

1.53

99.92

* Enrichment broths and incubation temperature : TTB, Tetra-

thionate Brilliant Green Broth, 37C; SC, Selenite Broth (with

brilliant green and sulphapyridine), 37C and 43C; RV,

Rappaport-Vassiliadis Broth (RV lo), 42C.

t

Numb er and percentage of positive isolation.

of the enrichment and isolation media and detection of

salmonellas were as already described in Exp erim ent 1 .

R E S U L T S

Exper iment1

Of the 264 lamb and chicken organ meats examined, 83

(31.43%) tested positive for Salmonel la . A total of 260

strains isolated were confirmed to be on e of

14

serotypes of

995

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4/8

M E T H O D S F O R

S A L M O N E L L A

D E T E C T I O N

363

Table

3

Efficiency of enrichment and

isolation procedures in terms of positive

strains of Salmonella detected

Isolation procedu re7

Enrichment procedure BGA DCA HEA

SSA

No. ( )

TTB/37 C* 7

(2.69)

SC/37 C 14

(5.38)

SC/43 C 13

(5)

RV/42 C 59

(22.69)

No. 93

( /.I (35.76)

15

(5.76)

27

(10.38)

15

(5.76)

57

(21.9)

15 10

(5.76) (3.84)

28 27

(10.76) (10.38)

12 18

(4.61) (6.92)

55 55

(21.13) (21.13)

47

(18.05)

96

(36.9)

58

(22.29)

59

(22.69)

260

(99.93)

Enrichment broths and incubation temperature : TTB, Tetrathionate Brilliant Green

Broth, 37C; SC, Selenite Broth (with brilliant green and sulphapyridine), 37C and 43C;

RV, Rappaport-Vassiliadis Broth (RV lo), 42C.

t

Isolation media: BGA, Brilliant Green Agar; DCA, Deoxycholate Citrate Agar; HEA,

Hektoen Enteric Agar; SSA Salmonella-Shigella Agar.

No.,

Number of strains detected.

Percentage

of

positive isolations given in paren theses.

Table

4 Serotypes detected according to the enrichment and isolation procedures used

Isolation procedure

Enrichment

procedure BGA DCA HEA

SSA

TTB/37 C

Sal m typhimurium Sal m enteritidis

virchow typhimurium

virchow

worthington

infantis

give

Salm

autoagglut.

SC/37 C

SC/43 C

RV/42 C

enteritidis

virchow

worthington

give

arizona

virchow

worthington

Salm autoagglut.

enteritidis

typhimurium

virchow

worthington

kapemba

infantis

brandenburg

anatum

agona

virchow

worthington

arizona

enteritidis

virchow

worthington

Salm enteritidis Sa lm typhimurium

typhimurium virchow

virchow kapemba

worthington infantis

in antis give

give

cubana

Salm

autoagglut.

virchow

worthington

kapemba

typhimurium

virchow

worthington

kapemba

enteritidis

virchow

worthington

kapemba

give

havana

arizona

enteritidis

virchow

worthington

For abbreviations see Table 3.

995

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Applied

Bacteriology,

Journal of Applied Bacteriology

78

360-367


5/8

364

G .

A R R O Y O A N D J . A . A R R O Y O

Salmonella. The distribution of serotypes, including

autoagglutinable strains, appears in T able 1 .

Table

2

shows that

96 Salmonella

strains

(36.92%)

were

detected using the

SC

enrichment medium incubated at

37C. Incubation of this same medium at 43C resulted in

only

58 Salmonella

strains

(22.30%).

Enrichment in TTB

incubated at

37C

yielded

47

positive strains

(18.07%),

while enrichment in RV 10 incubated at 42C yielded 59

(22.69%) positive isolations. Table 2 also lists the different

serotypes detected using each of the media. TTB/37 C and

RV/42 C

produced nine

of

the

14

serotypes detected and

SC/37 C

produced eight.

SC/43 C

yielded the fewest

serotypes (5).

Salmonella virchow

was the most abundant serotype and

was detected with

all

four of the enrichment methods used.

On the other hand, not all the minor serotypes present were

detected by all the different methods.

Table

3

gives the number and percentage of positive iso-

lations on the solid media for each of the selective enrich-

ment media employed. BGA yielded

93

positive isolations

(35.76%). Detection rates differed substantially, with TTB/

37C

yielding only seven positive isolations

(2.69%)

and

RV/42 C

yielding

59 (22.69%).

The results obtained using

enrichment in SC/37 C and SC/43 C were quite similar, 14

(538%)

and

13 ( 5 )

positive isolations, respectively. The

results obtained using

DCA,

HEA and SSA were more

uniform. T h e best results, 27 (10.38%) and 28 (10.76%)

positive isolations, were achieved following enrichment in

SC/37 C; results were not as good when enrichment was

carried out in

SC/43 C.

In terms of the variety

of

serotypes detected (Table

4),

the best results were obtained by BGA after enrichment in

RV/42 C (9

serotypes) and

HEA

after enrichment in TT B /

37C (8

serotypes). On the whole, interference by com-

peting organisms on the isolation media was lower

following enrichment in T T B and RV.

