沙门菌的分离鉴定及血清学分型
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吲哚、VP试验阴性
H2S producing and urea nagative
脲酶试验阴性，产H2S。 Methyl red and Simmons citrate positive
甲基红、枸橼酸盐试验阳性
Facultative anaerobe
兼性厌氧菌
Motile with peritrichous flagellae
Salmonella may have been injured or decrease in amount by heat process or food preservation.
So that should be add Pre–enrichment step and Selective-enrichment step before the Selective solid media.
subspecies indica
种II. Salmonella bongori
总计 (沙门菌属)
1998
2,443
1,454 489 94 324 70 12 20
2,463
2001
2,480
1,478 498 94 327 71 12 21
2,501
2007
2,557
1,531 505 99 336 73 13 22
第三步 选择性固体培养基培养 (选择性鉴定培养基 )
Step 4 Identification step 第四步 鉴定
Step 5 Confirmation step 第五步 确证
Enrichment medium 增菌培养基
1. Pre-enrichment: BPW = Buffered Peptone Water
Isolation, Identification Serotyping of Salmonella 沙门菌的分离鉴定及血清学分型
Aroon Bangtrakulnonth
Aroon @dmsc. moph. go.th
Genus Salmonella 沙门菌属
1884 :
The typhoid bacteria was cultivated.
Genus Salmonella 沙门菌属
Family : Enterobacteriaceae 科 : 肠杆菌科
Gastroenteric found in humans and animals 存在于人体与动物体内的胃肠道中 Gram nagative bacteria 革兰阴性 Short rod (0.7 – 1.5 um X 2.0 – 5.0 um)短杆状 Non spore forming 无芽胞 Oxidase negative 氧化酶阴性
第一步 损伤菌的复苏(前增菌)
Step 2 Selection of interested cell group (Selective enrichment step)
第二步 目的菌群的选择性增菌
Step 3 Select by specific solid media (Selective – Differential Plating Media )
MSRV media = Modified semi-solid Rappaport Vassiliadis
3.选择性鉴别培养基
XLD 培养基 = 木糖赖氨酸脱氧胆酸盐培养基
BG 培养基 = 煌绿培养基
(或其他的选择性培养基)
MSRV 培养基 = 改良的Rappaport Vassiliadis半固体培养基
animals
(a) = d-tartrate
(*) = Typhimurium d, Dublin -.
+ = 90% or more positive reaction
- = 90% or more negative reaciton
d = different reactions given by different serovars
8,20
y
1,5
S. Tananarive
6,8
y
1,5
O : 9 (D1)
S. Typhi
9,12(Vi)
d
-
S. Enteritidis
1,9,12
g,m
-
S. Gallinarum
1,9,12
-
-
O : 3,10 (E1) S. Weltevreden
3,10,(15) r
z6
O : 38 (P)
days)
优点 1. 除无鞭毛的沙门菌外，所有非伤寒沙门菌的血清型均可检测。 2. 操作简便:在DSM-1上加一滴前增菌的肉汤即可。 3. 价格比国外的培养基要少50%。 4. 便捷:可以在48h得到基本结果(2天)。
5. 99% sensitivity and 100% specificity 6. Easy evaluated: Salmonella on DSM-1 will change medium colour from green to red 7.No need expert Biologist to evaluated result 8. No need expensive complicate instrument and chemical substance.
Enrichment medium 增菌培养基
3.Selective – Differential Plating Media
XLD agar = Xylose Lysine Deoxycholate agar
BG agar
= Brilliant Green agar
(or another selective agar media)
2,579
Differential characters of Salmonella species and subspecies
Species Subspecies Characters
S. enterica
S. bongori
enterica salamae arizonae diarizonae houtenae indica
-
+
+
+
+
β-glucuronidase
d
d
-
+
-
d
-
Mucate
+
+
+
-(70%)
-
+
+
Salicine
-
-
-
-
+
-
-
Lactose
-
- -(75%) +(75%) -
d
-
Lysis by phage O1
+
+
-
+
-
+
d
Usual habitat
Warm-blooded Cold-blooded animals and environment
Vi-抗原位于伤寒、副伤寒 , (都柏林)沙门菌的荚膜
O-antigens on the surface as polyO side chains. O-抗原形成聚合的支链位于表面.
