Development of Compact Dry SL

for Salmonella detection

S.Mizuochi１）,H.Teramura１）,S.Nirazuka１）,H.Kodaka１）,K.Inoue２）、
K.Tamura３）
1)Research Dept., Nissui Pharmaceutical, 2) Saitama Pref. Ranzango Hp.,
3)National Institute of Infectious Diseases
 Background
• Normally, Salmonella detection from food sample is processed
these following steps, Pre-culture, Selective enrichment culture,
Isolation and Confirmation/Identification. There are strong
customer demands to improve the test procedure for more simple
and rapid one, if it could have an equivalent result.
• We develop simple and easy medium for Salmonella detection
(Compact Dry SL, hereinafter CDSL) that detects Salmonella by
combination of biochemical reaction, specific enzyme reaction and
detection of motility of Salmonella. CDSL is screening method for
detecting Salmonella which inoculate pre-culture sample as a
specimen.
• Herewith we report CDSL evaluation results compared with
conventional culture methods using meats on the market,
① growth and color forming used by pure culture strains
② recovery of Salmonella from food materials
③ comparison (sensitivity/specificity/agreement) between
conventional method and CDSL
Package, CDSL
 Comparison of operation between conventional method
and CDSL
Conventional CDSL

Day 1 Specimen 25g Specimen 25g Pre-

culture Pre-culture
(BPW/EEM) (BPW/EEM)
↓ ↓
Day 2 Selective Enrichment Culture CDSL
(TT/RV/SBG/SC)
↓ ↓
Day 3 Isolation Presume, presence of Salmonella
(MLCB/DHL/XLD/Rambach) Isolation
↓ ↓
Day 4 Presume, presence of Salmonella Confirmation/Identification
Confirmation/Identification (Biochemical/Serological Tests)
(Biochemical/Serological Tests)
 Operating procedure of CDSL (1)
Pre-culture Medium
（BPW,EEM) Pre-incubation
20-24 hours at 35℃
Sample 25g

Inoculate 0.1mL of Pre-culture solution

on CDSL
(approx. 1cm far from the edge of plate)

SL
 Operating procedure of CDSL (2)

Drop 1mL of sterilized water at the opposite point

where specimen dropped.
↓
Confirm water diffused all over the plate and
turn over the plate capped.
× Incubate 20 – 24 hours at 41 - 43℃.
↓
Interpretation
SL
 Test Principle and Features of Compact Dry SL
• Base medium: DHL agar
• Additives: Pigment / Magnesium chloride / Novobiocin / Other additives

• Test principle:
1) Medium alkalization that changes medium color from blue-purple
to yellow by Salmonella’s lysine decarboxylase ability.
2) Colony color will be greening caused by decomposition of
chromogenic substrate with specific enzyme on Salmonella.
3) Salmonella produces hydrogen (not all strains) sulfide which
generate black colonies.
4) Detect motility of Salmonella.

• Features:
1) CDSL detects Salmonella one day earlier than conventional
culture methods.
2) Isolated colonies on the plate can be fished for further
identification tests.
3) Ready to use and portable plate which no need to special preparation.
 Chicken (42℃, 24 hours incubation)

Positive reaction Negative reaction

Motility, Yellow sheet, Greening colony, No color change
Black colonies are generated by hydrogen Red – Red purple
sulfide.
 CDSL reactions (1)
Standard strains

