07.100.01 微生物学综合 标准查询与下载

ASTM E2562-22 用CDC生物膜反应器在高剪切和连续流动条件下生长的绿脓杆菌生物膜定量的标准试验方法

1.1 This test method specifies the operational parameters required to grow a reproducible (1)2 Pseudomonas aeruginosa ATCC 700888 biofilm under high shear. The resulting biofilm is representative of generalized situations where biofilm exists under high shear rather than being representative of one particular environment. 1.2 This test method uses the Centers for Disease Control and Prevention (CDC) Biofilm Reactor. The CDC Biofilm Reactor is a continuously stirred tank reactor (CSTR) with high wall shear. Although it was originally designed to model a potable water system for the evaluation of Legionella pneumophila (2), the reactor is versatile and may also be used for growing and/or characterizing biofilm of varying species (3-5). 1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded as log10 colony forming units per surface area. 1.4 Basic microbiology training is required to perform this test method. 1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor

T/LTIA 13-2021 基因检测服务中微生物组数据共享规范

在本标准制定过程中，随着研究的深入，先后完成标准讨论稿、草案和标准征求意见稿，标准内容不断完善，并趋于科学合理。根据需要，本标准的主要技术内容确定为： 在术语和定义中，对元数据，微生物组，宏基因组学，扩增子，生物观测矩阵格式，数据生产，数据分析，分析级数据，数据存储，数据服务，基因丰度，分类学丰度，功能丰度，相对丰度，操作分类单元，FASTQ格式，TSV格式等进行了定义。 在微生物组数据共享和交换流程中，描述了微生物组数据的生产、传输、使用等过程。将此过程分为四个环节：数据生产、数据分析、数据存储和数据服务，并描述各环节细节及注意要点。 　在微生物组数据元数据中，标准根据数据产生的环节，将元数据分为：样本信息关联的元数据、测序数据关联的元数据和分析级数据关联的元数据三个部分，规范了各部分微生物组数据交换中的常用元数据和最低要素。 在微生物组数据格式中，标准规范了微生物组数据在各个过程环节中的数据格式，介绍了常用格式作为参考。根据产生的环节，微生物组数据分为原始数据和分析级数据。高通量测序方法的原始数据为FASTQ，分析级数据指原始数据经生物信息学方法分析所得的微生物组成分组成信息，并提出TSV（制表格分割文本）和BIOM格式可作为分析级数据文件存储使用的文件格式。此外，也提出了在网络交互场景下的建议数据结构。

Specification for the interoperability of microbiome data in genetic testing services

NY/T 3960-2021 水生外来入侵植物监测技术规程

Technical regulations for monitoring aquatic alien invasive plants

NY/T 3959-2021 农业外来入侵昆虫监测技术导则

Technical Guidelines for Monitoring Invasive Alien Insects in Agriculture

ASTM E3161-21 使用CDC生物膜反应器制备绿脓杆菌或金黄色葡萄球菌生物膜的标准实施规程

1.1 This practice specifies the parameters for growing a Pseudomonas aeruginosa (ATCC 15442) or Staphylococcus aureus (ATCC 6538) biofilm that can be used for disinfectant efficacy testing using the Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method (E2871) or in an alternate method capable of accommodating the coupons used in the CDC Biofilm Reactor. The resulting biofilm is representative of generalized situations where biofilm exist on hard, non-porous surfaces under shear rather than being representative of one particular environment. Additional bacteria may be grown using the basic procedure outlined in this document, however, alternative preparation procedures for frozen stock cultures and biofilm generation (for example, medium concentrations, baffle speed, temperature, incubation times, coupon types, etc.) may be necessary. 1.2 This practice uses the CDC Biofilm Reactor created by the Centers for Disease Control and Prevention (1).2 The CDC Biofilm Reactor is a continuously stirred tank reactor (CSTR) with high wall shear. The reactor is versatile and may also be used for growing or characterizing various species of biofilm, or both (2-4) provided appropriate adjustments are made to the growth media and operational parameters of the reactor. 1.3 Basic microbiology training is required to perform this practice. 1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this practice. 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Standard Practice for Preparing a Pseudomonas aeruginosa or Staphylococcus aureus Biofilm using the CDC Biofilm Reactor