LPS = lipid + core + poly-O side chain LPS = 脂质+ 核心 + O聚合支链
1884 :
培养出伤寒沙门菌.
1896 :
The serum of a typhoid patient agglutinated
the typhoid bacteria ~ serodiagnos沙门菌发生凝集反
应—血清学诊断.
1926 :
Analysis of O- and H-antigens were initiated
D-glucose is fermented with production of acid and usually gas 发酵葡萄糖，产酸，大多数产气。
Genus Salmonella 沙门菌属
Non-lactose fermenting organisms (NLFs) 不发酵乳糖
Indole and Voges Proskauer(VP) negative
Dulcitol
+
+
-
-
-
d
+
ONPG (2 h)
-
-
+
+
-
d
+
Malonate
-
+
+
+
-
-
-
Gelatinase
-
+
+
+
+
+
-
Sorbitol
+
+
+
+
+
-
+
CL(u+lt)u-traertwraitthe(aK) CN
+
-
-
-
+
-
+
-
-
-
Galacturonate
-
+
-
+
+
+
+
γ-glutamyltransferase +(*) +
by White and extended by Kauffmann.
1926 :
White最先开始 H抗原和O抗原分析，
Kauffmann对其进行进一步研究.
Serotyping is performed according to the
Kauffmann- White scheme. 根据Kauffmann- White 的设想，完成血清学分型.
S. Bangkok
38
z4z24
-
Conventional method for detecting Salmonella in food
食物中检测沙门菌的常规方法
Detection of Salmonella from Food sample 食品中沙门菌的检测
Export Frozen Food and Ready to eat Food
Group群 O : 2 (A)
ANTIGENIC FORMULAS
抗原的构型
Serovar血清型 O - antigen O – 抗原 H - antigen H – 抗原
S.Paratyphi A
phase 1
1,2,12
a
phase 2 (1,5)
S. Kiel
1,2,12
g,p
-
O : 4 (B)
Salmonella spp.
H – 抗原
Vi – 抗原 O – 抗原
Salmonella antigens
沙门菌抗原
H-antigens on the flagella H-抗原位于鞭毛上
Vi antigens on the capsule of S. Typhi, S. Paratyphi, (S. Dublin)
S.Paratyphi B
1,4,(5)12 b
-
S. Schwazengrund 1,4,12,27 d
1,7
S. Typhimurium
1,4,(5)12 i
1,2
O : 7 (C1)
S. Choleraesuis
6,7
c
1,5
S. Virchow
6,7
r
1,2
O : 8 (C2-C3) S. Brunei
出口冷冻食品和速食食品 沙门菌可能在食品加热或保藏过程中受损或数量减少.
因此在选择性固体培养基培养前，需要前增菌和选择性的增菌培 养.
Step by step of detection of Salmonella from fooี ้d
食品中沙门菌属的检验步骤ี ้
Step 1 Resuscitation of injured cell (Pre-enrichment step )
具有周生鞭毛，能运动。
表1. 目前各个种及亚种的血清学分型数量
年份 种 I. Salmonella enterica
subspecies enterica subspecies salamae subspecies arizonae subspecies diarizonae subspecies houtenae
5. 灵敏度和特异度分别高达99%和100%。 6. 判定结果简单: 沙门菌在DSM-1培养基上生长使 培养基颜色由绿变红。 7.不需要专业的人员来评定结果。 8. 不需要昂贵的复杂仪器或是化学试剂。
2. Selective enrichment : RVB = Rappaport - Vassiliadis soy peptone broth TTB = Tetrathionate Broth(Mueller – Kauffmann
1.前增菌: BPW = 缓冲蛋白胨水
2.选择性增菌: RVB = 大豆蛋白胨肉汤 TTB = 四硫酸盐肉汤
The new modified media DSM-1 = DMSc Salm Media-1
新的改良培养基
DSM-1 = DMSc沙门菌培养基-1
Benefit 1. Able to detect Salmonella non typhoidal group all serovar,
except non flagella Salmonella. 2. Easy operate: 1 Drop from Pre-enrichment broth on DSM-1 3. Economy less expensive 50% than foreigner medium. 4. Comfortable and more rapidly: Basically result in 48 hours (2
H2S producing and urea nagative
脲酶试验阴性，产H2S。 Methyl red and Simmons citrate positive
甲基红、枸橼酸盐试验阳性
Facultative anaerobe
兼性厌氧菌
Motile with peritrichous flagellae
Salmonella may have been injured or decrease in amount by heat process or food preservation.
