Biologicalsamplepreparationtechnique
EP2.6.12译文
微生物限度检查1.简介：该试验方法所讲的是在需氧条件下生长的嗜温性细菌和真菌类微生物的定量检测。
设计该试验的最初目的是为了确定一种物质或制备液是否符合微生物质量已制定的规定。
如果是这种目的，则需要遵从下面的说明，包括所需样品数，从而根据下面所提到的方法来解释结果。
该法不适用于包含微生物作为活性成分的产品。
可以使用自动收集法作为替代法，与药典所用方法具有同样效力。
2.一般操作过程设计方法时所用条件要避免外在微生物对产品的污染。
必须釆取措施避免污染以便它们不影响任何所测微生物。
如果所测产品有抗菌活性，必须将这种活性去除或中和掉。
如果因此使用了灭活剂，必须证明它们对微生物的高效性和无毒性。
如果在样品制备中加了表面活性剂，必须证明它们对微生物的无毒性和与灭活剂的兼容性。
3.举例方法按规定用微孔滤膜法或平板菌落计数法。
MPN法对微生物测定来说是最不精确的方法，但是，如果某些产品所含极少的微生物量，该法是最合适的方法。
方法选择基于下列因素：如产品的特性和所要求的微生物量。
所选方法必须能够测定充足的样品从而来判断该法的适应性。
所选方法的合适性必须确定。
4.促生长实验和计数法4-1 总则在不加产品的条件下该试验能够检测微生物的能力必须已确定。
如果测试条件改变或产品改变，必须证实方法的适应性，因为这些改变可能影响试验的最终结果。
4-2 试验菌株的准备使用测试菌株标准稳定的悬浮液或按下面方法制备。
批种子培养技术（seed lot culture maintenance techniques, seed-lot systems）的使用以至于用于接种的微生物从最初的主批种子转移走不多于5通道。
细菌和真菌试验株的生产分别见表格2.6.12-2。
使用氯化钠蛋白胨（pH 7.0）缓冲液或pH 7.2的磷酸缓冲液制作试验用悬浮液。
对于曲霉菌需要加入0.05％聚山梨醇酯80到缓冲液中。
如果在2-8℃下保存则该悬浮液要在2-24小时内使用。
生物提取技术工艺流程
生物提取技术工艺流程英文回答：Biological extraction technology is a process used to isolate and extract specific components or substances from biological materials such as plants, animals, or microorganisms. This technology is widely used in various industries, including pharmaceuticals, food and beverages, cosmetics, and biotechnology.The process of biological extraction typically involves several steps. Firstly, the biological material iscollected and prepared for extraction. This may include drying, grinding, or chopping the material to increase the surface area and facilitate the extraction process.Next, a suitable solvent or extraction medium is chosen based on the desired component to be extracted. Common solvents used in biological extraction include water, ethanol, methanol, and various organic solvents. The choiceof solvent depends on the solubility of the target component and the compatibility with the biological material.The extraction is then carried out by immersing the biological material in the solvent and allowing the components to dissolve or diffuse into the solvent. This can be done through various techniques such as maceration, percolation, or Soxhlet extraction. The extraction may be performed at ambient temperature or under specific conditions such as elevated temperature or pressure, depending on the nature of the target component.Once the extraction is complete, the solvent containing the extracted components is separated from the biological material. This can be achieved through filtration, centrifugation, or evaporation. The separated solvent may undergo further purification or concentration steps to obtain a more concentrated extract.Finally, the extracted components are analyzed and characterized to ensure their quality and purity. This mayinvolve techniques such as chromatography, spectroscopy, or biological assays. The extracted components can then be used for various applications such as drug development, flavoring agents, or active ingredients in cosmetics.To illustrate the process, let's consider theextraction of essential oils from plants. In this case, the plant material is collected and dried to remove moisture. It is then ground into smaller particles to increase the surface area. A suitable solvent, such as ethanol, is chosen to extract the essential oils.The plant material is immersed in the ethanol and allowed to soak for a certain period of time. The essential oils present in the plant cells dissolve into the ethanol. After the extraction, the ethanol is separated from the plant material using a filtration process. The ethanol extract containing the essential oils is then subjected to evaporation to remove the solvent and obtain a concentrated essential oil extract.The extracted essential oils can be further analyzedusing gas chromatography to identify the different compounds present in the extract. The quality and purity of the essential oils can be determined through sensory evaluation and chemical analysis.中文回答：生物提取技术是一种从植物、动物或微生物等生物材料中分离和提取特定成分或物质的过程。
自然界微生物筛选的一般流程
自然界微生物筛选的一般流程英文回答：General Procedure for Screening Microorganisms from Nature.The screening of microorganisms from nature is acritical step in the discovery and development of novel bioactive compounds, enzymes, and microorganisms forvarious industrial, biotechnological, and pharmaceutical applications. Here's a general outline of the typical workflow involved in natural product screening:1. Sample Collection:Collect samples from diverse environments, such as soil, water bodies, extreme habitats, animal/plant surfaces, or food sources.Employ appropriate sampling techniques to ensurerepresentation of the microbial diversity of the environment.2. Sample Preparation:Pre-treat samples to remove debris, concentrate microorganisms, and prepare them for cultivation.Techniques used can include filtration, centrifugation, homogenization, and selective enrichment.3. Cultivation and Isolation:Culture samples on various growth media under different conditions (temperature, pH, oxygen availability) to promote the growth of diverse microorganisms.Isolate individual colonies representing different morphologies or growth characteristics using techniqueslike streaking and single-colony isolation.4. Primary Screening:Screen isolated microorganisms for the desired activity or trait of interest.Use high-throughput screening methods, such asmicrotiter plate assays, to test for antimicrobial, antifungal, antioxidant, or enzyme activities.5. Bioactivity Confirmation and Characterization:Confirm the bioactivity of selected isolates through repeated testing and dose-response experiments.Characterize the bioactivity by determining its potency, spectrum of activity, and stability under different conditions.6. Strain Improvement:Optimize the production of the desired compound or enzyme by improving the growth and metabolic capabilitiesof the producing microorganism.Employ techniques like mutagenesis, genetic engineering, and fermentation optimization.7. Fermentation and Scale-up:Scale up the fermentation process to produce sufficient quantities of the bioactive compound or enzyme.Optimize fermentation parameters to maximize yield and minimize production costs.8. Downstream Processing:Extract the active compound or enzyme from the fermentation broth.Use purification techniques like chromatography, crystallization, and filtration to obtain pure and concentrated products.9. Characterization and Application:Characterize the structure and properties of the bioactive compound or enzyme.Explore potential applications in medicine, industry, agriculture, or environmental remediation.中文回答：自然界微生物筛选的一般流程。