Table 1: Gram Positive bacteria and Yeast (24 hours incubation at 42℃)
Tested strains CDSL
Growth Color
1 Bacillus cereus ATCC 19637 - -
2 Bacillus licheniformis ATCC 14580 - -
3 Bacillus subtilis ATCC 6633 - -
4 Corynebacterium minutissium ATCC 23348 - -
5 Corynebacterium renale ATCC 19412 - -
6 Corynebacterium xerosis ATCC 373 - -
7 Enterococcus avium ATCC 14025 - -
8 Enterococcus durans ATCC 19432 - -
9 Enterococcus faecalis ATCC 19433 - -
10 Enterococcus faecalis NIH 1001 - -
11 Enterococcus faecium ATCC 19434 - -
12 Enterococcus faecium NIH 2001 - -
13 Lactobacillus delbrueckii subsp. Lactis ATCC 12315 - -
14 Micrococcus luteus ATCC 9341 - -
15 Staphylococcus aureus ATCC 6538 - -
16 Staphylococcus epidermidis ATCC 14990 - -
17 Streptococcus thermophilus ATCC 14485 - -
18 Candida albicans ATCC 10231 - -
19 Saccharomyces cerevisiae NHL 10010 - -
 CDSL reactions (2)
Gram Negative Bacteria rather than Salmonella
Table 2: Gram Negative Bacteria rather than Salmonella
Tested strains Numbers Growth Color Tested strains Numbers Growth Color
(NIH, ATCC, JCM) (NIH, ATCC, JCM)
Cedecial davisae 2 - - Klebsiella pneumoniae subsp. rhinoscleromatis 2 -/+ - / purple
Cedecia lapageri 2 - - Klebsiella terrigena 2 - -
Citrocacter amalonaticus 3 + - Kluyvera ascorbata 3 - -
Citrobacter diversus 3(2) -/+ - / yellow Kluyvera clyocrescens 3 - -
Citrobacter freundii 5 -/+ - Leclercia adecarboxylata 2 - -
Cirobacter koseri 1 + - Morganella morganii 4 -/+ -
Edwardiella hoshinae 2 - - Proteus mirabilis 4 + -
Edwardsiella tarda 3 - - Proteus penneri 2 - -
Enterobacter aerogenes 1 + - Proteus vulgaris 4(1) -/+ - / yellow
Enterobacter amnigenus 1 - - Providencia alcalifaciens 3 - -
Enterobacter cloacae 3 + purple Providencia rettgeri 3(1) -/+ - / pale yellow
Enterobacter intermidius 1 + - Providencia stuartii 2 - -
Enterobacter sakazakii 3 + - / purple Pseudomonas aeruginosa 6(5) + - / yellow
Escherichia coli 9 + - / purple Pseudomonas putida 1(1) + yellow
Escherichia coli Serotype O157 2 -/+ - Serratia fonticola 1 + -
Escherichia fergusonii 1 + - Serratia liquefaciens 1 + -
Escherichia hermannii 3 -/+ - Serratia marcescens 4 -/+ -
Escherichia vulneris 2 - - Serratia rubidaea 2 -/+ -
Hafnia alvei 1 - - Rehnella aquatilis 1 - -
Klebsiella ornithinolytica 2 -/+ - Shigella boydii 1 - -
Klebsiella oxytoca 3 + - Shigella flexneri 2 - -
Klebsiella planticola 2(1) -/+ - / yellow Shigella sonnei 2 - -
Klebsiella pneumoniae subsp . Ozaenae 3 + purple Yersinia enterocolitica 2 - -
Klebsiella pneumoniae subsp . Pneumoniae 3 + - / purple Yokenella regensburgei 2 - -
Total number of strains tested 120(11)
Color changed to yellow around sample dropping spot (no green colonies identified)
 CDSL reactions (3)
Salmonella spp.- 1

Table 3-1: Salmonella spp. (24 hours incubation at 42℃)

Tested strains Numbers Growth Color
(NIH, ATCC, JCM) Inoculated point Surrounding Colonies
Salmonella Agona 1 + Yellow Yellow Blue Green
Salmonella Anatum 2 + Yellow Yellow/Green Blue Green
Salmonella Blockley 2 + Yellow Yellow Blue Green
Salmonella bongori 1 + Yellow Gray Blue Green
Salmonella Cerro 1 + Yellow/Green Yellow Green
Salmonella Chester 2 + Yellow Yellow Green
Salmonella Choleraesuis 1 + Yellow Yellow Blue Green
Salmonella Cubana, H2S- 1 + Yellow Yellow Green
Salmonella Derby 1 + Yellow Yellow Blue Green
Salmonella enterica subsp . arizonae 1 + Yellow Yellow Blue Green
Salmonella enterica subsp. diarizonae 1 + Yellow Gray small colonies
Salmonella enterica subsp . houtenae 2 + Yellow Yellow
Salmonella Enteritidis 9 + Yellow/Green Yellow Green
Salmonella Enteritidis, H2S- 5 + Yellow Yellow Green
Salmonella Fayed 1 + Yellow Yellow Green
Salmonella Hadar 1 + Yellow/Green Yellow Blue Green
Salmonella Havana 2 + Yellow/Green Yellow Green
Salmonella Heidelberg 4 + Yellow Yellow Blue Green
Salmonella Infantis 3 + Yellow Yellow Blue Green/Green
41
 CDSL reactions (3)
Salmonella spp.- 2