So that should be add Pre–enrichment step and Selective-enrichment step before the Selective solid media.
subspecies indica
种II. Salmonella bongori
总计 (沙门菌属)
1998
2,443
1,454 489 94 324 70 12 20
2,463
2001
2,480
1,478 498 94 327 71 12 21
2,501
2007
2,557
1,531 505 99 336 73 13 22
第三步 选择性固体培养基培养 (选择性鉴定培养基 )
Step 4 Identification step 第四步 鉴定
Step 5 Confirmation step 第五步 确证
Enrichment medium 增菌培养基
1. Pre-enrichment: BPW = Buffered Peptone Water
Isolation, Identification Serotyping of Salmonella 沙门菌的分离鉴定及血清学分型
Aroon Bangtrakulnonth
Aroon @dmsc. moph. go.th
Genus Salmonella 沙门菌属
1884 :
The typhoid bacteria was cultivated.
Genus Salmonella 沙门菌属
Family : Enterobacteriaceae 科 : 肠杆菌科
Gastroenteric found in humans and animals 存在于人体与动物体内的胃肠道中 Gram nagative bacteria 革兰阴性 Short rod (0.7 – 1.5 um X 2.0 – 5.0 um)短杆状 Non spore forming 无芽胞 Oxidase negative 氧化酶阴性
第一步 损伤菌的复苏(前增菌)
Step 2 Selection of interested cell group (Selective enrichment step)
第二步 目的菌群的选择性增菌
Step 3 Select by specific solid media (Selective – Differential Plating Media )
MSRV media = Modified semi-solid Rappaport Vassiliadis
3.选择性鉴别培养基
XLD 培养基 = 木糖赖氨酸脱氧胆酸盐培养基
BG 培养基 = 煌绿培养基
(或其他的选择性培养基)
MSRV 培养基 = 改良的Rappaport Vassiliadis半固体培养基
animals
(a) = d-tartrate
(*) = Typhimurium d, Dublin -.
+ = 90% or more positive reaction
- = 90% or more negative reaciton
d = different reactions given by different serovars
8,20
y
1,5
S. Tananarive
6,8
y
1,5
O : 9 (D1)
S. Typhi
9,12(Vi)
d
-
S. Enteritidis
1,9,12
g,m
-
S. Gallinarum
1,9,12
-
-
O : 3,10 (E1) S. Weltevreden
3,10,(15) r
z6
O : 38 (P)
days)
优点 1. 除无鞭毛的沙门菌外，所有非伤寒沙门菌的血清型均可检测。 2. 操作简便:在DSM-1上加一滴前增菌的肉汤即可。 3. 价格比国外的培养基要少50%。 4. 便捷:可以在48h得到基本结果(2天)。
5. 99% sensitivity and 100% specificity 6. Easy evaluated: Salmonella on DSM-1 will change medium colour from green to red 7.No need expert Biologist to evaluated result 8. No need expensive complicate instrument and chemical substance.
Enrichment medium 增菌培养基
3.Selective – Differential Plating Media
XLD agar = Xylose Lysine Deoxycholate agar
BG agar
= Brilliant Green agar
(or another selective agar media)
2,579
Differential characters of Salmonella species and subspecies
Species Subspecies Characters
S. enterica
S. bongori
enterica salamae arizonae diarizonae houtenae indica
-
+
+
+
+
β-glucuronidase
d
d
-
+
-
d
-
Mucate
+
+
+
-(70%)
-
+
+
Salicine
-
-
-
-
+
-
-
Lactose
-
- -(75%) +(75%) -
d
-
Lysis by phage O1
+
+
-
+
-
+
d
Usual habitat
Warm-blooded Cold-blooded animals and environment
Vi-抗原位于伤寒、副伤寒 , (都柏林)沙门菌的荚膜
O-antigens on the surface as polyO side chains. O-抗原形成聚合的支链位于表面.