空间代谢组操作流程
空间代谢组操作流程英文回答：Spatial Metabolomics Workflow.Spatial metabolomics is a powerful technique that allows researchers to investigate the spatial distribution of metabolites within a biological sample. This information can be used to understand the metabolic processes that occur within different regions of a tissue or organ, and to identify changes in metabolism that are associated with disease.The spatial metabolomics workflow typically involves the following steps:1. Sample preparation: The biological sample is prepared by sectioning it into thin slices. The slices are then fixed and embedded in a resin.2. Imaging: The resin-embedded slices are imaged usinga mass spectrometer. This generates a series of images that show the distribution of metabolites within the sample.3. Data analysis: The mass spectrometry data is analyzed to identify the metabolites that are present in the sample. The data can also be used to generate metabolic maps that show the spatial distribution of metabolites.Spatial metabolomics is a relatively new technique, but it has already been used to make a number of important discoveries. For example, spatial metabolomics has been used to identify changes in metabolism that are associated with cancer, neurodegenerative diseases, and obesity.中文回答：空间代谢组学操作流程。
生化标本样本前处理流程
生化标本样本前处理流程英文回答：Pre-processing of Biomarker Specimens.Pre-processing of biomarker specimens is a critical step in biomarker discovery and validation. The goal ofpre-processing is to prepare the specimens in a way that maximizes the yield and quality of the biomarkers while minimizing the introduction of bias.The pre-processing steps typically include:1. Collection: Specimens are collected from patients or healthy controls according to a specific protocol. The collection method and storage conditions must be carefully controlled to minimize degradation of the biomarkers.2. Processing: Specimens are processed to remove impurities and to concentrate the biomarkers. This mayinvolve centrifugation, filtration, or precipitation.3. Storage: Processed specimens are stored at a controlled temperature and humidity to prevent degradation.4. Transportation: Specimens are transported to the laboratory for analysis. The transportation conditions must be carefully controlled to minimize degradation of the biomarkers.The choice of pre-processing steps depends on the type of specimen and the biomarkers being analyzed. For example, plasma specimens may be centrifuged to remove cells and debris, while urine specimens may be filtered to remove particulates.Pre-processing is a critical step in biomarker discovery and validation. By carefully following the pre-processing steps, researchers can ensure that the specimens are of high quality and that the biomarkers are accurately measured.中文回答：生物标志物标本的预处理流程。
Sample preparation
期待本版这个方面话题的整理！！1. 样品均匀化对于生物样品，应在测定前混合均匀，以免造成测定误差。
可置涡流混合器上混匀，血浆样品往复振摇亦可达到均匀化的目的。
对粪便、肌肉和组织等固体样品都存在均匀化这一问题。
对含有不溶性组分的样品(例如组织和粪便)，须将样品进行匀浆处理，以保证样品的均匀性。
同时还应注意取样的代表性问题。
2. 去蛋白处理生物样品如血浆、血清等含有大量的蛋白质，它们能结合药物，因此对于某些药物的测定，必须先将与蛋白结合的药物游离之后再作进一步处理。
通常去蛋白过程是将蛋白质变性处理后，把与蛋白结合的药物解离出来，离心分离以除去蛋白。
目前已有很多去蛋白方法可供使用，但使用各种方法之前应了解该方法是否会导致生物样品中的药物发生分解或影响药物的提取等。
通常除蛋白的方法是在含蛋白样品中加入适当的沉淀剂或变性剂，使蛋白质脱水而沉淀(如有机溶剂、中性盐)，有的是由于蛋白质形成不溶性盐而析出(如高氯酸)，离心后取上清液用于分析。
甲醇、乙腈、丙酮和乙醇是沉淀蛋白常用的有机溶剂，中性盐可用氯化铵等。
无机盐沉淀蛋白是可逆的，即将蛋白稀释后仍具有生理活性，而有机溶剂和酸类沉淀的蛋白是不可逆的。
也可用透析法与超滤法除去样品中蛋白。
3. 被测组分的提取生物样品中被测组分一般需提取后才能进行色谱分析，这一步骤包含了样品的净化与浓缩。
提取方法和提取条件的选择是分析方法研究的重要内容之一，它与分析方法的选择性、精密度和准确度紧密相关。
3.1 液-液提取液-液提取是经典的提取方法之一，它基于被测组分在不相混溶的两种溶剂中的分配。
在提取过程中，水相的pH是重要的参数；一般弱酸性药物可加入一定量的酸，弱碱性药物可加入一定量的碱，使药物以分子状态存在，有利于有机溶剂的提取效果；在液质联用中，必须使用可挥发性的酸和碱，如盐酸、甲酸、乙酸、氨水等。
有时加入一些强离子的无机盐(如氯化钠)，利用盐析作用，能促进组分进入有机相。
通过选择不同的有机溶剂可提高选择性。
生物提取技术工艺流程
生物提取技术工艺流程英文回答：Biological extraction technology is a process used to isolate and extract specific components or substances from biological materials such as plants, animals, or microorganisms. This technology has various applications in fields such as pharmaceuticals, food processing, andbiofuel production.The process usually involves several steps, including sample preparation, cell disruption, extraction, and purification. Let me explain each step in detail.1. Sample Preparation: This step involves collecting the biological material and preparing it for extraction. For example, if we want to extract essential oils from plants, we would collect fresh plant material, clean it, and dry it to remove any moisture.2. Cell Disruption: In this step, the cells of the biological material are broken open to release the desired components. There are various methods to achieve cell disruption, such as mechanical disruption, enzymatic treatment, or ultrasonication. For example, if we want to extract proteins from microorganisms, we can use ultrasonication to break open the cells and release the proteins.3. Extraction: Once the cells are disrupted, the desired components are extracted from the biological material. This can be done using different solvents or extraction techniques, depending on the nature of the target component. For example, if we want to extract natural pigments from plants, we can use organic solvents like ethanol or hexane to dissolve the pigments.4. Purification: After extraction, the crude extract obtained may contain impurities or unwanted components. Purification is done to remove these impurities and obtain a pure form of the desired component. This can be achieved through techniques like filtration, chromatography, ordistillation. For example, if we want to purify a specific enzyme from a microbial extract, we can use chromatography techniques to separate and isolate the enzyme from other proteins.Once the purification process is complete, theextracted component can be further analyzed, characterized, and used for various applications.中文回答：生物提取技术是一种从植物、动物或微生物等生物材料中分离和提取特定成分或物质的过程。
微生物测试均质拍打法 英语
微生物测试均质拍打法英语Microbial Homogenization Tapping Method。
Introduction。
