抗生素的生物效价测定法(管碟法)

抗生素的生物效价测定法(管碟法) 抗生素的生物效价测定法 测定抗生素效价的方法比较多，一般可以分为物理学方法、化学方法、生物学方法、和两种方法配合等四大类，可根据具体情况选择使用。 生物学方法以抗生素的杀菌力作为衡量效价的 标准 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 ，其原理恰好和临床应用于的要求一致这是它的优点,又其灵敏度较高，需用检品的量较小，也是其它方法所不及的。 抗生素的生物效价测定法，常用的有稀释法、比浊法和扩散法,或称渗透法,。 稀释法 这一方法是用培养基将检品抗生素稀释到各种浓度，并依次分装到一系列的容器内，再加入等量“试验菌种”菌种菌液，并放在37 ? 保温箱内培养一定时间，观察何种稀释度适能抑制细菌的生长，该稀释度即为测定终点,或以细菌生长所引起的PH改变及溶血现象等生化反应作为测定终点,，再与同样处理的标准抗生素的终点作比较，即可求得检品的效价。这种方法可以使用液体培养基，也可以使用固体培养基。所用的 材料 关于××同志的政审材料调查表环保先进个人材料国家普通话测试材料农民专业合作社注销四查四问剖析材料 及培养基都必须严格无菌，并要注意无菌操作。由于测定终点是以有无细菌生长来判断的，因此所得结果公是一个范围。 比浊法 原理及操作大致与稀释法相同。比浊法也是将不同量的检品及标准品分别加入培养基中，观察其对“试验菌种”的效应——即细菌生长所引起的混浊。这一方法和稀释法的区别有二:a.比浊法的稀释间隔的密度比较近，准确度高一些,b.比浊法不以细菌有无生长的区分为终点，而是将标准品浓度和细菌生长所引起的混浊度求得一定的比例，再由检品的细菌生长混浊度推算检品的效价。这一方法易受杂质的影响，并且不适用于有色或混浊的检品。 扩散法 使用固体培养基，在培养基凝固以前将“试验菌种”混合进去，在这样备妥的培养 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 面，可以用种种 设计 领导形象设计圆作业设计ao工艺污水处理厂设计附属工程施工组织设计清扫机器人结构设计 使检品液或含有抗生素的物质与有菌种的培养基接触。经过培育后，由于抗生素向培养基中扩散，凡抑菌浓度所能达到之下细菌不能生长因而形成透明的抑菌范围，此种范围一般都呈圆形，称为“抑菌圈”。扩散法有几种，或中一种叫管碟扩散法,筒称管碟法,为国际上常用的方法，在我国也作为法定的抗生素检定法。 下面我们将详细地讨论管碟法的原理和实验方法。 一、 管碟法测定的设计原理及计算方法 管碟法所用的管子系用瓷、铝或玻璃制成，较好的用不锈钢制成，它是内径为6+0.1毫米、外径为8+0.1毫米、高10+0.1毫米的圆筒形管子，管子的重量尽可能相等。 摊布固体培养基的容器可用双碟，亦可用平底下班盘。管子放在混有菌种的固体培养基上时，可用人工放臵，也可用特定的管子放臵器放臵。 and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 操作步骤，一般是将固体培养基放在水浴上融化，倒在双碟上，待其凝固，然后在上面再倒一层混有菌种融化培养基。待菌层培养基凝固后，在表面上放臵管子，向管子中加满抗生素的稀释液，在37?培养16~18小时，然后量取抑菌圈并进行计算。 ,一, 抑菌圈的形式 小管中的抗生素向培养基中扩散,呈球面扩散,， 在此同时试验菌也开始生长。抗生素浓度高于抑菌浓度之处试验 菌不长，因此出现抑菌圈，其圈之边缘处恰好为抗生素的最低抑 菌浓度。 抑菌圈的半径,r,与下列因素有关:(1)抗生素在管中的量,M,,,2,抗生素的扩散系数,D,,,3,细菌生长达到肉眼可见的时间,T,,,4,培养基的厚度,H,。其中的定量关系可用分子扩散定律来推导，得到如下所示的公式: 2logM= (1/9.21DT)r + log(C?ЛTH) (11-1) 2式中 D —— 扩散系数，毫米/小时; T —— 抗生素扩散时间(接近细 菌生长到肉眼可见的时间)，小时; M —— 在管中抗生素总量，单位; r —— 管中心到抑菌圈边缘的距 离，毫米; L —— 管的高度，毫米; H —— 培养基的厚度，毫米; 最低抑菌浓度，单位/毫米。 C —— 由于此公式相当于直线方程式,y=ax+b,，,logM相当于y,1/9.21DT相当于斜率a,log(C〃4πDTH)相 2当于截矩b,，因此设logM为纵标，r为横坐标时可得截矩为log(C〃4πDTH),斜率为1/9.21DT的直线。 2,二, 测定设计原理与计算公式 由公式可知logM与r成直线关系， 即抗生素总量的对数值与抑菌圈半径的平方值成直线关系，因此 抗生素的量可从抑菌圈大小来推算。