CN102994388B - Strain preservation liquid and strain preservation method - Google Patents
Strain preservation liquid and strain preservation method Download PDFInfo
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- CN102994388B CN102994388B CN201210509349.9A CN201210509349A CN102994388B CN 102994388 B CN102994388 B CN 102994388B CN 201210509349 A CN201210509349 A CN 201210509349A CN 102994388 B CN102994388 B CN 102994388B
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Abstract
The invention discloses strain preservation liquid and a strain preservation method. The strain preservation liquid contains 0.2-0.6g of yeast powder, 0.4-1.0g of sodium chloride, 2-10mL of dimethyl sulfoxide, 10-30mL of glycerol and the balance of water based on 100mL of solution. The strain preservation method comprises the following steps of: adding a strain to be preserved into the strain preservation liquid, and mixing uniformly to obtain bacterial liquid to be preserved; absorbing the bacterial liquid to be preserved with sterile filter paper to obtain bacterium-containing filter paper; fixing the bacterium-containing filter paper onto sterile wax paper with a sterile transparent adhesive tape; and preserving in a refrigerator. The strain preservation liquid disclosed by the invention can maintain the activity of microorganisms for a long time, and the preservation period is relatively long. The strain preservation method disclosed by the invention has higher biological safety and storage efficiency, and is convenient to operate and transfer; and particularly when the number of strains is large, the storage space is small, and the search is convenient.
Description
Technical field
The invention belongs to microbial engineering field, be specifically related to a kind of culture presevation liquid and culture collection process.
Background technology
Culture presevation is essential in microbe research and practical application.Because microorganism has the characteristic of easy variation, therefore, in preserving process, must make the metabolism of microorganism in least active or relatively static state, could in long-time, it not morphed and keep viability.
Culture collection process mainly contains following several at present:
(1) the cultivation preserving process that goes down to posterity.The method comprises again that slant culture, stab culture, cooked meat medium are cultivated and is used as preservation anaerobic bacterium) etc. several.
(2) whiteruss covers preserving process.The method is the covert method that goes down to posterity and cultivate, can the proper extension preservation time.
(3) carrier preserving process.The method is that microorganism adsorption is upper at suitable carrier (as soil, sand, silica gel etc.), then carries out dry preserving process.
(4) host's preserving process.For the microorganism that still can not grow on artificial medium at present, as virus, Rickettsiae, spirochete etc., they must infect and go down to posterity in the animal living, insect, chicken embryo, and this method is equivalent to the cultivation preserving process that goes down to posterity of general microorganism.
(5) freezing method.Can be divided into cryogenic refrigerator method (20 ℃～-30 ℃ or-50 ℃～-80 ℃), dry ice alcohol quick freezing method (approximately-70 ℃) and liquid nitrogen method (196 ℃) etc.
Above-mentioned several method respectively has relative merits, but generally speaking still has following shortcoming: (1) can not maintain microbial activity for a long time, and preservation term is shorter.(2) biological safety is lower, and preservation usefulness is lower.(3) complex operation, preserves inconvenience, is difficult to transhipment.(4) when bacterial classification quantity is more, storage area is larger, and inconvenience is searched.
Summary of the invention
One of object of the present invention is for the deficiencies in the prior art, provides a kind of and can maintain for a long time microbial activity, culture presevation liquid that preservation term is longer.
Another object of the present invention is to for the deficiencies in the prior art, a kind of biological safety is provided and preserves usefulness all higher, easy to operate and transhipment is convenient, particularly when bacterial classification quantity, have the culture collection process that storage area is little and be convenient to search.
The technical scheme that realizes one of the object of the invention is: a kind of culture presevation liquid, the 100mL solution of take contains yeast powder 0.2g～0.6g, sodium-chlor 0.4g～1.0g, methyl-sulphoxide 2mL～10mL, glycerine 10mL～30mL, all the other are water.The 100mL solution of preferably take contains yeast powder 0.5g, sodium-chlor 0.6g, methyl-sulphoxide 5mL, glycerine 20mL, all the other are water.
This culture presevation liquid also contains Trypsin 0.8g～1.2g and glucose 0.2g～0.6g in 100mL solution.The 100mL solution of preferably take contains Trypsin 1.0g, yeast powder 0.5g, sodium-chlor 0.6g, glucose 0.5g, methyl-sulphoxide 5mL, glycerine 20mL, all the other are water.
