PDA試管培養(yǎng)基瓊脂不凝固原因分析及解決辦法
發(fā)布時間：
2022-12-27
作者：
PDA試管培養(yǎng)基瓊脂不凝固原因分析及解決辦法
瓊脂：英文名稱為agar，食用菌母種PDA培養(yǎng)基中的A指的就是瓊脂，是用海產(chǎn)的麒麟菜、石花菜、江蘺等制成的植物膠，因主要來自海南，故稱為“瓊脂”。瓊脂是食用菌母種培養(yǎng)基必須使用的一種物質(zhì)，起到固定培養(yǎng)基斜面的作用，菌絲并不能夠利用瓊脂的營養(yǎng)。
進入6月后，經(jīng)常有朋友問制作的PDA培養(yǎng)基，瓊脂的添加量和以前一樣，為什么現(xiàn)在不能完全凝固了？瓊脂在PDA培養(yǎng)基中添加量是2%，就是1000ml培養(yǎng)基加入瓊脂20克，我們分析一下有幾種原因?qū)е颅傊荒芡耆獭?/span>
1滅菌過度
滅菌效果由溫度和時間兩個參數(shù)決定，PDA試管培養(yǎng)基的滅菌溫度是121℃，時間是20-30min，如果兩個參數(shù)有一個超出，就很容易引起瓊脂不凝固。
解決辦法：PDA試管培養(yǎng)基滅菌時間121℃，30min即可。對于液體培養(yǎng)基的滅菌溫度121℃是足夠的，為了保證滅菌效果將滅菌溫度提高到125℃的做法是錯誤的。
2瓊脂添加量少
瓊脂添加量小于2%，或者稱量不準(zhǔn)確導(dǎo)致瓊脂不凝固。
解決辦法：保證瓊脂添加量2%，校正天平，保證稱量準(zhǔn)確。
3瓊脂質(zhì)量差
使用劣質(zhì)，或者長期存放的瓊脂可能會導(dǎo)致瓊脂不凝固。
解決辦法：使用優(yōu)質(zhì)瓊脂，有條件的一定要使用瓊脂粉，瓊脂條是食品添加劑，瓊脂粉是實驗試劑，質(zhì)量可靠。
4加水量不準(zhǔn)
配置培養(yǎng)基時，加水過多會導(dǎo)致瓊脂不凝固。
解決辦法：加水準(zhǔn)確，以及熬制后重新稱量，稱量要用量筒，或者校正過的燒杯。
5分裝時瓊脂沉淀
在分裝時，如果瓊脂沒有完全溶解，就很容易出現(xiàn)沉淀的情況，這樣先分裝的瓊脂濃度就低，出現(xiàn)不能完全凝固的情況。
解決辦法：做到完全溶解，同時邊攪拌，邊分裝，均勻一致。
6pH值過低
pH值低于4.5，培養(yǎng)基酸化，瓊脂會不凝固。
解決辦法：引起PDA培養(yǎng)基酸化的原因主要是水的ph，所以盡量使用桶裝水，桶裝水呈弱堿性，而且穩(wěn)定。
7環(huán)境溫度高
夏天，一些沒有空調(diào)的工廠，制作好的PDA培養(yǎng)基因環(huán)境溫度過高而導(dǎo)致不能完全凝固。
解決辦法：在夏季可以將1000ml培養(yǎng)基的添加比例提高到22克，如果需要添加到30克才能夠凝固，說明瓊脂質(zhì)量有問題了。有條件的可以安裝空調(diào)。
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.