culture medium

[péi yǎng jī]

Nutrient substrate for growth of microorganisms, plants and animal tissues

Collection

zero Useful+1

zero

This entry is made by Compilation and application of scientific encyclopedia entries of "Science Popularization China" to examine.

The term "culture medium" refers to a nutrient substrate prepared by combining different nutrients for the growth and reproduction of microorganisms, plants or animals (or tissues). Generally contains carbohydrate , nitrogen-containing substances, inorganic salts (including trace elements), vitamins and water. The culture medium provides cell nutrition and promotes cell proliferation It is also the living environment for cell growth and reproduction. There are many kinds of culture media, which can be divided into natural culture media Synthetic medium Semi synthetic medium; According to the physical state, it can be divided into solid culture medium Liquid medium 、 Semi solid medium ； According to the culture function, it can be divided into basic culture medium, selection culture medium, enrichment culture medium, identification culture medium, etc; According to the scope of use, it can be divided into bacterial culture medium, actinomycete culture medium, yeast culture medium, fungal culture medium, etc. After the culture medium is prepared, it is generally necessary to test and adjust the pH, and also to sterilize, usually including high-temperature sterilization and Filter sterilization 。 The culture medium is easy to be polluted or deteriorated due to its rich nutrients. It should not be stored for a long time after being prepared. It is better to prepare and use it now.^[1]

Chinese name: culture medium
Foreign name: Medium
Role: For growth of microorganisms, plant and animal tissues

Composition: Water, nitrogen source, inorganic salt, carbon source, etc
Saving method: Store in a damp, dark and cool place
Storage temperature: 2-25℃
Special nutrients: Vitamins, amino acids, bases, etc

catalog

brief introduction

Announce

edit

culture medium

Medium is a manually prepared nutrient for the growth and maintenance of microorganisms, plants and animal tissues, which generally contains water, nitrogen sources, inorganic salts (including trace elements), carbon sources, growth factors (vitamins, amino acids, bases antibiotics , pigment, hormone, serum, etc.).

Due to the different raw materials prepared, the use requirements of the culture medium are different, and the storage is slightly different. The general culture medium is easy to be contaminated by bacteria or degraded after being heated and damped, so the general culture medium must be stored in a damp proof, dark and cool place. For some culture media (such as tissue culture media) requiring strict sterilization, they must be stored in a refrigerator at 3-6 ℃ for a long time. because Liquid medium It is not easy to keep for a long time, and is changed into powder.

Preparation of culture medium

Announce

edit

1. Ingredients

Formula conversion → add a small amount of water (distilled water, natural water) into the container → weigh various drugs according to the formula (add in turn) → add the required amount of water (one medicine for one spoon, and cover the bottle immediately after taking the medicine).

2. Dissolution

Starch dissolution: a small amount of cold water is mixed into paste

Heat to dissolve, especially the medium added with agar, must be boiled. The melting temperature of agar is 95-97 ℃, and it needs to be heated while stirring to prevent burning.

3. Adjust PH

Adjust the medium to the required value with 1N hydrochloric acid or 1N NaOH.

4. Filtering

Filter with filter paper or cotton. (Sometimes it can be omitted)

5. Sub packaging

Generally, the culture medium is sterilized in a triangular bottle or test tube.

(1) Triangular flask

If static culture is used, the triangular flask with 100ml medium/250ml shall not exceed the triangular flask with 150ml medium/250ml at most, otherwise the boiling of the medium during sterilization may easily contaminate the cotton plug and cause bacteria contamination; In case of shake flask culture, 15-20ml culture medium/250ml triangular flask shall be used to ensure good ventilation.

(2) Test tube subpackage

Liquid medium Generally 4-5ml, about 1/4 of the height of the test tube; solid slope medium It is generally filled with 3-4ml, about 1/5 of the height of the test tube.

6. Binding

After subpackaging, plug the cotton plug, and wrap the cotton plug with kraft paper to prevent moisture from entering and wetting the cotton plug during sterilization.

7. Sterilization

Autoclave according to the temperature and pressure required on the formula. If the sterilization temperature is too high, the nutrients will be destroyed, and the sugar and amino acid in the medium will darken the color of the medium.

8. Pendulum

After sterilization, the test tube that needs to be placed with an inclined plane should be placed with an inclined plane when it is hot, so that it can solidify into an inclined plane, accounting for about 1/2 of the length of the test tube.

9. Storage

Keep the culture medium at 30 ℃ for one day, and use it without pollution. It is generally wrapped with kraft paper and stored in a 2-8 ℃ refrigerator for standby.

