Medium General Practice

Medium

Medium (Medium) is for microorganisms, plants and animal tissue growth and maintenance of artificial preparation of nutrients, generally contain carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements) and vitamins and water. Some media also contain antibiotics and pigments.

According to the different raw materials used, can be divided into two categories: the application of broth, potato juice and other natural ingredients, known as natural culture medium; the application of chemicals with and marked ingredients, known as synthetic culture medium or comprehensive culture medium. Chemical reagents in the culture medium, mostly synthetic medium. Since the liquid medium is not easy to keep for a long time, it is now converted into powder. Medium due to the preparation of different raw materials, the use of different requirements, and storage is also slightly different. The general culture medium is easy to be contaminated or decomposed by bacteria after being heated and absorbed moisture. Therefore, the general culture medium must be protected from moisture, light and shade. For some media that need to be strictly sterilized (such as tissue culture medium), storage for a long time must be placed in 2~6. C in the refrigerator.
Common media are:

1. Bacterial culture medium

Formula oneBeef extract agar medium
Beef extract 0.3g, peptone 1.0g, sodium chloride 0.5g, agar 1.5g,
Water 100 ml
 

Add 100 ml of water in the beaker, add beef extract, peptone and sodium chloride, mark the outside of the beaker with crayons, and heat on the fire. After the components in the beaker are dissolved, add agar and stir constantly to avoid sticking to the bottom. After the agar is completely dissolved, the water loss is replenished, the pH value is adjusted to 7.2~7.6 "with 10% hydrochloric acid or 10% sodium hydroxide, and the test tubes are divided into test tubes, cotton plugs are added, and the test tubes are sterilized with high pressure steam for 30 minutes.

Formula IIPotato Medium

Take 250 grams of fresh beef heart (remove fat and blood vessels), finely chop into minced meat with a knife, and add 500 ml of distilled water and 5 grams of peptone. Mark the beaker, bring to a boil and simmer for 2 hours. Filtration, filtration of minced meat drying treatment, filtrate pH value adjusted to about 7.5. Add 10 ml of broth and a small amount of crushed dried beef heart into each test tube, sterilize and set aside.
Formula threerhizobia medium
Glucose 10g dipotassium hydrogen phosphate 0.5g
Calcium carbonate 3 grams Magnesium sulfate 0.2 grams
Yeast powder 0.4g agar 20g
Water 1000 ml 1% crystal violet solution 1 ml



 

First, add water to boil and dissolve the agar, then add other components respectively, stir to dissolve, subpackage, sterilize and reserve.

2. Actinomycete culture medium

Formula oneStarch agar medium (Gao's medium)

