CN113308510A - Culture medium for rapidly detecting fungi in cell product and preparation method and application thereof - Google Patents
Culture medium for rapidly detecting fungi in cell product and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 title description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 36
- 239000000843 powder Substances 0.000 claims abstract description 28
- 210000002966 serum Anatomy 0.000 claims abstract description 26
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- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 21
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 19
- 239000008103 glucose Substances 0.000 claims abstract description 19
- 239000001888 Peptone Substances 0.000 claims abstract description 18
- 108010080698 Peptones Proteins 0.000 claims abstract description 18
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- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 18
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
- G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
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Abstract
The invention discloses a culture medium for rapidly detecting fungi in cell products, and a preparation method and application thereof. Potato soup, peptone and yeast extract powder are used for providing a carbon source, a nitrogen source and part of growth factors for fungus growth; the glucose provides a carbon source for fermentation or growth of the fungi, and the growth of the fungi is facilitated; potassium dihydrogen phosphate is used as a buffering agent; magnesium sulfate is used to maintain osmotic pressure, while magnesium ions play an extremely important role in the metabolism of sugars and proteins; the serum contains plasma protein, polypeptide, fat, carbohydrate, growth factor, hormone, inorganic matter, etc., and especially rich growth factor and low molecular weight nutrient matter can promote the growth of microbe, raise the sensitivity of microbe detection in cell product and raise the accuracy of fungus detection.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a culture medium for rapidly detecting fungi in cell products, and a preparation method and application thereof.
Background
Cell culture is the beginning of biological experiments and is the basis of biotechnology, and therefore, when biological experiments are carried out, it is ensured that the cultured cells are sterile and clean. If the cultured cells are contaminated with microorganisms, they are found at a later stage of contamination. If the fungus infection is caused, small black spots can be seen under a macroscopic view at the later stage of the infection; the cells are polluted, the growth of the cells is abnormal, and the cells can be distinguished under a high power microscope, and the cells are in the later stage of culture. However, the growth cycle of fungi is slower than that of bacteria, and fungal infection is usually found in one week or more, and at this time, the later period of the experiment is usually carried out, resulting in waste of a large amount of data and time.
However, there is no product for rapidly and highly sensitively checking whether cells are contaminated by fungi, especially stem cell products. In view of the above, it is an urgent technical problem to be solved by those skilled in the art to develop a product capable of rapidly and highly sensitively checking whether a cell culture process is contaminated.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a culture medium for rapidly detecting fungi in cell products, a preparation method and application thereof, and the condition of fungal contamination of the cell products can be rapidly and highly sensitively detected through the culture medium.
The invention discloses a culture medium for rapidly detecting fungi in cell products, which comprises potato soup, glucose, peptone, yeast extract powder, potassium dihydrogen phosphate, magnesium sulfate and serum.
Preferably, the culture medium contains the following components per liter: 40-60g of potato soup, 3-6g of glucose, 2-4g of peptone, 1-2g of yeast extract powder, 0.3-1g of monopotassium phosphate, 0.1-0.5g of magnesium sulfate and serum.
Preferably, the culture medium contains the following components per liter: 50g of potato soup, 5g of glucose, 3g of peptone, 1-2g of yeast extract powder, 0.5g of monopotassium phosphate, 0.2g of magnesium sulfate and serum.
Preferably, the serum comprises fetal calf serum, and the mass ratio of the fetal calf serum to the culture medium is 200:1-1000: 1.
Preferably, the culture medium also comprises antibacterial antibiotics and agar powder.
Preferably, the antibacterial antibiotic comprises chloramphenicol, penicillin or cephalosporin; the content of the agar powder is 15-20 g/L.
The invention also provides a preparation method of the culture medium, which comprises the following steps:
step S1: mixing glucose, peptone, potassium dihydrogen phosphate, yeast extract powder, magnesium sulfate and water to obtain a premix;
step S2: adding potato soup to the premix to obtain a mixture;
step S3: serum was added to the mixture to obtain a culture medium.
Preferably, step S1 further includes: after adjusting the pH, the premix is filtered and sterilized;
step S2 includes: adding potato soup into the premix, adjusting the pH value, sterilizing, adding serum, and preparing a culture medium for detecting fungi;
wherein the pH value is 6.8-7.2.
Preferably, agar powder is also added in step S2.
The culture medium of the invention is used for detecting fungal contamination of cell preparations.
Preferably, the fungus is any one or a combination of the following species: aspergillus niger, Mucor, Aspergillus fumigatus, Sporomyces, Candida albicans, and yeast.
