Experiment four Enterobacteria,Words:,Enterobacteria 肠杆菌 E. coli 大肠埃希氏菌 S. dysenteriae 痢疾志贺氏菌 S. typhi 伤寒沙门菌 S. paratyphi 副伤寒沙门菌 Bile broth media 胆汁肉汤培养基 Rectal swab 肛拭 EMB agar plate 伊红美兰琼脂平板 KI agar medium 铁质双糖琼脂基 Glucose 葡萄糖 Mannitol 甘露醇 Lactose 乳糖 Indole 吲哚 Methyl red 甲基红 Citrate 枸橼酸盐 Urease 尿素酶 Eosin 伊红 Methylene blue 美兰 sodium thiosulfate 硫代硫酸钠 Neutral red 中性红 phenol red 酚红,Objectives:,1. Master the point of identifications between pathogen andnonpathogen. 2. Master the method of the specimens collection. 3. Master the principle and significance of the selective media anddifferential media. 4. Master the principle and significance of Widals test. 5. Know the characteristics of pathogen and nonpathogen coloniesin EMB and SS agar plate. 6. Know the test procedures, important biochemical reaction andthe motility test.,Contents:,1. Biochemical tests of Enterobacteria 2.The characteristics of pathogen and nonpathogen colonies in EMB and SS agar plate 3. Serological reaction (Agglutination test and Widals test),Biochemical reaction and motility test,1. Indole test 2. Sugar fermentation test 3. Motility test,Left Center Right,Indole reagent,Indole test,Principle: The indole test is based on the formation of a red color complex when indole reacts with indole reagents.,Sugar fermentation test:,All the enzyme of bacteria is different and the ability to ferment sugar is different too. Some produce acid only, while others produce acid and gas both. According to these, the type of bacteria can be identified, especially in enterobacteria.,Observe the resultsFirstly, observe whether the bacteria growth. If these bacteria reproduce, the medium is clouding. If these produce acid, the indicator in medium turns yellow. While these produce both acid and gas, the medium turns yellow and there is bubble in inverted tube. If the bacteria cannot ferment sugar, the medium appears purple still, without the bubble.,Materials: 1. Bacteria strain: E.coli, S.dysenteriae, S.typhi. 2. Medium: glucose or lactose-fermenting tube,Motility test:,.,The test is based on the flagellum of the bacteria. If the bacteria have flagellum, the semisolid medium will be clouding and the test will be positive. .,Semisolid medium,Special selective and differential agar medium:,EMB agar plate SS agar plate KI agar medium,EMB agar plate:,Ingredients: Nutrient agar (PH7.6) 100 mlLactose 1.00g2Eosin- y 2ml0.5methylene blue 1ml,E.coli on EMB,Principle: EMB is one of the usual media that is used to separate the pathogen and non-pathogen of the Enterobacteria. In EMB plate, the eosin and the methylene blue are the indicator. In acid condition, eosin and methylene blue combine into mauve (紫红色)or black production. The nonpathogen colony (e.g.E.coli) that can ferment lactose takes on mauve or black with metallic luster. The pathogen colony that cannot ferment lactose displays translucence.,SS agar plate:,Ingredients:Nutrient agar 100mlLactose 1.00gBile salts 0.85g inhibitorSodium citrate 0.85g inhibitorSodium thiosulfate 0.85g10% ferric citrate 1ml1% Neutral red 0.25ml indicator1% brilliant green 0.033ml inhibitor,Principle: SS agar is often used to help isolate salmonellae and shigellae from other enteric gram-negative bacteria. Neutral red is the indicator. In acid condition it turns red, and it turns yellow in base condition.,E.coli that ferment lactose can produce acid so the colonies turns red . while other enteropathogen cannot ferment lactose, but can use peptone to produce base, so the colonies turns yellow.,KI agar medium:,Ingredients:Peptones 2.00 gNaCl 0.50gGlucose 0.10gLactose 1.00 gferrous sulfate 0.05gSodium thiosulfate 0.075gAgar 0.50g0.2 phenol red 1.25mlDistilled water 100ml,The medium can be used to obtain pure culture and primarily identify the suspect colonies of enteric bacteria. Phenol red is the indicator. When the colonies produce lots of acid from the fermentation of lactose, the media appear yellow completely. If the colonies only produce few of acid form the fermentation of glucose, only the bottom of the media appear yellow .For the combination of ferrous sulfate with H2S (sulfureted hydrogen) produced by the H2S-produced enterobacteria, the color becomes black. And if the colonies produce gas, there is bubble in the bottom of the tube.,