CN113684152A - Bacteria preservation culture medium and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of strain preservation, in particular to a bacterium preservation culture medium and a preparation method thereof. The culture medium comprises the following components: contains, in 1000 ml: 8-13g of beef extract, 2-6g of peptone, 0.2-0.5g of bile salt, 0.2-0.3g of aluminum potassium sulfate, 2-5g of malt extract powder, 3-7g of yeast extract powder, 0.3-0.6g of sodium chloride, 5-10g of agar, 0.2-0.5g of soluble iron salt, 0.01-0.05g of calcium supplement, 0.02-0.05g of D-cycloserine, 0.001-0.004g of maozolone, 10.01-0.02g of vitamin K, 0.1-0.3g of complex protein phosphopeptide, 3-5g of glucose, 1-2.5g of lanthanum nitrate, 3-5g of erythritol, 18-23ml of glycerol, 0.1-0.2mg of warfarin sodium tablet, 8-12mg of isoniazide and 75-85ml of aseptic defibrinated sheep blood. The nutrient substances in the formula are rich and comprehensive, the survival of the strains can be ensured, and even if the preservation environment temperature is greatly changed, the culture medium can still ensure the effectiveness of the strains.

Description

Bacteria preservation culture medium and preparation method thereof

Technical Field

The invention relates to the technical field of strain preservation, in particular to a bacterium preservation culture medium and a preparation method thereof.

Background

Bacteria are one of the major biological resources and are frequently used in medical experiments. After a good strain is selected and bred, the good property of the strain must be kept unchanged or slowly changed as little as possible, so that the performance of the strain is not reduced, and the strain can be applied in production for a long time. The strain preservation method has a plurality of methods, the cryopreservation is an effective way for solving the germplasm degradation and preventing the naturally accumulated mutation, but the traditional cryopreservation needs expensive program cooling instruments and has complicated steps. Complete vitrification is a new method for low-temperature preservation, namely, under the condition of high-concentration protective agent, the cells and the protective agent are all in a vitrification state in the process of rapid cooling, the formation of ice crystals in cells is avoided, and all parts of organs and tissues are all in the same state.

In the prior art, the use amount of agar in the bacteria preservation culture medium is large, the restriction effect on strains is poor, the activity of the strains is reduced after preservation, and the long-term preservation of the strains is not facilitated.

Disclosure of Invention

Based on the above problems, the present invention provides a bacteria preservation medium and a method for preparing the same, which can improve the activity in the preservation of strains. The specific technical scheme is as follows:

a bacteria preservation medium comprises the following components:

contains, in 1000 ml: 8-13g of beef extract, 2-6g of peptone, 0.2-0.5g of bile salt, 0.2-0.3g of aluminum potassium sulfate, 2-5g of malt extract powder, 3-7g of yeast extract powder, 0.3-0.6g of sodium chloride, 5-10g of agar, 0.2-0.5g of soluble iron salt, 0.01-0.05g of calcium supplement, 0.02-0.05g of D-cycloserine, 0.001-0.004g of maozolone, 10.01-0.02g of vitamin K, 0.1-0.3g of complex protein phosphopeptide, 3-5g of glucose, 1-2.5g of lanthanum nitrate, 3-5g of erythritol, 18-23ml of glycerol, 0.1-0.2mg of warfarin sodium tablet, 8-12mg of isoniazide and 75-85ml of aseptic defibrinated sheep blood.

Preferably, the bacteria preservation medium comprises the following components:

in 1000 ml: 10g of beef extract, 5g of peptone, 0.3g of bile salt, 0.25g of potassium aluminum sulfate, 4g of malt extract powder, 5g of yeast extract powder, 0.5g of sodium chloride, 8g of agar, 0.3g of ferric citrate, 0.03g of calcium lactate, 0.03g of D-cycloserine, 0.003g of maozolone, 10.01g of vitamin K, 0.1g of complexing protein phosphopeptide, 5g of glucose, 2g of lanthanum nitrate, 4g of erythritol, 20ml of glycerol, 0.1mg of warfarin sodium tablets, 10mg of isoniazide, 80ml of sterile defibrinated sheep blood, and distilled water added to 1000 ml.