The technical scheme of another object of the present invention is: a kind of culture collection process, has following steps: 1. will treat that preservation of bacteria strain adds in above-mentioned culture presevation liquid, and mix, and obtain treating preservation bacterium liquid.That 2. with aseptic filter paper, draws that 1. step obtain treats preservation bacterium liquid, obtains containing bacterium filter paper; What 3. with aseptic scotch tape, 2. step is obtained is fixed on aseptic paraffin paper containing bacterium filter paper, is then placed in refrigerator preservation.
When treating that preservation of bacteria strain quantity is more, make in the more situation of aseptic filter paper (or containing bacterium filter paper), multiple can be to the one side that array is fixed on aseptic paraffin paper containing bacterium filter paper, and every is provided with one as the paster of sign containing bacterium filter paper one side is corresponding, be used for recording strain name, preservation date and identify number etc., thereby play clearly mark action, be convenient to search.
When treating that preservation of bacteria strain quantity is more, make in situation that the one side of an aseptic paraffin paper used not, can will containing bacterium filter paper and paster, be arranged on the another side of same aseptic paraffin paper.
And in the situation that the two sides that makes an aseptic paraffin paper when treating that preservation of bacteria strain quantity is more is all used not, can adopt multiple aseptic paraffin paper, and be clamped by folder, and this document is folded in to preservation in refrigerator.Folder title page is provided with Biosafety sign, and homepage indicates catalogue and index, thereby further play, knows mark action, is convenient to search.
The positively effect that the present invention has: the avidity that the methyl-sulphoxide in (1) culture presevation liquid of the present invention and glycerine can produce water and cell by hydrogen and ionic linkage is carried out the configuration of stabilized cell composition, to prevent because of the freezing or constantly infringement of distillation to cell of moisture.Yeast powder and sodium-chlor can be bacterium energy are provided, and prevent its death.Thereby make this culture presevation liquid can maintain for a long time microbial activity, greatly extended preservation term.(2) Trypsin in culture presevation liquid of the present invention and glucose may further be bacterium provides energy, thereby further maintains microbial activity.Use method of the present invention making bacterial classification-18 ℃ of preservations 2 years and in a monthly survival of 25 ℃ of recoveries.(3) culture collection process of the present invention adopts and has good water-absorbent and adhering aseptic filter paper as adhering to medium, and adopt paraffin paper and the scotch tape with good isolated air effect, can guarantee the anaerobic state of institute's preservation of bacteria strain like this, biological safety and preservation usefulness are all higher.(4) culture collection process of the present invention is easy and simple to handle, and transhipment is convenient, and the bacteria live phase is long, can be stored in general refrigerator cold storage chamber, is applicable to very much daily preservation and the transhipment of bacterium, yeast and the filamentous fungus of the laboratory separation of Clinical microorganism bacterial classification.(5) one aspect of the present invention makes treating that preservation of bacteria strain is more more containing bacterium filter paper in the situation that, multiple can be to array containing bacterium filter paper is arranged on aseptic paraffin paper, and adopt paster record strain name, preservation date and identify number etc., thereby play clearly mark action, be convenient to search.The in the situation that of making aseptic paraffin paper more treating that preservation of bacteria strain on the other hand, in folder mode, store bacterial classification, its title page is provided with Biosafety sign, and homepage indicates catalogue and index, thereby further play, knows mark action, is convenient to search.Particularly also greatly reduced storage area.
Accompanying drawing explanation
Fig. 1 is arranged on aseptic paraffin paper schematic diagram simultaneously for multiple are array containing bacterium filter paper.
Embodiment
(embodiment 1)
The present embodiment is the preparation of culture presevation liquid.
The sodium-chlor of the yeast powder of the Trypsin of 1.0g, 0.5g, 0.6g, 0.5g glucose, the methyl-sulphoxide of 5.0mL, the glycerine of 20mL are joined in the flask of 150mL, then add water to 100mL, fully mix after dissolving, at the temperature of the pressure of 0.1MPa and 121 ℃, carry out the sterilizing of 20min, obtain culture presevation liquid.
(embodiment 2～embodiment 5)
The preparation of the culture presevation liquid of each embodiment is substantially the same manner as Example 1, and difference is in Table 1.
Table 1
| ? | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Trypsin | 1.0g | 0.8g | 1.2g | - | - |
| Yeast powder | 0.5g | 0.2g | 0.6g | 0.5g | 0.4g |
| Sodium-chlor | 0.6g | 1.0g | 0.4g | 0.6g | 0.7g |
| Glucose | 0.5g | 0.6g | 0.2g | - | - |
| Methyl-sulphoxide | 5mL | 10mL | 2mL | 5mL | 8mL |
| Glycerine | 20mL | 12mL | 30mL | 20mL | 10mL |
| Water | Supply 100mL | Supply 100mL | Supply 100mL | Supply 100mL | Supply 100mL |
(Preparation Example)
First prepare aseptic filter paper, aseptic scotch tape, aseptic paraffin paper and aseptic nipper.
