CN1900266A - Preparing and using method of special culture medium capable of killing and controlling 'black bug' - Google Patents
Preparing and using method of special culture medium capable of killing and controlling 'black bug' Download PDFInfo
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- CN1900266A CN1900266A CN 200510085462 CN200510085462A CN1900266A CN 1900266 A CN1900266 A CN 1900266A CN 200510085462 CN200510085462 CN 200510085462 CN 200510085462 A CN200510085462 A CN 200510085462A CN 1900266 A CN1900266 A CN 1900266A
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Abstract
The present invention discloses the preparation process and usage of a kind of special culture medium. The special culture medium is prepared on the basis of common tissue culture medium, and through adding putrescine, transferin, thymine, urease pyridine,gentamycin, and other substances, and 0.22 micron filtering to sterilize. The special culture medium has killing and inhibiting effect on 'black bug' as cell pollutant, and may be used in rescuing polluted cell in scientific research.
Description
Technical field the invention belongs to the preparation of special culture medium, and the technology that is specifically related to is for removing and suppress the medium preparation and the using method of " black glue worm " pollution in the tissue culture.
The background technology cell culture technology is founded development so far from 20 beginnings of the century, has been widely used in every field such as virusology, immunology, genetics, oncology, clinical medicine and biotechnology, becomes important biological study instrument.Therefore, a successful research relevant with cell cultures is except having good experimental design and technological method, and one of key factor is to avoid polluting.
" black glue worm " is a kind of cell contamination thing of just finding the nearly more than ten years, and the classification of " black glue worm " does not still have the affirmation of evaluation at present.
" black glue worm " can parasitize zooblast, also can survive in substratum, relies on the nutrition in cell and the substratum to make a living, and goes down to posterity with passage." black glue worm " and cell competition growth, what influence pair cell does not have during beginning, but when the quantity of black glue worm how time to a certain degree, cell is grown and will be affected until death, has had a strong impact on carrying out and carrying out of scientific research activity.Culture condition changes, cell inoculation density reduces, cell state manifests when not good so the pollution of " black glue worm " is everlasting and experiment is interrupted, and especially can cause a large amount of necrocytosiss when the freeze-stored cell recovery.The pollution of " black glue worm " has following characteristics:
1. black glue worm presents tangible autokinetic movement, gathering together property under high power lens.Can be clearly seen that under the high power lens more than 400 times the glue worm has two kinds of different shapes, a kind of bigger, it is slower to move, general ruddiness; Another kind of less, to be with in red greenly, motion is very fast.
2. can pass through 0.22 μ m filter membrane, it is invalid to it that routine is changed liquid or added antibiotic commonly used.
3. compare with the pollution of other biological body, it does not make the nutrient solution muddiness, does not change its pH value yet.
4. the tetrazolium salts (MTT) that can reduce forms blue crystallisate, illustrates that it has plastosome.The viewpoint of relatively generally acknowledging thinks that " black glue worm " is a kind of microorganism at present, and propagation is slow but pair cell has infringement, and quantity acquires a certain degree and can cause necrocytosis, and its source may be the pollution of cell itself or pollute when animal is drawn materials and cause.This pollutes ubiquity in whole world Cell Lab, and addressing this problem is a global problem.
The special culture medium that the present invention is prepared has and well kills and restraining effect " black glue worm ".By using this kind special culture medium 2-3 time, the pollution of " black glue worm " can be controlled effectively and prevent.The present invention provides strong technique means for preventing to pollute and rescue valuable cell, is the important means of carrying out smoothly that uses manpower and material resources sparingly, reduces unnecessary funds expenditure and guarantee scientific effort.
Summary of the invention the objective of the invention is to by cell being used in combination of necessary nutritive substance and microorganism growth inhibitor of growing, remove and the control cell in the pollution of " black glue worm ", to ensure carrying out smoothly of every research work.
The present invention realizes as follows:
1. general tissue culture medium (TCM) is added in the deionized water, magnetic agitation is until dissolving fully.
2. in above-mentioned solution, add following material:
(1) putrescine (2) Transferrins,iron complexes (3) gsh (4) thymus pyrimidine (5) uracil (6) gentamicin (7) tsiklomitsin (8) Ceftriaxone (9) amikacin (10) amphotericin B (11) nystatin (12) Sodium desoxycholate (13) new-born calf serum (14) sodium bicarbonate
3.0.22 μ m filtration sterilization, packing.
4. remove the culture supernatant of contaminated cell, add the special culture medium of step 3 gained, 37 ℃, 5%CO
2Cultivate.
5. as step 4, use special culture medium at interval repeatedly.
Embodiment
1. get general tissue culture medium (TCM) 1 bag (1L/ bag), join in the 900ml deionized water, magnetic agitation makes it to dissolve fully.
2. adding putrescine, making its final concentration is 5-36 μ g/ml.
3. adding Transferrins,iron complexes, making its final concentration is 1-40 μ g/ml.
4. adding gsh, making its final concentration is 10-50mg/L.
5. adding thymus pyrimidine, making its final concentration is 2-8.3mg/L.
6. adding uracil, making its final concentration is 1-2.5mg/L.
7. adding gentamicin, making its final concentration is 20-300 μ g/ml.
8. adding tsiklomitsin, making its final concentration is 5-200 μ g/ml.