ASTM E3273-21 使用空气生物室评估室内空气微生物净化的标准实施规程

1.1 This practice is to assess technologies for microbial decontamination of indoor air using a sealed, room-sized chamber (~24 m3 ) as recommended by the U.S. Environmental Protection Agency (3). The test microbe is aerosolized inside the chamber where a fan uniformly mixes the aerosols and keeps them airborne. Samples of the air are collected and assayed, firstly to determine the rates of physical and biological decay of the test microbe, and then to assess the air decontaminating activity of the technology under test as log10 or percentage reductions in viability per m3 (1). The air temperature and relative humidity (RH) in the chamber are measured and recorded during each test. 1.2 The chamber can be used to assess microbial survival in indoor air as well as to test the ability of physical (for example, ultraviolet light) and chemical agents (for example, vaporized hydrogen peroxide) to inactivate representative pathogens or their surrogates in indoor air. 1.3 This practice does not cover testing of microbial contamination introduced into the chamber as a dry powder. 1.4 This practice does not cover work with human pathogenic viruses, which require additional safety and technical considerations. 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Standard Practice to Assess Microbial Decontamination of Indoor Air using an Aerobiology Chamber

KS J 0012-2021 II级（层流）生物安全柜

Class II(laminar flow) biosafety cabinetry

ASTM E3321-21 用于评估用于预防大肠杆菌生物膜生长的抗菌尿管的腔内导管模型的标准试验方法

1.1 This test method specifies the operational parameters required to assess the ability of antimicrobial urinary catheters to prevent or control biofilm growth. Efficacy is reported as the log reduction in viable bacteria when compared to a repeatable (1)2 Escherichia coli biofilm grown in the intra-lumen of a urinary catheter under a constant flow of artificial urine. 1.2 The test method is versatile and may also be used for growing and/or characterizing biofilms and suspended bacteria of different species, although this will require changing the operational parameters to optimize the method based upon the growth requirements of the new organism. 1.3 This test method may be used to evaluate surface modified urinary catheters that contain no antimicrobial agent. 1.4 This test method describes how to sample and analyze catheter segments and effluent for viable cells. Biofilm population density is recorded as log colony forming units per surface area. Suspended bacterial population density is reported as log colony forming units per volume. 1.5 Basic microbiology training is required to perform this test method. 1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard 1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Standard Test Method for Intraluminal Catheter Model used to Evaluate Antimicrobial Urinary Catheters for Prevention of Escherichia coli Biofilm Growth

ASTM F1608-21 多孔包装材料微生物分级的标准试验方法（暴露室法）

1.1 This test method is used to determine the passage of airborne bacteria through porous materials intended for use in packaging sterile medical devices. This test method is designed to test materials under conditions that result in the detectable passage of bacterial spores through the test material. 1.1.1 A round-robin study was conducted with eleven laboratories participating. Each laboratory tested duplicate samples of six commercially available porous materials to determine the Log Reduction Value (LRV) (see calculation in Section 12). Materials tested under the standard conditions described in this test method returned average values that range from LRV 1.7 to 4.3. 1.1.2 Results of this round-robin study indicate that caution should be used when comparing test data and ranking materials, especially when a small number of sample replicates are used. In addition, further collaborative work (such as described in Practice E691) should be conducted before this test method would be considered adequate for purposes of setting performance standards. 1.2 This test method requires manipulation of microorganisms and should be performed only by trained personnel. The U.S. Department of Health and Human Services publication Biosafety in Microbiological and Biomedical Laboratories (CDC/NIH-HHS Publication No. 84-8395) should be consulted for guidance. 1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Standard Test Method for Microbial Ranking of Porous Packaging Materials (Exposure Chamber Method)

ASTM E3286-21 非化学抗菌处理技术杀菌性能评估用玻璃表面细胞单层制备的标准实施规程

1.1 This practice provides a protocol for creating bacterial cell monolayers on a flat surface. 1.2 The cultures used and culture preparation steps in this Practice are similar to AOAC Method 961.02 and US EPA MB-06. However, test bacteria are applied to the carrier using an automated deposition device (6.2) rather than as a suspension droplet. 1.3 The carrier inspection protocol is similar to US EPA MB-03 except that carrier surfaces are inspected microscopically rather than visually, unaided. 1.4 A monolayer of cells eliminates the confounding effect caused by the shadowing effect of outer layers of bacteria stacked upon other bacteria on test specimens – thereby attenuating directed energy beams (that is, ultraviolet light, high-energy electron beams) before they can reach underlying cells. 1.5 An asperity-free surface eliminates the shadowing effect of specimen surface topology that can block direct exposure of target bacteria to non-chemical antimicrobial treatments. 1.6 This practice provides a reproducible target microbe and surface specimen to minimize specimen variability within and between testing facilities. This facilitates direct data comparisons among various non-chemical antimicrobial technologies. 1.6.1 Antimicrobial pesticides used in clinical and industrial applications are expected to overcome shadowing effects. However, this practice meets a need for a protocol that facilitates relative comparisons among non-chemical antimicrobial treatments. 1.6.2 This practice is not intended to satisfy or replace existing test requirements for liquid chemical antimicrobial treatments (for example Test Methods E1153 and E2197) or established regulatory agency performance standards such as US EPA MB-06. 1.7 This practice was validated using Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442) using a protocol based on AOAC Method 961.02. If other cultures are used, the suitability of this practice must be confirmed by inspecting prepared surfaces, by using scanning electron microscopy (SEM) or comparable high-resolution microscopy. 1.8 The specimens prepared in accordance with this practice are not meant to simulate end-use conditions. 1.8.1 Non-chemical technologies are only to be used on visibly clean, non-porous surfaces. Consequently, a soil load is not used. 1.9 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.10 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.11 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies

T/CI 011-2021 细菌生物被膜的培养、观测和耐药性评测技术指南

细菌生物被膜培养方法与技术标准 以下生物被膜培养方法与技术标准均以铜绿假单胞菌为模式菌株撰写，其他菌种培养以及实验条件可按需求适当调整。 铜绿假单胞菌（Pseudomonas aeruginosa）是一种常见的革兰氏阴性条件致病菌，在正常人皮肤、呼吸道、肠道以及多种自然环境如水、土壤中广泛分布。此菌为杆状单鞭毛形态，适宜生长温度为25-37℃，在42℃也可生长，于LB培养基上呈青绿色圆形菌落且极易附着在不同基质表面形成生物被膜。铜绿假单胞菌是医院内感染的主要致病菌之一，可在免疫力低下的患者不同部位形成生物被膜引起长期慢性感染，包括烧伤伤口感染、呼吸道感染和尿路感染等，并可引发菌血症和败血症等致命并发症。此菌对多种抗生素耐药，并且耐药机制非常复杂，包括形成生物被膜、加强外排泵系统、改变细胞膜通透性与药物作用靶点、以及分泌抗菌酶等。铜绿假单胞菌生物被膜的形成机制也相当复杂，例如通过过量分泌胞外多糖、积累胞外DNA、降低运动能力、调节群体感应系统等。作为研究生物被膜的模式菌株，铜绿假单胞菌早已被广泛应用于构建生物被膜、检测其耐药性并开发用药方案的研究中，是以本指南以铜绿假单胞菌为模式菌株撰写，其他菌种的培养及实验条件可按需适当调整以达到最佳实验结果。 4.1 体外静态生物被膜培养  本指南囊括四种常用的体外静态生物被膜培养方法，包括孔板培养法、玻璃珠培养法、玻片培养法和Peg-lid培养法。 （1）孔板培养法（4.1.1）将稀释菌液在96孔板或24孔板中培养，生物被膜附着于孔壁上，移除悬浮液并洗脱浮游菌体可得到生物被膜用于后续实验。孔板培养法一般用于检测菌种生物被膜形成能力、生物被膜定量、生物被膜耐药性和药物最小抑菌浓度等实验。此培养法可与多种染色法结合用于定量生物被膜，培养出的生物被膜结构可用不同显微镜观察。此培养法具有通量高、培养时间短和操作简单等优势，但培养的生物被膜厚度与硬度较低，浮游菌体不易洗脱造成平行间差异较大。 （2）玻璃珠培养法（4.1.2）在24孔板中将玻璃珠置于稀释菌液内培养，生物被膜附着于玻璃珠表面，洗脱浮游菌体可得到生物被膜用于后续实验。此培养法与孔板培养法类似，一般用于检测菌种生物被膜形成能力、生物被膜定量、生物被膜耐药性和药物最小抑菌浓度等试验。此培养法可与多种染色法结合用于定量生物被膜，但不易在显微镜下观察。此培养法具有通量高、培养时间短等优势，但操作略复杂。此方法培养的生物被膜厚度与硬度较高，浮游菌体较易清洗，平行间差异较小。 （3）玻片培养法（4.1.3）将玻片插入菌液培养，生物被膜附着于玻片表面，去除拨片并洗脱浮游菌体得到生物被膜用于后续实验。此方法一般用于检测菌种生物被膜形成能力、观察生物被膜结构与形态，可与多种染色方法和多种显微镜结合使用观测生物被膜构态。此方法通量高、培养时间短，但操作略复杂。此方法培养的生物被膜量较大、厚度与硬度高，浮游菌体易清洗，平行间差异较小。 （4）Peg-lid培养法（4.1.4）将带有凸起楔子（peg）的96孔板盖置于装有菌液的96孔板中培养，生物被膜附着于peg中下端，取下盖子并洗脱peg上的浮游菌体可得到生物被膜用于后续实验。此方法与孔板培养法类似，一般用于检测菌种生物被膜形成能力、生物被膜定量、生物被膜耐药性和药物最小抑菌浓度等试验。此培养法可与多种染色法结合用于定量生物被膜，但培养出的生物被膜由于在楔子顶端，不易于显微镜观察。此培养法具有通量高、培养时间短和操作简单等优势，培养的生物被膜厚度与硬度较高，浮游菌体较易洗脱、平行间差异较小。 每种培养与观测方法细节请见下文。 4.1.1 孔板培养法 适用范围：孔板生物被膜培养法通常适用于以下场景：（1）需要检测不同菌种（野生菌种和突变菌种）间生物被膜形成能力，（2）需要检测不同菌株在生物被膜状态下对药物的敏感/耐受性。