CN110862953B - Preparation and germination method and application of geobacillus stearothermophilus spores - Google Patents
Preparation and germination method and application of geobacillus stearothermophilus spores Download PDFInfo
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Abstract
The invention discloses a preparation and germination method and application of geobacillus stearothermophilus spores, and belongs to the technical field of applied microorganisms. The preparation and germination method comprises the following steps: (1) activating strains; (2) preparing spores; (3) and (4) germinating spores. The invention further comprises a germination medium for rapid spore recovery and application of the spores. The Geobacillus stearothermophilus spores prepared by the method have high spore yield, short preparation period and high resistance regulation, and the liquid induction culture medium has the advantages of simple spore collection, large-scale preparation and the like compared with solid plate induction, and is suitable for working bacteria of space sterilization indicators, such as pressure steam sterilization, hydrogen peroxide low-temperature plasma sterilization, antibiotic residue detection in fresh milk and the like.
Description
Technical Field
The invention belongs to the technical field of applied microorganisms, and particularly relates to a preparation and germination method and application of geobacillus stearothermophilus spores.
Background
Geobacillus stearothermophilus (Geobacillus stearothermophilus) Belongs to thermophilic aerobic bacillus, but has the characteristic of anaerobism, the blue staining of a bacterial propagule G is purple, and bacterial spore malachite green is colored.
Bacillus stearothermophilus is relatively easy to identify and harmless to the human body and is generally used as an indicator organism for space disinfection. Geobacillus stearothermophilus spores are the spores with the strongest resistance to moist heat pressure steam sterilization. That is, the preparation of the pressure steam sterilization biological indicator requires geobacillus stearothermophilus spores as the contaminating species.
However, in industrial production, Geobacillus stearothermophilus has low sporulation rate and long preparation period, and the quantity of spores required by the preparation of the biological indicator is difficult to meet.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a technical scheme for rapidly preparing Geobacillus stearothermophilus spores. In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a preparation method of geobacillus stearothermophilus spores, which comprises the following steps:
(1) activating strains: preparing geobacillus stearothermophilus strain G, stearothermophileus ATCC 7953 freeze-dried powder into a bacterial suspension by using PBS buffer solution, and coating the bacterial suspension on an activation culture medium for activation;
(2) preparing strains: inoculating the activated strain to a proliferation culture medium, and performing shake culture for 10-20 h;
(3) spore induction: inoculating the strain into an induction culture medium for induction culture, collecting the thalli and spores of Geobacillus stearothermophilus, and removing the thalli to obtain Geobacillus stearothermophilus spores.
In some embodiments of the invention, the PBS buffer 1000mL system is as follows: 2.83g of anhydrous disodium hydrogen phosphate and 1.36g of dipotassium hydrogen phosphate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2 +/-0.2.
In some embodiments of the invention, the activation medium is TSA medium, and the 1000mL system is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride and 16g of agar, preparing 1000mL of purified water, and adjusting the pH value to 7.2 +/-0.2; the proliferation culture medium is a TSB culture medium, and a 1000mL system comprises the following components: 17g of tryptone, 3g of soybean peptone, 5g of sodium chloride, 2.5g of glucose and 2.5g of anhydrous dipotassium hydrogen phosphate, wherein the total volume of the solution is 1000mL by using purified water, and the pH value is adjusted to 7.2 +/-0.2.
In some embodiments of the invention, the induction medium 1000mL system is as follows: 3.0-12g of tryptone, 1.0-6.0g of acid hydrolyzed casein, 1.0-6.0g of yeast extract powder, 1.0-6.0g of beef extract powder, 10.0-60.0mg of potassium chloride, 0.1-0.6g of calcium chloride, 2.0-20.0mg of manganese chloride and 0.2-0.8mg of magnesium sulfate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2 +/-0.2.
In some embodiments of the invention, the induction medium 1000mL system is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.25g of calcium chloride, 10.0mg of manganese chloride and 0.41g of magnesium sulfate, preparing 1000mL of purified water, and adjusting the pH value to 7.2-7.4.
In some preferred embodiments of the present invention, step (1) is specifically: selecting lyophilized powder, placing in PBS buffer solution, shaking, mixing, and culturing at 58 deg.C for 20-30h to obtain 100 μ L of TSA coated on 90mm dish; the colonies were picked up and placed in a 2mL EP tube containing 1mL PBS buffer, and after shaking and mixing well, 100. mu.L of the mixture was applied to 90mm TSA medium and cultured overnight at 58 ℃ (20-30 h) to form colonies, which were stored in a 4 ℃ refrigerator for later use.
In some preferred embodiments of the present invention, step (2) is specifically: the activated strain was inoculated into a 500mL conical flask containing 200mL of TSB medium and placed in a 58 ℃ shaking incubator at 180rpm overnight for 10-20 h.
Further, a step of proliferating the strain further is further included between the step (2) and the step (3).
In some embodiments of the invention, the step of repopulating is: inoculating the strain obtained in the step (2) into a proliferation culture medium for proliferation culture for 10-20 h. Preferably, the proliferation medium is the TSB medium described above.
In other embodiments of the invention, the step of proliferating is: inoculating the strain obtained in the step (2) into an induction culture medium for proliferation culture for 10-20 h.