Configuration principle

Announce

edit

1. Select appropriate nutrients

In general, all microbial growth and reproduction require culture media containing carbon source, nitrogen source, inorganic salt, growth factor, water and energy Microbial nutrition type Complex, different microorganisms have different requirements for nutrients, so it is necessary to prepare targeted culture media according to the nutritional requirements of different microorganisms. Autotrophic microorganisms can synthesize complex organics such as sugars, lipids, proteins, nucleic acids, vitamins, etc. from simple inorganic substances, so the culture medium for cultivating autotrophic microorganisms can (or should) consist of simple inorganic substances. For example, cultivating chemoautotrophic Thiobacillus thiooxidans (Thiobacillus thiooxdans) is shown in Table 3.9. During the preparation of the culture medium, no other carbon source materials were specially added, but CO dissolved in water was neutralized by air two Provide carbon source for Thiobacillus thiooxidans.

As far as the main types of microorganisms are concerned, there are bacteria, actinomycetes, yeasts, molds, protozoa, algae and viruses. The culture media required for cultivating them are different. Beef extract peptone medium (or common for short) is commonly used in the laboratory Broth culture medium ）Cultivate bacteria, and cultivate actinomycetes with Gaoshi I synthetic medium, and generally cultivate yeast Wort medium The culture of mold is generally made of Cha's synthetic medium.

2. The concentration and proportion of nutrients are appropriate

When the concentration of nutrients in the culture medium is appropriate, microorganisms can grow well. When the concentration of nutrients is too low, it can not meet the needs of normal growth of microorganisms. When the concentration is too high, it may inhibit the growth of microorganisms. For example, high concentrations of sugars, inorganic salts, heavy metal ions, etc. can not maintain and promote the growth of microorganisms, but instead play a role of bacteriostasis or sterilization. In addition, the concentration ratio of various nutrients in the culture medium also directly affects the growth and reproduction of microorganisms and (or) the formation and accumulation of metabolites, of which the carbon nitrogen ratio (C/N) has a greater impact. Strictly speaking, the carbon nitrogen ratio refers to the ratio of carbon to nitrogen in the medium, and sometimes also refers to the ratio of reducing sugar to crude protein in the medium. For example, in the process of microbial fermentation to produce glutamic acid, when the carbon nitrogen ratio of the culture medium is 4/1, the bacteria multiply in large numbers and the accumulation of glutamic acid is less; When the C/N ratio of the culture medium was 3/1, the cell reproduction was inhibited, and the glutamic acid production increased significantly. For another example, in the process of antibiotic fermentation and production, the coordination of bacterial growth and antibiotic synthesis can be controlled by controlling the ratio between the available nitrogen (or carbon) source and the slow available nitrogen (or carbon) source in the culture medium.

3. Control pH conditions

The pH of the culture medium must be controlled within a certain range to meet the growth and reproduction of different types of microorganisms or produce metabolites. The optimal pH conditions for the growth and reproduction of various microorganisms or the production of metabolites are different. Generally speaking, bacteria and actinomycetes are suitable for growth within the range of pH7-7.5, while yeast and mold usually grow within the range of pH4.5-6. It is worth noting that in the process of microbial growth, reproduction and metabolism, due to the decomposition and utilization of nutrients and the formation and accumulation of metabolites, the pH of the culture medium will change. If the pH condition of the culture medium is not controlled, the growth rate of microorganisms or (and) the production of metabolites will often decrease. Therefore, in order to maintain the relatively constant pH of the culture medium, pH buffers are usually added to the culture medium. The commonly used buffers are monohydrogen and dihydrophosphate (such as KH two PO four And K two HPO four ）A mixture of components. K two HPO four The solution is alkaline, and the KH2PO4 solution is acidic. The pH of the mixed solution of the same amount of the two substances is 6.8. When the concentration of H+increases due to the accumulation of acidic substances in the culture medium, H+combines with weakly basic salts to form weakly acidic compounds, and the pH of the culture medium will not be excessively reduced; If the concentration of OH - in the culture medium increases, OH - will combine with weak acidic salt to form weak alkaline compounds, and the pH of the culture medium will not increase excessively.

But KH two PO four And K two HPO four The buffer system can only regulate within a certain pH range (6.4-7.2). Some microorganisms, such as lactic acid bacteria, can produce a large amount of acid, so the above buffer system is difficult to play a buffer role. At this time, insoluble carbonate (such as CaCO3) can be added to the culture medium for adjustment. CaCO3 is insoluble in water, which will not cause excessive increase in the pH of the culture medium. However, it can continuously neutralize the acid produced by microorganisms, release CO2, and control the pH of the culture medium within a certain range.

There are also some natural buffer systems in the culture medium, such as amino acids, peptides and proteins Amphoteric electrolyte It can also act as a buffer.

4. Control Redox potential （redox potential）

Different types of microorganisms have different requirements for redox potential (F). Generally, aerobic microorganisms can grow normally when the F value is above+0.1V, and it is better to use+0.3 -+0.4V. Anaerobic microorganisms can only grow when the F value is below+0.1V, Facultative anaerobe Aerobic respiration is carried out when F value is above+0.1V, and fermentation is carried out when F value is below+0.1V. F value is related to partial pressure of oxygen and pH, and also affected by some microbial metabolites. Under the condition of relatively stable pH, the oxygen partial pressure of the culture medium can be increased by increasing the ventilation volume (such as shaking culture, stirring), or by adding oxidants, so as to increase the F value; Add to the culture medium ascorbic acid , hydrogen sulfide Cysteine 、 glutathione , dithiothreitol and other reducing substances can reduce the F value.

