犬C-反應(yīng)蛋白定量檢測卡CRP

犬C-反應(yīng)蛋白定量檢測卡CRP

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【犬C-反應(yīng)蛋白定量檢測卡CRP】

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1.2.3.接種物制備 將用生長法或直接菌懸液法制備的濃度相當于0.5麥氏比濁標準的菌懸液，經(jīng)MH肉湯1∶1000稀釋后，向每孔中加100μl，密封后置35℃普通空氣孵箱中，孵育16~20h判斷結(jié)果。當試驗嗜血桿菌屬，鏈球菌屬時，孵育時間為20~24h，試驗葡萄球菌和腸球菌對苯唑西林和萬古霉素的藥敏試驗時孵育時間必須滿24h。此時，第1孔至第11孔藥物濃度分別為128、64、32、16、8、4、2、1、0.5、0.25、0.125μg/ml。
1.2.4.結(jié)果判斷 以在小孔內(nèi)*抑制細菌生長的zui低藥物濃度為MIC。當陽性對照孔（即不含抗生素）內(nèi)細菌明顯生長試驗才有意義。當在微量肉湯稀釋法出現(xiàn)單一的跳孔時，應(yīng)記錄抑制細菌生長的zui高藥物濃度。如出現(xiàn)多處跳孔，則不應(yīng)報告結(jié)果，需重復(fù)試驗。通常對革蘭陰性桿菌而言，微量肉湯稀釋法測得的MIC與常量肉湯稀釋法測得的結(jié)果相同或低一個稀釋度（1孔或2倍）。
2.瓊脂稀釋法 瓊脂稀釋法是將不同劑量的抗菌藥物，加入融化并冷至50℃左右的定量MH瓊脂中，制成含不同遞減濃度抗菌藥物的平板，接種受試菌，孵育后觀察細菌生長情況，以抑制細菌生長的瓊脂平板所含zui低藥物濃度為MIC。本法優(yōu)點是可在一個平板上同時作多株菌MIC測定，結(jié)果可靠，易發(fā)現(xiàn)污染菌；缺點是制備含藥瓊脂平板費時費力。
2.1.培養(yǎng)基制備 使用MH瓊脂，按商品說明書進行配制，pH7.2~7.4。淋病奈瑟菌使用GC瓊脂基礎(chǔ)加1%添加劑；其它鏈球菌使用含5%（V/V）綿羊血的MH瓊脂（當試驗磺胺藥時，使用溶解的馬血）。
2.2.含藥瓊脂平板制備 根據(jù)實驗設(shè)計，將已倍比稀釋的不同濃度的抗菌藥物分別加入已加熱溶解，并在45~50℃水浴中平衡的MH瓊脂中，充分混勻傾倒滅菌平皿，瓊脂厚度3~4mm。通常按1∶9比例配制藥物瓊脂平板，根據(jù)需要來選擇藥物濃度范圍。配制好的含藥瓊脂平板應(yīng)裝入密封塑料袋中，置2~8℃冰箱可貯存5天。
2.3.接種物制備與接種 制備濃度相當于0.5麥氏標準比濁管的菌懸液，再1∶10稀釋，以多點接種器吸取制備好菌液（約1~2μl）接種于瓊脂平板表面，每點菌數(shù)約為104CFU，形成直徑為5~8mm的菌斑。接種好后置35℃孵育16~20h（甲氧西林耐藥葡萄球菌、萬古霉素耐藥腸球菌孵育時間應(yīng)滿24h），觀察結(jié)果。奈瑟菌屬、鏈球菌屬細菌置5%二氧化碳、幽門螺桿菌置微需氧環(huán)境中孵育。
2.4.結(jié)果判斷 將平板置于暗色、無反光物體表面上判斷試驗終點，以抑制細菌生長的zui低藥物濃度為MIC。在含甲氧芐胺嘧啶或磺胺瓊脂平板上可見輕微細菌生長，與生長對照比較抑制80%以上細菌生長的zui低藥物濃度作為終點濃度。
如果出現(xiàn)有2個以上菌落生長于含藥濃度高于終點水平的瓊脂平板上，或低濃度藥物瓊脂平板上不長而高濃度藥物瓊脂平板上生長現(xiàn)象，則應(yīng)檢查培養(yǎng)物純度或重復(fù)試驗。