soluble starch 2g potassium nitrate 0.1g

Dipotassium hydrogen phosphate 0.05g Sodium chloride 0.05g

Magnesium sulfate 0.05g Ferrous sulfate 0.001g

Agar 2g water 100 ml first put starch in a beaker, use 5 ml water to make paste, pour 95 ml water, stir well and add other drugs to dissolve it. Mark the outside of the beaker, add agar when heated to boiling, keep stirring, and make up for water loss after the agar is completely dissolved. Adjust the pH value to 7.2~7.4, and sterilize after repackaging for standby.
Formula IIflour agar medium
Flour 60g agar 20g
Water 1000 ml
Mix the flour with water to a paste, add water to 500 ml and cook on a gentle heat for 30 minutes. Another 500 ml of water, into the agar, heating and boiling until dissolved, the two liquid mix, add water, adjust the pH value to 7.4, sub-packaging, sterilization, standby.
3. Fungal culture medium
Formula oneSabouraud's Medium
Peptone 10g agar 20g
Maltose 40g water 1000 ml
First, add peptone and agar to water, heat and stir continuously. After the agar is dissolved, add 40 grams of maltose (or glucose), stir to dissolve it, then subpackage, sterilize and set aside.
This culture is commonly used for the cultivation of many types of fungi.
Formula IIPotato Sugar Agar Medium
Wash and peel the potatoes, cut 200g into small pieces, add 1000 ml of water, boil for half an hour, and make up the water. Add 10 grams of agar to the filtrate, boil and dissolve, add 20 grams of sugar (add sucrose for mold culture, add glucose for yeast culture), make up the water, sub-pack, sterilize and spare.
The pH value of the medium is adjusted to 7.2~7.4, and the sugar in the formula, such as glucose, can also be used to culture actinomycetes and bacillus.
Formula threesoybean sprout juice medium
Soybean sprouts 100g agar 15g
Glucose 20g water 1000 ml
Wash bean sprouts, add water and boil for 30 minutes. Filter with gauze, add agar to the filtrate, heat and dissolve, add sugar, stir to dissolve it, make up the water to 1000 ml, sub-pack, sterilize and set aside.
The pH value of this medium is adjusted to 7.2~7.4, which can be used to cultivate bacteria and actinomycetes.
Formula fourPea agar medium
Pea 80 grains agar 5g
Water 200 ml
Add water to 80 dried peas, boil for 1 hour, filter with gauze, add agar to the filtrate, boil until dissolved, sub-pack, sterilize and reserve.
4. Edible Fungi Culture Medium
Formula onePotato-sucrose-agar medium
20% Potato Boiling Juice 1000 ml
Sucrose 20g agar 18g
Wash and peel the potatoes and cut them into small pieces. Weigh 200g of potato pieces, add 1000 ml of water, boil for 20 minutes, and filter. Make up 1000 ml of water in the filtered juice to make 20% boiled potato juice. Add agar and sucrose to the potato juice, boil it, make it dissolved, make up the water, sub-package, sterilization, standby. The use of this medium does not require a strict pH value and may not be measured.
Formula IIIntegrated Potato Medium
20% Potato Boiling Juice 1000 ml
Potassium dihydrogen phosphate 3 grams Magnesium sulfate 1.5 grams
Glucose 20g Vitamin 10 mg
Agar 18g
First prepare 20% potato juice, the method is the same as above. The above components are added to the boiled juice, and the water is added after heating and dissolving, and the pH value is adjusted to 6. Sub-packaging, sterilization, standby. The culture medium is used for the cultivation and preservation of edible fungi such as Ganoderma lucidum, Pleurotus ostreatus and Lentinus edodes.
5. Tobacco culture medium
































 

In plant tissue culture, the ratio of IAA to CTK can affect the differentiation of roots or buds from callus. CTK/IAA high '''' callus differentiation buds

'''' differentiates roots when CTK/IAA is low; CTK/IAA ratio is moderate to maintain callus undifferentiated

Preparation of callus induction medium

Based on the mother liquor of MS culture medium, a large number of elements 20 × mother liquor 100 mL''' trace elements 100 × mother liquor 20 mL''' iron salt 100 × mother liquor 20mL mL''' vitamin 100 × mother liquor 20 mL''' inositol 200 × mother liquor 10 mL''' glycine 200 × mother liquor 10 mL''' to prepare MS culture medium, and then add 0.5mg of NA0 mg · L-1. L-1. Then add distilled water with a volume of about 2/3-3/4 of the actual preparation medium. ''Add 40g sucrose and stir to dissolve.'' Adjust the pH value to 5.8-6.0 with 0.5 mol L-1 NaOH and 0.5 mol L-1 HCl. Add 14g agar. '''' Place an aluminum pan on an electric furnace. ''Stir and heat to completely dissolve the agar.'' Then use distilled water to fix the volume to a final volume of 2L. '''' in a triangular bottle.

Callus Induction in Tobacco Leaves

Take a sterile petri dish, cut 1-2 pieces of sterile seedling leaves with a scalpel and place them in a sterile petri dish, cut the leaves into small pieces of about 2mm2, and then inoculate them on the prepared medium, inoculate 5 pieces per bottle, inoculate a total of 6 bottles. After inoculation, the triangular flat was placed under 24 conditions and cultured in the dark for 1 week '''' and then in the same
The sample temperature was cultured in light and full darkness for 3 weeks until callus formation (3 bottles in each case). Observe the callus induction results'''' Statistical callus induction rate.