Table 3-2: Salmonella spp. (24 hours incubation at 42℃)

Tested strains Numbers Growth Color
(NIH, ATCC, JCM) Inoculated point Surrounding Colonies
Salmonella Lexington 1 + Yellow Yellow Green
Salmonella Livingstone 1 + Yellow Yellow Green
Salmonella Manhattan 1 + Yellow Yellow Blue Green
Salmonella Mbandaka 1 + Yellow Yellow Blue Green
Salmonella Montevideo 1 + Yellow/Green Yellow Blue Green
Salmonella Muenchen 1 + Yellow Yellow Green
Salmonella Muenster 1 + Yellow Yellow Black
Salmonella Newport 1 + Yellow Yellow Blue Green
Salmonella Paratyphi B 1 + Yellow Yellow Blue Green
Salmonella Schleissheim 1 + Yellow Yellow Blue Green
Salmonella Schwarzengrund 1 + Yellow Yellow Blue Green
Salmonella Senftenberg 1 + Yellow Yellow Blue Green
Salmonella Solna 2 + Yellow/Green Yellow Blue Green
Salmonella Thompson 3 + Yellow Yellow Blue Green/Green
Salmonella Tshiongwe 1 + Yellow Yellow Green
Salmonella Typhi 1 + Yellow Yellow
Salmonella Typhimurium 17 + Yellow/Green Yellow Blue Green/Green
Salmonella Virchow 1 + Yellow Yellow Green

37
 Recovery of Salmonella from inoculated Food samples (1)

Method Flow Chart Food SPC

1. Ground Chicken Breast 5.7 x 105 CFU/mL
2. Dried whole Egg 6.0 x 10 CFU/mL
Food Sample 25g + Buffer Peptone Water 225mL
3. BPW ----

Dispense 9mL each into sterile test tube

Dilute S. Enteritidis (5.1 x 108 CFU/mL) from 10-1 to 10-8 Dilute and inoculate 1mL each into above tube

Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth

Inoculation: 0.1 mL 0.1 mL 0.1 mL
Incubate 24 hours at 42C°

Confirmation of typical Salmonella colonies

MLCB Rambach XLD

Confirmation: TSI agar / LIM Medium

Biochemical Tests: PYR / NPA
Serological Test
 Recovery of Salmonella from inoculated Food samples (1)
5
Table 4: Recovery of Salmonella from inoculated Food sample (Ground Chicken Breast Meat: 5.7 x 10 CFU/mL)
Inoculated amount of Salmonella (CFU/mL)
7 6 5 4 3 2 1
5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 None
CDSL ++ ++ ++ ++ ++ ++ + + -
DIASALM + + + + + + + +W -
RV + + + + + + + - -
+ : Potitive ++ : Whole Yellowed Positive +W : Weak Postive

Table 5: Recovery of Salmonella from inoculated Food sample (Dried Whole Egg : 6.0 x 10 CFU/mL)
Inoculated amount of Salmonella (CFU/mL)
7 6 5 4 3 2 1
5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 None
CDSL ++ ++ ++ ++ ++ ++ + 1 -
DIASALM + + + + + + - - -
RV + + + + + + + - -
+ : Potitive ++ : Whole Yellowed Positive +W : Weak Postive

Table 6: Recovery of Salmonella from inoculated Food sample (BPW)

Inoculated amount of Salmonella (CFU/mL)
7 6 5 4 3 2 1
5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 x 10 5.1 None
CDSL ++ ++ ++ ++ ++ ++ + - -
DIASALM + + + + + + - - -
RV + + + + + + + - -
+ : Potitive ++ : Whole Yellowed Positive +W : Weak Postive
 Recovery of Salmonella from inoculated Food samples (2)
(Sensitivity test for pre-cultured sample)
Method Flow Chart

Chicken Breast 25g Egg Yolk 25g

(SPC 8.3 x 103 CFU/g) (SPC < 10 CFU/g)

Inoculate 1.0mL of Pre-cultured Salmonella Typhimurium 4-207B in TSB (over night) to each food sample
(Controlled 101 to 104 CFU/mL)

Chicken Breast Egg Yolk

EEM 225mL (Incubate 24 hours at 35C°) BPW 225mL

Inoculate to below media (incubate 24 hours at 42C°)

Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth
Inoculation: 0.1 mL 0.1 mL 0.1 mL

Confirmation of typical Salmonella colonies

MLCB Rambach XLD
(Incubate 24 hours at 35C°)

Confirmation: TSI agar / LIM Medium

Biochemical Tests: PYR / NPA
Serological Test
 Recovery of Salmonella from inoculated Food samples (2)
(Sensitivity test from pre-cultured sample)

Table 7: Comparison of Salmonella detection from each medium (Chicken Breast meat)
Inoculate CDSL DIASALM TT RV
(CFU/25g) 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1 92000 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +
2 9200 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +
3 920 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +
4 92 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +
5 7 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +
6 - - - - - - - - - - - - - - - - - - - - -
++ : Whole reaction are confirmed
+ : Partial reaction are confirmed

Table 8: Comparison of Salmonella detection from each medium (Egg Yolk)

Inoculate CDSL DIASALM TT RV
(CFU/25g) 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
1 410 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +
2 41 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +
3 - - - - - - - - - - - - - - - - - - - - -
++ : Whole reaction are confirmed
+ : Partial reaction are confirmed
 Comparison of Salmonella detection by methods from
Selective Enrichment Media by meat products (1)

Method Flow Chart

Food Specimen 25g + EEM Broth 225mL (Meat products, 60 samples)

Pre-Enrichment culture <24 hours at 35C°>

Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth Tetrathionate Broth

Inoculation: 0.1 mL 0.1 mL 0.1 mL 1.0mL

Incubate 24 hours at 42C°

MLCB agar / Rambach agar / XLD agar

Incubate 24 hours at 35C°

Confirmation: TSI agar / LIM Medium

Biochemical Tests: PYR / NPA
Serological Test
 Comparison of Salmonella detection by methods from
Selective Enrichment Media by meat products (2)
Name of Product Number of Compact Dry DIASALM Positives RV Positives TT Positives
(selected unintentionally) specimen SL Positives
Chicken (breast/ground) 8 8 8 8 8
Chicken (dark/sliced) 4 4 4 4 4
Chicken (breast) 4 4 4 4 4
Chicken (breast white) 6 4 3 4 4
Chicken (leg dark) 6 4 3 2 4
Chicken (ground) 4 4 4 4 4
Chicken (wing) 4 2 2 2 2
Chicken (skin) 2 2 2 2 2
Chicken (gizzard) 2 2 2 0 0
Chicken (steamed) 2 2 2 0 0
Bacon 4 0 0 0 0
Sausage 6 0 0 0 0
Processed Ham 2 0 0 0 0
Fried Chicken (Frozen) 4 0 0 0 0
Japanese Style Fried 2 0 0 0 0
Chicken (Frozen)
TOTAL 60 36 34 30 32
 Comparison of Salmonella detection methods from
Selective Enrichment Media by meat products (3)

Comparison of Sensitivity, Specificity and Agreement between Compact Dry SL and Conventional method

CDSL CDSL vs DIASALM

DIASALM + - Sensitivity 34 / 34 x 100 = 100%
+ 34 0 34 Specificity 24 / 26 x 100 = 92.3%
- 2 24 26 Agreement 58 / 60 x 100 = 96.7%
36 24 60

CDSL CDSL vs RV Broth

RV + - Sensitivity 30 / 30 x 100 = 100%
+ 30 0 30 Specificity 24 / 30 x 100 = 80%
- 6 24 30 Agreement 54 / 60 x 100 = 90%
36 24 60

CDSL CDSL vs Tetrathionate Broth

TT + - Sensitivity 32 / 32 x 100 = 100%
+ 32 0 32 Specificity 24 / 28 x 100 = 85.7%
- 4 24 28 Agreement 56 / 60 x 100 = 93.3%
36 24 60
 Conclusion

• All Salmonella samples are being positive on Compact

Dry SL. Several high concentration of Proteus and
Pseudomonas strains showed “yellowed” reaction, but
other Gram negatives were negative or inhibited its
growth.
• Sensitivity and specificity of CDSL were higher than
conventional methods when detected Salmonella from
meat products on the market.
• Preparation of medium is not necessary for CDSL and
the presumption of test result may receive one day
earlier than conventional enrichment culture methods
that will be useful for food manufacturers.