土霉素微生物效價測定法培養(yǎng)基的改進
發(fā)布時間：
2022-12-27
作者：
干粉培養(yǎng)基固體粉末培養(yǎng)基
摘要：本文改進了測定土霉索生物教價所用的培養(yǎng)基，有教地解決了原用較易產(chǎn)生抑菌圈模糊的問題。試驗結(jié)果表明，改用培養(yǎng)基IIpH70，在不改變其他檢定條件下，可獲得清晰且無雙圈的抑菌圈，所得測定結(jié)果符合生物榆定的要求。
土霉素(0xytetracychne)含量測定在文獻中采用的是微生物教價測定法(管碟法)，此法以藤黃微球菌[CMCC(B)28001]為試驗菌，用培養(yǎng)基IIpH65-6．6：所得結(jié)果并不理想．容易產(chǎn)生抑菌圈模糊,雙圈等現(xiàn)象．使卡尺測囂誤差增太，用zY一300A型抑菌圈測量儀直接測定，重現(xiàn)性差，困而影響了測定結(jié)果的準確性。為此，本文從培養(yǎng)基方而對本品的告量測定進行研究，結(jié)果表明，改用培養(yǎng)基pH7.0，能有效解決上述問題：
材料與方法：
ZY-300A型抑菌圈測量議；游標卡尺。精密度0.02mrn。氯化鈉磷酸氫二鉀,磷酸二氫鉀均為分析純。
試驗菌：藤黃徽球菌[CMCC(B)28001]培養(yǎng)基：①培養(yǎng)基pH6.5—6.6，簡稱培養(yǎng)基I1；②培養(yǎng)基T1．(配方為：培養(yǎng)基Tl26g，氯化鈉35g．磷酸氫二鉀3.6g，磷酸二氫鉀1.4g，加水1000m,[)：③培養(yǎng)基IT：(配方為：培養(yǎng)基I126g，氯化鈉3.5g，加水1000m1)；@培養(yǎng)基II_(配方為：培養(yǎng)基I126g．氯化鈉l0g，加水I000mi)；⑤培養(yǎng)基II(配方為：將培養(yǎng)基II的pH值調(diào)至7.0.
士霉素標準品:
方法：緩沖液,標準品溶液,試驗菌菌懸液,雙碟的制備均按文獻制備～
供試品溶液：用于標準曲線測定濃度為采用土霉素標準品分別配成998,1218,1485,18112209,2694,3285,40,20u/mL【的溶液；用于樣品教價測定的溶液按文獻”制備，濃度為200u／ml(高劑量),100u~ml(低劑量)；用于培養(yǎng)基對照試驗的溶液濃度為200ll／ml。
結(jié)果與討論
培養(yǎng)基對照試驗結(jié)果(見表1)表明，
在培養(yǎng)基中加人一定量的氯化鈉．可提高土霉紊在培養(yǎng)基中的擴散速度，但抑菌圈模糊：而將培養(yǎng)基Il的pI-I值調(diào)至7．0，可獲得較清晰的抑菌圈，但因擴散慢，要使高劑量抑菌圈的直徑達到18～22nwn的要求，如降低苗層培養(yǎng)基內(nèi)的加菌量．則菌層生長不好，與抑菌圈反差不夠明顯；而在培養(yǎng)基Il中加人氯化鈉35g．磷酸氧二鉀36g．磷酸二氫鉀14g，則培養(yǎng)基的pH剛好為70(II)。采用培養(yǎng)基I1，苗層生長密集，抑菌圈與菌層反差明顯，邊緣清晰且無雙圈．易于卡尺及儀器直接測量.
在培養(yǎng)基中加人一定量的氯化鈉．可提高土霉紊在培養(yǎng)基中的擴散速度，但抑菌圈模糊：而將培養(yǎng)基Il的pI-I值調(diào)至7．0，可獲得較清晰的抑菌圈，但因擴散慢，要使高劑量抑菌圈的直徑達到18～22nwn的要求，如降低苗層培養(yǎng)基內(nèi)的加菌量．則菌層生長不好，與抑菌圈反差不夠明顯；而在培養(yǎng)基Il中加人氯化鈉35g．磷酸氧二鉀36g．磷酸二氫鉀14g，則培養(yǎng)基的pH剛好為70(II)。采用培養(yǎng)基I1，苗層生長密集，抑菌圈與菌層反差明顯，邊緣清晰且無雙圈．易于卡尺及儀器直接測量.Improvement of culture medium for determination of oxytetracycline by microbial potency assay
In this paper, the medium used to determine the biological value of terrestrial moulds was improved, and the problem of ambiguity of bacteriostatic circle in the original medium was solved. The results showed that the medium IIpH70 could obtain a clear and non-double circle bacteriostatic circle without changing other test conditions, and the results were in accordance with the requirements of bio-elution.
Oxytetracycline (0xytetracychne) content in the literature is determined by microbial teaching method (tube-dish method), this method with Micrococcus luteus [CMCC (B) 28001] as the test bacteria, medium IIpH65-6.6: The results are not ideal. Prone to produce the phenomenon of ambiguous bacteriostatic circle, double circles and so on. The inhibition zone measuring instrument is directly measured, with poor reproducibility and difficulty, which affects the accuracy of the determination results. In this paper, the determination of the product was studied from the culture medium. The results showed that the above problems could be effectively solved by changing the medium pH to 7.0.
Materials and methods:
ZY-300A bacteriostatic circle measurement; vernier caliper. Precision 0.02mrn. Sodium chloride hydrogen phosphate two potassium and potassium dihydrogen phosphate are analytically pure.
The test bacteria: CMCC (B) 28001 culture medium: (1) Medium pH 6.5-6.6, abbreviated as Medium I 1; (2) Medium T1. (Formula: Medium Tl26g, Sodium Chloride 35g, Potassium Dihydrogen Phosphate 3.6g, Potassium Dihydrogen Phosphate 1.4g, Water 1000m, [): Medium IT: (Formula: Medium I126g, Sodium Chloride 3.5g, Water 1000m1) _Medium II_ (formula: medium I126g. sodium chloride l0g, water I000mi); _Medium II (formula: the pH value of medium II was adjusted to 7.0.
Standard materials:
Methods: buffer solution, standard solution, bacteria suspension, and double dish preparation were prepared according to literature.
Sample solution: For the determination of concentration in standard curve, oxytetracycline standard was used to prepare 998, 1218, 1485, 18112209, 2694, 3285, 40, 20u/mL [solution; for the determination of sample price according to the literature], the concentration was 200 u/ml (high dose), 100 u~ml (low dose); for the medium control test, the concentration was 200 ll. / ml.
Results and discussion
The results of medium control test (see Table 1) showed that adding a certain amount of sodium chloride in the medium could increase the diffusion rate of soil mold turbulence in the medium, but the bacteriostatic circle was vague. When the pI-I value of medium Il was adjusted to 7.0, a clearer bacteriostatic circle could be obtained, but the diameter of high dose bacteriostatic circle could reach 18-2 because of slow diffusion. 2 NWN requirements, such as reducing the amount of bacteria in the seedling layer culture medium, the growth of the bacterial layer is not good, and the contrast with the bacteriostatic circle is not obvious; and in the medium Il with human sodium chloride 35g. potassium dioxide phosphate 36g. potassium dihydrogen phosphate 14g, the pH of the medium is just 70 (II). With medium I1, the growth of seedling layer was dense, the contrast between bacteriostatic zone and bacterium layer was obvious, the edge was clear and there was no double circle.
孫俊軍龍綺群