Microbial homogenization tapping method is a technique used to obtain a representative sample of microorganisms from a larger population. It involves the use of tapping or shaking to evenly distribute the microorganisms in a sample, ensuring accurate and reliable test results. This method is commonly used in microbiology laboratories for various applications, including environmental monitoring, food safety testing, and clinical diagnostics. In this article, we will discuss the principles, procedure, and advantages of the microbial homogenization tapping method.Principles。
The microbial homogenization tapping method is based on the principle that microorganisms tend to settle or clump together, leading to uneven distribution in a sample. This uneven distribution can result in biased test results, as some areas of the sample may contain higher or lower concentrations of microorganisms. By tapping or shaking the sample, the microorganisms are dislodged from their settled positions and distributed more evenly, providing a representative sample for testing.Procedure。
生物样品分析方法验证指导 英文
Guidance for Industry Bioanalytical Methods Validationfor Human StudiesDRAFT GUIDANCEThis guidance document is being distributed for comment purposes only. Comments and suggestions regarding this draft document should be submitted within 60 days of publication in the Federal Register of the notice announcing the availability of the draft guidance. Submit comments to Documents Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. All comments should be identified with the docket number listed in the notice of availability that publishes in the Federal Register.For questions on the content of the draft document contact Vinod Shah, (301) 594-5635.U.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)December 1998BP #j:\!guidance\2578dft.wpd12/14/98Guidance for Industry Bioanalytical Methods Validationfor Human StudiesAdditional copies are available from:Drug Information Branch (HFD-210)Center for Drug Evaluation and Research (CDER)5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573Internet at /cder/guidance/index.htmU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)December 1998BP #j:\!guidance\2578dft.wpd12/14/98Table of ContentsI. INTRODUCTION (1)II. BACKGROUND (1)III. REFERENCE STANDARD (2)IV. PRE-STUDY VALIDATION (3)A. Specificity (3)B. Calibration Curve (4)C. Precision, Accuracy, and Recovery (5)D. Quality Control Samples (6)E. Stability (7)F. Acceptance Criteria (9)V. IN-STUDY VALIDATION (9)VI. DOCUMENTATION (10)REFERENCES (13)j:\!guidance\2578dft.wpd12/14/98 iThis guidance has been prepared by the Biopharmaceutics Coordinating Committee and the Clinical1Pharmacology Section of the Medical Policy Coordinating Committee in the Center for Drug Evaluation and Research (CDER) at the Food and Drug Administration. This guidance document represents the Agency’s current thinking on validation of analytical methods for human studies based on drug or metabolite assay in a biological matrix. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of the applicable statute, regulations, or both.j:\!guidance\2578dft.wpd12/14/98GUIDANCE FOR INDUSTRY 1Bioanalytical Methods Validation for Human StudiesI.INTRODUCTIONThis guidance provides assistance to sponsors and applicants of investigational new drug applications (INDs), new drug applications (NDAs), abbreviated new drug applications(ANDAs), and supplements, in developing validation information for bioanalytical methods used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies. The guidance does not address analytical methods used for nonhuman pharmacology/toxicology studies, CMC information, or in vitro dissolution studies.The information in this guidance is generally applicable to gas chromatography or high-pressure liquid chromatography analytical methods performed on drugs and metabolites obtained from biological matrices such as blood, serum, plasma, or urine. This guidance should also apply to other analytical techniques such as immunological and microbiological methods or otherbiological matrices, such as tissue samples including skin samples, although in these cases a higher degree of variability may be observed.II.BACKGROUNDThis guidance is based primarily on a conference on Analytical Methods Validation:Bioavailability, Bioequivalence and Pharmacokinetic Studies, which was held on December 3 - 5,1990, and sponsored by the American Association of Pharmaceutical Scientists, U.S. Food and Drug Administration, Federation Internationale Pharmaceutique, the Canadian Health Protection Branch, and the Association of Official Analytical Chemists (Shah 1992).Selective and sensitive analytical methods for the quantitative determination of drugs and their metabolites (analytes) are critical for successful performance of clinical pharmacology, BA, and BE studies. Analytical method validation includes all of the procedures recommended todemonstrate that a particular method for the quantitative measurement of an analyte in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible. The parameters essential to this validation include (1) accuracy, (2) precision, (3) sensitivity, (4) specificity, (5) linearity, and (6) reproducibility. In addition, the stability of the analyte in the matrix under study storage conditions should be determined. Validation involves documenting through the use of specific laboratory investigations that the performance characteristics of the method are suitable and reliable for the intended analytical applications (Shah 1992, Taylor 1983). The acceptability of analytical data corresponds directly to the criteria used to validate the method.Published methods of analyte analysis are often modified to suit the requirements of the laboratory performing the assay. These modifications should be validated to ensure suitable performance of the analytical method. When changes are made to a previously validated method, the analyst should exercise judgment as to how much additional validation is needed. For minor modifications, such as a change in the ratio of solvents for elution, a change in buffer system, the number of extractions of the biological matrix, or a small change in column temperature to obtain better separation, only limited validation may be recommended. For