LPS = lipid + core + poly-O side chain LPS = 脂质+ 核心 + O聚合支链
1884 :
培养出伤寒沙门菌.
1896 :
The serum of a typhoid patient agglutinated
the typhoid bacteria ~ serodiagnos沙门菌发生凝集反
应—血清学诊断.
1926 :
Analysis of O- and H-antigens were initiated
D-glucose is fermented with production of acid and usually gas 发酵葡萄糖，产酸，大多数产气。
Genus Salmonella 沙门菌属
Non-lactose fermenting organisms (NLFs) 不发酵乳糖
Indole and Voges Proskauer(VP) negative
Dulcitol
+
+
-
-
-
d
+
ONPG (2 h)
-
-
+
+
-
d
+
Malonate
-
+
+
+
-
-
-
Gelatinase
-
+
+
+
+
+
-
Sorbitol
+
+
+
+
+
-
+
CL(u+lt)u-traertwraitthe(aK) CN
+
-
-
-
+
-
+
-
-
-
Galacturonate
-
+
-
+
+
+
+
γ-glutamyltransferase +(*) +
by White and extended by Kauffmann.
1926 :
White最先开始 H抗原和O抗原分析，
Kauffmann对其进行进一步研究.
Serotyping is performed according to the
Kauffmann- White scheme. 根据Kauffmann- White 的设想，完成血清学分型.
S. Bangkok
38
z4z24
-
Conventional method for detecting Salmonella in food
食物中检测沙门菌的常规方法
Detection of Salmonella from Food sample 食品中沙门菌的检测
Export Frozen Food and Ready to eat Food
Group群 O : 2 (A)
ANTIGENIC FORMULAS
抗原的构型
Serovar血清型 O - antigen O – 抗原 H - antigen H – 抗原
S.Paratyphi A
phase 1
1,2,12
a
phase 2 (1,5)
S. Kiel
1,2,12
g,p
-
O : 4 (B)
Salmonella spp.
H – 抗原
Vi – 抗原 O – 抗原
Salmonella antigens
沙门菌抗原
H-antigens on the flagella H-抗原位于鞭毛上
Vi antigens on the capsule of S. Typhi, S. Paratyphi, (S. Dublin)
S.Paratyphi B
1,4,(5)12 b
-
S. Schwazengrund 1,4,12,27 d
1,7
S. Typhimurium
1,4,(5)12 i
1,2
O : 7 (C1)
S. Choleraesuis
6,7
c
1,5
S. Virchow
6,7
r
1,2
O : 8 (C2-C3) S. Brunei
出口冷冻食品和速食食品 沙门菌可能在食品加热或保藏过程中受损或数量减少.
因此在选择性固体培养基培养前，需要前增菌和选择性的增菌培 养.
Step by step of detection of Salmonella from fooี ้d
食品中沙门菌属的检验步骤ี ้
Step 1 Resuscitation of injured cell (Pre-enrichment step )
具有周生鞭毛，能运动。
表1. 目前各个种及亚种的血清学分型数量
年份 种 I. Salmonella enterica
subspecies enterica subspecies salamae subspecies arizonae subspecies diarizonae subspecies houtenae
5. 灵敏度和特异度分别高达99%和100%。 6. 判定结果简单: 沙门菌在DSM-1培养基上生长使 培养基颜色由绿变红。 7.不需要专业的人员来评定结果。 8. 不需要昂贵的复杂仪器或是化学试剂。
2. Selective enrichment : RVB = Rappaport - Vassiliadis soy peptone broth TTB = Tetrathionate Broth(Mueller – Kauffmann
1.前增菌: BPW = 缓冲蛋白胨水
2.选择性增菌: RVB = 大豆蛋白胨肉汤 TTB = 四硫酸盐肉汤
The new modified media DSM-1 = DMSc Salm Media-1
新的改良培养基
DSM-1 = DMSc沙门菌培养基-1
Benefit 1. Able to detect Salmonella non typhoidal group all serovar,
except non flagella Salmonella. 2. Easy operate: 1 Drop from Pre-enrichment broth on DSM-1 3. Economy less expensive 50% than foreigner medium. 4. Comfortable and more rapidly: Basically result in 48 hours (2