Specifically, the soluble ferric salt is ferric ammonium citrate.

Specifically, the calcium supplement is calcium lactate.

The preparation method of the bacteria preservation culture medium comprises the following steps:

(1) according to the raw material proportion, taking beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride, agar, soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazid and sterile defibrinated sheep blood for later use;

(2) uniformly mixing beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride and agar, pouring soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazide and sterile defibrinated sheep blood, adding distilled water to 1000ml, and heating to 50-60 ℃ to dissolve all substances;

(3) sterilizing the above solutions, and subpackaging in culture vessels for cooling.

Specifically, the sterilization condition is that the temperature is 130-150 ℃ and the time is 3-5 h.

Specifically, the culture vessel is any one of a test tube or a culture vessel.

Specifically, the refrigeration temperature is 2-4 ℃.

Compared with the prior art, the invention has the beneficial effects that:

the formula of the invention contains peptone, bile salt, potassium aluminum sulfate, malt extract powder, yeast extract powder, sodium chloride, agar, ferric ammonia, calcium lactate, D-cycloserine, maozolone, vitamins, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol and distilled water, and the nutrient substances are rich and comprehensive. The contained components of ferric citrate, calcium lactate, complex protein phosphopeptide, glucose, vitamin, lanthanum nitrate and the like can ensure the survival of strains. Wherein, the bile salt, D-cycloserine, maozolone and other substances have the function of inhibiting the growth of bacteria and are beneficial to the long-term survival of the bacteria. The warfarin sodium tablet and isoniazid can reduce the metabolism of bacteria and inhibit the growth of bacteria. Erythritol, glycerol and other substances have the moisturizing effect and meet the requirement of sufficient water content of the culture medium. The agar powder has low content, is semisolid in culture medium, and is beneficial for bacteria preservation. When the culture medium is used for preserving bacteria, the growth period of the bacteria is slow, mixed bacteria can not grow, the activity of the bacteria can be kept and the bacteria can not die easily, and even if the preservation environment is changed greatly, the culture medium can still ensure the effectiveness of the strains. The culture medium has simple preparation equipment, low cost and high practical value.

Detailed Description

Example 1:

a bacteria preservation medium comprises the following components:

contains, in 1000 ml: 8g of beef extract, 2g of peptone, 0.2g of bile salt, 0.2g of potassium aluminum sulfate, 2g of malt extract powder, 3g of yeast extract powder, 0.3g of sodium chloride, 5g of agar, 0.2g of soluble ferric salt, 0.01g of calcium supplement, 0.02g of D-cycloserine, 0.001g of maozolone, 10.01g of vitamin K, 0.1g of complex protein phosphopeptide, 3g of glucose, 1g of lanthanum nitrate, 3g of erythritol, 18ml of glycerol, 0.1mg of warfarin sodium tablets, 8mg of isoniazide and 75ml of sterile defibrinated sheep blood; the soluble ferric salt is ferric ammonium citrate; the calcium supplement is calcium lactate.

The bacteria preservation medium described in this example was prepared as follows:

(1) according to the raw material proportion, taking beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride, agar, soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazid and sterile defibrinated sheep blood for later use;

(2) uniformly mixing beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride and agar, pouring soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazide and sterile defibrinated sheep blood, adding distilled water to 1000ml, and heating to 50 ℃ to dissolve all substances;

(3) sterilizing the solution, and subpackaging in culture vessels for cooling for later use; the sterilization condition is that the temperature is 130 ℃ and the time is 3 h; the culture vessel is a test tube; the refrigeration temperature is 2 ℃.

Example 2

A bacteria preservation medium comprises the following components:

in 1000 ml: 10g of beef extract, 5g of peptone, 0.3g of bile salt, 0.25g of potassium aluminum sulfate, 4g of malt extract powder, 5g of yeast extract powder, 0.5g of sodium chloride, 8g of agar, 0.3g of ferric citrate, 0.03g of calcium lactate, 0.03g of D-cycloserine, 0.003g of maozolone, 10.01g of vitamin K, 0.1g of complexing protein phosphopeptide, 5g of glucose, 2g of lanthanum nitrate, 4g of erythritol, 20ml of glycerol, 0.1mg of warfarin sodium tablets, 10mg of isoniazide, 80ml of sterile defibrinated sheep blood, and distilled water added to 1000 ml.