major modifications, such as change of an instrument, solvent system, detector, or temperature, full validation of the modified method should be performed.The analytical laboratory conducting BA and BE studies should closely adhere to FDA’s Good Laboratory Practices (GLPs) (21 CFR Part 58) and to sound principles of quality assurance throughout the testing process. In addition, the analytical methods for in vivo bioavailability studies must meet the criteria in 21 CFR 320.29. The analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a complete system of quality assurance. The SOPs should cover all aspects of analysis from the time the sample is collected and reaches the laboratory until the results of analysis are reported. They also should include record keeping, security and chain of sample custody (accountability systems that ensure integrity of test articles), sample preparation, and analytical tools, such as methods, reagents, equipment, instrumentation, and procedures for quality control and verification of results.The process by which a specific analytical method is validated may be divided into (1) reference standard preparation, (2) pre-study validation for analytical method development and method establishment, and (3) in-study validation to include study performance, drug analysis, and acceptance criteria (Shah 1992, Brooks 1985). These three processes are described in the following sections of the guidance.III.REFERENCE STANDARDj:\!guidance\2578dft.wpd12/14/982Analysis of drugs and their metabolites in a biological matrix is invariably carried out using samples spiked with calibration standards and quality control (QC) samples. The quality of the reference standard used to prepare spiked samples can affect study data. For this reason, an authenticated analytical reference standard should be used to prepare solutions of known concentrations. If possible, the reference standard should be identical to the analyte. When this is not possible, an established chemical form (free base or acid, salt or ester) of known purity can be used as a surrogate. Three types of reference standards are usually used: (1) certified reference standards (e.g., USP compendial standards); (2) commercially supplied reference standards obtained from a reputable commercial source; and/or (3) other materials of documented purity custom-synthesized by an analytical laboratory or other noncommercial establishment. The source and lot number, certificates of analyses when available, and/or internally or externally generated evidence of identity and purity should be furnished for each reference standard. A master standard (a synthetic batch for which identity and purity are clearly established and acceptable) should be maintained for each reference standard. All subsequently synthesized batches are to be compared chromatographically with that master standard. All reference materials should be checked prior to use to determine if there are significant interfering chromatographic peaks at the retention time of the analyte and/or the internal standard, using the analytical procedure to be used in the study.IV.PRE-STUDY VALIDATIONPre-study validation should include analytical method development and documentation. Validation should be performed for each biological matrix and for each chemical species to be measured in the biological matrix (Shah 1992, Buick 1990). In addition, the stability of quality control samples and the analyte in spiked samples should be determined. Typical performance parameters that should be assessed during pre-study validation include (1) specificity, (2) calibration curve and its linearity, (3) precision, accuracy, recovery, (4) quality control samples,(5) stability of analyte in spiked samples, and (6) acceptance criteria.A.SpecificitySpecificity is the ability of an analytical method to differentiate and quantitate the analyte in the presence of other constituents in the sample and refers directly to the ability of themethod to produce a response for a single analyte (Karnes 1991). For specificity, analyses of blank samples of the appropriate biological matrix (plasma, urine, or other matrix)should be obtained from six individuals under controlled conditions, with reference to time of day, food ingestion, and other factors considered important in the intended study. Each blank sample should be tested for interference using the proposed extraction procedureand chromatographic or spectroscopic conditions. The results should be compared toj:\!guidance\2578dft.wpd12/14/983those obtained with an aqueous solution of the analyte at a concentration near the limit of quantitation (LOQ).Any blank sample with significant interference at the retention time of the drug,metabolites, or internal standard should be rejected. If more than 10% of the blanksamples exhibit significant interference at these retention times, additional matrix blanksamples should be tested. If more than 10% of this subsequent group of blank samplesstill shows interference, the method should be changed to eliminate the interference.Potential interfering substances in a biological matrix include endogenous matrixcomponents, metabolites, decomposition products, and, in the actual study, concomitantmedication. Potential interference from nicotine and common OTC drugs and metabolites, such as caffeine, aspirin, acetaminophen, and ibuprofen should be routinely tested. If the method is intended to quantitate more than one analyte, each analyte should be injectedseparately to determine its retention time and to ensure that impurities from one analyte do not have the same retention time as another analyte.B.Calibration CurveCalibration is the relationship between instrument response and known concentrations of the analyte. A calibration (standard) curve should be generated for each analyte in thesample. A sufficient number of standards should be employed to adequately define therelationship between concentration and response. A calibration curve should be prepared in the same biological matrix as the samples in the intended study by spiking with known concentrations of the analyte. Precautions should be taken to avoid precipitation whilespiking the biological matrix. The number of standards used in constructing a calibration curve will be a function of the anticipated range of analytical values and the nature of the analyte/response relationship. Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study. A calibration curve shouldconsist of a blank sample (matrix sample processed without internal standard), a zerosample (matrix sample processed with internal standard), and five to eight non-zerosamples covering the expected range, including lower LOQ. Blank and standard zerosamples should not be used in the calibration function, but should only serve to evaluateinterference. Additional factors in developing a calibration curve relate to LOQ andlinearity.1.Limit of Quantitation (LOQ)The lowest standard on the calibration curve should be accepted as the limit ofquantitation if the following conditions are met:j:\!guidance\2578dft.wpd12/14/984C No interference present in blanks at the retention time of the analyte at thisconcentration, or typical response at this concentration at least five timesgreater than any interference in blanks at the retention time of the analyteC Analyte peak (response) identifiable, discrete, and reproducible with aprecision of 20% and accuracy of 80-120% (Shah 1992).2.LinearityThe simplest workable regression equation should be used with minimal or noweighting. Selection of weighting and use of a complex regression equationshould be justified. Four factors should be met in developing a calibration curve:C#20% deviation of the LOQ from nominal concentration (Shah 1992)C#15% deviation of standards other than LOQ from nominal concentration (Shah 1992)C At least four out of six non-zero standards meeting the above criteria,including the LOQ and the calibration standard at the highest concentration C0.95 or greater correlation coefficient (r)C.Precision, Accuracy, and RecoveryThe precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a singlehomogeneous volume of biological matrix. Precision should be measured using aminimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% coefficient of variation (CV) except for theLOQ where it should not exceed 20% CV. Precision is further subdivided into within-day, intra-batch precision or reproducibility, which assesses precision during a single analytical run, and between-day, inter-batch precision or reproducibility, which measures precisionwith time and may involve different analysts, equipment, reagents, and laboratories (Shah 1992, USP XXII 1990, Brooks 1985).The accuracy of an analytical method describes the closeness of test results obtained bythe method to the true value of the analyte. Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. A minimum of five determinations j:\!guidance\2578dft.wpd12/14/985per concentration should be conducted for a minimum of three concentrations in the range of expected concentrations. The mean value should be within 15% of the actual valueexcept at LOQ, where it should not deviate by more than 20%. The deviation from thetrue value serves as the measure of accuracy (USP XXII 1990, Brooks 1985).The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and recovered from the biological matrix, compared to thedetector response obtained for the pure authentic standard (Brooks 1985, Mehta 1989).Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Although recoveries close to 100% are desirable, the extent of recovery of an analyte and/or the internal standard may be as low as 50 to 60% if the recovery is precise, accurate, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high)with unextracted standards that represent 100% recovery.D.Quality Control SamplesPre-study validation of an analytical method should be carried out using at least threebatches of biological matrix, where each batch is collected from a different source. Each batch should contain (1) a calibration curve constructed using a blank sample, zerosample, and five to eight non-zero standards, (2) LOQ quality control (QC) samples, (3) low QC samples, (4) medium QC samples, (5) high QC samples, (6) a matrix blanksample, and (7) a reference standard. Quality control samples at concentrations notedbelow should be made from a stock solution separate from that used to prepare thestandards.LOQ QC sample:Same concentration as the lowest non-zero standardLow QC sample:#3 x LOQMedium QC sample:Approximately midway between the high and low QCconcentrationsHigh QC sample:75 to 90% of highest calibration standardThe accuracy of preparation of calibration and QC samples should be checked with thefirst batch. The data from replicate analyses of QC samples and duplicate analyses ofreference standards should be used to obtain the intra-day (within batch) precision, inter-day (between batch) precision, accuracy, and recovery.j:\!guidance\2578dft.wpd12/14/986j:\!guidance\2578dft.wpd12/14/987To obtain within-batch data, the mean, standard deviation, and CV of each QCconcentration in each batch should be calculated. The global (overall) mean, standard deviation, and CV for each QC concentration from the three batches should be calculated to obtain between-batch data. Precision is indicated by the %CVs. Percent accuracy is determined by dividing the mean concentration of a QC by its nominal concentration, and multiplying by 100.E.StabilityDrug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system. The stability of an analyte in a particular matrix and container system is relevant only to that matrix and container system and should not be extrapolated to other matrices and container systems. Stability procedures should evaluate the stability of the analytes in biological fluids after long-term (frozen at the intended storage temperature and conditions) and short- term (bench top,room temperature and conditions) storage, and after going through freeze and thaw cycles and the analytical process. The procedure should also include an evaluation of analyte stability in stock solution (Buick 1985, Pachla 1989).All stability determinations should use a set of standard samples prepared from a freshly made stock solution of the analyte in the appropriate analyte-free, interference-free biological matrix. Stock solutions of the analyte for stability evaluation should beprepared in an appropriate