Filter paper is cut into little of several 0.3 * 1.4cm, packs in ampoul tube, plug is with tampon, under the pressure of 0.1MPa and the sterilizing of carrying out 20min at the temperature of 121 ℃, obtains several aseptic filter papers, standby.
Scotch tape and paraffin paper (15cm * 10cm) are used ethylene oxide sterilizing before use, obtain aseptic scotch tape and aseptic paraffin paper, standby.
Tweezers use the calcination of spirit lamp flame to obtain aseptic nipper, standby.
(embodiment 6)
The present embodiment is the method for preserving of single bacterial classification, has following steps:
1. Acinetobacter bauamnnii is cultivated to 18h in 37 ℃ of blood agars, obtain treating preservation of bacteria strain.3 bacterium colonies of picking splash in the culture presevation liquid that 0.5mL embodiment 1 makes, and mix, and obtain treating preservation bacterium liquid.
That 2. with aseptic filter paper of aseptic nipper gripping, draws that 1. step obtain treats preservation bacterium liquid, obtains containing bacterium filter paper 2.
What 3. with aseptic scotch tape, 2. step is obtained is fixed on aseptic paraffin paper 1 containing bacterium filter paper 2, then immediately this aseptic paraffin paper 1 is placed in to the refrigerator preservation of-18 ℃.
During recovery, only need first aseptic paraffin paper 1 to be placed on 20min under envrionment temperature (0～40 ℃), can be by aseptic scotch tape together with taking off containing bacterium filter paper 2, one side containing the aseptic paraffin paper 1 of bacterium filter paper 2 contact is applied to blood agar one district, line subregion, in 37 ℃ of cultivation 18h, can obtain the single bacterium colony of Acinetobacter bauamnnii.
In addition, for experiment or research purpose, as needing transhipment, only aseptic paraffin paper 1 need be placed in to thick transparent plastic bag, is Transshipment Permitted (fresh bacterial classification can reach the destination in one month and without low temperature transhipment) after sealing.
(embodiment 7)
The present embodiment is the method for preserving of a plurality of bacterial classifications:
See Fig. 1, the common microorganism of the clinical separation such as streptococcus aureus, escherichia coli, Klebsiella Pneumoniae, enterobacter cloacae, enteroaerogen, Pseudomonas aeruginosa, saccharomyces albicans, aspergillus tubigensis, mucormycosis is separately fixed to the one side of aseptic paraffin paper 1 according to the culture collection process of embodiment 6, and is array placement.Every is equipped with one and puts up paper 3 containing bacterium filter paper 2 one sides, for recording the signs such as strain name, preservation date, evaluation number, thereby plays clearly mark action, is convenient to search.
If treat, the quantity of preservation of bacteria strain is more and make in situation that the one side of an aseptic paraffin paper 1 used not, can will containing bacterium filter paper 2 and paster 3, be arranged on the another side of the aseptic paraffin paper 1 of same.
In the situation that the two sides that the quantity of preservation of bacteria strain is more if treat and make an aseptic paraffin paper 1 is all used not, can adopt multiple aseptic paraffin paper 1, and be clamped by folder, and this document is folded in to preservation in the refrigerator of-18 ℃.The title page of folder is provided with Biosafety sign, and the homepage of folder is provided with catalogue and index, thereby further play, knows mark action, is convenient to search.
(experimental example)
To part bacterial classification use method preservation of the present invention in two years and recovery one month in viability detect, the results are shown in Table 2.
The different preservation of table 2 spawn activity in period detects
| Bacterial classification | Acinetobacter bauamnnii | Streptococcus aureus | Enteroaerogen | Aspergillus tubigensis | Mucormycosis |
| Preservation three months (18 ℃) | Survival | Survival | Survival | Survival | Survival |
| Preservation six months (18 ℃) | Survival | Survival | Survival | Survival | Survival |
| Preservation 1 year (18 ℃) | Survival | Survival | Survival | Survival | Survival |
| Preservation 2 years (18 ℃) | Survival | Survival | Survival | Survival | Survival |
| Recovery two weeks (25 ℃) | Survival | Survival | Survival | Survival | Survival |
| Recover one month (25 ℃) | Survival | Survival | Survival | Survival | Survival |
As shown in Table 2, use method for preserving of the present invention-18 ℃ of preservations 2 years and 25 ℃ of recoveries one month, all bacterial classifications are all survived.
