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
Pre-culture
(BPW/EEM)
↓
Specimen 25g
Pre-culture
(BPW/EEM)
↓
Day 2
Selective Enrichment Culture
CDSL
Day 3
(TT/RV/SBG/SC)
↓
Isolation
(MLCB/DHL/XLD/Rambach)
↓
Day 4
Presume, presence of Salmonella
Confirmation/Identification
(Biochemical/Serological Tests)
↓
Presume, presence of Salmonella
Isolation
↓
Confirmation/Identification
(Biochemical/Serological Tests)
Operating procedure of CDSL (1)
Pre-culture Medium
（BPW,EEM)
Sample 25g
Pre-incubation
20-24 hours at 35℃
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,
Black colonies are generated by hydrogen
sulfide.
No color change
Red – Red purple
CDSL reactions (1)
Standard strains
Table 1: Gram Positive bacteria and Yeast (24 hours incubation at 42℃)
CDSL
Tested strains
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
Growth
Color
-
-
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
Klebsiella pneumoniae subsp. rhinoscleromatis
2
-/+
- / purple
Klebsiella terrigena
2
Kluyvera ascorbata
3
-
(NIH, ATCC, JCM)
(NIH, ATCC, JCM)
Cedecial davisae
2
Cedecia lapageri
2
Citrocacter amalonaticus
3
+
-/+
-/+
+
Citrobacter diversus
3(2)
Citrobacter freundii
5
Cirobacter koseri
1
Edwardiella hoshinae
2
Edwardsiella tarda
3
-
Enterobacter aerogenes
1
+
Enterobacter amnigenus
1
-
Enterobacter cloacae
3
Enterobacter intermidius
1
Enterobacter sakazakii
3
Escherichia coli
9
Escherichia coli Serotype O157
2
Escherichia fergusonii
1
Escherichia hermannii
3
+
+
+
+
-/+
+
-/+
Escherichia vulneris
2
Hafnia alvei
1
Klebsiella ornithinolytica
2
Klebsiella oxytoca
3
2(1)
Klebsiella planticola
Klebsiella pneumoniae
subsp . Ozaenae
3
Klebsiella pneumoniae
subsp . Pneumoniae
3
-/+
+
-/+
+
+
-
Leclercia adecarboxylata
2
Morganella morganii
4
Proteus mirabilis
4
-/+
+
Proteus penneri
2
-
-
Proteus vulgaris
4(1)
-/+
- / yellow
3
-
-
- / yellow Kluyvera clyocrescens
-
Providencia alcalifaciens
3
purple
Providencia rettgeri
3(1)
-/+
- / pale yellow
-
Providencia stuartii
2
-
-
- / purple Pseudomonas aeruginosa
6(5)
- / yellow
- / purple Pseudomonas putida
1(1)
+
+
+
+
-/+
-/+
-
Serratia fonticola
1
Serratia liquefaciens
1
Serratia marcescens
4
Serratia rubidaea
2
Rehnella aquatilis
1
Shigella boydii
1
Shigella flexneri
2
- / yellow Shigella sonnei
2
purple
Yersinia enterocolitica
- / purple Yokenella regensburgei
Total number of strains tested
Color changed to yellow around sample dropping spot (no green colonies identified)
2
2
120(11)
-
yellow
-
CDSL reactions (3)
Salmonella spp.- 1
Table 3-1: Salmonella spp. (24 hours incubation at 42℃)
Tested strains
Numbers
Growth
(NIH, ATCC, JCM)
Salmonella Agona
1
Salmonella Anatum
2
Salmonella Blockley
2
Salmonella bongori
1
Salmonella Cerro
1
Salmonella Chester
2
Salmonella Choleraesuis
1
Salmonella Cubana, H2S-
1
Salmonella Derby
1
Salmonella enterica subsp . arizonae
1
Salmonella enterica subsp. diarizonae
1
Salmonella enterica subsp . houtenae
2
Salmonella Enteritidis
9
Salmonella Enteritidis, H2S-
5
Salmonella Fayed
1
Salmonella Hadar
1
Salmonella Havana
2
Salmonella Heidelberg
4
Salmonella Infantis
3
41
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Color
Inoculated point
Surrounding
Colonies
Yellow
Yellow
Yellow
Yellow
Yellow
Blue Green
Yellow/Green
Blue Green
Yellow
Gray
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Gray
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Blue Green
Yellow/Green
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow/Green
Yellow
Yellow
Yellow/Green
Yellow/Green
Yellow
Yellow
Blue Green
Green
Green
Blue Green
Green
Blue Green
Blue Green
small colonies
Green
Green
Green
Blue Green
Green
Blue Green
Blue Green/Green
CDSL reactions (3)
Salmonella spp.- 2
Table 3-2: Salmonella spp. (24 hours incubation at 42℃)
Tested strains
Numbers
Growth
(NIH, ATCC, JCM)
Salmonella Lexington
1
Salmonella Livingstone
1
Salmonella Manhattan
1
Salmonella Mbandaka
1
Salmonella Montevideo
1
Salmonella Muenchen
1
Salmonella Muenster
1
Salmonella Newport
1
Salmonella Paratyphi B
1
Salmonella Schleissheim
1
Salmonella Schwarzengrund
1
Salmonella Senftenberg
1
Salmonella Solna
2
Salmonella Thompson
3
Salmonella Tshiongwe
1
Salmonella Typhi
1
Salmonella Typhimurium
Salmonella Virchow
17
1
37
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Color
Inoculated point
Surrounding
Colonies
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Green
Yellow/Green
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow/Green
Yellow
Yellow
Yellow
Yellow/Green
Yellow
Green
Blue Green
Blue Green
Blue Green
Green
Black
Blue Green
Blue Green
Blue Green
Blue Green
Blue Green
Blue Green
Blue Green/Green
Green
Blue Green/Green
Green
Recovery of Salmonella from inoculated Food samples (1)
Method Flow Chart
Food
Food Sample 25g + Buffer Peptone Water 225mL
SPC
1.