Compared with the prior art, the invention has the beneficial effects that: potato soup, peptone and yeast extract powder are used for providing a carbon source, a nitrogen source and part of growth factors for fungus growth; the glucose provides a carbon source for fermentation or growth of the fungi, and the growth of the fungi is facilitated; potassium dihydrogen phosphate is used as a buffering agent; magnesium sulfate is used to maintain osmotic pressure, while magnesium ions play an extremely important role in the metabolism of sugars and proteins; the serum contains plasma protein, polypeptide, fat, carbohydrate, growth factor, hormone, inorganic matter, etc., and especially rich growth factor and low molecular weight nutrient matter can promote the growth of microbe, raise the sensitivity of microbe detection in cell product and raise the accuracy of fungus detection.
Drawings
FIG. 1 is a flow chart of a method for preparing a medium of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention is described in further detail below with reference to the attached drawing figures:
a culture medium for rapidly detecting fungi in cell products comprises potato soup, glucose, peptone, yeast extract powder, potassium dihydrogen phosphate, magnesium sulfate and serum.
Wherein, the potato soup, the peptone and the yeast extract powder are used for providing a carbon source, a nitrogen source and part of growth factors for fungus growth; the glucose provides a carbon source for fermentation or growth of the fungi, and the growth of the fungi is facilitated; potassium dihydrogen phosphate is used as a buffering agent; magnesium sulfate is used to maintain osmotic pressure, while magnesium ions play an extremely important role in the metabolism of sugars and proteins; the serum contains plasma protein, polypeptide, fat, carbohydrate, growth factor, hormone, inorganic matter, etc., and especially rich growth factor and low molecular weight nutrient matter can promote the growth of microbe, raise the sensitivity of microbe detection in cell product and raise the accuracy of fungus detection.
The culture medium contains the following components per liter: 40-60g of potato soup, 3-6g of glucose, 2-4g of peptone, 1-2g of yeast extract powder, 0.3-1g of monopotassium phosphate, 0.1-0.5g of magnesium sulfate and serum. In one embodiment, the following components are present per liter of medium: 50g of potato soup, 5g of glucose, 3g of peptone, 1-2g of yeast extract powder, 0.5g of monopotassium phosphate, 0.2g of magnesium sulfate and serum. Wherein the serum comprises fetal calf serum, and the mass ratio of the fetal calf serum to the culture medium is 200:1-1000: 1. However, the serum may be newborn bovine serum or calf serum.
Wherein the culture medium may further comprise an antibacterial antibiotic, such as chloramphenicol, penicillin or cephalosporin, for inhibiting the growth of bacteria. The growth speed of bacteria is high, the detection period is short, and the growth speed of bacteria is far higher than that of fungi under the condition of bacterial pollution, so that the fungi cannot be correctly detected. The accuracy of fungus detection is improved by antibacterial antibiotics.
Wherein agar powder can be added into the culture medium, for example, agar powder with the final content of 15-20g/L can be added. Is beneficial to preparing a culture plate.
As shown in FIG. 1, the preparation method of the culture medium comprises the following steps:
step S1: glucose, peptone, potassium dihydrogen phosphate, yeast extract powder, magnesium sulfate and water were mixed to obtain a premix. In step S1, the premix may be filtered and sterilized after pH adjustment. Wherein, the adjusted pH value is 6.8-7.2, and the water can adopt deionized water.
Step S2: adding potato soup to the premix to obtain a mixture. Specifically, potato soup is added into the premix, the pH value is adjusted, sterilization treatment is carried out, and then serum is added to prepare the fungus detection culture medium. Agar powder may also be added in step S2 to prepare a fungal culture plate, i.e., a solid culture medium.
Step S3: serum was added to the mixture to obtain a culture medium. Antibacterial antibiotics may be added in step S3.
Wherein the sterilization treatment comprises any one of the following methods or a combination thereof: boiling for sterilization, filtering for sterilization, microwave sterilization, ray sterilization or autoclaving. The cell product refers to cells obtained by applying common or biological technologies such as genetic engineering, cell engineering, protein engineering, fermentation engineering and the like; and various animal and human tissues and liquids, and can be used for preventing, treating and diagnosing human diseases. However, the medium of the present invention may be a medium for detecting cells, a frozen medium for detecting cells, a cell culture apparatus, or a cell culture tool. The cell culture apparatus may be a carbon dioxide incubator or the like, and the cell culture tool may be a tip for cells, a culture dish for cells, or the like.