The bacteria preservation medium described in this example was prepared as follows:

(1) according to the raw material proportion, taking beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride, agar, soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazid and sterile defibrinated sheep blood for later use;

(2) uniformly mixing beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride and agar, pouring soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazide and sterile defibrinated sheep blood, adding distilled water to 1000ml, and heating to 55 ℃ to dissolve all substances;

(3) sterilizing the solution, and subpackaging in culture vessels for cooling for later use; the sterilization condition is that the temperature is 140 ℃ and the time is 4 hours; the culture vessel is a test tube; the refrigeration temperature is 3 ℃.

Example 3

A bacteria preservation medium comprises the following components:

contains, in 1000 ml: 13g of beef extract, 6g of peptone, 0.5g of bile salt, 0.3g of aluminum potassium sulfate, 5g of malt extract powder, 7g of yeast extract powder, 0.6g of sodium chloride, 10g of agar, 0.5g of soluble ferric salt, 0.05g of calcium supplement, 0.05g of D-cycloserine, 0.004g of maozolone, 10.02g of vitamin, 0.3g of complexing protein phosphopeptide, 5g of glucose, 2.5g of lanthanum nitrate, 5g of erythritol, 23ml of glycerol, 0.2mg of warfarin sodium tablets, 12mg of isoniazide and 85ml of sterile defibrinated sheep blood; the soluble ferric salt is ferric ammonium citrate; the calcium supplement is calcium lactate.

The bacteria preservation medium described in this example was prepared as follows:

(1) according to the raw material proportion, taking beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride, agar, soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazid and sterile defibrinated sheep blood for later use;

(2) uniformly mixing beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride and agar, pouring soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazide and sterile defibrinated sheep blood, adding distilled water to 1000ml, and heating to 60 ℃ to dissolve all substances;

(3) sterilizing the solution, and subpackaging in culture vessels for cooling for later use; the sterilization condition is that the temperature is 150 ℃ and the time is 5 h; the culture vessel is a culture vessel; the refrigeration temperature was 4 ℃.

To verify the inventive scheme, the following comparative examples were set up:

comparative example 1	The difference from example 1 is that maozolone is not used in the manufacturing raw material;
		comparative example 2	The difference from the embodiment 1 is that the ferric ammonium citrate is not used in the preparation raw material;
comparative example 3	The difference from example 1 is that lanthanum nitrate is not used in the preparation raw material;
		comparative example 4	The difference from example 1 is that D-cycloserine was not used in the starting material;
comparative example 5	The difference from example 1 is that isoniazid was not used as a starting material.

Test examples

Respectively preparing culture media according to examples 1-3 and comparative examples 1-5, pouring the culture media into culture dishes in an aseptic environment, adding 2ml of bacterial liquid into each culture dish, and storing in a refrigerator at minus 50 ℃; the test strains are respectively staphylococcus aureus, escherichia coli and proteus, the culture dish inoculated by each bacterium is independently preserved, and the recovery is carried out after 5 months and 10 months of preservation, and the activity of the bacteria is counted.

When the strains are revived, hot water with the temperature of 35 ℃ is selected for heating and unfreezing in water bath, the most suitable culture medium of each strain is selected for culture, and the revival quantity is counted.

The test results are shown in the following table.

As can be seen from the table, the bacteria preservation effect of the bacteria preservation medium of the embodiment 1-3 is excellent, the preservation survival rate of the staphylococcus aureus, the escherichia coli and the proteus can reach 100% within five months, the preservation time is prolonged to 1 month, the survival rate of the staphylococcus aureus can reach more than 98.94%, the survival rate of the escherichia coli can reach more than 98.41%, and the survival rate of the proteus can reach more than 98.31%, so that the remarkable effect of the bacteria preservation effect of the medium is achieved.

