According to the different raw materials used, it can be divided into two categories: natural culture medium prepared with natural ingredients such as broth and potato juice; The medium prepared with chemicals and marked with ingredients is called synthetic medium or comprehensive medium. Most of the media in chemical reagents are synthetic media. Because the liquid medium is not easy to keep for a long time, it is now converted into powder. Because of different raw materials, the storage requirements of culture medium are different, and the storage and preservation are slightly different. Generally, the culture medium is easy to be contaminated or decomposed by bacteria after being heated and absorbing moisture, and must be kept in a damp-proof, dark and cool place. Some of them need strict disinfection.
Common media are:
1, bacterial culture medium
Formula 1 beef paste agar medium
0.3g of beef sauce, 0.0g of peptone/kloc-0, 0.5g of sodium chloride and 0.5g of agar/kloc-0,
Water 100 ml
Add 100 ml water to the beaker, add beef paste, peptone and sodium chloride, mark the beaker with crayons, and heat it on the fire. After all the ingredients in the beaker are dissolved, add agar and stir constantly to avoid sticking to the bottom. When the agar is completely dissolved, make up for the water loss, and adjust the pH value to 7.2 ~ 7.2 with 10% hydrochloric acid or 10% sodium hydroxide.
Formula II potato culture medium
Take 250g fresh beef heart (excluding fat and blood vessels), finely chop it into minced meat with a knife, add 500ml distilled water and 5g peptone, mark it on a beaker, boil it, and simmer for 2 hours. Filtering, drying the filtered minced meat, and adjusting the pH value of the filtrate to about 7.5. Add 10ml broth and a small amount of bovine heartbreak to each test tube and sterilize for later use.
Preparing rhizobia culture medium
Glucose 10g dipotassium hydrogen phosphate 0.5g
3 grams of calcium carbonate and 0.2 grams of magnesium sulfate.
0.4g yeast powder and 20g agar.
Water 1000ml 1% crystal violet solution 1ml.
Firstly, the agar is boiled and dissolved in water, then other ingredients are added, stirred and dissolved, and packaged and sterilized for later use.
2. Actinomycete culture medium
Formula 1 starch agar medium (Gaussian medium)
2g of soluble starch and 0. 1g of potassium nitrate.
0.05g dipotassium hydrogen phosphate and 0.05g sodium chloride.
0.05g of magnesium sulfate and 0.00 1 g of ferrous sulfate.
Agar 2g water 100ml.
First, put the starch into a beaker, make it into paste with 5 ml of water, then pour in 95 ml of water, stir it evenly, and then add other drugs to dissolve it. Mark the outside of the beaker, add agar when it is heated to boiling, and keep stirring until the agar is completely dissolved to make up for the loss of water. Adjust the pH value to 7.2 ~ 7.4, and package and sterilize for later use.
Formula II flour agar medium
Flour 60g, agar 20g.
Water 1000 ml
Mix water into batter, add water to 500 ml, and simmer for 30 minutes. Take another 500 ml of water, add agar, heat and boil until dissolved, mix the two liquids evenly, replenish water, adjust the pH to 7.4, subpackage and sterilize for later use.
3. Fungal culture medium
Sabouraud medium's formula
Peptone 10g agar 20g
Maltose 40g water 1000ml.
First, add water to peptone and agar, then heat and stir constantly. After the agar is dissolved, 40g of maltose (or glucose) is added, stirred and dissolved, and then packaged and sterilized for later use.
This culture strain is usually used to cultivate a variety of fungi.
Potato sugar agar medium of formula II
Wash and peel potatoes, cut 200 grams into small pieces, add 1000 ml of water, and cook for half an hour to make up the water. Add10g agar to the filtrate, boil and dissolve, then add 20g sugar (sucrose for mold culture and glucose for yeast culture), make up water, package and sterilize for later use.
Adjust the pH value of this culture medium to 7.2 ~ 7.4, and the sugar in the formula, such as glucose, can also be used to cultivate actinomycetes and Bacillus.
Formula bean sprout juice medium
Soybean sprouts 100g agar 15g.
Glucose 20g, water 1000ml.