Experlment

2

Table

5

presents the recovery of

Sal m. enteritidis Salm.

kapemba

and

Salm. virchow

in the different samples. The

results show that direct inoculation with

1000

cells in T S B

yielded a recovery rate of 100% positive samples, irrespec-

tive

of

the enrichment and isolation method used and the

serotype inoculated.

Inoculation of a foodstuff devoid

or

with negligible levels

of contamination, namely, pasteurized eggs, with 1000 cells

and homogenization with T S B also produced a

100%

recovery rate.

As

in the preceding case, the recovery rate

was the sam e for all the serotypes inoculated.

Table 5 Recovery of Salmonella

Sample Enrichment procedure enteritidis Salm. kapemba and

Salm.

No. TTB/37OC

sc-37 ~ S C / ~ ~ O C

V/42OC virchow using different procedures

of

Inocula Nature

enrichment

Salm. enteritidis TSB

Eggs

Lamb liver

Chicken liver

Salm.

kapemba

TSB

Eggs

Lamb liver

Chicken liver

Salm. virchow TSB

Eggs

Lamb liver

Chicken liver

4 4 4 4

(100)

4 4 4 4

(loo)

44 38

44 36

4 4 4 4

(loo)

4 4 4 4

(100)

44 38

44 38

4 4 4 4

(100)

4 4 4 4

(100)

44 39

44 39

(86.4)

(81.8)

(86.4)

(86.4)

(88.6)

(88.6)

44

44

39

(88.6)

38

(86.4)

44

(100)

44

(100)

40

(90.9)

39

(88.6)

44

(loo)

44

(100)

40

(90.9)

41

(92.3)

(100)

(100)

44

(100)

44

(loo)

37

(84.1)

36

(81.8)

44

(100)

44

(100)

37

(84.1)

38

(86.4)

44

(loo)

44

(loo)

38

(86.4)

36

(81.8)

44

(100)

44

(1W

40

(90.9)

37

(84.1)

44

( 1 0 )

44

( loo )

39

(88.6)

41

(92.3)

44

W

44

41

(92.3)

39

(88.6)

Percentage

of

positive samples in parentheses.

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M E T H O D S F O R

S L M O N E L L

D E T E C T I O N 365

In contrast, inoculation of the lamb and chicken livers,

foodstuffs with normally high levels of contamination

(Enterobacteriaceae counts of up to lo7), with 1000 cells in

TSB homogenate yielded more uneven results, ranging

from

a

low of

81.81%

positive isolations to a high of

92.27% positive isolations and a slight difference in favour of

RV/42 C. SC/43 C

produced the lowest recovery rates.

Again, the results were independent of the salmonella

serotype inoculated.

The results obtained for the selective isolation media

tested in turn depended upon the enrichment broth

employed. On the whole, interference by competing

organisms was high, particularly on the BGA, making

detection of

Salmonella

difficult even though the samples

had been inoculated with that bacterium.

D I S C U S S I O N

The success of enrichment and isolation media is based on

the presence of inhibitors intended to act against Gram-

positive contaminating microflora and Gram-negative com-

petitive flora, normally Enterobacteriaceae.

A

study of 12

inhibitors

a t

similar concentrations (Arroyo and Arroyo

1995) demonstrated that the level of inhibition on salmon-

ellas was the same as on the othe r Enterobacteriaceae exam-

ined and accordingly that inhibition of the multiplication of

such organisms during enrichment was difficult. Direct

counts of total Enterobacteriaceae in lamb and chicken

organ meats on Violet-Red-Bile-Glucose-Agar (VRB G)

reached up to lo7 cfu

g- '

(Arroyo and Arroyo 1995). T h e

high initial levels of Enterobacteriaceae and their further

growth in the enrichment media masked the presence of

Salmonella

giving rise to false positives (Watson and

Walker 1978; Rhodes and Quesnel 1986). Salmonellas

cannot begin to multiply during enrichment until the

number of competitors has fallen (Van Schothorst and

Renaud 1983; Rhodes and Quesnel 1986). This decrease is

brought about by a fall in pH and depends upon the food-

stuff in question, bacterial metabolism, and the presence of

large numbers of Gram-positive bacteria, including lactic

acid bacteria (Van Schothorst and Renaud

1985).

In the

samples examined by the authors, there were rather high

numbers of Enterobacteriaceae with low numbers of Gram-

positive organisms, and t he p H did not fall below 6.2

(unpublished data).

The results for the enrichment media in Experiment

1

varied. The greatest variety of different

Salmonella

serotypes were detected using RV 10 (Vassiliadis et al.

1981

;

Vassiliadis 1983) and the largest number of positive

isolations using SC/37 C, though it should be noted that

most of these belonged to the two most ab undan t serotypes,

Salm. virchow

and

Salm. worthington

and that detection of

the minor serotypes using this latter medium was more dif-

ficult. Fagerberg and Avens (1976) reported that certain

serotypes were easier to detect in certain media than in

others.