The above refrigeration operation of the bacteria was repeated in a refrigerator at a refrigeration temperature of-30 ℃ to count the survival rates of the bacteria after 5 months and 10 months of refrigeration.

The test results are shown in the following table.

As can be seen from the table, when the bacteria were preserved using the culture media of examples 1 to 3 of the present invention, the survival rate of Staphylococcus aureus was more than 99.50%, the survival rate of Escherichia coli was more than 99.70%, and the survival rate of Proteus bacteria was more than 99.65% in the 5-month preservation; the survival rate of staphylococcus aureus exceeds 96.50 percent, the survival rate of escherichia coli exceeds 96.81 percent and the survival rate of proteus bacteria exceeds 96.62 percent in the preservation of 10 months. The culture medium prepared by the method can ensure the activity of the strains even under the condition that the strain preservation temperature is increased, and if the preservation equipment has problems in actual use and causes great change of the environment, the method can still ensure the effectiveness of the strains, and the practical value is obvious.

Claims (8)

1. A bacteria preservation medium is characterized by comprising the following components:

contains, in 1000 ml: 8-13g of beef extract, 2-6g of peptone, 0.2-0.5g of bile salt, 0.2-0.3g of aluminum potassium sulfate, 2-5g of malt extract powder, 3-7g of yeast extract powder, 0.3-0.6g of sodium chloride, 5-10g of agar, 0.2-0.5g of soluble iron salt, 0.01-0.05g of calcium supplement, 0.02-0.05g of D-cycloserine, 0.001-0.004g of maozolone, 10.01-0.02g of vitamin K, 0.1-0.3g of complex protein phosphopeptide, 3-5g of glucose, 1-2.5g of lanthanum nitrate, 3-5g of erythritol, 18-23ml of glycerol, 0.1-0.2mg of warfarin sodium tablet, 8-12mg of isoniazide and 75-85ml of aseptic defibrinated sheep blood.

2. A bacterial preservation medium according to claim 1, comprising the following components:

in 1000 ml: 10g of beef extract, 5g of peptone, 0.3g of bile salt, 0.25g of potassium aluminum sulfate, 4g of malt extract powder, 5g of yeast extract powder, 0.5g of sodium chloride, 8g of agar, 0.3g of ferric citrate, 0.03g of calcium lactate, 0.03g of D-cycloserine, 0.003g of maozolone, 10.01g of vitamin K, 0.1g of complexing protein phosphopeptide, 5g of glucose, 2g of lanthanum nitrate, 4g of erythritol, 20ml of glycerol, 0.1mg of warfarin sodium tablets, 10mg of isoniazide, 80ml of sterile defibrinated sheep blood, and distilled water added to 1000 ml.

3. The bacteria preservation medium of claim 1, wherein the soluble iron salt is ferric ammonium citrate.

4. A preservation medium according to claim 1, wherein said calcium supplement is calcium lactate.

5. A preservation medium for bacteria according to claim 1, prepared by the following method:

(1) according to the raw material proportion, taking beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride, agar, soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazid and sterile defibrinated sheep blood for later use;

(2) uniformly mixing beef extract, peptone, bile salt, aluminum potassium sulfate, malt extract powder, yeast extract powder, sodium chloride and agar, pouring soluble iron salt, a calcium supplement, D-cycloserine, maozolone, vitamin K1, complex protein phosphopeptide, glucose, lanthanum nitrate, erythritol, glycerol, warfarin sodium tablets, isoniazide and sterile defibrinated sheep blood, adding distilled water to 1000ml, and heating to 50-60 ℃ to dissolve all substances;

(3) sterilizing the above solutions, and subpackaging in culture vessels for cooling.

6. The bacteria preservation medium according to claim 5, wherein the sterilization conditions are a temperature of 130 ℃ and a temperature of 150 ℃ for a period of 3-5 h.

7. A bacteria preservation medium according to claim 5, wherein said culture vessel is any one of a test tube or a culture dish.

8. A preservation medium for bacteria according to claim 5, wherein the refrigeration temperature is 2-4 ℃.