针对孔板培养的生物被膜的分析方法主要包括：生物被膜定量（章节5.1.2a）、生物被膜耐药性和药物最小抑菌浓度（章节6.1）。以下示意装置和操作均以铜绿假单胞菌（PA）为模式菌株，在耗氧培养条件下进行示例：   图1. 体外静态生物被膜培养方法（孔板培养法） 4.1.1.1 实验材料： a. 无菌96孔板（或24孔板, Nest/Corning）； b. 无菌储液器； c. 100 μL排枪移液器（或1 mL移液器）； d. 15 mL无菌摇菌管； e. 100 μL（或1 mL）移液器枪头； f. LB培养基（可按需使用其他培养基）； g. 所需菌株； h. 废液桶； i. 75%酒精； j. 无菌ddH2O； k. 无菌生理盐水。 4.1.1.2 培养步骤【附录以protocol/SOP模式进行详细阐述】 首先将目标菌株接种至LB培养基中培养过夜。在无菌操作台中，用新鲜培养基将菌液按一定比例稀释，随后将稀释好的菌液倾倒入无菌储液器，再用排枪移液器将稀释菌液加入96孔板或24孔板孔洞中，并置于所需温度下静置培养生物被膜。实验菌种和对照菌种均按照附录Xa的培养步骤进行接种、培养（附录Xa），随后对生物被膜进行分析（章节5）。 4.1.1.3 技术要点 a. 所有操作须严格遵守无菌操作流程，在操作过程中注意须使用灭菌器具并用酒精消毒操作台以及所使用的器具，避免交叉污染； b. 在将菌液加入孔板前，轻轻摇晃储液器将菌液混合均匀，并用排枪反复轻轻吹打菌液，以减小实验误差； c. 孔板培养生物被膜需保证培养后孔洞内剩余菌液体积基本一致，培养时需用透气封口膜密封孔板防止液体蒸发； d. 移除废液以及清洗生物被膜时须注意不可用枪头触碰生物被膜，以保证生物被膜完整性。 4.1.2玻璃珠培养法 适用范围：玻璃珠生物被膜培养法通常适用于以下场景：（1）用于循环培养生物被膜进行压力条件进化实验，（2）需要检测不同菌株在生物被膜状态下对药物的敏感/耐受性。针对玻璃珠培养的生物被膜的分析方法主要为：CFU计数法（章节5.3.2b）。以下示意装置和操作均以铜绿假单胞菌（PA）为模式菌株，在耗氧培养条件下进行示例：   图2. 体外静态生物被膜培养方法（玻璃珠培养法） 4.1.2.1 实验材料： a. 无菌24孔板； b. 无菌储液器； c. 1 mL移液器 d. 15 mL无菌摇菌管； e. 1 mL移液器枪头； f. LB培养基（可按需使用其他培养基）； g. 无菌ddH2O（可按需使用PBS溶液或生理盐水）； h. 所需菌株； i. 废液桶； j. 75%酒精； k. 镊子； l. 3-5 mm玻璃珠。 4.1.2.2 培养步骤【附录以protocol/SOP模式进行详细阐述】 首先将目标菌株接种至LB培养基中培养过夜，用新鲜培养基将菌液按一定比例稀释，随后将稀释好的菌液转移至24孔板孔洞中，并加入两颗无菌玻璃珠，37℃ 100 rpm转速摇晃培养过夜。实验菌种和对照菌种均按照附录Xa的培养步骤进行接种、培养（附录Xb），随后对生物被膜进行分析（章节5）。 4.1.2.3 技术要点 a. 所有操作须严格遵守无菌操作流程，在操作过程中注意须使用灭菌器具并用酒精消毒操作台以及所使用的器具，避免交叉污染； b. 在将菌液加入孔板前，轻轻摇晃储液器将菌液混合均匀，并用移液器反复轻轻吹打菌液，以减小实验误差； c. 孔洞内菌液不可过多，以免摇晃培养时菌液溅出造成交叉污染； d. 用无菌镊子转移玻璃珠时，需尽量减少镊子与玻璃珠接触面，以保证生物被膜完整性； e. 孔板培养生物被膜需保证培养后孔洞内剩余菌液体积基本一致，培养时需用透气封口膜密封孔板防止液体蒸发。 4.1.3 玻片培养法 适用范围：玻片生物被膜培养法通常适用于以下场景：（1）需要检测不同菌种（野生菌种和突变菌种）间生物被膜形成能力，（2）需要检测不同菌株在生物被膜状态下对药物的敏感/耐受性。