Wash soybean sprouts and add water to boil for 30 minutes. Filtering with gauze, adding agar into the filtrate, heating to dissolve, adding sugar, stirring to dissolve, supplementing water to 1000 ml, and packaging and sterilizing for later use.
Adjusting the pH value of this culture medium to 7.2 ~ 7.4 can be used to cultivate bacteria and actinomycetes.
Formula four pea agar medium
Pea 80 agar 5g
Water 200 ml
Add water to 80 dried peas, boil for 65438 0 hours, filter with gauze, add agar to the filtrate, boil until dissolved, package and sterilize for later use.
4. Edible fungi culture medium
Formula 1 potato-sucrose-agar medium
20% potato juice 1000 ml
20g sucrose, agar18 g.
Wash and peel the potatoes and cut them into small pieces. Weigh 200 grams of potato pieces, add 1000 ml of water, boil for 20 minutes, and then filter. Make up the water in the filtered juice to 1000 ml to make 20% boiled potato juice. Adding agar and sucrose into potato juice, boiling, dissolving, adding water, packaging, and sterilizing.
Formula II Comprehensive Potato Culture Medium
20% potato juice 1000 ml
Potassium dihydrogen phosphate 3g magnesium sulfate1.5g.
Glucose 20g, vitamin 10 mg.
Agar 18g
First, prepare 20% boiled potato juice. The method is as above. Adding the above ingredients into the boiled fruit juice, heating to dissolve, adding water, and adjusting the pH value to 6. Packaging, and sterilizing. The culture medium is used for cultivating and preserving edible fungi such as Ganoderma lucidum, Pleurotus ostreatus and Lentinus edodes.
5. Tobacco culture medium
In plant tissue culture, callus can be differentiated into roots or buds by adjusting the ratio of IAA to CTK. When CTK/IAA is high, the callus can differentiate into buds.
When CTK/IAA is low, the root system differentiates; Moderate CTK/IAA ratio maintained the undifferentiated callus.
Preparation of callus induction medium
Based on the mother liquor of MS culture medium, macro-element 20× mother liquor 100mL, micro-element 100× mother liquor 20mL, iron salt 100× mother liquor 20mL, vitamin 100× mother liquor 20mL and inositol 200× were added into clean aluminum pot in turn. After preparing MS culture medium, add 0.5 mg L- 1 Ba8ml and 0.5 mg L- 1 NAA8mL, then add distilled water about 2/3-3/4 of the actual volume of the prepared culture medium, add 40g sucrose and stir to dissolve it. Adjust the pH value to 5.8-6.0 with 0.5 mol of L- 1 NaOH and 0.5 mol of L- 1 HCl. Add 14g agar, put aluminum pot in an electric furnace, stir and heat to completely dissolve the agar, then use distilled water to make the volume reach the final volume of 2L, continue heating for a few minutes to mix it evenly, and then sub-package it in triangular bottles.
Callus induction of tobacco leaves
Take a sterile Petri dish, cut 1-2 sterile seedling leaves with a scalpel, put them into the sterile Petri dish, and use the solution.
Cut the leaves into small pieces of about 2mm2 with a cutting knife, then inoculate them on the prepared culture medium, and inoculate each bottle.
Five small pieces, one * * * inoculated with 6 bottles. Inoculated triceratops was cultured in the dark for 65438 0 weeks under 24 conditions, and then cultured under the same conditions.
The samples were cultured in light and complete darkness for 3 weeks until callus was formed (3 bottles in each case).
Observe the callus induction results and count the callus induction rate.
Organ differentiation and plant regeneration culture
The induced calli were transferred to differentiation medium according to their types, and cultured for 3 weeks under continuous illumination and temperature of 20-22℃, and the plant regeneration of calli was counted.
General situation of callus induction
After 4 weeks of tobacco callus induction culture, callus basically formed, which ruled out that callus was caused by insufficient growth time.
No callus was formed. See table 1 for details. Callus was formed in all six culture bottles, but none of them developed.
Pollution, but the induction rate is almost the same. Among them, the average callus induction rate of three bottles cultured under light condition was 100%.