这就是说，抗生素总量的多 少受最低抑菌浓度(C)、培养基厚度(H)、扩散系数(D)、和细菌生 长时间(T)的影响，例如培养基厚度减少到H/2时则抑菌圈增大， 可计算如下: 培养基厚度为H时 2 logM= (1/9.21DT)r + log(C〃4πDTH) 培养基厚度为H/2时 and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 2 logM= (1/9.21DT)r + log(C〃4πDTH/2) 两式相减，化简可得: 22 r=9.21DT〃log2+r 1 2 由于(9.21πDT)为斜率的倒数、r为原抑菌圈半径的平方值，均为已知，则增大的圈,r,即可算出。 1 同理，若知细菌最低抑菌一半时，则抑菌圈的半径,r,也将增大: 2 22 r=(9.21DT)log2+r 2 从上可知，抗生素总量的对数值与抑菌圈的平方成直线关系，因此可作定量测定。但由于抑菌圈的大小还受C、H、D、T的影响， 所以应消除这些影响才能准确测定。要消除这些影响， 可用标准品同时对照测定,即所谓相对效价设计法,， 计算方法如下。 设:标准品的高单位抑菌圈为S H 标准品的低单位抑菌圈为S L 样品高单位的抑菌圈为U H 样品低单位的抑菌圈为U L 则小管听标准品总量,M,与样品总量,M’,分别为: 因log(C〃4πDTH)为截距，在同一双碟上其数值是相等的，因此两者相减，它就被消去了。 因,1/9.21DT,为直线的斜率，所以样品与标准品的效价比即等于斜率乘样品的抑菌圈的平方值与标准品抑菌圈平方值之差数。此即管碟法的标准曲线法的基础，如要消除斜率,1/9.21DT,的影响，可用二剂量法，即标准品用二剂量,S及S,,样品也用二剂 量,U及U,代入公式并计算之，即可消除斜HLHL 率的影响。计算方法如下: and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 在这个分式中无斜率因素，即在样品与标推品对比测定时斜率可被消除。此即为管碟法的二剂量法的计算公式,应用详见二剂量法,。由于(U)2、(S)2、HH(U)2、(S)2计算太麻烦，经实验证明也可近似地用,U,、,S,、,U,、,S,TTHHTT来代替。 ,三, 图解法计算原理及作计算图的方法管碟测定时常用图解法来计算 结果。 用图解法计算结果就是把公式用图来表示，其原理如下: and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 即当V=ob、W=ab及logK=cd时，则,即od线段表示效对数值,. 在方格纸上在W坐标上取logK×100值,例如logK为log2时，其值为0.301×100,，平行于V坐标划一平行线。然后在此线段上取0.0043×100介于，此点b值即为效价101%值。因log(101%)为0.0043，为了便于划线，故乘100。同理C点为效价102%值，d点为99%值，依此类推。为了便于观察，也可延长ob或oc或od线段把效价值表示在图的上方。如此可作成一放射图形。当logK=log4时，同理可在W坐标上取log4×100值，然后平行于V划平行线，取0.0043×100点，即为效价101%值，取0.0086×100点，即为102%，依此类推，也同样可能表示在图表的上方，成放射图形,图11-3,.当K=4时，二剂量效价计算放射图见(11-4). and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 二、 管碟法的实验设计方法 根据上述原理，可知效价的对数值与抑菌圈关径的平方值,抑菌圈直径,成直线关系，其直级方程式为logy=ax+b.当与标准品对照测定时，b可消除成为logθ=a(x-x’),也即效价的对数值等于斜率乘样品与标准品的抑菌圈差数，可以此作为标准曲线法测定效价。当样品与标准品各用二剂量对比测定时，则斜率因数也可以消除，成为logθ=V/W×logK而求得效价比值,θ,。现将此两种方法分述于下。 ,一,标准曲线法 材料 双碟直径为90毫米,不锈钢小管外径为8+0、1毫米，内径为6+0、1毫米，高为10+0.1毫米。钢管重量差异不得相差+0.05克。 培养基 各种抗生素测定效价所用的培养基不完全相同，《中国药典》中均有规定。 菌种 各种抗生素测定效价所用的菌种也不完全相同，《中国药典》中均有规定。 标准品 由药品生物制品检定所统一分发。每一种抗生素标准品的单位也由该所规定一般都以容易精制达到高纯度的衍生物作为标准品。