5. Selection of raw material sources

When preparing the culture medium, we should try to use cheap and easily available raw materials as the ingredients of the culture medium. Especially in the fermentation industry, the amount of culture medium is large, and the use of low-cost raw materials reflects its economic value. For example, in microorganisms Single cell protein In the industrial production process of. For another example, industrial methane fermentation mainly uses wastewater and waste residue as raw materials. In rural areas of China, it has been promoted to use human and livestock manure and grass as raw materials to produce methane as fuel. In addition, a large number of agricultural and sideline products or products, such as hulls, rice bran, corn milk, yeast extract, distiller's grains, bean cakes, peanut cakes, peptone, etc., are commonly used as raw materials for fermentation industry.

6. Sterilization treatment

In order to obtain pure microbial culture, it is necessary to avoid contamination by miscellaneous bacteria. Therefore, all equipment and workplaces should be disinfected and sterilized. For the culture medium, strict sterilization is required. The culture medium is generally sterilized by high-pressure steam, and the general culture medium can be sterilized by 1.05kg/cm2121.3 ℃ for 15-30min. In the process of high-pressure steam sterilization, high temperature for a long time will damage some heat resistant substances, such as the formation of amino sugar and caramel from sugar substances. Therefore, sugar containing media are usually sterilized at 0.56kg/cm2112.6 ℃ for 15-30 minutes. For some media with high requirements for sugar, sugar can be filtered or intermittently sterilized first, and then mixed with other sterilized ingredients; High temperature for a long time will also cause phosphate, carbonate and some cations (especially calcium, magnesium, iron ions) to combine to form insoluble complexes and produce precipitation. Therefore, when preparing Synthetic medium It is often necessary to add a small amount of chelating agent in the culture medium to avoid precipitation in the culture medium. The commonly used chelating agent is EDTA （EDTA）。 The components containing calcium, magnesium and iron plasma can also be sterilized separately with phosphate and carbonate, and then mixed to avoid precipitation; After high-pressure steam sterilization, the pH of the culture medium will change (generally reducing the pH), which can be adjusted before and after the sterilization of the culture medium according to the requirements of the cultured microorganisms.

In the process of preparing the culture medium, the existence of foam is extremely unfavorable to the sterilization treatment, because the air in the foam forms a thermal insulation layer, making it difficult to kill the microorganisms in the foam. Therefore, it is sometimes necessary to add defoamer in the culture medium to reduce the generation of foam, or appropriately increase the sterilization temperature.

classification

Announce

edit

Chemical classification

1. Natural culture medium: Natural culture medium refers to a kind of culture medium made from animals, plants or micro biological objects, including their extracts. For example. Beef extract peptone medium, malt juice medium, LB medium, etc. Common natural organic nutrients include bean sprout juice, corn flour, soil extract, bran, milk, serum, coconut juice, etc. The natural culture medium has low cost and is suitable for large-scale industrial Microbial fermentation Production.

2. Combination medium Synthetic culture medium is a kind of culture medium prepared by artificial design and simulated synthesis with chemical substances according to the composition of natural culture medium. For example, Gaoshi No.1 medium And Cha's culture medium, etc. Synthetic culture medium has a certain formula, which is highly repeatable when preparing synthetic culture medium, but its cost is higher than that of natural culture medium, and the growth rate of microorganisms in it is slower. It is generally suitable for research work on microbial nutrition requirements, metabolism, classification and identification, biomass measurement, strain breeding and genetic analysis in the laboratory.^[2]

3. Semi combination medium : Refers to a class of Chemical Reagents Prepare the culture medium with some natural ingredients. For example, potatoes Sucrose medium 。

Physical classification

Solid medium

1. Liquid culture medium : 80%~90% is water, in which the medium is equipped with soluble or insoluble nutrients.

2. Solid medium : A kind of prepared matrix in solid state. According to the nature, it can be divided into solidified medium, non reversible solidified medium, natural solid medium and filter membrane.

3. Semi solid medium : refers to Liquid medium A semisolid medium prepared by adding a small amount of coagulant into the medium.

4. Dehydrated medium : also called prefabrication Dry medium It refers to commercial culture medium containing all ingredients except water.

Culture function classification

Selective medium : A kind of culture medium designed according to the special nutritional requirements of a microorganism or its resistance to certain chemical and physical factors, which has the function of turning the inferior bacteria in the mixed bacteria sample into the superior bacteria, and is widely used strain Screening and other fields.

Identification medium : One kind of ingredients added with colorless metabolites that can occur with the target bacteria Chromogenic reaction So that it is easy to identify the color with the naked eye colony of bacteria Find out the culture medium of the target bacterial colony. For example, Eosin Methylene blue lactose Culture medium (EMB).