Organ differentiation and plant regeneration culture

The induced callus was transferred to the differentiation medium according to the type of callus, ''' was cultured for 3 weeks under the condition of continuous light and temperature of 20-22 C. ''' The regenerated plants of the callus were counted.

The general situation of callus induction

After 4 weeks of induction and culture of tobacco callus, the callus was basically formed, that is, the situation that the callus was not formed due to insufficient growth time was excluded. The specific situation is shown in Table 1. ''''' 6 bottles of cultures all have callus formation'''' and no contamination'''' but the induction rate is almost different. Among them, the average callus induction rate of the three bottles cultured in the light was 60.0. The average callus induction rate of the three bottles cultured in the dark was 46.7. Due to the small size of the explants, I .e. tobacco leaves, during the experiment, most of the cells of some of the explants may have dehydrated and died during the inoculation'', the callus induction rate of the whole experiment is low.

The culture medium also includes: 1640 culture medium
Special media:
a 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 0.01% yeast extract 0.05% Bengal red 0.003% pH4.5
2 Ashby nitrogen-free medium enriched with autotrophic nitrogen-fixing bacteria
Mannitol 1% potassium dihydrogen phosphate 0.02 magnesium sulfate heptahydrate 0.02 sodium chloride 0.02
Calcium sulfate dihydrate 0.01% Calcium carbonate 0.5%
II Identification Medium
EMB medium, commonly used to identify E.coli
Peptone 10g lactose 5g sucrose 5g dipotassium hydrogen phosphate 2g eosin Y 0.4g methylene blue 0.065g
Distilled water 1000g pH7.2
Medium Formulation for Separation of Marine Microorganisms
2216E medium formulation (solid medium)
Peptone 5g
Yeast extract 1g
High iron phosphate 0.01g
Agar 15 ----- 20g
Chen seawater 1000 ml
Boiling sodium hydroxide (5%) solution to adjust PH value 7.6-7.8


















 

Other media:
1. Gao's No.1 Medium
KNO3:1g''''
NaCl:0.5g''''
K2HPO4:0.5g
MgSO4·7H2O：0.5g
FeSO4·7H2O：0.01g
Soluble starch 20g
Agar 20g
Distilled water 1000ml
Adjust pH to 7.2~7.4, sterilize at 121°C for 30min
Method: First, mix starch into paste with a small amount of water, then heat and boil 700ml of water on an electric stove, then pour the starch paste while stirring, while keeping boiling. Then add other ingredients, dissolve and make up the water to 1000ml










 

broth peptone bevel:
Peptone 10g
Beef paste 5g
Distilled water 1000ml
NaCl：5g
pH:7.0~7.2,121 ℃ sterilization for 20min
If the solid medium needs to be prepared, 1.5 ~ 2% agar can be added to the semi-solid medium, 0.5~0.8 agar can be added to the semi-solid medium.





 

Different cell culture media
Natural Cell Culture Medium
 

Natural culture medium refers to a kind of culture medium extracted from animal body fluid or tissue separation, such as plasma, serum, lymph, chicken embryo extract, etc. In the early days of the establishment of tissue culture technology, cells cultured in vitro used natural medium. However, due to the complexity of the production process of natural medium and the large differences between batches, it is gradually replaced by synthetic medium. At present, the widely used natural medium is serum. In addition, various tissue extracts and collagen substances that promote cell adhesion are also essential in the culture of some special cells.
Type of serum

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 to culture cells are: sufficient sources, mature preparation technology, and people have a deeper understanding of it after a long time of application. Bovine serum is suitable for most mammalian cells, but it is not excluded that the use of other animal serum is more suitable in the culture of certain cells.