solvent at concentrations defined in the method SOP. Further information about validation for these factors appears in the following five sections of the guidance.1.Freeze and Thaw StabilityTesting for freeze and thaw analyte stability should be determined during three freeze and thaw cycles. At least three aliquots at each of the low and highconcentrations should be stored at -20C, or the intended storage temperature, for 024 hours and thawed unassisted at room temperature. When completely thawed,the samples should be transferred back to the original freezer and kept refrozen for 12 to 24 hours. The cycle of thawing and freezing should be repeated two more times, then analyzed on the third cycle. If an analyte is unstable at -20C, the0stability sample should be frozen at -70C during the three freeze and thaw cycles 0(Shah 1992, Buick 1990).2.Short-Term Room Temperature Stabilityj:\!guidance\2578dft.wpd12/14/988Three aliquots of each of the low and high concentrations should be thawed atroom temperature and kept at this temperature from 4 to 24 hours (based on theexpected duration that samples will be maintained at room temperature in theintended study) and analyzed (Buick 1990).3.Long-Term StabilityThe storage time in long-term stability evaluation should exceed the time betweenthe date of first sample collection and the date of last sample analysis. Long-termstability should be determined by storing at least three aliquots of each of the lowand high concentrations under the same conditions as the study samples. Asuggested storage temperature for the majority of drugs and metabolites in a biological matrix is -20C, but lower temperatures (e.g., -70C) may be 0 0recommended to prevent degradation problems observed at higher temperatures.The volume of samples should be sufficient for analysis on three occasions. Theconcentrations of all the stability samples should be compared to the mean of backcalculated values for the standards at the appropriate concentrations from the firstday of long-term stability testing (Buick 1990).4.Stock Solution StabilityThe stability of stock solutions of drug and the internal standard should beevaluated at room temperature for at least 6 hours. The stability samples shouldthen be refrigerated or frozen for 7 to 14 days or other relevant period. Aftercompletion of the desired storage time, the stability should be tested by comparingthe instrument response with that of freshly prepared solutions (Buick 1990).5.Autosampler StabilityThe stability of processed samples in the autosampler should be determined at theautosampler temperature that will be used during analysis, which is usually roomtemperature, but may sometimes be a lower temperature (e.g., when a refrigeratedautosampler is used). Stability should be assessed over the anticipated run time forthe batch size to be used in studies. The stability of both the drug and the internalstandard should be evaluated in validation samples under these conditions bydetermining concentrations on the basis of original calibration standards.Although the traditional approach of comparing analytical results for storedsamples with those for freshly prepared samples has been referred to in thisguidance, other statistical approaches based on confidence limits are also availablefor the development of SOPs for evaluation of an analyte’s stability in a biologicalmatrix (Timm 1985). Whatever approach is used, the SOPs should clearlydescribe the statistical method and rules employed. Additional validation mayinclude investigation of samples from dosed subjects.F.Acceptance CriteriaAn analytical method is considered fully validated when it meets the following criteria:Precision:The between-batch CVs for low, medium, and high concentrations should be #15%, and #20% for the LOQ QC, using a minimum of three batches.Accuracy: The between-batch mean value should be within ±15% of the nominal value at low, medium, and high QC concentrations and should not deviate by more than ±20% at the LOQ.Sensitivity: The lowest standard should be accepted as the limit of quantitation of themethod if the between-batch CV at the LOQ QC is #20%.Specificity: The responses of interfering peaks at the retention time of the analyte should be less than 20% of the response of an LOQ standard. Responses of interfering peaks at the retention time of the internal standard should be #5% of the response of theconcentration of the internal standard to be used in studies.Stability: Long-term, short-term, freeze and thaw, stock solution, and autosamplerstability data should meet the criteria specified in the SOP.V.IN-STUDY VALIDATIONAssays of all samples of an analyte in a biological matrix should be completed within the time period for which stability data are available. In general, analysis of biological samples can be done with a single determination without duplicate or replicate analysis if the assay method has acceptable variability as defined by validation data. This is true for procedures where precision and accuracy variabilities routinely fall within acceptable tolerance limits. For a difficult procedure with a labile analyte, where high precision and accuracy specifications may be difficult to achieve, duplicate or even triplicate analyses may be recommended for better estimate of analyte.j:\!guidance\2578dft.wpd12/14/989A calibration curve should be generated for each analyte to assay samples in each analytical run and it should be used to calculate the concentration of the analyte in the unknown samples in the run. The spiked samples may contain more than one analyte. An analytical run could consist of either all the processed samples to be analyzed as one batch or a batch composed of processed unknown samples of one or more volunteers in a study, QC samples, and calibration standards. The calibration (standard) curve should cover the expected unknown sample concentration range in addition to a calibrator sample at LOQ. Estimation of concentration in unknown samples by extrapolation of standard curves below LOQ or above the highest standard is not recommended. Instead, the standard curve should be redefined or samples with higher concentration should be diluted and assayed (Shah 1992). All study samples from