培養(yǎng)基對(duì)藥敏試驗(yàn)結(jié)果的影響因素
發(fā)布時(shí)間：
2022-12-27
作者：
國(guó)藥標(biāo)準(zhǔn)微生物培養(yǎng)基
藥敏試驗(yàn)培養(yǎng)基的選擇具有一定的原則和要求，不以個(gè)人經(jīng)驗(yàn)或愛好而更改。為了使臨床試驗(yàn)和醫(yī)療保健服務(wù)水平具備一致的標(biāo)準(zhǔn)，許多國(guó)家或機(jī)構(gòu)制定了不同的參照標(biāo)準(zhǔn)。由于參照標(biāo)準(zhǔn)有所差異，故藥敏培養(yǎng)基的要求也有所不同。我國(guó)藥敏試驗(yàn)結(jié)果判斷標(biāo)準(zhǔn)參照美國(guó)CLSI標(biāo)準(zhǔn)文件，則要求除了特殊菌群，K-B法所用的培養(yǎng)基必須是M-H培養(yǎng)基；英國(guó)參照的標(biāo)準(zhǔn)BSAC則要求培養(yǎng)基為ISO培養(yǎng)基，而M-H營(yíng)養(yǎng)瓊脂只適用于葡萄球菌的藥敏試驗(yàn)。因此在工作中，我們要嚴(yán)謹(jǐn)?shù)膮⒄誄LSI，根據(jù)不同菌群的需求選擇合適、正確的藥敏培養(yǎng)基。
培養(yǎng)基的成分和質(zhì)量是保證藥敏結(jié)果準(zhǔn)確性的先決條件。培養(yǎng)基對(duì)藥敏結(jié)果的影響因素具體如下：
1、pH：pH的變化可導(dǎo)致抑菌圈直徑變大或變小。
2、培養(yǎng)基中含有能拮抗菌藥活性的物質(zhì)：如鈣、鎂離子能降低氨基糖苷類藥物的抗菌活性。
3、培養(yǎng)基的厚度：標(biāo)準(zhǔn)厚度為4mm,厚度>4mm，抑菌圈則會(huì)變小，<4mm,則會(huì)變小。
4、培養(yǎng)基的濕度：使用藥物平皿前一般先在室溫平衡半個(gè)小時(shí)，使培養(yǎng)基表面干燥，培養(yǎng)基濕度過高不利于細(xì)菌的生長(zhǎng)或?qū)咕幤鸬较♂屪饔谩?/span>
5、培養(yǎng)基的硬度：硬度過高不利于抗菌藥在瓊脂中的擴(kuò)散，可導(dǎo)致抑菌圈直徑變小。
6、營(yíng)養(yǎng)條件：營(yíng)養(yǎng)條件太差不利于細(xì)菌的生長(zhǎng)，抑菌圈直徑變大；反之，培養(yǎng)及營(yíng)養(yǎng)條件太好會(huì)讓抑菌圈直徑變小。
將這些原理和原則解說后，針對(duì)他們對(duì)話中的問題，我一一進(jìn)行了剖析：
根據(jù)CLSI，絕大多數(shù)細(xì)菌藥敏試驗(yàn)瓊脂均為標(biāo)準(zhǔn)的M-H瓊脂，M-H瓊脂基本不含有抑制磺胺類藥物和四環(huán)素類藥物抗菌活性的物質(zhì)，可滿足大多數(shù)細(xì)菌的生長(zhǎng)需要，腸球菌對(duì)營(yíng)養(yǎng)要求不高且生長(zhǎng)速度較快，因此可選用M-H瓊脂（MHA），而不是血平皿。對(duì)于大多數(shù)細(xì)菌，MHA是不需要添加羊血，血液或血清中的蛋白會(huì)不同程度結(jié)合抗菌藥分子，影響了抗菌藥物的活性，如羊血中的某些成分可抑制磺胺類藥物和四環(huán)素類藥物抗菌活性，同時(shí)也可影響新生霉素、萘夫西林及頭孢菌對(duì)腸球菌的藥敏結(jié)果。
溶血鏈球菌、肺炎鏈球菌等營(yíng)養(yǎng)要求高的苛養(yǎng)菌則需要使用M-H瓊脂+5%脫纖維羊血培養(yǎng)基，也叫M-H藥敏試驗(yàn)血瓊脂平板（MHBA）。MHBA與普通的分離細(xì)菌的血平板或者哥倫比亞血平板（CNA）雖然長(zhǎng)相相似，但配方及產(chǎn)業(yè)標(biāo)準(zhǔn)（如pH、濕度等）均不一樣，是不能相互代替使用的。為了比較肺炎鏈球菌ATCC49619在MHBA和 CNA培養(yǎng)基上的藥敏結(jié)果，在其他條件均一致的情況下我們分別用MHBA和CNA做了藥敏試驗(yàn)，結(jié)果發(fā)現(xiàn)，CNA的藥敏結(jié)果抑菌圈直徑偏大，細(xì)菌生長(zhǎng)情況較差，從而導(dǎo)致“假敏感”結(jié)果。
因此，作為一名合格的微生物檢驗(yàn)人員，我們應(yīng)該時(shí)刻遵守操作規(guī)程，牢記標(biāo)準(zhǔn)要求，熟讀規(guī)則原理，杜絕含糊不清、似是而非，才能確保結(jié)果的準(zhǔn)確性。
"Teacher, I remember that the Department used the blood plate to make Streptococcus drug sensitivity test. Does the enterococcal susceptibility test also use blood plate?" Intern A's face to ask
I haven't had time to respond, the intern B immediately replied: "I think the nutritional requirements of the streptococcal culture are high, then the blood plate should be selected and the Enterococcus grows well on the common nutrition base, so it is possible to use the MH tablet to do the drug sensitivity test."
