PDA试管培养基琼脂不凝固原因分析及解决办法

发布时间:

2022-12-27

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PDA试管培养基琼脂不凝固原因分析及解决办法
 
 
琼脂:英文名称为agar,食用菌母种PDA培养基中的A指的就是琼脂,是用海产的麒麟菜、石花菜、江蓠等制成的植物胶,因主要来自海南,故称为“琼脂”。琼脂是食用菌母种培养基必须使用的一种物质,起到固定培养基斜面的作用,菌丝并不能够利用琼脂的营养。
 
进入6月后,经常有朋友问制作的PDA培养基,琼脂的添加量和以前一样,为什么现在不能完全凝固了?琼脂在PDA培养基中添加量是2%,就是1000ml培养基加入琼脂20克,我们分析一下有几种原因导致琼脂不能完全凝固。
 
1灭菌过度
 
灭菌效果由温度和时间两个参数决定,PDA试管培养基的灭菌温度是121℃,时间是20-30min,如果两个参数有一个超出,就很容易引起琼脂不凝固。
 
解决办法:PDA试管培养基灭菌时间121℃,30min即可。对于液体培养基的灭菌温度121℃是足够的,为了保证灭菌效果将灭菌温度提高到125℃的做法是错误的。
 
2琼脂添加量少
 
琼脂添加量小于2%,或者称量不准确导致琼脂不凝固。
 
解决办法:保证琼脂添加量2%,校正天平,保证称量准确。
 
3琼脂质量差
 
使用劣质,或者长期存放的琼脂可能会导致琼脂不凝固。
 
解决办法:使用优质琼脂,有条件的一定要使用琼脂粉,琼脂条是食品添加剂,琼脂粉是实验试剂,质量可靠。
 
4加水量不准
 
配置培养基时,加水过多会导致琼脂不凝固。
 
解决办法:加水准确,以及熬制后重新称量,称量要用量筒,或者校正过的烧杯。
 
5分装时琼脂沉淀
 
在分装时,如果琼脂没有完全溶解,就很容易出现沉淀的情况,这样先分装的琼脂浓度就低,出现不能完全凝固的情况。
 
解决办法:做到完全溶解,同时边搅拌,边分装,均匀一致。
 
6pH值过低
 
pH值低于4.5,培养基酸化,琼脂会不凝固。
 
解决办法:引起PDA培养基酸化的原因主要是水的ph,所以尽量使用桶装水,桶装水呈弱碱性,而且稳定。
 
7环境温度高
 
夏天,一些没有空调的工厂,制作好的PDA培养基因环境温度过高而导致不能完全凝固。
 
解决办法:在夏季可以将1000ml培养基的添加比例提高到22克,如果需要添加到30克才能够凝固,说明琼脂质量有问题了。有条件的可以安装空调。
 
 
 

Causes of agar-agar noncoagulation in PDA test tube medium and its solutions
 
 
AGAR: English name of AGAR, edible fungus mother refers to A kind of PDA medium AGAR, is made of seafood eucheuma, AGAR weed, gracilaria made of vegetable gum, mainly from hainan, is called "AGAR. Agar-agar is a kind of substance that must be used in the culture medium of edible fungi, which plays the role of fixing the slant of the culture medium.
 
At the beginning of June, a friend often asked the PDA media, the amount of AGAR added is the same as before, why can't it be completely solidified now? The amount of AGAR added in PDA medium was 2%, that is, 1000ml medium added with 20 grams of AGAR. We analyzed several reasons that the AGAR could not completely solidify.
 
1 excessive sterilization
 
Sterilization effect is determined by the temperature and time of two parameters, PDA in vitro culture medium sterilization temperature is 121 ℃, and time is 20 to 30 min, if there is a more than two parameters, it is easy to cause AGAR solidification.
 
Solution: PDA in vitro culture medium sterilization time is 121 ℃, 30 min. For the sterilization of liquid medium temperature 121 ℃ is enough, in order to ensure the sterilization effect to increase the sterilization temperature to 125 ℃ is wrong.
 
The amount of AGAR added is low
 
Agar-agar addition is less than 2%, or the inaccuracy of weighing results in agar-agar non-coagulation.
 
Solution: to ensure agar-agar addition of 2%, balance correction, to ensure accurate weighing.
 
AGAR is of poor quality
 
The use of poor quality or long-term storage of agar-agar may cause agar-agar not to solidify.
 
Solution: use high quality agar-agar, the condition must use agar-agar powder, agar-agar strip is food additive, agar-agar powder is experimental reagent, quality is reliable.
 
4. Improper water addition
 
When the culture medium is prepared, too much water will cause AGAR to not solidify.
 
Solution: add water accurately, and re-weigh after boiling, using a measuring cylinder, or calibrated beaker.
 
Agar-agar precipitation during the separation
 
If the agar-agar is not completely dissolved, it is easy to precipitate. In this way, the concentration of the agar-agar is low and cannot be completely solidified.
 
Solution: dissolve completely, stir and divide evenly.
 
6 low ph
 
The pH value is less than 4.5, the culture medium is acidified, AGAR will not solidify.
 
Solution: the main reason for the acidification of PDA medium is the ph of water, so try to use water in barrels, which are weak alkaline and stable.
 
7 high ambient temperature
 
In the summer, some factories without air conditioning, the PDA culture gene environment produced is too hot to coagulate completely.
 
Solution: the addition ratio of 1000ml medium can be increased to 22 grams in summer. If it needs to be added to 30 grams to solidify, the quality of agar-agar is problematic. Air conditioning can be installed if conditions permit.