Claims (6)
1. a culture presevation liquid, is characterized in that: the 100mL solution of take consists of Trypsin 0.8g～1.2g, yeast powder 0.2g～0.6g, sodium-chlor 0.4g～1.0g, glucose 0.2g～0.6g, methyl-sulphoxide 2mL～10mL, glycerine 10mL～30mL, all the other are water.
2. culture presevation liquid according to claim 1, is characterized in that: the 100mL solution of take consists of Trypsin 1.0g, yeast powder 0.5g, sodium-chlor 0.6g, glucose 0.5g, methyl-sulphoxide 5mL, glycerine 20mL, all the other are water.
3. a culture collection process, is characterized in that having following steps:
1. will treat that preservation of bacteria strain adds in culture presevation liquid, mix, obtain treating preservation bacterium liquid; Described culture presevation liquid take that 100mL solution consists of Trypsin 0.8g～1.2g, yeast powder 0.2g～0.6g, sodium-chlor 0.4g～1.0g, glucose 0.2g～0.6g, methyl-sulphoxide 2mL～10mL, glycerine 10mL～30mL, all the other are water; Describedly treat that preservation of bacteria strain is a kind of in Acinetobacter bauamnnii, streptococcus aureus, enteroaerogen, aspergillus tubigensis, mucormycosis;
That 2. with aseptic filter paper, draws that 1. step obtain treats preservation bacterium liquid, obtains containing bacterium filter paper;
What 3. with aseptic scotch tape, 2. step is obtained is fixed on aseptic paraffin paper containing bacterium filter paper, is then placed in the refrigerator preservation of-18 ℃.
4. culture collection process according to claim 3, is characterized in that: described aseptic filter paper is according to treating that preservation of bacteria strain quantity is at least provided with one; Every aseptic filter paper is drawn containing of obtaining after preservation bacterium liquid, and bacterium filter paper one side is corresponding is provided with one as the paster of sign.
5. culture collection process according to claim 4, is characterized in that: described aseptic paraffin paper is according to treating that preservation of bacteria strain quantity is at least provided with one; Described aseptic paraffin paper during more than one, is clamped by folder, and this document is folded in to preservation in refrigerator.
6. culture collection process according to claim 3, is characterized in that: described culture presevation liquid take that 100mL solution consists of Trypsin 1.0g, yeast powder 0.5g, sodium-chlor 0.6g, glucose 0.5g, methyl-sulphoxide 5mL, glycerine 20mL, all the other are water.
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| CN201210509349.9A CN102994388B (en) | 2012-11-23 | 2012-11-23 | Strain preservation liquid and strain preservation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103667061A (en) * | 2013-11-21 | 2014-03-26 | 宁夏启元药业有限公司 | Strain preservation method |
| CN105420110A (en) * | 2015-12-29 | 2016-03-23 | 广西大学 | Pestalotiopsis bacterial strain preservation method |
| CN106342493B (en) * | 2016-08-24 | 2019-01-22 | 安徽中草香料股份有限公司 | The preparation method of balm preservative fluid |
| CN106544275A (en) * | 2016-10-11 | 2017-03-29 | 明德松 | The liquid composite protectant for being preserved for strain for a long time and its preparation, using method |
| CN107699491B (en) * | 2017-11-21 | 2021-01-15 | 江西省科学院微生物研究所 | Sample total microorganism and protective agent and preservation method of total DNA thereof |
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| WO2005072523A2 (en) * | 2004-02-02 | 2005-08-11 | I.M.T. Interface Multigrad Technology Ltd. | Biological material and methods and solutions for preservation thereof |
| CN101012439A (en) * | 2006-09-28 | 2007-08-08 | 中南大学 | Acidithiobacillus ferrooxidans freezing-preservation protective agent |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005072523A2 (en) * | 2004-02-02 | 2005-08-11 | I.M.T. Interface Multigrad Technology Ltd. | Biological material and methods and solutions for preservation thereof |
| CN101012439A (en) * | 2006-09-28 | 2007-08-08 | 中南大学 | Acidithiobacillus ferrooxidans freezing-preservation protective agent |
Non-Patent Citations (2)
| Title |
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| 农业植物病害研究中可培养性病原菌的常用保存方法;汪进仕等;《现代农业科技》;20081031(第20期);154-155 * |
| 汪进仕等.农业植物病害研究中可培养性病原菌的常用保存方法.《现代农业科技》.2008,(第20期), |
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