"Is this blood plate the Columbia blood agar plate (CNA) that we usually cultivate bacteria?" Intern A continues to ask.
"It seems to be!" Intern B is not sure to answer.
When I heard the dialogue between the two interns, I expressed appreciation and support for their knowledge and interrogative spirit, but they were also worried that their basic knowledge of the most conventional drug sensitivity test of clinical microbiology - the basic knowledge of the paper diffusion method (K-B method) was not firmly consolidated; the recognition and selection of drug sensitive Petri dishes was in a confusing state of confusion. It leads to inaccurate results of drug sensitivity and delays the correct treatment of patients. After that, I explained the requirements and principles of the K-B drug sensitive plate for them.
The selection of medium for drug sensitivity test has certain principles and requirements, and is not changed by personal experience or hobbies. In order to have consistent standards for clinical trials and health care services, many countries or institutions have drawn up different reference standards. Because of the difference in reference standard, the requirements for drug sensitive medium are also different. The criteria for judging the results of drug sensitivity test in our country refer to the CLSI standard documents in the United States. In addition to the special flora, the culture medium used by the K-B method must be the M-H medium; the standard BSAC of the British reference requires the medium as the ISO medium, and the M-H nutritious agar is only suitable for the drug sensitivity test of Staphylococcus. Therefore, in our work, we should carefully consult CLSI and select suitable and correct drug sensitive medium according to the needs of different microbiota.
The composition and quality of the medium are prerequisites for ensuring the accuracy of the results. The factors affecting the sensitivity of the medium are as follows:
1, the change of pH:pH can lead to larger or smaller diameter of bacteriostatic circle.
2, the medium contains substances that are antagonistic to antibacterial activity: calcium and magnesium ions can reduce the antibacterial activity of aminoglycoside drugs.
3, the thickness of the medium: the standard thickness is 4mm, the thickness is >4mm, the inhibition zone will be smaller, and the <4mm will be smaller.
4, the humidity of the culture medium: it is generally balanced at room temperature for half an hour before using the medicine plate, which makes the medium surface dry, and the high humidity of the medium is not conducive to the growth of bacteria or the dilution effect on the antimicrobial agents.
5. Hardness of the medium: excessive hardness is not conducive to the diffusion of antibiotics in agar, which can lead to a smaller diameter of the bacteriostatic circle.
6, nutritional conditions: poor nutrition conditions are not conducive to the growth of bacteria, the diameter of the bacteriostasis circle becomes larger; on the contrary, the cultivation and nutrition conditions will make the diameter of the bacteriostasis circle smaller.
After explaining these principles and principles, I analyze them in the light of their dialogues.
According to CLSI, the overwhelming majority of bacterial susceptibility tests are standard M-H agar, M-H agar does not contain substances that inhibit the antibacterial activity of sulfonamides and tetracycline, and can meet the growth needs of most bacteria. Enterococcus is not high in nutrition and fast in growth. Therefore, M-H agar (MHA) can be used. Not a Petri dish. For most bacteria, MHA does not need to add sheep blood, and the proteins in blood or serum combine with antimicrobial molecules in varying degrees and affect the activity of antibiotics. For example, some of the ingredients in the sheep blood can inhibit the antibacterial activity of sulfonamides and tetracycline, and can also influence the new mycophenycin, naphthalene and cephalosporin. The drug sensitivity of Enterococcus.
The high nutritive bacteria, such as Streptococcus hemolytic streptococcus and Streptococcus pneumoniae, need to use the M-H agar +5% defibrous sheep blood culture medium, and also called the M-H drug sensitive test blood agar plate (MHBA). MHBA is similar to the ordinary separate bacterial blood plate or the Columbia blood plate (CNA), but the formula and industrial standards (such as pH, humidity, etc.) are not the same, and can not be replaced with each other. In order to compare the drug sensitivity results of Streptococcus pneumoniae ATCC49619 on MHBA and CNA medium, we used MHBA and CNA for drug sensitivity test under the same conditions. The results showed that the diameter of the bacteriostasis circle of the drug sensitivity of CNA was large and the growth of the bacteria was poor, thus leading to the "false sensitivity" result.
Therefore, as a qualified microbial inspector, we should always abide by the rules of operation, keep in mind the standard requirements, read the rules of the rules well, avoid ambiguity and specious, so as to ensure the accuracy of the results.