60.0℅, the average callus induction rate of three bottles cultured in dark condition was 46.7%. Because of the explants in the experiment,
The volume of tobacco leaves is too small, and most cells of some explants may have been dehydrated and died when inoculated, making the whole plant
The callus induction rate in this experiment is low and the callus is small.
The culture medium also includes: 1640 culture medium.
Special medium:
Selective medium
1 yeast enrichment medium
Glucose 5% urea 0. 1% ammonium sulfide 0. 1% potassium dihydrogen phosphate 0.25% disodium hydrogen phosphate 0.05% magnesium sulfate heptahydrate 0. 1% ferric sulfate heptahydrate 0.0 1% yeast extract 0.05%.
Bengal red 0.003% pH4.5
Enrichment and culture of autotrophic nitrogen-fixing bacteria in Ashby nitrogen-free medium
Mannitol 1% potassium dihydrogen phosphate 0.02% magnesium sulfate heptahydrate 0.02% sodium chloride 0.02%
Calcium sulfate dihydrate 0.0 1% calcium carbonate 0.5%
Two kinds of differential media
EMB culture medium, which is often used to identify Escherichia coli.
Peptone 10g lactose 5g sucrose 5g dipotassium hydrogen phosphate 2g eosin Y 0.4g methylene blue 0.065g
Distilled water 1000g pH7.2
Formula of culture medium for separating marine microorganisms
22 16E medium formula (solid medium)
Peptone 5g
Yeast mud 1 g
0.0 1 g iron phosphate
Agar 15-20g
Chenhaishui 1000ml
Boil the sodium hydroxide solution (5%) to adjust the PH value to 7.6-7.8.
Other media:
1. high first medium
KNO3: 1g，
Sodium chloride: 0.5g,
Potassium dihydrogen phosphate: 0.5g.
Magnesium sulfate ·7H2O:0.5g
Ferrous sulfate heptahydrate: 0.0 1 g.
Soluble starch 20g
Agar 20g
Distilled water 1000 ml
Adjust the pH to 7.2 ~ 7.4, and sterilize at 12 1℃ for 30 minutes.
Methods: First, starch was mixed into paste with a little water, and then 700ml of water was heated and boiled in an electric furnace.
Then pour the starch paste while stirring, while keeping boiling, and then add other ingredients.
After dissolution, add water to 1000 ml.
Broth peptone bevel:
Peptone 10g
Beef sauce 5g
Distilled water 1000 ml
Sodium chloride: 5g
Ph: 7.0 ~ 7.2, 12 1℃ for 20min.
If agar 1.5%~2% is needed to prepare solid medium, 0.5%~0.8% agar can be added to semi-solid medium.
Different cell culture media
Natural cell culture medium
Natural culture medium refers to a kind of culture medium derived from animal body fluids or extracted by tissue separation, such as plasma, serum, lymph, chicken embryo extract, etc. In the early stage of tissue culture technology, cells were cultured in vitro with natural medium. However, due to the complex production process and large difference between batches, natural culture medium is gradually replaced by synthetic culture medium. At present, the widely used natural culture medium is serum, and various tissue extracts and collagen substances that promote cell adhesion are also essential in the culture of some special cells.
Serum type
At present, the serum used for tissue culture is mainly bovine serum, and some special cells are also cultured with human serum and horse serum. The reasons for choosing bovine serum for cell culture are: sufficient sources, mature preparation technology, and people have a deeper understanding of it after long-term application tests. Bovine serum is suitable for most mammalian cells, but it is not excluded that it is more suitable to use other animal serum when cultivating some cells.
Bovine serum is the most widely used natural medium in cell culture, which contains rich nutrients necessary for cell growth and has extremely important functions. Bovine serum can be divided into calf serum, newborn bovine serum and fetal bovine serum. Fetal calf serum should be taken from fetal calf by caesarean section. The serum of newborn cows was taken from newborn cows within 24 hours after birth; Calf serum was obtained from calves born at 10-30 days. Obviously, the quality of fetal calf serum is the highest, because the fetal calf has no contact with the outside world, and the serum contains the least harmful components to cells such as antibodies and complements.