例如青霉素以青and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 霉素G钠盐作为标准品,链霉素以链霉素盐酸盐的氯化钙复盐作为标准品,卡那霉素以含一分子结晶水的一硫酸卡那霉素作为标准品等。当然如抗生素本身能达到较高纯度，就即可以作为标准品，如制霉菌素等。 方法 在双碟上量入加热融化的底层培养基21毫升，并在碟底平均摊布，放臵使其凝固 ，即为底层。另取上层培养基加热融化后，冷到50?加入适量菌液，摇匀后，于每碟中各加4毫升，使在底层上平均摊布，即为菌层。冷却后，适当安臵小管六枚，用陶瓦盖覆盖备用。 标准曲线的制法 用缓冲液,各种抗生素所用缓冲液不完全相同,将标准品稀释为多种浓度，在备妥之双碟上各放适度间隔六枚小管，三枚装中心点浓度之标准液，另三枚空间管，使装液管与空间管互相间隔，以三碟为一组，在三个双碟的九个空间管中各装入一种浓度的标准液，其它浓度的标准液也如此装入。培养后量取抑菌圈之直径，求取每组三碟的中心点浓度的抑菌圈直径平均值、各组抑菌圈直径的平均值和所有中心点浓度的抑菌圈直径的平均值。依下法求得各种浓度之坐标，试以某一浓度的坐标为例:设中心点浓度的所有直径平均值为20毫米，而某一组碟中心点浓度的九个抑菌圈直径平均为19.8毫米，则20-19.8=0.2，即为“更正数”,若某一浓度之九个直径平均为19.0毫米，则19.0+0.2=19.2毫米作为某一浓度之坐标值。依同法求得各种浓度之数值后，取双周半对数图纸，以浓度为纵坐标，将各点贯串即成标准曲线，在应用时取直线范围内的各点。 样品效价的求法 先估计效价。按估计效价用缓冲液稀释到与标准液的中心点相同的浓度。在备妥已放六枚小管之双碟上装满其中三管，另三管装满中心点浓度的标准液，令样品与标准液互相间隔。共用三碟。装妥后全部放入培养，然后量取抑菌圈直径，求9个标准液抑菌圈直径的平均值及9个样品液抑菌圈的平均值。同样找出“更正数”，将样品抑菌圈直径的平均值进行校正，即自标准曲线读取样品的单位。 为了精确起见，也可用公式计算。设样品的直线关系为logy’=ax’+b;标准品的直线关系为logy=ax+b,则两式相减即得: log(y’/y)=a(x’-x) 即log效价=,斜率,×,样品抑菌圈直径,标准品抑菌圈直径, 在上式中，只要斜率已知，则log效价即可求出。斜率可从标准曲线上直接用量角仪量出直线的倾角，其正切即为斜率。 ,二,二剂量法,即四点法, 材料 培养基、菌种、标准品及双碟制备同标准曲线法。 方法 取备妥的双碟四点，各于碟底用蜡笔作S、S、U及U四个标志。HLHLand special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 将标准液稀释为每毫升含某一浓度,此浓度应取在标准曲线的直线范围内,，及为浓度1/3或1/4的浓度,均应取在标准曲线的直线范围内,。于碟内S管量H入高单位标准液，于碟内S管量入低单位标准液。在U管内量入高单位的样品LH 液，在U管内量入低单位的样品液。经培养后量取抑菌圈直径。 L 样品效价的计算，可用前述的放线图或用前述的式,26-14,求得θ值，然后按下式计算即得。 样品效价=θ×估计效价 三、 管碟法精确测定抗生素效价的基本条件 抗生素效价测定的及方法已见上述。如要精确测定抗生素的效价，必须具备 下列几项条件。 ,1, 抑菌圈要圆而边缘清晰, ,2, 抗生素抑菌圈直径的直线关系范围应宽, ,3, 标准品所作的直线应互相平行, ,4, 直线的斜率应小,log效价坐标，抑菌圈直径为横坐标时,，直线 的截距应小 达到上述几点要求，就能精确测定抗生素的效价，这也是建立一个良好的测定方法必须具备的基本条件。怎样才能达到这些基本要求呢,现逐点讨论于下。 ,一,抑菌圈圆与清晰的条件 抑菌圈常有破裂现象，有时甚至无圈，或有圈而不圆，或呈馒头形、鸡蛋形等，其原因有几种:1. 在加小管时抗生素溅出，或小管底不平漏出，或工作服上 有抗生素粉末飞入，以及双碟未洗去残余的抗生素、或小 管污染抗生素时，均会发生圈破裂及奇形怪状的现象,抗 生素测定时抗生素是最主要的东西，查也是最应防止污染 的东西，务必建立“无抗生素操作”的观念，当培养基污 染抗生素时，甚至可能全部无抑菌圈现象,2.菌种太老， 或污染杂菌，或在倒上层培养基时温度太高，或温度正常而放臵时间太久等等， 也均可使抑菌圈破裂或有的好有的坏，甚至有完全 无圈等现象产生,特别是以霉菌及细菌作试验菌 时，很容易因上层培养基温度过高而烫死，以致造 成抑菌圈破裂或无圈现象,3.