The cells can be stem cells, the culture medium of the stem cells or the fungi of a stem cell culture tool can be detected while the stem cells are cultured, and whether fungal contamination occurs can be detected without waiting until the stem cells have the contamination characteristics.
Example 1
A culture medium configured for rapid detection of fungi in a cell preparation, the composition of the culture medium being as follows:
| name (R) | Content (wt.) |
| Glucose | 5g |
| Peptone | 3g |
| Yeast extract powder | 1g |
| Potassium dihydrogen phosphate | 0.5g |
| Magnesium sulfate | 0.2g |
| Potato soup | 50g |
| Water (W) | 1L |
| Serum | 2.5mL |
The specific preparation method comprises the following operation steps:
step S11: preparing a culture medium according to the components in the table, wherein glucose, peptone, potassium dihydrogen phosphate, yeast extract powder, magnesium sulfate and water are mixed to obtain a premix; the premix was adjusted to pH, boiled and filtered (to remove impurities) through a 70 micron screen.
Step S12: adding potato soup into the sterilized premix obtained in step S11, adjusting pH to 7.0, boiling to obtain a fungus detection medium, and sterilizing.
Step S13: after completion of step S12, sterile serum was added to obtain medium a.
And (3) detection of a culture medium:
the sensitivity verification and comparison test is carried out on the culture medium A of the example 1 and a common trypticase soytone liquid culture medium, and the specific operation steps are as follows:
step S31: the following strains were obtained: aspergillus niger, Mucor, Aspergillus fumigatus, Sporomyces, Candida albicans, yeast (isolated from a laboratory environment).
Step S32: preparing a bacterial liquid from the strain obtained in the step S31, and specifically comprising the following steps:
step S321: inoculating yeast into wort liquid culture medium, culturing at 35 deg.C for 24 hr, and making into bacterial suspension containing bacteria less than 100CFU (colony forming unit) per 1ml with 0.9% sterile sodium chloride solution.
Step S322: inoculating mould to a glucose saxifrage liquid culture medium, culturing at 25 deg.C for 5 days, and making the culture solution into bacterial suspension containing bacteria less than 100CFU (colony forming unit) per 1ml with 0.9% sterile sodium chloride solution.
Step S33: inoculating the bacterial liquid obtained in the step S32 with the culture medium A and the common thioglycollate fluid culture medium, and specifically comprising the following steps:
step S331: taking 610 common thioglycollate fluid culture media with the volume of 9ml per tube, taking 100 common thioglycollate fluid culture media as a group, respectively inoculating aspergillus niger, mucor, aspergillus fumigatus, sporoderm, candida albicans and saccharomycetes with the number of less than 100CFU in each group, and culturing for 2 days without inoculating 10 common thioglycollate fluid culture media as blank controls. The results were observed day by day.
Step S332: the medium of example 1 was cultured in a tube of 9ml in a set of 100 medium 510, each set was inoculated with less than 100CFU of aspergillus niger, mucor, aspergillus fumigatus, sporoderm, candida albicans, yeast, and 10 medium-free medium as a control for 5 days. The results were observed day by day.
Step S333: and (4) judging a result: the blank control tube has no microorganism growth, the common fungus culture medium and the culture medium tube added with the culture medium A of the embodiment 1 have good growth, and the sensitivity check is judged to be in accordance with the regulation. The experimental results are as follows:
the sensitivity of medium A of example 1 and the general fungal medium was judged based on the percentage growth of each species in the table above. The percentage of good growth of microorganisms in the culture medium A is more than 97 percent, which is higher than that of a common fungus liquid culture medium, particularly on the detection of candida albicans and yeast. The above table shows that medium a has better sensitivity to 6 different species than the common fungal liquid medium. Therefore, if the cell product contains the above bacterial species, the above bacterial species will grow rapidly in the culture medium A, resulting in turbidity of the detection medium, and it can be judged that the cell product is contaminated with microorganisms.
Example 2
A culture medium configured for rapid detection of fungi in a cell preparation, the composition of the culture medium being as follows:
| name (R) | Content (wt.) |
| Glucose | 5g |
| Peptone | 3g |
| Yeast extract powder | 1g |
| Potassium dihydrogen phosphate | 0.5g |
| Magnesium sulfate | 0.2g |
| Potato soup | 50g |
| Water (W) | 1L |
| Agar powder | 1.5g |
| Serum | 2.5mL |
Step S21: preparing a culture medium according to the components in the table, wherein glucose, peptone, potassium dihydrogen phosphate, yeast extract powder, magnesium sulfate and water are mixed to obtain a premix; the premix was adjusted to pH, boiled and filtered (to remove impurities) through a 70 micron screen.
