神奈川實(shí)驗(yàn)（Kanagawa phenomenon)培養(yǎng)基和試劑
發(fā)布時(shí)間：
2022-12-27
作者：
新鮮平皿和一次性培養(yǎng)基廠家
副溶血性弧菌有845個(gè)血清型，主要通過(guò)十三種耐熱的菌體抗原（即O抗原）鑒定，而七種不耐熱的包膜抗原（即K抗原）可以用來(lái)輔助鑒定。其致病力可用神奈川試驗(yàn)來(lái)區(qū)分。該菌能使人或兔的紅細(xì)胞發(fā)生溶血，在瓊脂培養(yǎng)基上出現(xiàn)β溶血環(huán)，稱為“神奈川試驗(yàn)”陽(yáng)性。神奈川試驗(yàn)陽(yáng)性菌的感染能力強(qiáng)，多數(shù)毒性菌株為神奈川試驗(yàn)陽(yáng)性（K+），多數(shù)非毒性菌株為神奈川試驗(yàn)陰性（Kˉ）。引起食物中毒的副溶血性弧菌90%神奈川試驗(yàn)陽(yáng)性。
洗滌血紅細(xì)胞方法：
取一定量的新鮮的（24h以內(nèi)）人或兔血加入到離心管中，加入等量的無(wú)菌生理鹽水，4℃離心15min(4000 x g)。吸出上層液體，再加入現(xiàn)在所剩液體量等量的無(wú)菌生理鹽水，再次離心洗滌。重復(fù)3次。第3次結(jié)束后，加入無(wú)菌生理鹽水，使其恢復(fù)至最初的原體積。
神奈川試驗(yàn)：
將測(cè)試菌株接種3%氯化鈉胰蛋白胨大豆瓊脂斜面，36℃±1℃培養(yǎng)18h～24h。用接種針挑取培養(yǎng)物點(diǎn)種表面干燥的我妻氏血瓊脂平板。每個(gè)平板上可以點(diǎn)種幾個(gè)菌。36℃±1℃培養(yǎng)不超過(guò)24h，并立即觀察。
陽(yáng)性結(jié)果為菌落周?chē)拾胪该鞯?nbsp;β-溶血環(huán)。
神奈川試驗(yàn)即食品中副溶血性弧菌檢驗(yàn)方法，屬于食品中微生物的檢驗(yàn)，標(biāo)準(zhǔn)是GB/T 4789.7-2008-副溶血性弧菌檢驗(yàn)。神奈川試驗(yàn)是在我妻氏瓊脂上測(cè)試是否存在特定溶血素。神奈川實(shí)驗(yàn)陽(yáng)性結(jié)果與副溶血性弧菌分離株的致病性顯著相關(guān)。用接種環(huán)將測(cè)試菌株的3%氯化鈉胰蛋白胨大豆瓊脂18h培養(yǎng)物點(diǎn)種表面干燥的我妻氏血瓊脂平板。每個(gè)平板可以環(huán)狀點(diǎn)種幾個(gè)菌。35士1攝氏度培養(yǎng)不超過(guò)24h，并立即觀察。陽(yáng)性結(jié)果為菌落周?chē)拾胪该鳝h(huán)的β溶血。