Common culture medium

Announce

edit

Bacterial culture medium

(1) Beef paste agar culture medium

Beef paste 0.3g, 1.0g peptone, sodium chloride 0.5g, agar 1.5g, water 100ml

Add 100ml of water into the beaker, add beef extract, peptone and sodium chloride, mark the beaker with a crayon, and heat it on the fire. After each component in the beaker is dissolved, add agar and keep stirring to avoid sticking to the bottom. Make up the water loss after the agar is completely dissolved, and use 10% hydrochloric acid or 10% sodium hydroxide Adjust the pH value to 7.2-7.6, sub pack in each test tube, add cotton plugs, and use High pressure steam sterilization : 1.05 kg/cm2, maintained at 121 ℃ for 15-30 minutes.

(2) Bovine heart culture medium

Bacterial culture medium

Take 250g fresh beef heart (excluding fat and blood vessels), finely chop it into minced meat with a knife, and then add 500ml distilled water And 5 grams of peptone. Mark the beaker, boil it, and then simmer it for 2 hours. Filter, dry the filtered meat powder, and adjust the pH value of the filtrate to about 7.5. Add 10ml broth and a small amount of crushed dried beef heart into each test tube, sterilize and reserve.

(3) Rhizobium culture medium

Glucose 10g, Dipotassium hydrogen phosphate 0.5g， calcium carbonate 3g， magnesium sulphate 0.2g, yeast powder 0.4, agar 20g, water 1000ml, 1% crystal violet Solution 1ml.

First boil the agar with water to dissolve it, then add other components separately, stir to dissolve it, then repack, sterilize and reserve.

Actinomycete culture medium

(1) Starch Nitrate medium (Gaoshi No.1 medium)

Soluble starch 2.0g， potassium nitrate 0.1g, dipotassium phosphate 0.05g, sodium chloride 0.05g, magnesium sulfate 0.05g, ferrous sulfate 0.001g, 2g agar, 100ml water.

Put the starch in a beaker, mix it into a paste with 5ml of water, pour 95ml of water, mix it well, and then 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, sterilize after subpackaging, and reserve.

(2) Flour agar medium

60g flour, 20g agar, 1000ml water

Mix the flour into a paste with water, add water to 500ml, and boil it for 30 minutes over a mild fire. Take another 500ml of water, put it into agar, heat and boil it until it is dissolved, mix the two solutions evenly, add water, adjust the pH value to 7.4, repackage, sterilize and standby.

Microalgae culture medium

BG-11 medium

NaNO3 1.5g, K2HPO4 · 3H2O0.04g, MgSO4 · 7H2O0.075g, CaCl2 · 2H2O0.036g Citric Acid 0.006g, Ferric ammonium chloride（ Ammonium ferric citrate 0.006g, EDTA (dinatrium salt) 0.001g, Na2CO3 0.02g, A5+Co solution * (A5 solution 1000 ×) 1ml Distilled Water 919ml

SE Medium

NaNO3	0.25g
K2HPO4 . 3H2O	0.075g
MgSO4 . 7H2O	0.075g
CaCl2 . 2H2O	0.025g
KH2PO4	0.175g
NaCl	zero point zero two five
Soil extract	40ml
FeCl3·6H2O	zero point zero zero five
Fe—EDTA	1ml
A5 solution 1000×	1ml
distilled water	958ml

Preparation method of Soil Extract

Take 0.5kg of unfertilized garden soil and put it into a beaker or flask, add 1000ml of distilled water, seal the mouth of the flask with a vent plug, heat it with boiling water in a water bath for 2h, cool it for several hours, filter it under sterile conditions, take the supernatant, and add the sterilized distilled water into the supernatant to a total volume of 1000ml. The soil extract is stored at 4 ℃ for standby.

Composition of the A5 solution

Add to 100 ml of distilled water: (add 100ml of distilled water)

H3BO3	286 mg
MnCl2 . 4H2O	181 mg
ZnSO4 . 7H2O	22 mg
CuSO4 . 5 H2O	7.9 mg
（NH4）6Mo7O24 .4H2O	3.9 mg

Configuration method of EDTA Fe:

Dissolve EDTA and FeCl3 · 6H2O in water and HCl (0.1N) respectively, and mix well.

Na2EDTA	1g
distilled water	50ml
FeCl3·6H2O	81 mg
HCl（0.1N）	50ml

Pr Medium

Preparation: to 997 mL of glass-distilled water, add 1.0 g proteose peptone, 15.0 g agar, and the following stock solutions:

working solution	g/1000 mL H2O
NaNO3	zero point two five
CaCl2. 2H2O	zero point zero two five
MgSO4. 7H2O	zero point zero seven five
K2HPO4	zero point zero seven five
KH2PO4	zero point one seven five
NaCl	zero point zero two five
A5 solution	1ml
EDTA-Fe	1ml
FeCl3 (0.05%)	1ml

The preparation method of A5 solution and EDTA Fe solution is the same as above.