Step S22: adding potato soup into the sterilized premix obtained in step S11, adjusting pH to 7.0, boiling to obtain a fungus detection medium, and sterilizing.
Step S23: after completion of step S12, sterile serum was added to obtain medium B.
And (3) detection of a culture medium:
performing a plate counting method test on the culture medium B and a common fungus culture medium (PDA culture medium), and quantitatively investigating the growth capacities of strains of Aspergillus niger, Mucor, Aspergillus fumigatus, spore mold, Candida albicans and yeast on the fungus detection culture medium and the common fungus agar culture medium in the embodiment 2 of the invention according to a plate counting method in a microorganism limit inspection method of Chinese pharmacopoeia (2010 version), wherein the specific operation steps are as follows:
step S41: ordinary fungal agar medium and fungal detection agar medium suitable for counting the number of fungi were prepared in ordinary fungal medium and medium B of example 2.
Step S42: the bacterial liquids of aspergillus niger, mucor, aspergillus fumigatus, sporotrichum, candida albicans and yeast were prepared according to the method for preparing bacterial liquids of example 2.
Step S43: inoculating and culturing, comprising the following specific operation steps: respectively inoculating the bacterial liquids of aspergillus niger, mucor, aspergillus fumigatus, sporotrichum, candida albicans and saccharomycetes on a common fungus agar culture medium and a fungus detection agar culture medium, wherein each experimental bacterium is 4 parallel plates, pouring the plates, solidifying and then placing in an incubator at 28 ℃ for 5 days. Colony growth was observed daily and the number of colonies was recorded. Comparing the growth promoting capacity of the common fungus agar culture medium and the fungus detection agar culture medium by the colony recovery rate, and recording the result. Wherein, the colony recovery rate is the average colony number on the culture medium plate to be tested/the average colony number on the control culture medium plate. The results are shown in the following table:
from the above table, the colony recovery rates of the fungus detection agar medium are all higher than 100%, which indicates that the medium B has the effect of promoting the growth of various strains, especially sporotrichum and candida albicans, and the colony recovery rates are as high as 120.45% and 128.73%. Therefore, if the cell product contains the above bacterial species, the above bacterial species will grow rapidly in the medium B, resulting in the growth of colonies on the plate of the fungus detection agar medium, and the cell product is known to be contaminated by microorganisms.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A culture medium for rapidly detecting fungi in cell products is characterized by comprising potato soup, glucose, peptone, yeast extract powder, potassium dihydrogen phosphate, magnesium sulfate and serum.
2. The culture medium according to claim 1, wherein the following components are contained per liter of the culture medium: 50g of potato soup, 5g of glucose, 3g of peptone, 1-2g of yeast extract powder, 0.5g of monopotassium phosphate, 0.2g of magnesium sulfate and serum.
3. The culture medium according to claim 2, wherein the serum comprises fetal bovine serum, and the mass ratio of the fetal bovine serum to the culture medium is 200:1-1000: 1.
4. The culture medium of claim 1, further comprising an antibacterial antibiotic and agar powder.
5. The culture medium of claim 4, wherein the antibacterial antibiotic comprises chloramphenicol, penicillin, or cephalosporin;
the content of the agar powder is 15-20 g/L.
6. A method for preparing a culture medium according to any one of claims 1 to 5, comprising:
step S1: mixing glucose, peptone, potassium dihydrogen phosphate, yeast extract powder, magnesium sulfate and water to obtain a premix;
step S2: adding potato soup to the premix to obtain a mixture;
step S3: serum was added to the mixture to obtain a culture medium.
7. The method according to claim 6, wherein step S1 further includes: after adjusting the pH, the premix is filtered and sterilized;
step S2 includes: adding potato soup into the premix, adjusting the pH value, sterilizing, and adding serum to obtain a culture medium for detecting fungi;
wherein the pH value is 6.8-7.2.
8. The method according to claim 6, wherein agar powder is further added in step S2.
9. Use of a culture medium according to any one of claims 1 to 5 for detecting fungal contamination of a cell preparation.
10. Use according to claim 9, wherein the fungus is of any one of the following species or combinations thereof: aspergillus niger, Mucor, Aspergillus fumigatus, Sporomyces, Candida albicans, and yeast.
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