针对玻片培养的生物被膜的分析方法主要包括：生物被膜定量（章节5.1.2c）和生物被膜耐药性（章节5.3.2c）。以下示意装置和操作均以铜绿假单胞菌（PA）为模式菌株，在耗氧培养条件下进行示例：   图3. 体外静态生物被膜培养方法（玻片培养法） 4.1.3.1 实验材料： a. 无菌培养皿（静置培养需准备6孔板）； b. 15 mL无菌摇菌管； c. LB培养基（可按需使用其他培养基）； d. 无菌ddH2O（可按需使用PBS溶液或生理盐水） e. 所需菌株； f. 废液桶； g. 75%酒精； h. 镊子； i. 载玻片。 4.1.3.2 培养步骤【附录以protocol/SOP模式进行详细阐述】 首先将目标菌株接种至LB培养基中培养过夜，用新鲜培养基将菌液按一定比例稀释，随后将稀释好的菌液转移至培养皿，并加入无菌载玻片，37 ℃ 100 rpm转速摇晃培养过夜。实验菌种和对照菌种均按照附录Xa的培养步骤进行接种、培养（附录Xc），随后对生物被膜进行分析（章节5）。 4.1.3.3 技术要点 a. 所有操作须严格遵守无菌操作流程，在操作过程中注意须使用灭菌器具并用酒精消毒操作台以及所使用的器具，避免交叉污染； b. 培养皿内菌液不可过多，以免摇晃培养时菌液溅出造成污染； c. 用无菌镊子转移载玻片时，需尽量减少镊子与玻璃珠接触面，以保证生物被膜完整性； d. 摇晃培养时需用透气封口膜密封培养皿防止液体蒸发。 4.1.4 Peg-lid 培养法 适用范围：Peg-lid生物被膜培养法通常适用于以下场景：（1）需要检测不同菌种（野生菌种和突变菌种）间生物被膜形成能力，（2）需要检测不同菌株在生物被膜状态下对药物的敏感/耐受性。针对Peg-lid培养的生物被膜的分析方法主要包括：生物被膜定量（章节5.1.2c）和生物被膜耐药性（章节5.3.2c）。以下示意装置和操作均以铜绿假单胞菌（PA）为模式菌株，在耗氧培养条件下进行示例：   图4. 体外静态生物被膜培养方法（peg lid培养法） 4.1.4.1 实验材料： a. 96孔板； b. Peg lid盖子（见附录图三）; c. 无菌储液器； d. 100 μL排枪移液器； e. 15 mL无菌摇菌管； f. 100 μL（或1 mL）移液器枪头； g. LB培养基（可按需使用其他培养基）； h. 所需菌株； i. 废液桶； j. 75%酒精； k. 无菌ddH2O（可按需使用PBS溶液或生理盐水）； l. 无菌生理盐水。 4.1.4.2 培养步骤【附录以protocol/SOP模式进行详细阐述】 首先将目标菌株接种至LB培养基中培养过夜，用新鲜培养基将菌液按一定比例稀释，随后将稀释好的菌液转移至96孔板中，再将peg lid小心盖入菌液中。37℃ 100 rpm转速摇晃培养过夜。实验菌种和对照菌种均按照附录Xa的培养步骤进行接种、培养（附录Xd），随后对生物被膜进行分析（章节5）。 4.1.4.3 技术要点 a. 所有操作须严格遵守无菌操作流程，在操作过程中注意须使用灭菌器具并用酒精消毒操作台以及所使用的器具，避免交叉污染； b. 在将菌液加入孔板前，轻轻摇晃储液器将菌液混合均匀，并用排枪反复轻轻吹打菌液，以减小实验误差； c. 孔洞内菌液不可过多，以免置入peg lid或震荡培养时菌液溅出造成污染； d. 需保证培养后孔洞内剩余菌液体积基本一致，培养时需用透气封口膜密封孔板防止液体蒸发； e. 移除废液以及清洗生物被膜时须注意不可触碰生物被膜，以保证生物被膜完整性。 4.2 体外动态生物被膜培养  本指南囊括两种常用的体外静态生物被膜培养方法，包括管式培养法和池式培养法，详细介绍如下： （1）管式培养法将菌液注入硅胶管内静置附着后，将培养基以匀速注入硅胶管，流动培养生物被膜，生物被膜附着于管壁上。