Fungal culture medium

(1) Sabouraud's culture medium

Peptone 10g, agar 20g, malt dust 40g, water 1000ml

First, add water to peptone and agar, heat them, and stir them continuously. After the agar is dissolved, add 40g maltose (or glucose), stir them to dissolve them, and then repackage them, sterilize them, and reserve them.

This culture is commonly used to cultivate many kinds of fungi.

(2) Potato sugar agar medium

Wash and peel the potatoes, cut 200g into small pieces, add 1000ml of water, boil for half an hour, and replenish the water. Add 10g agar to the filtrate, add 20g sugar after boiling and dissolving (add sucrose for cultivating mold, for cultivating Yeast Add glucose), replenish water, repackage, sterilize, and reserve.

Adjust the pH value of this medium to 7.2-7.4, and the sugar in the formula, such as glucose, can also be used for culture Actinomycetes and Bacillus 。

(3) Bean sprout juice culture medium

Soybean sprout 100g, agar 15g, glucose 20g, water 1000ml

Wash the bean sprouts, add water and boil for 30 minutes. Filter with gauze, add agar to the filtrate, heat and dissolve it, add sugar, stir to dissolve it, replenish water to 1000ml, repackage, sterilize and reserve.

Adjust the pH value of this medium to 7.2-7.4, which can be used to culture bacteria and actinomycetes.

(4) Pea agar medium

80 peas, 5g agar, 200ml water

agar

Take 80 dried peas, add water, boil for 1 hour, filter with gauze, add agar to the filtrate, boil until dissolved, repackage, sterilize, and standby.

Edible fungi culture medium

(1) Potato sucrose agar medium

20% boiled potato juice 1000ml, sucrose 20g, agar 18g

Wash and peel the potatoes and cut them into small pieces. Weigh 200g of potato chips, add 1000ml of water, boil for 20 minutes, and filter. Add enough water to 1000ml in the filtered juice to make 20% boiled potato juice. Add agar and sucrose into the boiled potato juice, boil it, make it dissolve, replenish water, repackage, sterilize and reserve. The pH value is not strictly required when using this medium, so the determination is unnecessary.

(2) Comprehensive Potato culture medium

20% boiled potato juice 1000ml, Potassium dihydrogen phosphate 3g, 1.5g magnesium sulfate, 20g glucose, 10mg vitamin, 18g agar

First prepare 20% potato boiling juice in the same way as above. Add the above components into the boiling juice, heat and dissolve them to supplement the water, and adjust the pH value to 6. Sub packaging, sterilization and standby. This medium is used for culture and preservation Ganoderma lucidum 、 Oyster mushroom , mushroom, etc Edible fungi 。

Tobacco culture medium

stay plant tissue culture By adjusting the ratio of IAA and CTK Callus Differentiate into roots or buds. When CTK/IAA was high, the callus differentiated into buds.

When CTK/IAA was low, the root differentiated; The moderate ratio of CTK/IAA kept the callus undifferentiated.

Preparation of callus induction medium

In MS medium mother liquor On the basis of, add in order to the clean aluminum pot A large number of elements 20 × mother liquor 100mL, trace element 100 × mother liquor 20mL, iron salt 100 × mother liquor 20mL, vitamin 100 × mother liquor 20mL, inositol 200 × mother liquor 10mL, glycine 200 × 10 mL of mother liquid, after preparing MS culture medium, add 0.5 mg · L-1 BA8 mL and 0.5 mg · L-1 NAA8 mL. Then add distilled water with the volume of about 2/3-3/4 of the actually prepared medium, add 40g sucrose, stir to dissolve it, and adjust the pH value to 5.8-6.0 with 0.5mol · L-1 NaOH and 0.5 mol · L-1 HCl. Add 14g agar, put the aluminum pan on the electric furnace, stir and heat to make the agar completely dissolved, then use distilled water to fix the volume to 2L, continue heating for several minutes to make it mixed evenly, and then sub pack it into a flask.

tobacco

Aseptic induction of tobacco leaf callus Petri dish Use a scalpel to cut 1-2 sterile seedling leaves into a sterile culture dish, and use a scalpel to cut the leaves into small pieces of about 2mm2, and then inoculate them on the prepared culture medium. Each bottle is inoculated with 5 small pieces, and a total of 6 bottles are inoculated. The inoculated Sanjianping was cultured under 24 conditions in darkness for one week, and then cultured under the same temperature with light and full darkness for three weeks until callus formation (3 bottles in each case). The results of callus induction were observed and the callus induction rate was counted.

Organ differentiation and plant regeneration culture

The induced calli were transferred into differentiation medium according to their types, and cultured for 3 weeks under the condition of continuous light and temperature of 20-22 ℃, and the regenerated plants from the calli were counted.