TTB, which Beckers

et al. (1987a,

b) recommended

replacing with RV in the I S 0 standard (Anon. 1990), pro-

duced the fewest positive isolations, but the number of dif-

ferent serotypes detected was higher than using SC/37 C.

This suggests that certain inhibitors may affect the growth

and multiplication of some of the minor serotypes present.

However, according to Van Schothorst et al. (1977), inhibi-

tion depends less upon the serotype than upon the sensi-

tivity of certain cells to the inhibitor. Patil and Parhad

(1986) reported that T T B was the best enrichment medium

when E.

coli

and other lactose-positive Enterobacteriaceae

were the predominant competing organisms, whereas

SC

yielded better results when organisms of the genus Proteus

predominated.

Raising the incubation temperature may increase the

number of positive isolations (Carlson and Snoeyenbos

1972). However, tetrathionate with brilliant green at 43C

is toxic to many salmonellas (Harvey and Price

1979;

Arroyo

1990).

Both the number of positive isolations and

the variety of serotypes detected decreased using SC/43 C.

Studies with pure cultures have indicated that certain Sal-

monella serotypes cannot multiply at high incubation tem-

peratures (Carlson and Snoeyenbos 1974; Van Schothorst

et al. 1977). On the other hand, survival of most of the

Enterobacteriaceae at 43C and at 44C (e.g. E.

coli

whose

presence as a faecal coliform is detected in lactose broth

incubated at

44 C),

made isolation of salmonellas extremely

difficult. Van Schothorst and Renaud (1983) reported that

isolation of salmonellas was virtually impossible when the

number of lactose-positive Enterobacteriaceae was

10'

g '

or m l-' , hence raising the temperature would appear to be

unnecessary when using th e SC medium.

Isolation on BGA following enrichment in SC/37 C and

SC/43 C

was quite difficult because of the large numbers

of competing bacteria present.

For

the salmonellas

to

be

detectable on that medium, their concentration in the

enrichment m edium after incubation m ust reach a level of

lo3

m l -

'

(Van Leusden

et al.

1982);

at lower levels they

remain undetectable, even if the level of the competing

flora drops to below lo 3 ml

.

Detection on BGA is facilitated by greater inhibition of

competing organisms in RV

10

(Vassiliadis

1983).

Com-

peting bacteria belong mainly to the genera

Proteus Morga-

nella and Shigella. The presence of Yersinia is important,

because the Rappaport-Wauters medium is used for enrich-

ment of that genus. T ha t medium is similar to RV, and the

colonies of that genus on BGA do not differ in appearance

from Salmonella colonies.

Isolation o n HEA and DCA after enrichment in T T B

yielded the most positive isolations, and anaerobic culture is

R ? 1995 The Society for Applied Bacteriology.

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366 G . A R R O Y O A N D J . A . A R R O Y O

not required when using DCA (Edgar and Soar 1979).

However, contradicting the findings of Moriiiigo

e t

al.

(1989),

these media did not inhibit the growth of

Pseudo-

monas aeruginosa

or other Pseudomonadaceae and Vibriona-

ceae. Based on these results, RV would appear to be the

most appropriate enrichment medium, but complete rejec-

tion of T T B would appear to be premature, since that

medium yielded the greatest variety in the number of

serotypes and lower interference by competing organisms

than SC.

Th e results of Experiment 2 corroborated the results of

Experiment 1 . In the TSB and commercial pasteurized egg

samples, in which there was no interference by competing

organisms,

100%

of the samples tested positive, irrespective

of the serotype inoculated and the method employed, even

when lower concentrations of inocula were used (results not

presented here). Previously, inoculation with small

numbers of

Salm. montevideo

and

Salm. heidelberg

yielded

excellent recoveries using Selenite Broth, whereas the con-

centration of the inoculum had to be increased to 500 cells

when using Tetrathio nate B roth (Bailey

e t

al.

1981).

In the

present experiment the pre-enrichment broth was inocu-

lated to facilitate the recovery of salmonellas in the T T B

(Taylor and Silliker

1962).

The level of competing organisms was high in the lamb

and chicken liver samples, and the percentage of positive

isolations was lower, though the results did not vary much

with the method used and the serotype inoculated. Accord-

ingly, successful detection of salmonellas in those organ

meats was dependent upon interference by competitors.

The similar behaviour exhibited by

Salm. k a p emb a

a minor

serotype in Experiment

1,

indicates that it is able to tolerate

the same inhibitor concentrations as

S a l m. en t er i t i d i s

and

S a l m . virchow

and provides further confirmation that sal-

monella concentrations must be

l o3

ml - after enrichment.

Difficulties in detecting salmonellas are heightened when

there are cross-reactions with other Enterobacteriaceae,

such as

Proteus Morganel la

and

Citrobacter freundii.

Increasing interest in developing fast methods of analysis

and future application of those methods may help to miti-

gate these difficulties.

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Efficiency of Different Enrichment and Isolation Procedures For