离体培养中两种脱鞘方法和几种不同营养成份对蒙古马歇尔线虫L_3发育为L_4的影响

IN VITRO CULTIVATION OF L3 AND L4 OF MARSHALLAGIA MONGOLICA (NEMATODA. TRICHOSTRONGYLOIDEA)

本文研究了六个不同pH梯度;四种人工综合培养基(M_(199),EMEM,F_(10),F_(12));YE_(25)(酵母提取物),CLB_(33)(牛肝汤),RLE_(50)(兔肝提取物);Filde's试剂对离体培养中蒙古马歇尔线虫L_3发育为L_4的影响;比较了次氯酸钠脱鞘和MRF(改良法配制的RFN)85分钟脱鞘效果及对L_3发育为L_4的影响。 较早出现脱皮的培养基为:M_(1(?))+CEE_(50)+CBS,pH5.2(脱皮出现于培养后第8天)。L_4出现率较高的培养为:(1)MRF85分钟脱鞘后移入MRF内培养2天;(2)以M_(199)为主配制而成的M_9加谷胱苷肽、Filde's试剂,pH4.5～5,培养8天;(3)M_9加Filde's试剂,pH6,培养40天。L_4的出现率达46.7％。

The influence of (l)the pH value of the media, (2)commercial media EMEM,Ml99, F10,Fl2, (3)some additives as yeast extract, cattle liver broth and raw rabbit liver extract,and (4)Filde's reagent on the development of the exsheathed L3 and L4 of Marshallagia mongolica in vitro has been investigated. Comparison of two methods of exsheath-ment showed different exsheath rate and different effects on the development of L3 to L4. 1 . In medium M199 + CEE + CBS, pH value 5.2-7, L3 developed and moulted into L4, and the results showed that pH value 5.2 got the best development rate; on the other hand, when pH value were 7.2 and 7.61,L3 could not develop to L4. 2 . Four commercial media supplemented with CEE & CBS have been tested for cultivation under the same condition. The results showed that M3 only occurred in Ml99 and larvae died on 13th day post-inoculation in media F10 &F12. In medium EMEM only a few survived on day 13 post-inoculation. 3. The medium Ml99 +CEE50 + CBS supplemented with additives of 2%.Filde's reagent can increase the survival rate of L3 and L4,and might prolong the survival time of L4. 4 . Comparisons of the influence of yeast extract,raw rabbit liver extract and cattle liver broth on the development of L3 to L4 in vitro showed that raw rabbit liver extract and cattle liver broth could enhance the survival rate of L3 and L4, and rabbit raw liver extract played the best role; yeast extract appeared noneffective. 5. A 85-min. exsheathing process in MRF(modified RFX,group a) and 0.06% sodium hypochlorite exsheathing technique (group b) were carried out, and then the exsheathed L3 were cultured in (l)MRF with gas phase of 10%carbon dioxide/5%oxygen/85%nitrogen for 2 days; (2)M9 plus additives of 20mM reduced glutathione and 2%Filde's reagent, pH value 4.5-5 and with the same gas phase to (1)for 8 days; and(3)in M9 plus additives of 2%Filde's reagent, with the same gas phase as described above in pH 6 for 40 days. The results were as follows: the exsheath rate was 98% in group a, and 80% in group b, the yield of L4 was 33.3% in group a and 46.7% in group b.