General situation of callus induction

After 4 weeks of induction and culture of tobacco callus, the callus was basically formed, that is to say, the situation that callus was not formed due to insufficient growth time was excluded. See Table 1 for details. All the six culture bottles had callus formation and no pollution, but the induction rates were almost different. Among them, the average callus induction rate of three bottles cultured under light conditions was 60.0%, and the average callus induction rate of three bottles cultured under dark conditions was 46.7%. Because during the experiment, Explant That is to say, the tobacco leaves obtained are too small, and most of the cells of some explants may have dehydrated and died during inoculation, so that the callus induction rate of the whole experiment is low, and the callus is too small.

Animal cell culture medium

RPMI1640 medium is a full nutrient medium, widely used in Mammalian cell and Hybridoma cell Training, such as people Myeloma cells , mouse hybridoma cell, human white blood cell as well as B cell and T cell 。 Originally designed for suspension cell culture and human leukemia cells Monolayer cell culture 。

Special medium

Announce

edit

Selective medium

Selective culture medium refers to the culture medium designed according to the special nutritional requirements of certain microorganisms or their resistance to certain chemical and physical factors. Its function is to make the inferior bacteria in the mixed bacteria sample become the superior bacteria, so as to improve the screening efficiency of the bacteria.

Yeast Enrichment medium

Glucose 5%, urea 0.1%, Ammonium sulfide 0.1%， Potassium dihydrogen phosphate 0.25%， Disodium hydrogen phosphate 0.05%, seven Hydrated magnesium sulfate 0.1%, heptahydrate ferric sulfate 0.01%, yeast extract 0.05%, Bengal red 0.003%，pH4.5

Ashby nitrogen free medium

Enriched aerobic autogenesis Azotobacter

Mannitol 1%, Potassium dihydrogen phosphate 0.02%, seven Hydrated magnesium sulfate 0.02%, sodium chloride 0.02%, Calcium sulfate dihydrate 0.01%, calcium carbonate 0.5%

Identification medium

Announce

edit

EMB medium

Commonly used for identification E.coli

Peptone 10g, lactose 5g, sucrose 5g, dipotassium phosphate 2g, eosin Y0.4g, methylene blue 0.065g, distilled water 1000g, pH7.2

2216E medium formula

separate Marine microorganism Culture medium formula of

Peptone 5g, yeast extract 1g, ferric phosphate 0.01g, agar 15-20g, Chen Haihai 1000ml, boiled sodium hydroxide Adjust the pH of (5%) solution to 7.6-7.8

eosin methylene blue medium

Can be used to detect water Escherichia coli Content of

First prepare according to the following composition Basic culture medium 。

Peptone 10g, NaCl5g

Agar 15-20g

After dissolving the above substances, use distilled water to fix the volume to 1000mL, and adjust the pH to 7.6. After sterilization, cool it to about 60 ℃, and then add sterilized lactose solution, eosin solution and methylene blue solution in the following proportion under aseptic operation conditions. After shaking well, pour the plate immediately. The lactose in the solution will be destroyed under high temperature, so the pressure of 70kPa and the temperature of 115 ℃ are generally used for sterilization for 20 minutes.

Basic culture medium 100mL, 20% lactose solution 2mL

2% eosin solution 2mL, 0.5% methylene blue solution 1mL

Natural culture medium

Announce

edit

Natural culture medium refers to the automatic liquid or Tissue segregation A kind of culture medium extracted, such as plasma, serum, lymph, chicken embryo extract, etc. Tissue culture technology Early establishment, in vitro culture Cells are used Natural culture medium 。 However, due to the complex production process of natural culture medium and large differences between batches Synthetic medium Is replaced. The widely used natural culture medium is serum. In addition, various tissue extracts and collagen substances that promote cell adhesion are also essential for cultivating some special cells.

Serum type

The serum used for tissue culture is mainly bovine serum, and some special cells are also cultured with human serum, horse serum, etc. The reasons for choosing to use bovine serum to culture cells are that the source is sufficient, the preparation technology is mature, and people have a deep understanding of it after a long application test. Bovine serum for most Mammalian cell All are suitable, but it is not ruled out that it is more suitable to use other animal serum when cultivating certain cells.

Bovine serum is the most used in cell culture Natural culture medium , rich in Cell growth Essential nutrients have extremely important functions. Bovine serum is divided into calf serum, new bovine serum fetal bovine serum 。 The fetal bovine serum should be taken from the fetal bovine delivered by caesarean section; New bovine serum is taken from newborn cattle within 24 hours of birth; Calf serum is taken from calves born 10-30 days. Obviously, the fetal bovine serum is of the highest quality, because the fetal bovine serum has not been exposed to the outside world, and the serum contains the least components harmful to cells, such as antibodies and complements.