此方法一般应用于对生物被膜样品需求量大的实验，如生物被膜样品收集，胞外多糖收集生物转化（biotransformation）等。此方法可形成大量生物被膜，生物被膜结构厚，通量较高，但培养时间较长，操作较复杂，并不易用显微镜观察生物被膜结构。 （2）池式培养法将菌液注入培养池通道内，翻转静置待细菌附着与盖子上之后，将培养基以匀速注入培养通道，流动培养生物被膜，生物被膜附着于三通道池盖上。此方法一般应用于生物被膜形态观察、药物对生物被膜形态的影响等实验，此方法形成生物被膜形态与结构完整，可结合有荧光标记的菌株或不同染色法，直接在显微镜下观测生物被膜形态与结构，但通量较低，操作复杂，培养时间较长。 4.2.1 管式培养法（tubing-biofilm） 适用范围：管式生物被膜培养法通常适用于以下场景：（1）需要模拟管道内生物被膜生长环境的研究，（2）需要相对大量生物被膜样品的研究，（3）生物被膜对药物/化合物代谢（biotransformation）的相关研究等。针对管式培养的生物被膜的分析方法主要包括：干重/湿重分析（章节5.4）、基因组/转录组/蛋白组提取和测序分析、电子显微镜观察（章节5.7）、氧气探针/化合物探针监测等。以下示意装置和操作均以铜绿假单胞菌（PA）为模式菌株，在耗氧培养条件下进行示例： 管式生物被膜培养法具有较高通量，可以一次性培养多通道生物被膜样品；该方法能够一次性产出大量生物被膜样品，常用于干重/湿重分析、基因样本提取，EPS等代谢产物提取、电子显微镜观察和氧气探针/化学探针观察等。   图5. 体外动态生物被膜培养方法（管式培养法） 4.2.1.1实验材料： a. 15 mL无菌摇菌管； b. 长50 cm，内直径1.0 mm 硅胶管（润泽）； c. 长2520 cm，内直径 1.0 mm 硅胶泵管（润泽）； d. 长15 cm，内直径1.0 mm 硅胶管（润泽）； e. 长10 cm，内直径3.2 mm 硅胶管（润泽）；  f. 长30 cm，内直径 1.0 mm 硅胶泵管（润泽）； g. 等径直通接头（润泽）； h. 变径直通接头（润泽）； i. 鲁尔接头（润泽）； j. 2个2L玻璃培养瓶； k. 10% LB培养基（可按需使用其他培养基）； l. 过滤器（过滤孔0.22 μm）； m. 蠕动泵（aperistaltic pump，MasterFlex）； n. 所需菌株； o. 医用尖锐物处理容器； p. 75%酒精； q. 镊子； r. 5 mL及1 mL无菌针管； s. 无菌针头； t. 移液器； u. 无菌移液管。 4.2.1.2培养步骤【附录以protocol/SOP模式进行详细阐述】 首先将装有培养基的玻璃培养瓶、硅胶管、除泡器和镊子在121℃高温高压灭菌15分钟，取出并将硅胶管、除泡器和镊子干燥备用。如图一所示，在无菌操作台中按顺序用安装双向接头的硅胶管b连接装有培养基的玻璃培养瓶、无阻泵、过滤器、硅胶管c、d、e、f，2L废液罐，同个装置内至少三个平行（图内展示一个平行），将整个系统置于恒温培养箱内。 将目标菌株接种至适当体积的LB培养基中，在所需温度下200 rpm摇晃培养过夜。在无菌操作台中，用新鲜LB培养基将菌液稀释至OD600nm=0.1，用无菌针管吸入2 mL稀释菌液，并将稀释菌液分别注入硅胶管b平行装置中，用硅胶封口后静置培养2小时使细菌细胞粘附于管壁, 用10% LB流动培养基培养3至7天，建立生物被膜。 首先将生物被膜管式培养装置进行灭菌和组装，在用药组的培养基玻璃瓶中加入工作浓度的目标抗生素并混合均匀；在阳性对照组的培养基玻璃瓶中加入已知具有抑菌抑生物被膜浓度的粘菌素colistin并混合均匀；在阴性对照组的培养基玻璃瓶中加入等体积的用药组抗生素的溶剂并混合均匀。