Bovine serum is the largest amount of natural culture medium in cell culture, which is rich in nutrients necessary for cell growth and has extremely important functions. Bovine serum is divided into calf serum, new bovine serum, fetal bovine serum. Fetal bovine serum should be taken from caesarean-section fetal cattle; new bovine serum from newborn cattle within 24 hours of birth; calf serum from calves 10-30 days of birth. Obviously, fetal bovine serum is of the highest quality, because fetal cattle have not been exposed to the outside world, and the serum contains the least amount of antibodies, complement and other harmful components to cells.
Main components of serum

Serum is a very complex mixture formed by the removal of fibrin from plasma. Although most of its components are known, some are still unclear, and the composition and content of serum often vary with the sex, age, physiological conditions and nutritional conditions of the donor animals. Serum contains a variety of plasma proteins, peptides, fats, carbohydrates, growth factors, hormones, inorganic substances, these substances to promote cell growth or inhibit growth activity is to achieve physiological balance.
The main role of serum
1. Provide basic nutrients: amino acids, vitamins, inorganic substances, lipids, nucleic acid derivatives, etc., are necessary 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, platelet growth factor, etc.
3. Provide binding protein: binding protein is important to carry low molecular weight substances, such as albumin carrying vitamins, fats, and hormones, transferrin carrying iron. Binding proteins play an important role in cellular metabolic processes.
4. Providing contact-promoting and stretching factors to protect cell attachment from mechanical damage.
5. Some protective effects 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 to neutralize. This effect was discovered by accident, and serum is now used purposeful to stop the trypsin digestion. Because trypsin has been widely used for the digestion of adherent cells. Serum proteins form the viscosity of the serum, which can protect the cells from mechanical damage, especially when the suspension culture is stirred, the viscosity plays an important role. Serum also contains some trace elements and ions, they play an important role in metabolic detoxification, such as SeO3'''' selenium and so on.




 

The main problem of serum medium
1. There may be hundreds of serum components, but the exact composition, content and mechanism of its action are still unclear, especially some of the 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 vary greatly from batch to batch, and the storage period of serum is up to one year. Therefore, it is extremely difficult to ensure the similarity of each batch of serum, thus limiting the standardization and continuity of experiments.
3. For most cells, in vivo, serum is not the physiological fluid they contact, only in the healing of the injury and blood coagulation process, so the use of serum may change the normal state of certain cells in the body, serum may promote the growth of certain cells (fibroblasts) and inhibit the growth of another type of cell (epidermal cells).
4. Serum contains some substances that are toxic to cells, such as polyamine oxidase, which can react with polyamines from highly propagated cells (e. g. spermine, spermidine) to form polyspermine with cytotoxic effects. Complement, antibodies, bacterial toxins, etc. can affect cell growth and even cause cell death.
5. Different animal individuals, different serum origin, batch number, the quality of each batch is very different, its composition can not be consistent.
6. Mycoplasma and virus may be brought into the material, which may have a potential impact on cells and may lead to experimental failure or unreliable experimental results.
7. In large-scale production, serum sources are increasingly difficult and expensive, and constitute one of the main parts of the production cost of animal cell culture.
Quality standards for serum







 

The quality of serum depends on two factors: one is the object of sampling, and the other is the process of sampling. The animals used for sampling should be healthy and disease-free and within the specified number of days of birth. The sampling process should be strictly implemented in accordance with the operating procedures, and the prepared serum should undergo strict quality identification. The requirements in the "Regulations for the production of biological products by in vitro culture of animal cells" published by WHO:
1. Bovine serum must be from a herd or country documented as free of bovine spongiform encephalopathy. An appropriate monitoring system should be in place.
Some countries also require that bovine serum be derived from herds that have not been fed ruminant protein.
3. It is proved that the bovine serum used does not contain inhibitors to the vaccine virus produced.
4. The serum should be filtered through the filter membrane to ensure sterility.
5. No bacteria, mold, mycoplasma and virus contamination, some countries require no bacteriophage contamination.
6. Good support for cell reproduction.




 

China has put forward relatively strict standard requirements for the quality of bovine serum in the 2000 edition of "Quality Control Standards for Main Raw Materials of Biological Products in China. Including protein content, bacteria, fungi, mycoplasma, bovine virus, Escherichia coli phage, bacterial endotoxin, support cell proliferation test.