Ground Chicken Breast
5.7 x 105 CFU/mL
2.
Dried whole Egg
6.0 x 10
3.
BPW
----
CFU/mL
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
Inoculation: 0.1 mL
DIASALM (MERCK)
0.1 mL
Rappaport-Vassiliadis Broth
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
++
+
++
+
++
+
++
+
++
+
+
+
+
DIASALM
++
+
+W
RV
+
+
+
+
+
+
+
-
-
CDSL
+ : Potitive
+W : Weak Postive
++ : Whole Yellowed Positive
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
+W : Weak Postive
++ : Whole Yellowed Positive
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
(SPC 8.3 x 103 CFU/g)
Egg Yolk 25g
(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
(CFU/25g) 1
1
2
3
4
5
6
92000
9200
920
92
7
-
++
++
++
++
++
-
2
3
DIASALM
4
5
1
2
3
4
TT
5
++ ++ ++ ++
++ ++ ++ ++ ++
++ ++ ++ ++
++ ++ ++ ++ ++
++ ++ ++ ++
++ ++ ++ ++ ++
++ ++ ++ ++
++ ++ ++ ++ ++
++ ++ ++ ++
++ ++ ++ ++ ++
- - - - - - - ++ : Whole reaction are confirmed
+ : Partial reaction are confirmed
RV
1
2
3
4
5
1
2
3
4
5
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
Table 8: Comparison of Salmonella detection from each medium (Egg Yolk)
Inoculate
CDSL
(CFU/25g) 1
1
2
3
410
41
-
2
3
DIASALM
4
5
1
2
3
4
TT
5
++ ++ ++ ++ ++
++ ++ ++ ++ ++
++ ++ ++ ++ ++
++ ++ ++ ++ ++
- - - - - - - - ++ : Whole reaction are confirmed
+ : Partial reaction are confirmed
1
RV
2
3
4
5
1
2
3
4
5
+ +
+ +
- -
+
+
-
+
+
-
+
+
-
+
+
-
+
+
-
+
+
-
+
+
-
+
+
-
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
Inoculation: 0.1 mL
DIASALM (MERCK)
0.1 mL
Rappaport-Vassiliadis Broth Tetrathionate Broth
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
(selected unintentionally)
Number of
specimen
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
Chicken (Frozen)
2
0
0
0
0
60
36
34
30
32
TOTAL
Compact Dry
SL Positives
DIASALM Positives
RV Positives
TT Positives
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
DIASALM
+
-
+
34
2
36
0
24
24
34
26
60
CDSL
RV
+
-
+
30
6
36
0
24
24
30
30
60
CDSL
TT
+
-
+
32
4
36
0
24
24
32
28
60
CDSL vs DIASALM
Sensitivity 34 / 34 x 100 = 100%
Specificity 24 / 26 x 100 = 92.3%
Agreement 58 / 60 x 100 = 96.7%
CDSL vs RV Broth
Sensitivity 30 / 30 x 100 = 100%
Specificity 24 / 30 x 100 = 80%
Agreement 54 / 60 x 100 = 90%
CDSL vs Tetrathionate Broth
Sensitivity 32 / 32 x 100 = 100%
Specificity 24 / 28 x 100 = 85.7%
Agreement 56 / 60 x 100 = 93.3%
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.