a subject should be analyzed in a single run.Once the analytical method has been validated for routine use, its accuracy and precision should be monitored regularly to ensure that the method continues to work satisfactorily. To achieve this objective, a number of separately prepared QC samples should be analyzed with processed test samples at intervals based on the total number of samples. The QC samples in duplicate at three concentrations (one near the LOQ (i.e., #3 x LOQ), one in midrange, and one close to the high end of the range) should be incorporated in each assay run. The results of the QC samples provide the basis of accepting or rejecting the run. At least four of the six QC samples should be within ±20% of their respective nominal value. Two of the six QC samples may be outside the±20% of their respective nominal value, but not both at the same concentration (Shah 1992, Brooks 1985, Buick 1990, Mehta 1989, Ayers 1981).VI.DOCUMENTATIONThe validity of an analytical method should be established and verified by laboratory studies. Documentation of successful completion of such studies should be provided in the assay validation report. Protocols that define a set of specific directions that must be followed are important if the analytical results are useful for a given purpose.General and specific SOPs and good record keeping are essential parts of a validated analytical method. The analytical protocols and SOPs should be signed and dated by the laboratory director and updated regularly. The SOP should state situations under which reassay of samples is permitted. Reassays should be done in triplicate.The pre-study validation experiments, the data generated from them, and the assay quality control data should be recorded in a bound laboratory notebook. The entries should be signed by the chemist and witnessed by the laboratory supervisor. All records should be available for data audit and inspection.Documentation for pre-study validation should include:! A description of the analytical method! A description of stability studies and supporting data! A description of experiments conducted to determine accuracy, precision, recovery, specificity, linearity, limit of quantitation, and relevant data obtainedfrom these studies!Tables of intra- and inter-day precision and accuracy!Evidence of purity of drug standards, metabolites, and internal standards used in validation experiments!Deviations from SOP, if any, and justification for deviationDocumentation for in-study validation should include:!Calibration curves used in analyzing samples and intra-day accuracy and precision data!Information on inter-day values of QC samples and data on inter-day accuracy and precision from calibration curves and QC samples used for accepting the analyticalrun! A protocol for reassay of samples that describes the reasons for reassay and acceptance criteria for reassayed samples!Reasons for missing samples!Acceptance criteria for reported values when all unknown samples are assayed in duplicate!Deviations from the protocol or SOP, with reasons and justifications for the deviationsDocumentation for submission to the Agency should include:!Pre-study validation data!Calibration curves, equations, and weighting factors used, if any!In-study validation data。
生物组织样品的处理流程
生物组织样品的处理流程Processing biological tissue samples is a critical step in various scientific research and medical diagnostics. The process involves a series of steps to ensure the preservation and preparation of the tissue for analysis. 生物组织样品的处理流程是科学研究和医学诊断中非常关键的一步。
这个过程包括一系列步骤，以确保组织的保存和准备用于分析。
First and foremost, it is important to properly collect the tissue sample. This may involve a surgical procedure to obtain the tissue from a specific area of the body. The collection process must be meticulously executed to avoid contamination or damage to the tissue. 首先并且最重要的是正确地收集组织样品。
这可能涉及手术程序，从身体特定部位获取组织。
收集过程必须精心执行，以避免污染或损坏组织。
Once the tissue sample is collected, it needs to be preserved to maintain its integrity and prevent degradation. This is typically done through a process of fixation, where the tissue is treated with chemicals to stabilize its structure. Proper preservation is crucial for ensuring accurate analysis of the tissue. 一旦收集到组织样本，就需要进行保护，以保持其完整性并防止降解。
生物样品的预处理
蛋白酶抑制剂的添加
保护剂的添加(β-巯基乙醇、EDTA、辅酶等)
提取物缓冲液的选择(中性)
提取液的澄清( 高速离心、沉淀、吸附等)
匀浆化前的预处理(冷冻)
1.2 动物组织预处理注意事项
各种发酵产品，由于菌种不同和发酵液特性不同，其预处理方法的选择也有所不同。
01
大多数发酵产物存在于发酵液中，也有少数产物存在于菌体中，或发酵液和菌体中都含有。
螯合剂法
叁
贰
壹
酚类化合物的干扰及对策
牛血清白蛋白（BSA）法 原花色素类物质与BSA间可产生类似于抗原－抗体间的相互作用，形成可溶性的或不溶性的复合物，减小了原花色素类物质与RNA结合的机会
丙酮法 用-70℃的丙酮抽提冷冻研磨后的植物材料,可有效从云杉、松树、山毛榉等富含酚类化合物的植物材料中分离到高质量的RNA
高速匀浆机
高速匀浆法为大规模细胞破碎的常用方法，设备由高压泵和匀浆阀组成。
研磨法与珠磨法 研磨法(实验室规模) 由陶瓷的研钵和研杆组成，加入少量研磨剂（如精制石英砂、玻璃粉、硅藻土） 珠磨法(工业规模) 进入珠磨机的细胞悬浮液与极细的玻璃小珠、石英沙、氧化铝等研磨剂（直径<1 mm）一起快速搅拌或研磨，研磨剂、珠子与细胞之间的互相剪切、碰撞，使细胞破碎,释放出内含物。在珠液分离器的协助下，珠子被滞留在破碎室内，浆液流出从而实现连续操作。 适用：微生物（细菌）与植物细胞
通过Li+或Ca2+沉淀RNA的方法可以将未被氧化的酚类化合物去除。
01
03
02
植物组织往往富含多糖，而多糖的许多理化性质与RNA很相似，较难将它们分开。含多糖的RNA沉淀难溶于水，或溶解后产生粘稠状的溶液，对RNA提取造成较大的干扰。
Biolog微生物鉴定步骤
Biolog微生物鉴定步骤一检测原理Biolog微生物鉴定系统测试的是微生物在鉴定板中利用或氧化化和物的能力。
测试会产生特征性的紫色孔模式，组成代谢指纹。
所有必需的营养物质和生化试剂都预先加进96孔板中，四唑紫是一种氧化还原染料，指示碳源的利用情况。
.鉴定步骤非常简单，纯化分离到的菌株经扩大培养，再制成接种液加到鉴定板中。
在培养过程中，一些孔中的化学物质能被氧化并将显色物质成紫色，对照孔（A-1）和阴性孔仍然为无色。
鉴定板在相应的培养条件下培养4-6小时或16-24小时即可形成代谢模式。
系统软件自动和数据库对比，如果能找到合适的匹配，就可以得出一个鉴定结果。
二所需器材和消耗品：培养基、接种液、巯基乙酸钠、长棉签、接种棒、储液槽、八道移液器、移液器头、浊度仪、浊度标准品、控温培养箱和相应的鉴定板。
其中接种液自行配制，接种棒、储液槽可选用国产品牌代替。
三鉴定步骤：Biolog微生物鉴定样品处理步骤分离纯化培养基BUG+B通用培养基加羊血BUA+B厌氧培养基加羊血BUY酵母培养基2%ME2%的麦芽汁提取物革兰氏染色和菌落菌株形态观察革兰氏染色结果革兰氏阴性革兰氏阳性厌氧菌酵母菌丝状真菌确认实验氧化酶反应阳性氧化酶反应阴性、三糖铁实验A/A或K/A需在巧克力培养基上或需要6.5%CO2培养确认实验氧化酶反应阴性、三糖铁实验K/K或K/A w第一步：在用户自己的培养基上纯化菌株，如果菌株为冻干或冷冻样品，需要传代培养2-3代，让菌株恢复活力。
对纯化好的菌株做革兰氏染色，确定菌株是革兰氏阴性还是阳性。
观察菌落外部形态或用显微镜观察菌株形态，确定是酵母还是丝状真菌，是球菌还是杆菌。
如果是革兰氏阴性菌，还需要最终确认是肠道菌、非肠道菌或苛生菌。
方法是，氧化酶阳性或氧化酶阴性但三糖铁实验为K/K或K/A w，则该菌株为非肠道菌(GN-NENT)，氧化酶阴性以及三糖铁实验为A/A或K/A，则该菌株为肠道菌(GN-ENT)。
细菌学采集送检流程