用药组、阳性对照组和阴性对照组均按照附录X的培养步骤进行接种、培养（附录Xe），后收集生物被膜进行观察。 4.2.1.3技术要点 a. 所有操作须严格遵守无菌操作流程，在操作过程中注意须使用灭菌器具并用酒精消毒操作台以及所使用的器具，避免交叉污染; b. 使用针管与针头吸取菌液后，针头不可朝向操作人，不可盖回针头盖子，以免误伤操作人员； c. 将使用过的针管与针头丢弃入医用尖锐物处理容器统一处理，避免误伤操作人员； d. 保证无阻泵运行速度一致，避免液体回流造成污染； e. 保证硅胶管管壁厚度适宜，造成管壁破裂，影响生物被膜形成； f. 在培养过程中如需添加培养基，须用酒精消毒瓶口与邻近装置表面，避免污染； g. 培养瓶口须用透气封口膜密封防止培养基蒸发。 4.2.2 池式培养法（flow-cell biofilm） 适用范围：池式生物被膜培养法通常适用于以下场景：（1）需要实时观察生物被膜形态的相关研究，（2）需要实时观察生物被膜中标记蛋白/基因表达及分布的相关研究，（3）需要实时观察生物被膜群落中不同菌种相对位置关系的研究等。针对池式培养的生物被膜的分析方法主要包括：死活菌细胞染色法（章节5.2）、激光共聚焦荧光显微镜观测法（章节5.6）、3D荧光成像定性定量法（章节5.5）等。以下示意装置和操作均以铜绿假单胞菌（PA）为模式菌株，在耗氧培养条件下进行示例：池式生物被膜培养法通常适用于以下场景：（1）   图6. 体外动态生物被膜培养方法（池式培养法） 4.2.2.1　实验材料： a. 15 mL无菌摇菌管； b. 三通道培养池（3-channel flow-cell）与树脂盖； c. 盖玻片； d. 硅胶胶水; e. 长50 cm，内直径1.0 mm硅胶管； f. 长25 cm长20 cm，内直径 1.0 mm硅胶泵管，双端安装； g. 长15 cm，内直径1.0 mm硅胶管； h. 长30 cm，内直径1.0 mm硅胶泵管； i. 等径直通接头； j. 鲁尔接头； k. 2个2 L玻璃培养瓶； l. 10% LB培养基（可按需使用其他培养基）； m. 过滤器（过滤孔0.22 μm）； n. 蠕动泵（aperistaltic pump）； o. 所需菌株； p. 医用尖锐物处理容器； q. 75%酒精； r. 镊子； s. 5 mL及1 mL无菌针管； t. 无菌针头； u. 移液器； v. 无菌移液管。 4.2.2.2 培养步骤【附录以protocol/SOP模式进行详细阐述】 首先将生物被膜池式培养装置进行灭菌和组装，在用药组的培养基玻璃瓶中加入工作浓度的目标抗生素并混合均匀；在阳性对照组的培养基玻璃瓶中加入已知具有抑菌抑生物被膜浓度的粘菌素colistin并混合均匀；在阴性对照组的培养基玻璃瓶中加入等体积的用药组抗生素的溶剂并混合均匀。用药组、阳性对照组和阴性对照组均按照附录X的培养步骤进行接种、培养（附录Xf），后收集生物被膜进行观察。 4.2.2.3 技术要点 a. 所有操作须严格遵守无菌操作流程，在操作过程中注意须使用灭菌器具并用酒精消毒操作台以及所使用的器具，避免交叉污染; b. 使用针管与针头吸取菌液后，针头不可朝向操作人，不可盖回针头盖子，以免误伤操作人员； c. 将使用过的针管与针头丢弃入医用尖锐物处理容器统一处理，避免误伤操作人员； d. 保证无阻泵运行速度一致，避免液体回流造成污染； e. 保证培养池翻转状态，以免影响生物被膜形成； f. 黏结培养池与池盖时须小心不可将池盖压破，并须保证培养池与池盖完全密封，避免菌液与培养基溢出造成污染； g. 保证培养池所有通道畅通，避免堵塞造成液体回流与污染； h. 在培养过程中如需添加培养基，须用酒精消毒瓶口与邻近装置表面，避免污染； i. 培养瓶口须用透气封口膜密封防止培养基蒸发。