副溶血性弧菌有845個(gè)血清型，主要通過(guò)十三種耐熱的菌體抗原(即O抗原)鑒定，而七種不耐熱的包膜抗原(即K抗原)可以用來(lái)輔助鑒定。其致病力可用神奈川(kanagawa)試驗(yàn)來(lái)區(qū)分。該菌能使人或家兔的紅細(xì)胞發(fā)生溶血，在瓊脂培養(yǎng)基上出現(xiàn)β溶血帶，稱為"神奈川試驗(yàn)"陽(yáng)性。神奈川試驗(yàn)陽(yáng)性菌的感染能力強(qiáng)，多數(shù)毒性菌株為神奈川試驗(yàn)陽(yáng)性(K+)，多數(shù)非毒性菌株為神奈川試驗(yàn)陰性(Kˉ)。引起食物中毒的副溶血性弧菌90%神奈川試驗(yàn)陽(yáng)性，通常在12h內(nèi)出現(xiàn)癥狀。K+菌株能產(chǎn)生一種耐熱型直接溶血素，Kˉ菌株能產(chǎn)生一種熱敏型溶血素，而有些菌株能產(chǎn)生這兩種溶血素。
神奈川試驗(yàn)的培養(yǎng)基和試劑
硫代硫酸鹽檸檬酸鹽-膽鹽-蔗糖（TCBS）瓊脂 擴(kuò)展閱讀>> 弧菌在TCBS培養(yǎng)基上的顯色原理
3%氯化鈉胰蛋白胨大豆〔TSA）瓊脂
3%氯化鈉三糖鐵（TSI）瓊脂
3%氯化鈉甘露醇試驗(yàn)培養(yǎng)基
3%氯化鈉賴氨酸脫狡酶試驗(yàn)培養(yǎng)基
3%氯化鈉MR-VP培養(yǎng)基
我妻氏血瓊脂
氧化酶試劑
革蘭氏染色液
ONPG試劑
3%氯化鈉溶液
Voges-Proskauer（V-P）試劑
API20E生化鑒定試劑盒或VITEKNFC生化鑒定卡
相關(guān)產(chǎn)品：一次性顯色平板
神奈川試驗(yàn)的操作步驟編輯
1.樣品制備
1.1冷凍樣品應(yīng)在45℃以下不超過(guò)15min或在2-5攝氏度不超過(guò)18小時(shí)解凍，若不能及時(shí)檢驗(yàn)應(yīng)放于--15攝氏度左右保存；非冷凍而易腐蝕的樣品應(yīng)盡可能及時(shí)檢驗(yàn)，若不能及時(shí)檢驗(yàn)，應(yīng)置2-5攝氏度冰箱保存，在24h內(nèi)檢驗(yàn)。
1.2魚(yú)類和頭足類動(dòng)物取表面組織、腸或鰓。貝類取全部?jī)?nèi)容物，包括貝肉和體液；甲殼類取整個(gè)動(dòng)物，或者動(dòng)物的中心部分，包括腸和鰓，如為帶殼貝類或甲殼類則應(yīng)先在自來(lái)水中洗刷外殼并甩干表面水分，然后以無(wú)菌操作打開(kāi)外殼，按照上述要求取相應(yīng)部分。
1.3以無(wú)菌操作取檢樣25g（ml），加人3%氯化鈉堿性蛋白胨水225mL，用旋轉(zhuǎn)刀片式均質(zhì)器以8000r/min均質(zhì)1min，或拍擊式均質(zhì)器拍擊2min，制備成1：10的均勻稀釋液。如無(wú)均質(zhì)器，則將樣品放人無(wú)菌乳缽中磨碎，然后放在500mL的滅菌容器內(nèi)，加225ml3%氯化鈉堿性蛋白胨水，并充分振蕩。
2增菌
2.1定性檢測(cè)
將上述1:10稀釋液于36℃士1℃培養(yǎng)8h～18h。
2.2定量檢測(cè)
2.2.1用滅菌吸管吸取1:10稀釋液1mL，注入含右9mL3%氯化鈉堿性蛋白胨水的試管內(nèi)，振搖試管混勻，制備1：100的稀釋液
2.2.2另取1mL滅菌吸管，按上述操作依次制備10倍遞增稀釋液每遞增稀釋一次，換用一支lmL滅菌吸管.