当双碟上的小管彼此 间隔太小而抑菌圈较大时，则圈与圈之间彼此影 响，形成馒头形抑菌圈，如图11-5所示，由于在 a处有低于抑菌浓度之抗生素，当二个抑菌圈太近时，则低于抑菌浓度的抗生素浓度加大，使该处之圈放大而呈为馒头形,有时二and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 个抑菌圈并不太近，也会发生这种现象，例如四环类抗生素，当PH过低或过高时，圈外的抗生素较多，因此彼此影响较大，也呈馒头形,4.当抗生素中含盐浓度太高或PH太低时，有的抗生素如新霉素A、链霉素等就无抑菌圈，或抑菌圈呈鸡蛋形，如图11-6中a处的盐较浓或PH较低，故呈鸡蛋形,5.有时因双碟接近温箱底部温度较高，细菌生长较快，也会使温度较高处的抑菌圈变小而不圆,6.有时因培养基厚薄不匀，也会影响抑菌圈不呈圆形。 抑菌圈的边缘应清晰，不然会影响结果。但有时抑菌圈不清晰，原因很多，概括起来可以认为是抑菌圈形成时的动力学现象不均一引起的。例如当菌种中有二种最低抑菌浓度不同的菌种时，则形成了双圈。较耐药的菌种抑菌圈较小，敏感的菌种抑菌圈较大，并使抑菌圈边缘形成不清晰的锯齿形，其范围的大小可由前述的动力学公式推算求得。 设一种菌株的最低抑菌浓度为C，另一种菌株的最低抑菌浓度为C’，，则代入动力学公式中成为: 2 r/9.21DT=logM-logM(C?4πDTH) (11-15) 2 r/9.21DT=logM-logM(C’?4πDTH) (11-16) 由此即能求出二种菌所形成的抑菌圈不清晰地带的宽度为 9,21DT(logc'-logc)。此式中9.21DT为斜率的倒数，为已知数。 又如培养基中含有杂质，会影响扩散，其扩散系数不只一个而是二个或二个以上，则同理可计算出抑菌圈不清晰地带为9,21DT(logD’—logD)。 又如抗生素有二种组分，含量较多的组分具有抑菌作用(无杀菌作用)，含量少的具有杀菌作用，则能形成二圈，外圈模糊，呈模糊边缘。这种模糊边缘的宽度也可以同理算出为9.21DT(logM—logM')。 抗生素抑菌圈边缘不清晰与抗生素种类有关。有的抗生素如竹桃霉素，对不少试验菌主要为抑菌作用(不是杀菌作用)，其抑菌圈边缘因杀菌作用弱抑菌作用强而不清晰，也即最低抑菌浓度(C)不均一，其中以抑菌作用为主，杀菌作用为次，同理可以动力学公式计算其不清晰边缘的宽度。 由抑菌圈边缘不清晰的原因，即可找出使抑菌圈清晰的 办法 鲁班奖评选办法下载鲁班奖评选办法下载鲁班奖评选办法下载企业年金办法下载企业年金办法下载 。为了避免有二种最低抑菌浓度的菌种，则可用分离单菌落的方法纯化菌种，使其成为只有一种最低抑菌浓度的菌种。为了避免培养基不均一，特别是琼脂的不均一，则应把琼脂适当纯化(如多洗几次，或换批号或换产地)。为了避免抗生素的抑菌作用大于杀菌作用，则可更换试验菌，因为某一抗生素对某一试验菌有较高的杀菌作用。例如竹桃霉素对腊状芽孢杆菌或蕈状杆菌有较高的杀菌作用，抑菌圈就非常清晰。抗生素的杀菌作用与抑菌作用也可因培养基的成分不同而异，例如氯霉素，当培养基中含有维生素B族时，则杀菌作用较强，抑菌圈就较清晰。总之，抑and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 菌圈不清晰的问题可通过纯化菌种、改进培养基、更换菌种、改变稀释抗生素用的缓冲液pH及浓度来解决。 (二)抗生素浓度与抑菌圈的直线关系 抗生素浓度的对数值与抑菌圈半径的平方值之间的关系是直线关系。如果为了计算方便起见以抑菌圈直径来计算，这样直线关系的范围就较窄，但不妨碍一般成品的测定。若样品的效价很难估计，则仍以抑菌圈半径的平方值来计算较为合适，因这样做直线范围较宽。直线范围有时较窄，其原因是抗生素受到破坏或者被菌种或培养基所吸附，应考虑去除这种影响因素，使直线范围增长。 (三)标准品与样品所作的直线平行问题 标准品与样品所作的直线应互相平行，计算公式是在假定两者平行而斜率相等的前提下推算出来的，若斜率不相等就不能这样计算。所以实验得出的直线应互相平行才能得到精确的数据。 直线不平行除操作误差外，主要是标准品的"质"不相同而造成的。若标准品中有生物活性的物质或影响生物活性的物质与样品中所含者不同时，直线就不平行。若用来稀释标准品的缓冲液(如pH、浓度)与样品不同时，直线也不平行。所以要直线平行，就要考虑这些因素，并尽可能品质一致、条件一致。若样品中含有维生素C，则在标准晶中也应加入等量的维生素C。若样品(如四环素等)稀释液放臵过程中能转化成差向异构体(只有原抗生素5,活性)，则标准品稀释液的放臵时间也应相同，如此等等。 (四)直线的斜率与截距问题 标准直线的斜率当log效价为纵座标、抑菌圈直径为横座标时，斜率愈小愈好。因为斜率愈小，则效价差别很小时抑菌圈大小的差别却很大，这样测定结果就较为精确。 斜率减小的条件可从动力学公式中斜率为1/9.21DT来推论,若扩散快，即扩散系数D值大，则斜率小，若细菌生长时间T值大，则斜率小。