9. adding Ceftriaxone, making its final concentration is 10-500 μ g/ml.
10. adding amikacin, making its final concentration is 5-150 μ g/ml.
11. it is 1-25 μ g/ml that the adding amphotericin makes its final concentration.
12. it is 2-50 μ g/ml that adding nystatin makes its final concentration.
13. it is 2-50 μ g/ml that the adding Sodium desoxycholate makes its final concentration.
14. it is 12% that new-born calf serum makes its final concentration.
15. it is 3g/L that sodium bicarbonate makes its final concentration.
16. magnetic agitation is dissolved each material fully, regulates PH to 7.0-7.1, constant volume is to 1000ml.
17.0.22 μ m filtration sterilization, packing, 100ml/ bottle, 2-8 ℃ of refrigeration.
18. remove contaminated cell conditioned medium, add the as above special culture medium of gained, every 25cm
2Culturing bottle adds the 5-8ml special culture medium.37 ℃, 5%CO
2Remove supernatant after the cultivation, add conventional substratum.
19. the interval is repeating step 18 repeatedly.
The special culture medium of the above-mentioned gained of measurement result shows that through cell growth curve mensuration the nutritional support of pair cell is identical with ordinary culture medium, satisfies the needs of growth and proliferation of cell fully; The cell that " black glue worm " pollutes, through the processing of special culture medium, " black glue worm " is killed and suppresses, and disappears in culture supernatant, and cell can continue to cultivate, and is used for every experimental study work; Stability experiment shows that 2-8 ℃ of stored refrigerated time of this product is 6 months.
Claims (11)
1. kill and suppress the preparation and the using method of the special culture medium of " black glue worm " in the tissue culture, it is characterized in that:
Kill medicine 1.1 add " black glue worm " in the general tissue culture medium (TCM):
(1) putrescine (2) Transferrins,iron complexes (3) gsh (4) thymus pyrimidine (5) uracil (6) gentamicin (7) tsiklomitsin (8) Ceftriaxone (9) amikacin (10) amphotericin B (11) nystatin (12) Sodium desoxycholate (13) new-born calf serum (14) sodium bicarbonate
1.2 0.22 μ m filtration sterilization, packing 2-8 ℃ preservation.
1.3 remove the substratum of contaminated cell, add step 2 gained special culture medium, 37 ℃, 5% CO
2Cultivate.
But 1.4 can not kill repeating step 1.3 fully.
2. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (5) add in the step 1.1 is a uracil, and final concentration is 2.5mg/L.
3. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (6) add in the step 1.1 is a gentamicin, and final concentration is 20-300 μ g/ml.
4. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (7) add in the step 1.1 is a tsiklomitsin, and final concentration is 5-200 μ g/ml.
5. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (8) add in the step 1.1 is a Ceftriaxone, and final concentration is 10-500 μ g/ml.
6. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (9) add in the step 1.1 is an amikacin, and final concentration is 5-150 μ g/ml.
7. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (10) add in the step 1.1 is an amphotericin B, and final concentration is 1-25 μ g/ml.
8. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (11) add in the step 1.1 is a nystatin, and final concentration is 2-50 μ g/ml.
9. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: the material that (12) add in the step 1.1 is a Sodium desoxycholate, and final concentration is 2-50 μ g/ml.
10. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: 37 ℃, 5%CO in the step 1.3
2Incubation time is 24 hours.
11. as killing and suppress the preparation method of the special culture medium of " black glue worm " as described in claims, it is characterized in that: use be 24-48 hour the pitch time of specific culture in the step 1.4, the number of times that uses repeatedly is as 2-3 time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510085462 CN1900266A (en) | 2005-07-21 | 2005-07-21 | Preparing and using method of special culture medium capable of killing and controlling 'black bug' |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510085462 CN1900266A (en) | 2005-07-21 | 2005-07-21 | Preparing and using method of special culture medium capable of killing and controlling 'black bug' |
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| Publication Number | Publication Date |
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| CN1900266A true CN1900266A (en) | 2007-01-24 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510085462 Pending CN1900266A (en) | 2005-07-21 | 2005-07-21 | Preparing and using method of special culture medium capable of killing and controlling 'black bug' |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320386A (en) * | 2013-06-25 | 2013-09-25 | 新乡医学院 | Method for saving cells from slightly polluted cell fluid |
| CN112626004A (en) * | 2021-02-03 | 2021-04-09 | 姜云瀚 | Black glue worm remover and application thereof |
| CN113652387A (en) * | 2021-07-16 | 2021-11-16 | 上海佐润生物科技有限公司 | Hemicellae removing reagent and preparation method thereof |
-
2005
- 2005-07-21 CN CN 200510085462 patent/CN1900266A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320386A (en) * | 2013-06-25 | 2013-09-25 | 新乡医学院 | Method for saving cells from slightly polluted cell fluid |
| CN112626004A (en) * | 2021-02-03 | 2021-04-09 | 姜云瀚 | Black glue worm remover and application thereof |
| CN112626004B (en) * | 2021-02-03 | 2024-05-17 | 姜云瀚 | Black beetle remover and application thereof |
| CN113652387A (en) * | 2021-07-16 | 2021-11-16 | 上海佐润生物科技有限公司 | Hemicellae removing reagent and preparation method thereof |
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