在生化科研的过程中，使用标准受体细胞系（如：大肠杆菌、酵母菌、CHO细胞系等等）进行研究已经是非常常规的手段。在使用各种细胞进行实验的时候，保存细胞样本就成了必不可少的一步。一般来说保存细胞是为了能够再次使用，所以使用什么方法保存、能保存多长时间也就至关重要。当再次使用自己保存的细胞时，我们肯定不想发现长满杂菌的样本，或是都死了的样本、亦或者蛋白表达不出来的样本。那今天就来说说常见的一些保存细菌细胞样本的方法和时限。
几种保存方法
一个最基本的概念：保存温度越低，保存时限更长；温度越低，对细胞伤害越大；低温也不怕，我有防冻剂（e.g. 甘油）。下面的表格就是五种常见保存细菌细胞的方法和大约时限。
| 保存条件 | 温度°C | 大约时间 |
| 琼脂平板
（Agar Plate） |
4 | 4-6周 |
| 穿刺培养基
（Stab culture） |
4 | 3周-1年 |
| 冰冻 | -20 | 1-3年 |
| 超低温冰冻 | -80 | 1-10年 |
| 冻干 | ≤4 | 15年+ |
但是，具体到你用的那一种菌用什么方法保存能够保存多长时间，就要视情况而定了，并没有一个唯一答案，上表中所列出来的时限一般来说是适用大部分细菌样本的。小编如果要保存一个细胞样本，一般不会费时费力来查这个表的，那怎么办？问师兄，找师姐咯~
短期保存
上表中的琼脂平板和穿刺培养基都适合于短期保存。这两种方法都保存在4度冰箱，为避免杂菌，最好用塑料或封口膜（parafilm）对平板或培养瓶进行封口，这样可以拒绝冰箱内本身存在的杂菌、霉菌入侵样本，也可以防止自己的培养基过快失水而变得干燥。小编就曾有一盘放在4度冰箱的菌落样本，忘记了封口，一个月后，菌落和琼脂都变成了木乃伊T_T
长期保存
长期低温冰冻必然会导致部分细胞死亡，而且很多时候我们要把细胞样本拿来重新接种做实验，所以解冻再冻的过程也会导致细胞死亡。那么起始样本的细胞浓度就要达到一定的高度，这样在以后保存的过程中就算死亡一些细胞，我们也还有很多有效细胞可以用，一般来说这个起始细胞密度要达到10的七次方的样子（单位：细胞/mL）。
除了常见抗冻剂甘油外，我们也会添加多糖（polysaccharide）或者葡聚糖（dextran）或某些蛋白质用来吸附在细胞表面形成保护薄膜，防止在冷冻过程中对细胞的破坏。
另外在解冻细胞样本的时候，最好在低温条件下，不要让整个细胞样本解冻成液体，只要解冻到少量液态样本出现就好。这样也可以帮助更多的样本存活过一次解-再冻的过程，方便下次或多次使用。
关于冻干保存，其实并不是所有的细菌都适合冻干，有的细菌没法在冻干过程中存活。所以如果你实在需要超长时间保存（超过十年）你的样本，一定要自己验证一次，看冻干细胞再培养能不能成功。
Reference
Lee J J. ATCC Preservation Methods: Freezing and Freeze‐Drying[J]. Journal of Eukaryotic Microbiology, 1992, 39(5): 651-651.
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