即斜率大小主要是D、T这二个因素决定的，加大D值或增长T值，斜率就可减小。 D值与抗生素的分子大小，及培养基或菌种是否吸附抗生素有关。抗生素分子的大小不能改变，只有在吸附上多加考虑。吸附与培养基及稀释用的缓冲液的pH及盐浓度很有关系。改变缓冲液的pH及盐浓度，常可解决扩散太慢的问题。如加吐温80等扩散剂，则可使有些多肽类抗生素的扩散速度增快。 有的菌种生长慢，有的菌种(如腊状芽孢杆菌)生长虽快，但当培养基的pH调到4.5时也生长缓慢，直线斜率就小。增加T值的方法较易，培养基的营养差、培养温度低等，均可使T值增加。但T值太大，得出结果太慢，抑菌圈的清晰度也受到影响。快速法时间很短，但斜率很大，精密度较差，这一对矛盾要正确处理解决。 直线的截距应小，因为同样浓度的抗生素其截距小的所显示的抑菌圈大。截and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 距的大小决定于动力学公式中log(C〃4πDTH)的数值。如培养基厚度减小一半时，截距就减小，抑菌圈就增大，计算方法见式(26—3)。即培养基厚度减小一半时，增大的抑菌圈半径的平方值为原抑菌圈半径的平方值加上斜率的倒数乘log2的值。 截距小，抑菌圈增大，利用这一原理设计的薄层法，可测定微量的抗生物质。如树叶中微量链霉素的测定，血液中微量抗生素的测定等。由于微量抗生素显示很大的抑菌圈，样品可高度稀释，这样，杂质的影响就大大减小。若该杂质在高度稀释后仍有抗菌性能，则此杂质也为抗生物质。 四、管碟法实验操作时应注意的事项 影响抗生素管碟测定的因素很多，例如影响菌生长时间(T)的因素有营养、pH、菌液数量、菌种性质等，影响扩散系数(D)的因素有琼脂品种及纯度、培养基成分、无机盐的量、细菌吸附情况、pH,温度及菌量，以及菌层的厚薄和菌层中含糖量(糖在培养时会产酸)、抗生素分子的大小等，影响最低抑菌浓度(C)的有菌的浓度、营养、温度，pH、菌种种类及类型(如粗糙型,光滑型)、菌种性能(如耐药株、敏感株)以及菌龄,影响培养基厚度的有双碟不平，桌子不平、小管深浅，培养基倒得不均匀，影响抗生素量(M)的有抗生素浓度、小管中的数量、辅料、抑菌与杀菌性能等。 虽然影响因素很多，而且这些因素彼此又互相影响。但是，应该看到，只有对标准品和样品有差异的一些因素(称为主要因素)才对测定结果影响较大。这样的主要因素并不多， 但要特别注意，它们是:1,加小管的时间及量不同,2,双碟的底不平而使培养基层厚度b不均匀，3,双碟中温度不均匀，4，小管的深浅不同，5,标准品与样品的“质”(例如样品中若含有影响效价的辅料，则与标准品有质的差异)，pH、盐浓度及稀释液放臵时间的不同。 另外，操作中还应注意下列各点。有些抗生素很易吸水，有时会使抗菌活性降低,例如四环类抗生素吸水后，很易差向异构化，因此若条件不一样，就会造成误差。测量抑菌圈及稀释用量器也应一致。标准品溶液若在冰箱放臵时冻结，则一定要完全融化后再稀释使用，否则融化部分的单位较高，会使测定结果偏低。链霉素标准品稀释液在pH6保存(因为在此pH时，较稳定)，而测定时稀释用的缓冲液pH为7.8—8.0(因为在此pH时抗菌作用强)。若链霉素标准品保存液浓度太稀，则用缓冲液稀释后，pH达不到7.8:8.0，测定结果就偏高。 管碟法是国际上常用的方法，也是所有生物测定中最简单、最准确的方法。但由于其原理是以扩散为基础的，所以只要有影响扩散的因素存在，就会出现假单位，这是它的一个很大的缺点。其次，有的抗生素的分解产物对治疗无意义而对试验菌却有更强的抑制作用，例如环丝氨酸的分解产物能抑制腊状芽孢杆菌，and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic 也应予以注意。 主要参考资料 (1) 上海化工学院等:《抗菌素生产工艺学》，1974。(内部教材) [2) 中华人民共和国卫生部药典委员会:《中华人民共和国药典》，人民卫生出版社，1977。 [3] 马誉徽主编:《抗菌素》，第3版，人民卫生出版社，1965。 [4] 张绪敏，《抗菌素微生物效价测定法》，人民卫生出版社，1963。 [5] 耶戈罗夫，H,C,著，冯明霞、周本励合译:《抗生菌的分离及其抗菌效能的生物学检定》，人民卫生出版社，1960。 