1.2.3. Inoculum preparation The growth suspension method or direct bacterial suspension concentration of the equivalent of 0.5 McNeil turbid standard bacterial suspension, diluted by 1: 1000 MH broth, to each well 100μl, Sealed at 35 ℃ normal air incubator, incubated 16 ~ 20h to determine the results. When testing Haemophilus, Streptococcus, incubation time is 20 ~ 24h, test staphylococcus and enterococci oxacillin and vancomycin susceptibility test incubation time must be full 24h. At this point, the first well to the 11th hole drug concentrations were 128,64,32,16,8,4,2,1,0.5,0.25,0.125μg / ml.
1.2.4. Results Judgment The minimum drug concentration to compley inhibit bacterial growth in the wells is MIC. When the positive control wells (that is, without antibiotics) significant bacterial growth test only makes sense. The highest drug concentration that inhibits bacterial growth should be recorded when there is a single perforated hole in the broth microdilution method. If there are multiple holes, you should not report the results, the need to repeat the test. For gram-negative bacilli, the MIC measured by the broth microdilution method is the same or a lower dilution (1 well or 2 times) as measured by the broth dilution method.
2. Agar dilution agar dilution method is to different doses of antibacterial drugs, added to melt and cooled to about 50 ° C quantitative MH agar, made with different decreasing concentrations of antibacterials of the plate, inoculated with test bacteria, bacteria were observed after incubation Growth conditions to inhibit the growth of bacteria in the agar plate containing the minimum concentration of MIC. The advantage of this method is that it can measure multiple strains of MIC at the same time on a flat plate, and the result is reliable and easy to find the contaminated bacteria. The disadvantage is that it takes time and effort to prepare the medicated agar plate.
2.1 Medium Preparation MH agar, formulated according to the product instructions, pH 7.2 ~ 7.4. Neisseria gonorrhoeae used GC agar base plus 1% additive; other streptococci used MH agar containing 5% (V / V) sheep blood (when sulfa drugs were tested, dissolved horse blood was used).
2.2. Drug-containing agar plate preparation According to the experimental design, different concentrations of antibacterial drugs that have been diluted to multiple dilutions were respectively added to MH agar which had been heated to dissolve and equilibrated in a water bath at 45-50 DEG C, Agar thickness of 3 ~ 4mm. The drug agar plate is usually formulated in a ratio of 1: 9, and the range of drug concentration is selected as needed. Prepared good containing agar plate should be sealed plastic bag, set 2 ~ 8 ℃ refrigerator can be stored for 5 days.
2.3 Inoculum preparation and inoculation Preparation of the equivalent of 0.5 Macrobrachium standard turbidimension bacterial suspension, and then 1:10 dilution, with a multi-point inoculum prepared bacteria (about 1 ~ 2μl) inoculated on the surface of the agar plate , The number of bacteria per point is about 104CFU, forming a diameter of 5 ~ 8mm plaque. After inoculation set 35 ℃ incubated 16 ~ 20h (methicillin-resistant staphylococcus, vancomycin-resistant enterococci incubation time should be full 24h), the observation results. Neisseria, Streptococcus bacteria set 5% carbon dioxide, Helicobacter pylori microaerophilic environment incubated.
2.4 determine the results of the plate placed on a dark, non-reflective surface to determine the end of the test to inhibit bacterial growth minimum drug concentration MIC. Slight bacterial growth was seen on trimethoprim or sulfanilamide agar plates, with the lowest drug concentration that inhibited the growth of more than 80% of the bacteria compared to the growth control as the endpoint concentration.
If more than 2 colonies grow on agar plates with drug concentrations above the end point or on long, high concentration drug agar plates on low concentration drug agar plates, check the culture for purity or repeat the test.