2.2.3根據(jù)對(duì)檢樣污染情況的估計(jì),選擇三個(gè)連續(xù)的適宜稀釋度，每個(gè)稀釋度接種三支含有9mL3%氯化鈉堿性蛋白胨水的試管，每管接種1mL。置36℃士1℃恒溫箱內(nèi)，培養(yǎng)8h--18h
3分離
3.1在所有顯示生長(zhǎng)的試管或增菌液中用接種環(huán)沾取一環(huán)，于TCBS平板或弧菌顯色培養(yǎng)基平板上劃線分離。一支試管劃線一塊平板，于36℃士1℃培養(yǎng)18h-24h。
5.3.2典型的副溶血性弧菌在TCBSL呈圓形、半透明、表而光滑的綠色菌落，用接種環(huán)輕觸，有類似口香糖的質(zhì)感，直徑2mm-3mm。從培養(yǎng)箱取出TCBS平板后，應(yīng)盡決（不超過(guò)1h）挑取菌落或標(biāo)記要挑取的菌落。典型的副溶血性弧菌在弧菌顯色培養(yǎng)基上呈圓形、半透明、表面光滑的粉紫色菌落，直徑2mm-3mm。
4純培養(yǎng)
挑取三個(gè)或以上可疑菌落，劃線3%氯化鈉胰蛋白胨大豆瓊脂平板，36℃土l℃培養(yǎng)18h-24h。
5初步鑒定
5.1氧化酶試驗(yàn)：挑選純培養(yǎng)的單個(gè)菌落進(jìn)行氧化酶試驗(yàn)，副溶血性弧菌為氧化酶陽(yáng)性。
5.2涂片鏡檢：將可疑菌落涂片，進(jìn)行革蘭氏染色．鏡檢觀察形態(tài)。副溶血性弧菌為革蘭氏陰性，呈棒狀、弧狀、卵圓狀等多形態(tài)，無(wú)芽胞，有鞭毛。
5.3挑取純培養(yǎng)的單個(gè)可疑菌落，接種3%氯化鈉三糖鐵涼脂斜面并穿刺底層，35℃士1℃培養(yǎng)24h觀察結(jié)果。副溶血性弧菌在3%氯化鈉三糖鐵瓊脂中的反應(yīng)為底層變黃不變黑，無(wú)氣泡，斜面顏色不變或紅色加深，有動(dòng)力。
5.4嗜鹽性試驗(yàn)：挑取純培養(yǎng)的單個(gè)可疑菌落．分別接種于不同氯化鈉濃度的胰胨水，36℃士1℃培養(yǎng)24h觀察液體混濁清況。副溶血性弧菌在無(wú)氯化鈉和10%氯化鈉的胰胨水中不生長(zhǎng)或微弱生長(zhǎng)，在7%氯化鈉的胰胨水中生長(zhǎng)旺盛 [2] 。
6確定鑒定
6.1生化試驗(yàn)：取純培養(yǎng)物分別接種含3%氯化鈉的甘露醇、賴氨酸、MR-VP培養(yǎng)基，36℃士1℃培養(yǎng)24h--18h后觀察結(jié)果。隔夜培養(yǎng)物進(jìn)行ONPG試驗(yàn)。
5.6.2API20E生化鑒定試劑盒或VITEK：刮取3%氯化鈉胰蛋白胨大豆瓊脂平板上的單個(gè)菌落，用生理鹽水制備成濁度適當(dāng)?shù)募?xì)胞懸浮液．使用API20E生化鑒定試劑盒或VITEK鑒定。
7報(bào)告
當(dāng)檢出的可疑菌落生化性狀符合表1要求時(shí)，報(bào)告25g(ml)樣品中檢出副溶血性弧菌。如果進(jìn)行定量檢測(cè)，根據(jù)證實(shí)為副溶血性弧菌陽(yáng)性的試管管數(shù)，查最可能數(shù)（MPN）檢索表，報(bào)告每克(毫升）副溶血性弧菌的MPN值。
Vibrio parahaemolyticus has 845 serotypes, mainly identified by thirteen kinds of heat-resistant bacterial antigen (O antigen), and seven kinds of heat-resistant envelope antigen (K antigen) can be used to assist identification. Its pathogenicity can be distinguished by Kanagawa test. The bacteria can cause hemolysis of human or rabbit erythrocytes, and a beta hemolytic ring appears on agar medium, which is called "Kanagawa test" positive. The infection ability of Kanagawa test positive bacteria was strong, most of the toxic strains were in Kanagawa test positive (K+), and most of the non toxic strains were in Kanagawa test negative (K). Vibrio parahaemolyticus causing food poisoning was positive in 90% Kanagawa test.
Washing blood red blood cell method:
A certain amount of fresh (24h) human or rabbit blood was added to the centrifuge tube, adding the same amount of sterile saline, and centrifugally 15min (4000 x g) at 4 C. Suck out the upper layer of liquid and add the same amount of sterile saline as the amount of liquid remaining now, then centrifuge again. Repeat 3 times. After the third end, sterile saline was added to restore the original volume.
Kanagawa test:
The test strains were inoculated with 3% sodium chloride, tryptone soy agar slope and 18h ~ 24h at 36 C and 1 C. Use the inoculation needle to pick up the culture to dry the surface of my wife's blood agar plate. Several bacteria can be found on each plate. The culture was not more than 24h at 36 and 1 C, and was observed immediately.
The positive result was a semi transparent beta hemolytic ring around the colony.