Main components of serum
Serum is a very complex mixture, which is formed by removing fibrin from plasma. Although most of its components are known, some of them are still unknown. The composition and content of serum often change with the gender, age, physiological status and nutritional status of blood donors. Serum contains various plasma proteins, peptides, fats, carbohydrates, growth factors, hormones, inorganic substances and so on. They are in physiological balance in promoting cell growth or inhibiting growth activity.
The main function of serum
1. Provide basic nutrients: amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives, etc. , is an essential substance for cell growth.
2. Provide hormones and various growth factors: insulin, adrenocortical hormones (hydrocortisone, dexamethasone), steroid hormones (estradiol, testosterone, progesterone), etc. Growth factors, such as fibroblast growth factor, epidermal growth factor and platelet growth factor.
3. Provide binding proteins: binding proteins are used to carry important low molecular weight substances, such as albumin carrying vitamins, fats and hormones, and transferrin carrying iron. Binding proteins play an important role in cell metabolism.
4. Provide contact promotion and stretching factors to prevent cell adhesion from being mechanically damaged.
5. It has a certain protective effect on cells in culture: some cells, such as endothelial cells and bone marrow-like cells, can release protease, and the serum contains anti-protease components, which play a neutralizing role. This effect was discovered by accident, and now serum is purposefully used to stop the digestion of trypsin. Because trypsin has been widely used in the digestion and passage of adherent cells. Serum protein forms the viscosity of serum, which can protect cells from mechanical damage. Especially in suspension culture, viscosity plays an important role. Serum also contains some trace elements and ions, which play an important role in metabolism and detoxification, such as SeO3 and selenium.
Main problems of serum culture medium
There may be hundreds of 1. serum components, but the exact composition, content and mechanism of action are not clear, especially some polypeptide growth factors, hormones and lipids are not fully understood, which brings many difficulties to the research work.
2. Serum is produced in batches, which varies greatly from batch to batch, and the storage period of serum is at most one year. Therefore, it is extremely difficult to ensure the similarity of each batch of serum, which limits the standardization and continuity of the experiment.
3. For most cells, in vivo, serum is not the physiological fluid they contact, but in the process of injury healing and blood coagulation, so the use of serum may change the normal state of some cells in vivo, and serum may promote the growth of some cells (fibroblasts) and inhibit the growth of another type of cells (epidermal cells).
4. Serum contains some substances that are toxic to cells, such as polyamine oxidase, which can react with polyamines (such as spermine and spermidine) produced by highly proliferative cells to form poly-spermine with cytotoxic effect. Complement, antibody and bacterial toxin will all affect cell growth and even lead to cell death.
5. Different animals have different serum sources and batch numbers, and the quality of each batch is very different, so its composition cannot be consistent.
6. Mycoplasma and virus may be brought into the sample, which may have a potential impact on cells and may lead to the failure of the experiment or the unreliable experimental results.
7. In mass production, the source of serum is becoming more and more difficult and expensive, which is one of the main parts of the production cost of animal cell culture.
Serum quality standard
The quality of serum depends on two factors: one is the sampling object, and the other is the sampling process. Animals used for sampling should be healthy and disease-free, and within the specified date of birth. The sampling process should be carried out in strict accordance with the operating procedures, and the prepared serum should undergo strict quality appraisal. Requirements in the "Regulations for the Production of Biological Products by Animal Cell Culture in Vitro" promulgated by WHO:
1. Bovine serum must come from cattle or countries with documented evidence that there is no bovine spongiform encephalopathy, and there should be an appropriate monitoring system.
2. Some countries also require bovine serum to come from cattle that have never used ruminant protein feed.
3. Prove that the bovine serum used does not contain inhibitors of the vaccine virus produced.
4. Serum should be sterilized by filtration membrane to ensure sterility.
5. No bacteria, mold, mycoplasma and virus pollution, and some countries require no phage pollution.
6. It has a good effect of supporting cell reproduction.
In the 2000 edition of Quality Control Standard for Main Raw Materials and Accessories of Biological Products in China, China put forward strict standards for the quality of bovine serum, including protein content, bacteria, fungi, mycoplasma, bovine virus, Escherichia coli phage and bacterial endotoxin, which supported cell proliferation inspection.