Technical guidelines for the cultivation, observation and drug resistance evaluation of bacterial biofilm

JIS K 3800:2021 二级生物安全柜

Class II biological safety cabinets

DB15/T 2072-2021 动物垫料中大肠埃希氏菌O157：H7/NM检测

Detection of Escherichia coli O157:H7/NM in animal litter

T/CIAA 103.1-2021 抗菌技术规范 第1部分：抗菌产品

T/CIAA 103的本部分规定了抗菌产品的术语和定义、技术要求、试验方法、检验规则、标志、包装、运输和贮存。

The antimicrobial technical specifications Part I: Antimicrobial pruducts

T/CIAA 104.1-2021 抗菌产品标注通用要求 第1部分：抗菌性能

T/CIAA 104的本部分规定了抗菌产品抗菌性能标注的术语和定义及标注要求。

General requirements for antimicrobial products labeling Part 1：Antimicrobial properties

T/SZAS 22-2020 基于全基因组测序的益生菌菌株分型鉴定指南

本文件提供了基于全基因组测序（WGS）的益生菌菌株分型鉴定的原理、实验条件、实验步骤、数据分析和报告的指南。 本文件用于指导食品、药品、保健品、化妆品和饲料中包含一种或多种不同的益生菌的菌株鉴定。适用于相关的检测机构或研究机构等。

The guide to genotyping of the probiotics at strain level by whole genome sequencing

DB45/T 2165-2020 树脂油质量要求

Resin oil quality requirements

ASTM E2149-20 用于确定动态接触条件下抗微生物剂抗菌活性的标准试验方法

1.1 This test method is designed to evaluate the antimicrobial activity of antimicrobial-treated specimens under dynamic contact conditions. This dynamic shake flask test was developed for routine quality control and screening tests in order to overcome difficulties in using classical antimicrobial test methods to evaluate substrate-bound antimicrobials. These difficulties include ensuring contact of inoculum to treated surface (as in AATCC TM100), flexibility of retrieval at different contact times, use of inappropriately applied static conditions (as in AATCC TM147), sensitivity, and reproducibility. 1.2 This test method allows for the ability to evaluate many different types of treated substrates and a wide range of microorganisms. Treated substrates used in this test method can be subjected to a wide variety of physical/chemical stresses or manipulations and allows for the versatility of testing the effect of contamination due to such things as hard water, proteins, blood, serum, various chemicals, and other contaminants. 1.3 Surface antimicrobial activity is determined by comparing results from the test sample to controls run simultaneously. 1.4 This test method may not be appropriate for all types of antimicrobial-treated articles or antimicrobial agents. The proper test methodology should be determined based on antimicrobial mode of action and end-use expectations (Guide E2922) 1.5 Proper neutralization of all antimicrobials must be confirmed using Test Methods E1054. 1.6 This test method should be performed only by those trained in microbiological techniques. 1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.8 This standard may involve hazardous materials, operations and equipment. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Standard Test Method for Determining the Antimicrobial Activity of Antimicrobial Agents Under Dynamic Contact Conditions

T/BPMA 0004-2020 新型冠状病毒肺炎样本采集包装运输及检测规范

对样本的采集、样本的包装与运输、样本的交接与辅助容器的开启、样本的灭活与分装、核酸提取、样本检测、样本保存、生物安全柜内清场及感染性废物处理等新型冠状病毒检测步骤中的安全防护、操作步骤、质量控制以及注意事项进行了具体要求，指导实验人员操作，降低实验人员的生物安全风险，同时提高检测结果的准确性，最大程度上为北京市的新型冠状病毒肺炎的防控提供数据支持。

The technical guide of coronavirus disease 2019 sample collection, packaging, transportation and testing

T/BPMA 0005-2020 新型冠状病毒肺炎样本意外溢洒事故处理规范

规定了新型冠状病毒核酸检测在进行样本采集、包装、运输等过程以及实验室进行样本处理、核酸提取、实时荧光逆转录聚合酶链式反应（Real time RT-PCR）检测时发生的新型冠状病毒样本包装不规范及检测中发生的样本溢洒、同一建筑物内样本运送溢洒的处理。特别对意外事故处理中人员的生物安全防护和事故发生后的报告程序进行了梳理和归纳。

The standard for handling of accidents of coronavirus disease 2019 sample