The Kanagawa test is a test method for Vibrio parahaemolyticus in foods. It belongs to the examination of microorganisms in food. The standard is GB/T 4789.7-2008- Vibrio parahaemolyticus. The Kanagawa trial tested whether hemolysin existed on my wife's agar. The positive results of Kanagawa experiment were significantly correlated with the pathogenicity of Vibrio parahaemolyticus isolates. A 3% sodium chloride tryptone soy agar 18h culture was used to inoculate the dried surface of my wife's blood agar plate with inoculation ring. Each plate can ring a few bacteria. 35 people 1 degrees Celsius cultivation does not exceed 24h, and immediately observed. The positive result was the beta hemolysis of semitransparent rings around the colony.
Vibrio parahaemolyticus has 845 serotypes, mainly identified by thirteen kinds of heat-resistant bacterial antigen (O antigen), and seven kinds of heat-resistant envelope antigen (K antigen) can be used to assist identification. Its pathogenicity can be distinguished by Kanagawa (Kanagawa) test. The bacteria can cause hemolysis of human or rabbit erythrocytes, and a beta hemolytic band appears on agar medium, which is called "Kanagawa test" positive. Most of the virulent strains were positive in Kanagawa test (K+), and most of the non toxic strains were negative in Kanagawa test (K). The infection rate of positive bacteria was strong in Kanagawa. Vibrio parahaemolyticus, which causes food poisoning, is 90% positive in Kanagawa test, usually presenting symptoms in 12h. K+ strain can produce a heat resistant direct hemolysin, K strain can produce a heat sensitive hemolysin, and some strains can produce these two hemolysin.
Culture medium and reagents in Kanagawa test
Thiosulfate citrate bile salt sucrose (TCBS) agar
3% Sodium Chloride Peptone Soybean [TSA] agar
3% sodium chloride three sugar iron (TSI) agar
Salt tolerance test medium
3% NaCl mannitol test medium
Experimental culture medium of 3% sodium chloride lysine deenzyme
3% sodium chloride MR-VP medium
My wife's blood agar
oxidase reagent
Gram staining
ONPG reagent
3% Sodium Chloride Solution
Voges-Proskauer (V-P) reagents
ChromID Vibrio
API20E biochemical identification kit or VITEKNFC biochemical identification card
The editing of the operation steps of the Kanagawa test
1. sample preparation
1.1 frozen samples should be below 45 degrees centigrade not more than 15min or at 2-5 degrees Celsius not more than 18 hours thawing, if not timely inspection should be placed at about --15 degrees centigrade; non frozen and easy to corrode samples should be as soon as possible inspection, if not timely inspection, the refrigerator should be stored at 2-5 degrees centigrade and tested within the 24h.
1.2 fish and cephalopods take surface tissues, intestines, or gills. Shellfish take all the contents, including shellfish and body fluids; crustaceans take the whole animal, or the central part of the animal, including the intestines and gills, such as shell shellfish or crustaceans, to wash the shell in tap water and dry the surface moisture first, then open the shell with aseptic operation, and take the corresponding part according to the requirements mentioned above.
1.3 25g (ML) was taken with aseptic operation, and 3% sodium chloride alkaline peptone water 225mL was added, and a rotating blade homogenizer was used for 8000r/min homogeneous 1min or slap homogenizer to beat 2min, and the 1:10 uniform diluent was prepared. If the homogenizer is no homogenizer, the sample is grinded in the aseptic bowl and then placed in the sterilizing container of 500mL, adding 225ml3% sodium chloride alkaline peptone water and fully oscillating.
2 increasing bacteria
2.1 qualitative detection
The above 1:10 diluent was cultured at 36 C 1 C for 8h ~ 18h.
2.2 quantitative detection
2.2.1 uses the sterilization pipette to absorb the 1:10 diluent 1mL and injections into the test tube containing the right 9mL3% sodium chloride alkaline peptone water. The shaking tube is mixed to prepare 1:100 diluent.
2.2.2 also takes 1mL sterilization pipette. According to the above operation, 10 times increasing diluent is prepared in turn, and each time the dilution is replaced by a lmL sterilization pipette.
According to the estimation of sample contamination, 2.2.3 selected three continuous suitable dilution degrees, each of which was inoculated with three 9mL3% NaCl alkaline peptone water for each tube, and each tube was inoculated with 1mL. In the incubator of 36 degrees centigrade 1 C, 8h--18h
3 separation
3.1 smear a ring in the growth tube or enrichment solution with TCBS ring or Vibrio.