细菌学采集送检流程英文回答：Bacteriological Specimen Collection and Submission Procedure.1. Specimen Collection.Swabs:Collect specimens from the appropriate anatomical site using a sterile swab.Rotate the swab gently over the surface of the lesion or discharge.Avoid contaminating the swab with surrounding skin or mucous membranes.Pus and exudates:Collect purulent material or exudates using asterile needle and syringe or a sterile swab.Aspirate the fluid from deep cavities or abscesses.Avoid aspirating from areas of obvious contamination.Blood:Collect blood samples according to thelaboratory's specific requirements.Urine:Collect a fresh, mid-stream urine sample in asterile container.Avoid collecting the first or last portion of urine.Stool:Collect stool specimens in a clean, dry container.Avoid collecting samples that are contaminated with urine or toilet paper.2. Specimen Transport.Place the collected specimen in a sterile container with appropriate preservation media, if necessary.Label the container with the patient's name, date of collection, specimen type, and site of collection.Transport the specimen to the laboratory promptly at the appropriate temperature.3. Specimen Submission.Submit the specimen to the laboratory along with a completed requisition form.Include relevant clinical information, such as symptoms, date of onset, and any known exposures.4. Quality Control.Ensure that the specimen is properly collected, labeled, and transported.Monitor the transport temperature and record any deviations.中文回答：细菌学标本采集送检流程。
生物医用材料评价流程
生物医用材料评价流程下载温馨提示:该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。
文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor. I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copy excerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!生物医用材料评价流程主要包括以下步骤：1. 材料收集：收集各种生物医用材料的相关信息，如材料的成分、结构、性能等。
微生物限度检查操作规程(中国药典2015版四部通则)
菌总数、控制菌的检查。
二、引用标准：《中国药典》2015年版(通则1105-1106)非(2)．目测霉变者以不合格论。
单）、分度小时，除再用刷和肥皂粉，内外刷洗，再用清水涮洗干净，晾干备用。
容量仪器宜用清洁液浸泡或涮洗，再用流试管及培养皿：先正放或直立于高压蒸汽灭菌器内，经121℃灭菌30 分钟。
趁热倾出培养物，再以清水或用毛刷及肥皂粉刷洗，最后以流水涮净。
吸管：全部直立浸没于清洁液的长筒形容器中，筒底应衬有棉或橡皮垫，以防管尖损坏。
24小时后，逐支用流水反复冲洗洁净，晾干后包扎灭菌备用。
载、盖玻片：应分别浸泡于清洁液12~24小时后，取出用流水冲洗，再放入3%~5%肥皂水或5%碳酸钠液内煮沸10~15 分钟，待自然冷却后，再用流水冲洗。
沥干后置95%乙醇中浸泡，晾干备用。
2，3，3玻璃器皿用前应洗涤干净，无残留抗菌物质。
吸管口上端距0.5 cm处塞入约2cm的适宜疏松棉花，置吸管桶内或牛皮纸袋中。
锥形瓶、量筒、试管均应加棉塞或硅胶塞，若用振荡器制备混悬液时，尚需用玻璃纸包裹瓶塞（以免振荡时供试液污染瓶塞），再用牛皮纸包扎。
玻璃器皿，均于160℃干热灭菌2小时或高压蒸汽121℃灭菌30 分钟，烘干备用。
2.4用具2.4.1大、小橡皮乳头（放于干净带盖的容器中，并应定期用70%~75%乙醇溶液浸泡）。
2.4.2无菌衣、帽、口罩、手套（洗净后配套，用牛皮纸包严）灭菌，备用。
也可用一次性无菌衣、帽、口罩、手套。
2.4.3接种环（白铱金或镍铬合金，环径4~5mm、长度6~10cm）、乙醇灯、乙醇棉球或碘伏棉球、。
500ml。
100ml；。
变色3.1.11．亚甲蓝指示液：取亚甲蓝0.5g，加水使溶解成100ml。
3.1.12．溴麝香草酚蓝指示液：取溴麝香草酚蓝0.4g，加1mol/L氢氧化钠溶液0.64ml使溶解，再加水至100ml。
变色范围 pH6.0~7.6（黄→蓝）。
3.1.13．酸性品红指示液：以酸性品红0.5g，加水100ml使溶解，再逐渐加1mol/L氢氧化钠溶液16ml，每加1滴均应将溶液充分摇匀后再加第二滴，直至溶液呈草黄色；于沸水内保持15分钟，再静置2小时，滤过，即得。
biological reference preparation 生物标准品
biological reference preparation 生物标准品
生物标准品（biological reference preparation）广泛应用于生物医学领域的质量控制和质量保证。
生物标准品是由已知浓度并经过特定处理的生物分子，如蛋白质、激素、细胞因子等，它们具有较高的稳定性和一致性。
这些生物标准品被广泛用于各种生物学试验和医学诊断、治疗、研究等领域中，以确保实验数据的准确性和可比性。
生物标准品的制备和检测需要经过严格的质量控制和验证，以确保产品的质量和安全性。
生物标准品在现代医学和生物学领域中有着重要的地位，对于研究和治疗人类疾病具有重要的推动作用。
第 1 页共 1 页。
生物样品预处理技术
取500g有代表性的猪肉可食部分，用组织捣碎机充 分捣碎均匀；
样品均匀化
取2g 捣碎样品，加入8ml乙酸钠缓冲液(pH=5.2)，再
• 固相萃取
• • • • • • • 操作： 活化 上样 淋洗 洗脱 收集 直接进样或者 浓缩后进样 与色谱理论相同
二、生物样品预处理方法
二、生物样品预处理方法
• 采用离心或者抽真空 的办法，以一定离心 力或者流速，进行上 样、淋洗和洗脱
二、生物样品预处理方法
• 固相萃取的优点：
• 无乳化现象 • 使用溶剂安全价格低廉
有机强酸
季铵碱、盐
有机酸、酚类
示例 不同生物样品中虎杖苷预处理方法
• A：虎杖苷标准对照品 由海王集团技术中心天然药物室提供 批号：03050803 • 3，4’，5-三羟基芪-3-β-D-葡萄糖苷 Mr390 • • B：二苯乙烯苷化学对照品 由中国药品生物制品检定所提供 • 2，3，5，4‘-四羟基二苯乙烯-β-D-葡萄糖苷 Mr406
• 萃取效率高
• 处理速度快，具有一定通量 • 易于同时萃取出目标药物及其代谢产物，包括极性 强的Ⅱ相代谢产物
• MCX固相萃取柱 • 反相作用和阳离子交换 作用
• 酸性环境中含氮碱性基 团与磺酸基结合 • 酸性药物呈分子状态发 生反相结合作用
对有机碱: 对有机酸、酚类 对有机强酸 对季铵碱、盐 pKa 2-8 pKa 2-10 pKa <1.0 pKa >10 选择 Oasis® MCX 选择 Oasis® WAX 选择 Oasis® WCX 选择 Oasis® MAX
• 储存条件
–20℃：大部分药物 –80℃：阿莫西林等抗生素 加稳定剂：卡托普利等
Biolog接种液、培养基制备方法
BIOLOG培养基制备方法一、BUG+B培养基的制备1. 取一个容器，按量称取BUG培养基，如需配制1000mL培养基，方法如下：57g BUG琼脂培养基950ml 纯净水、蒸馏水或去离子水2. 煮沸溶解。
3. 冷却至25℃调整pH值至7.3± 0.1。
4. 121℃灭菌15分钟。
5. 冷却至45-50℃。
6. 加50mL新鲜的脱纤羊血，摇匀。
7. 倒平板。
二、BUG+M培养基的制备1. 取一个容器，按量称取BUG培养基，如需配制1000mL培养基，方法如下：57g BUG琼脂培养基990ml 纯净水、蒸馏水或去离子水。
2. 煮沸溶解。
3. 冷却后调整pH值至7.3 ±0.1（25℃）。
4. 121℃灭菌15分钟。
5. 冷却至45-50℃。
6. 加10mL已灭菌的麦芽糖（浓度25%），混匀。
7. 倒平板。
三、BUG培养基的制备1. 取一个容器，按量称取BUG培养基，如需配制1000mL培养基，方法如下：57g BUG琼脂培养基1000 ml 纯净水、蒸馏水或去离子水。
2. 煮沸溶解。
3. 冷却后调整pH值至7.3 ±0.1（25℃）。
4. 121℃灭菌15分钟。
5. 冷却至45-50℃。
6. 倒平板。
四、BUA+B培养基的制备1. 取一个容器，按量称取BUA培养基，如需配制1000mL培养基，方法如下：51.7g BUA琼脂培养基950 ml纯净水、蒸馏水或去离子水。
2. 用无氧的氮气吹洗下，轻微煮沸，搅拌以溶解琼脂和其它组份。
3. 冷却后调整pH值至7.2 ±0.1（25℃）。
4. 121℃灭菌15分钟。
注意盖紧瓶盖，防止氧气进入。
5. 在无氧的氮气保护下，冷却至45-50℃。
6. 加50mL新鲜的脱纤羊血，摇匀。
7. 在厌氧环境中倒平板。
五、BUY培养基的制备1. 取一个容器，按量称取BUY培养基，如需配制1000mL培养基，方法如下：60g BUY琼脂培养基1000ml 纯净水、蒸馏水或去离子水。
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（二）四氧化锇 主要优点：是一种强氧化剂，对氮具有很大亲和力，能和 含氮的化合物蛋白质发生化学结合来稳定蛋白质，故而能 够近乎完满的保存生物的微细结构。具有“电子染色“作 用。
主要缺点：对于碳水化合物（糖类），核酸等的固定作用 差。
常规透射样品的制备流程
（1）取材 动物样品 植物样品 细胞样品 取材注意事项：低温操作，快，准，小
乙醇10-15分钟/次 （7）叔丁醇浸透2小时 （8）冷冻干燥 （9）离子溅射仪或喷镀仪喷金 （10）扫描电子显微镜观察
生物样品的其他制备技术
1.滴网、负染色技术； 2.细胞化学技术（酶细胞化学技术；免疫电镜技术 ；离子
细胞化学技术） 3.冷冻蚀刻技术； 4.核酸分子杂交技术（原位杂交免疫电镜技术）； 5.标本的能谱分析；
（一）戊二醛 主要优点： 1.对组织的穿透力比四氧化锇较强，较快，故组织块可适
当增大到数毫米而不影响固定效果。 2.组织在戊二醛固定液中保存较长时间（数周至数月），
对精细结构保存并无不利影响。 3.对糖原，核蛋白，尤其对微管，内质网等细胞膜系统和细
胞基质有较好的固定作用，从而弥补锇酸固定液之不足。 4.戊二醛不挥发，因此没有蒸发中毒的危险，但不要太多的
接近或接触. 5.适用于进行超微细胞化学研究。
• 主要缺点：
1.戊二醛对脂质不起固定作用，用它固定的一些微细结构 在脱水时可能被抽提而丢失，因此它也不适用于单独作电 镜技术的固定剂。最好作为四氧化锇的前固定剂使用。
2.戊二醛没有“电子染色”作用，用它固定的样品反差较 差。
3.戊二醛不影响细胞的渗透压，所以对缓冲液的渗透压， 以及其他条件要求较高，固定效果与缓冲液有很大关系。
超 薄 切 片 技 术
常规扫描样品制备流程
（1）取材 动物样品 植物样品 细胞样品 取材注意事项：低温操作，快，准，小
（2）3%戊二醛前固定 （3）1/15M PBS液漂洗3次 10-15分钟/次 （4）1%四氧化锇后固定1.5h （5）1/15M PBS液漂洗3次 10-15分钟/次 （6）梯度脱水 50% ， 70% ，80% ，90%， 100%
多聚甲醛-戊二醛混合固定液：1/15 PBS 50ML 10%多聚甲醛 20ML 25%戊二醛水溶液 10ML 双蒸水加至100ML
固定液中含2%多聚 甲醛 2.5%戊二醛
*配置10%的多聚甲醛溶液配置20ML
• 多聚甲醛粉末5g
• 双蒸水50ML
取5g多聚甲醛放进80ML烧杯内注入双蒸水50ML.必须在通 风厨内加热到60度左右（温度不可再高，以免煮沸溢出） 若用电炉加热，温度在60度左右可以拔掉插头，并且不停 用玻璃棒搅拌至溶液为乳白色，多聚甲醛粉末溶解时，用 滴管加1N NaoH，同时搅拌至溶液清亮，冷却待用。
生物样品制备技术
1、前期准备（试剂瓶的清洗，液体的配制）
(1)1/15M磷酸缓冲液
A液：KH2PO4
1.816g
100ML 双蒸水
B液： Na2HPO4 5.5g
双蒸水200ML
PH值7.4 1/15MPBS液：A液19ML B液81ML
(2)固定液的配制 戊二醛固定液：3%戊二醛（25%戊二醛水溶液 12ML 1/15MPBS液 88ML )
（2）3%戊二醛前固定 （3）1/15M PBS液漂洗3次 10-15分钟/次 （4）1%四氧化锇后固定1.5h （5）1/15M PBS液漂洗3次 10-15分钟/次 （6）梯度脱水 50% ， 70% ，80% ，90%， 100%
乙醇10-15分钟/次，100%纯丙酮2次，10-15分钟/次 （7）1:1丙酮:EPON-812包埋剂浸透2h （8）包埋，35℃，24h，45℃，24h,68℃，24h聚合 （9）半薄切片，甲苯胺蓝染色，光镜下定位 （10）60-80nm超薄切片，枸橼酸铅，醋酸柚双染
谢谢大家
病 毒
药 剂 颗 粒
材 料
酶 反 应 物 沉 积
冷 冻 蚀 刻
扫细 描胞 图器 （的 粗冷 面冻 内断 裂 网蚀 ）刻
线 粒 体 （
）
SEM
正常人体组织细胞结构超微结构和功能的研究
扫描电镜应用
昆 虫 表 面 形 貌
材料表面形貌的观察
细 菌 形 态 的 观 察
透射电镜应用
（3）锇酸固定液： 2%四氧化锇原液 四氧化锇1g 放入干净棕色瓶内 双蒸水50ML 4度冰箱保存
(3)脱水剂的配制：50% 70% 80% 90% 100%丙酮
(4)包埋剂的配制：EPON-812 14 ml DDSA 6.2ml MNA 8.9ml DMP-30 4滴
100% 乙醇
固定液特点