Serum samples

The main component of serum serum is removed by plasma fibrin Although most of the components of the complex mixture are known, some are still unknown, and the serum composition and content often vary with the sex, age, physiological conditions and nutritional conditions of the blood supplying animals. Serum contains various Plasma protein 、 polypeptide , fat carbohydrate 、 growth factor , hormones, inorganic substances, etc. These substances achieve physiological balance in promoting cell growth or inhibiting growth activity.

Main functions of serum

1. Provision Essential nutrients ： amino acid , vitamins Inorganic Lipids, nucleic acid derivatives, etc. are necessary for cell growth.

2. Provide hormones and various growth factors: insulin Adrenocortical hormone (hydrocortisone, dexamethasone) Steroid hormone (estradiol, testosterone, progesterone), etc. Growth factors such as Fibroblast growth factor 、 epidermal growth factor , platelet growth factor, etc.

3. Provision Binding protein : Binding protein function is to carry important low molecular weight substances, such as albumin Carry vitamins, fats, hormones, etc, transferrin Carry iron. Binding proteins play an important role in cell metabolism.

4. Provide contact promoting and stretching factors to prevent cell adhesion from mechanical damage.

5. It can protect the cells in culture: some cells, such as endothelial cells Bone marrow like cells can release protease, and serum contains anti protease components, playing a neutralizing role. If this effect is discovered accidentally, the serum will be used purposefully to terminate it Trypsin The digestive function of. Because trypsin has been widely used Adherent cell Digestion and passage. serum albumin It forms the viscosity of serum, which can protect cells from mechanical damage, especially in suspension culture Viscosity plays an important role in mixing. Serum also contains some trace elements and ions, which play an important role in metabolic detoxification, such as SeO3, selenium, etc.

Main problems of serum culture medium

1. There may be hundreds of components in serum, and the exact composition, content and mechanism of action are not clear, especially some polypeptide growth factors, hormones and lipids, which have not yet been fully understood, which brings many difficulties to the research work.

2. Serum is produced in batches, and there is a great difference between batches, and the retention 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 liquid they contact, but only in the process of injury healing and blood coagulation. Therefore, the use of serum may change the normal state of some cells in vivo, and serum may promote the growth of some cells（ Fibroblast ）Simultaneously inhibit the growth of another type of cells（ Epidermal cell ）。

4. Serum contains some substances that are toxic to cells, such as Polyamine Oxidase, which can react with polyamines from highly reproductive cells (such as Spermine 、 Spermidine ）Formed with Cytotoxicity Active spermidine. Complement, antibody Bacterial toxin It will affect cell growth and even cause cell death.

5. The individual animals are different, the origin and batch number of serum are different, the quality of each batch is very different, and the composition cannot be kept consistent.

6. May be brought in during material collection mycoplasma , virus, which has potential impact on cells, may lead to experimental failure or unreliable experimental results.

7. In large-scale production, the source of serum is becoming more and more difficult and expensive Animal cell culture One of the major components of production costs.

Quality standard of serum

The quality of serum depends on two factors: one is the object of sampling, the other is the process of sampling. The animals used for sampling shall be healthy, disease-free and within the specified birth days. The sampling process shall be strictly in accordance with the operating procedures, and the serum prepared shall be subject to strict quality identification. Production by Animal Cells in Vitro Biological products Requirements in the Procedure:

1. The bovine serum must be from a documented source Bovine spongiform encephalopathy Of cattle or countries. It shall also have an appropriate monitoring system.

2. Some countries also require cattle serum from cattle that have not used ruminant protein feed.

3. It is proved that the bovine serum used does not contain the inhibitor of the vaccine virus produced.

4. The serum should be filtered and sterilized through the filter membrane to ensure sterility.

5. No bacteria, mold, mycoplasma and virus pollution. Some countries require no Bacteriophage Pollution.

6. It has a good role in supporting cell reproduction.

China has put forward strict standard requirements for the quality of bovine serum in the Quality Control Standard for Main Raw and Auxiliary Materials of Biological Products in China (2000). Including protein content, bacteria, fungi, mycoplasma, bovine virus Escherichia coli bacteriophage , Bacteria endotoxin , support cell proliferation Check.

media preparation

Announce

edit

1、 Preparation water

Most of the medium is water, so the quality of water directly affects the quality of the medium. According to Waymouth standard, the resistance of water should be 2 million ohms, which is usually prepared in the laboratory with a glass distiller

Double distilled water or Triple distilled water It can meet the use conditions.

2、 Culture medium

There are natural and artificial culture media. Generally, RPMI-1640 powder culture medium is used in the laboratory, which is prepared with double distilled or triple distilled water. NaHCO three (or HCl) adjust the pH value to 7.2-7.4. If the pH value exceeds 7.6 or is far below 6.8, most cells cannot grow. RPMI-1640 is not resistant to high pressure sterilization, so it needs to be sterilized by 0.22 μ m pore size filter membrane before use.