and special tools for emergency response equipment and facilities. Emergency preparedness and response plan 1) emergency response (1) If a major accident occurs, the project should set up emergency response teams, chaired by the project manager, head of production and safety coordinators, professional foreman for the crew, primarily responsible for the orderly rescue when an emergency occurs or processing, outsourcing, team manager and support staff, assisting the Deputy Project Manager for related auxiliary work. (2) after the accident, the project manager is responsible for field commander, advance the establishment of emergency response team personnel, accountability, seriously injured officers sent out rescue, have procedures to deal with accidents, incidents, maximum reduction in casualties and property loss. (3) If an accident occurs, press Division in advance for emergency personnel cleared, concentrate manpower and material and financial resources to rescue the injured, decrease of the maximum loss. (4) after the accident processing work (5) identified the cause of the accident and the person responsible. (6) the written report to the supervisor in writing, including the time and place of the accident, injury (death) person's name, sex, age, trades, injuries, injuries. (7) the development of effective preventive measures to prevent such accidents from happening again. (8) education of all personnel of the organization. (9) informed criticism of those responsible. (10) all personnel read out the results, and responsible disposal. (11) maintenance of emergency supplies, maintenance and testing ? strengthening the day-to-day management of the various fire fighting equipment fire-fighting equipment, authorities should be staffed, equipped with fire extinguishers. Fire hydrants identified person responsible for regularly inspecting, testing, at any time in good condition. ? Guard monthly check-fire extinguishers and fire-fighting equipment. ? once every quarter, inspections and tests of a fire hydrant in good condition. 2), plans to develop (1) casualties occurred in construction, such as accidents or accidents of the direct economic