3、 Serum

Calf serum (or fetal bovine serum) is often used in culture. The serum cannot be autoclaved either. The sterile calf serum needs to inactivate the complement in a 56 ℃ water bath for 30 minutes, and store it in a 4 ℃ refrigerator for future use. The content in preparation depends on the type and time of cultured cells, generally 10-15%. Because the serum composition is complex and the conditions are difficult to control, it can be selected Serum free medium 。

4、 Antibiotics

In order to prevent bacterial contamination during the culture period, appropriate antibiotics can be added to the culture medium cell culture Same: Kanamycin 100 units/ml, or double antibody penicillin 100 units/ml, streptomycin 100 μ g/ml.

V Phytohemagglutinin （PHA）

Cells in non proliferative phase cannot prepare chromosomes, such as human peripheral blood lymphocytes, but during in vitro culture, they can be stimulated to transform into Lymphoblast And into mitosis.

The peak of division was measured at 44-48 hours and 68-72 hours after culture, respectively.

PHA consists of mucopolysaccharide and protein. Mucopolysaccharide Promotes mitosis and protein initiation Agglutination 。 The number of PHA activated cells increases with its concentration until all Immunocompetent cell They are activated, but if the concentration of PHA is too high, it will cause agglutination. Generally, 4% concentration is preferred.

Basic preparation method

Announce

edit

Selection of culture medium formula

The formula of the same culture medium often has some differences in different works. Therefore, in addition to the standard method used, which shall be prepared in strict accordance with its provisions, relevant data shall be collected as far as possible, compared and checked, and then selected according to their own use purpose, and their sources shall be recorded.

Preparation record of culture medium

Each preparation of culture medium shall be recorded, including the name of culture medium, formula and its source, and the brand of various ingredients. The final pH value, disinfection temperature and time, the preparation date and the preparer, etc., should be copied. The original record should be kept for future reference. The copied record should be stored together with the prepared culture medium to prevent confusion.

Weighing of culture medium components

The various components of the culture medium must be accurately weighed and attention should be paid to prevent confusion. It is best to complete it at one time without interruption. The formula can be placed on the side. Mark the formula after each ingredient is weighed, and take the medicine to be weighed at one time and place it on the left side. After each ingredient is weighed, move it on the right side. After complete weighing, another inspection shall be carried out.

Mixing and melting of various components of culture medium

All chemicals used in the culture medium shall be chemically pure. The cooking pan used shall not be copper or iron, so as to prevent trace copper or iron from mixing into the culture medium, making it difficult for bacteria to grow. Best use Stainless steel pot Heat and melt, or put it into a large beaker or flask High pressure steam sterilizer Or boiling and dissolving in a flowing steam sterilizer. When melting in the pot, it can be heated with warm water and disturbed at any time to prevent coking. If coking is found, the medium cannot be used and should be prepared again. After most of the solid components are dissolved, use small firepower to completely dissolve all the components. Till it boils down. If it is an agar culture medium, first use some water to dissolve the agar, and then use another part of water to dissolve other ingredients, and then fully mix the two solutions. In the process of heating and melting, the water lost due to evaporation must be supplemented finally.

Preliminary adjustment of medium pH

After all components of the culture medium are completely dissolved, the pH should be initially adjusted. Because the pH of the culture medium will change during the heating and disinfection process, for example, the beef extract can reduce the pH by about 0.2. However, the pH of intestinal extract will increase significantly. Therefore, for this step, the operator should always pay attention to the exploration experience, so as to grasp the final pH of the culture medium and ensure the quality of the culture medium. After the pH is adjusted, the culture medium should be boiled for several minutes to facilitate the precipitation of the precipitate of the culture medium.

matters needing attention

Announce

edit

Before use, the culture medium is subjected to high pressure or Filter sterilization 。 The following precautions shall be taken to prevent pollution:

Confirm whether the working cell bank is polluted, confirm whether the working seed batch of virus seed is polluted, and confirm the culture medium used and its added ingredients (such as calf serum, NaHCO three Whether it is polluted or not can be confirmed and eliminated by bacteria, fungi and mycoplasma sterility test. At the same time, the sensitivity of the sterile test medium should be verified. In actual production, a small amount of nutrient agar can be added from the prepared culture medium to Constant temperature incubator It can be cultured within 48 hours to observe whether there is pollution.

Others: High pressure sterilization equipment and filtration sterilization equipment shall be verified to ensure sterilization and sterilization effect; The former shall be verified before production and every 6 months thereafter, and the latter shall be verified before and after each sterilization (at least once after sterilization).

In addition, the toxic area should be strictly separated from the non-toxic area, and have their own independent air purification system and incubation room. The toxic area should maintain a relative negative pressure against the non-toxic area to prevent the virus from polluting the cultured cells (especially the cell bank).^[3]

Novice on the road

Growth task Getting Started with Editing Edit Rule Edit by myself

I have questions

Content query Online Service Official post bar Feedback

Complaints and suggestions

Report bad information Failed to appeal through entries Complaint of infringement information Blocking query and unblocking

Jinggong Network Anbei No. 1100000200001