In some embodiments of the invention, the induction medium 1000mL system is as follows: 3.0-12g of tryptone, 1.0-6.0g of acid hydrolyzed casein, 1.0-6.0g of yeast extract powder, 1.0-6.0g of beef extract powder, 10.0-60.0mg of potassium chloride, 0.1-0.6g of calcium chloride, 2.0-20.0mg of manganese chloride and 0.2-0.8mg of magnesium sulfate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2 +/-0.2.
In some embodiments of the invention, the induction medium 1000mL system is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.25g of calcium chloride, 10.0mg of manganese chloride and 0.41g of magnesium sulfate, preparing 1000mL of purified water, and adjusting the pH value to 7.2-7.4.
In other embodiments of the present invention, the induction medium 1000mL system is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.20g of calcium chloride, 6.0mg or 18.0mg of manganese chloride and 0.41g of magnesium sulfate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2-7.4.
In further specific embodiments of the present invention, the system of 1000mL of the induction medium is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.1g or 0.3g of calcium chloride, 10.0mg of manganese chloride and 0.41g of magnesium sulfate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2-7.4.
In some embodiments of the present invention, the induction culture in step (3) is 55-65 ℃ at 180rpm for 1.5-3.5 days.
In some embodiments of the present invention, the collecting of geobacillus stearothermophilus spores and spores in step (3) refers to collecting the spores and spores by differential centrifugation.
In some embodiments of the present invention, the step (3) of removing the bacteria comprises lysing the bacteria with 0.2mg/mL hen egg albumen lysozyme for 1-4 h.
The second aspect of the invention provides an induction medium for inducing geobacillus stearothermophilus to produce spores, which is characterized in that a 1000mL induction medium system comprises the following components: 3.0-12g of tryptone, 1.0-6.0g of acid hydrolyzed casein, 1.0-6.0g of yeast extract powder, 1.0-6.0g of beef extract powder, 10.0-60.0mg of potassium chloride, 0.1-0.6g of calcium chloride, 2.0-20.0mg of manganese chloride and 0.2-0.8mg of magnesium sulfate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2 +/-0.2.
In some embodiments of the invention, the induction medium 1000mL system is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.25g of calcium chloride, 10.0mg of manganese chloride and 0.41g of magnesium sulfate, preparing 1000mL of purified water, and adjusting the pH value to 7.2-7.4.
In other embodiments of the present invention, the induction medium 1000mL system is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.20g of calcium chloride, 6.0mg of manganese chloride and 0.41g of magnesium sulfate, preparing 1000mL of purified water, and adjusting the pH value to 7.2-7.4.
In other embodiments of the present invention, the induction medium 1000mL system is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.20g of calcium chloride, 18.0mg of manganese chloride and 0.41g of magnesium sulfate, preparing 1000mL of purified water, and adjusting the pH value to 7.2-7.4.
In further specific embodiments of the present invention, the system of 1000mL of the induction medium is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.1g of calcium chloride, 10.0mg of manganese chloride and 0.41g of magnesium sulfate, preparing 1000mL of purified water, and adjusting the pH value to 7.2-7.4.
In further specific embodiments of the present invention, the system of 1000mL of the induction medium is as follows: 6.0g of tryptone, 3.0g of acid hydrolyzed casein peptone, 3.2g of yeast extract powder, 2.5g of beef extract powder, 32mg of potassium chloride, 0.3g of calcium chloride, 10.0mg of manganese chloride and 0.41g of magnesium sulfate, preparing 1000mL of purified water, and adjusting the pH value to 7.2-7.4.
In some embodiments of the invention, the Geobacillus stearothermophilus isG. stearothermophilus ATCC 7953。
A third aspect of the present invention provides a germination method for Geobacillus stearothermophilus spores prepared by the preparation method of the first aspect of the present invention, comprising the step of adding the spores to a germination medium, wherein the germination medium 1000mL system is as follows: 5-20g of peptone, 0.5-7.0g of soluble starch, 0.2-5.0g of fructose, 0.2-5.0g of sucrose, 0.2-5.0g of maltose, 0.1-3.0g of L-alanine, 0.05-0.30g of L-asparagine, 0.01-0.10g of bromocresol purple and 0.25-2.5g of dipotassium hydrogen phosphate, wherein 1000mL of purified water is utilized for preparation, and the pH value is adjusted to 7.2-7.4.
In some embodiments of the invention, the 1000mL system of germination medium is as follows: 15g of peptone, 2.0g of soluble starch, 0.5g of fructose, 0.5g of sucrose, 0.2g of maltose, 0.2g of L-alanine, 0.3g of L-asparagine, 0.02g of bromocresol purple and 1.2g of dipotassium hydrogen phosphate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2-7.4.
The invention provides a culture medium for promoting germination of Geobacillus stearothermophilus spores, wherein a 1000mL culture medium system comprises the following components: 5-20g of peptone, 0.5-7.0g of soluble starch, 0.2-5.0g of fructose, 0.2-5.0g of sucrose, 0.2-5.0g of maltose, 0.1-3.0g of L-alanine, 0.05-0.30g of L-asparagine, 0.01-0.10g of bromocresol purple and 0.25-2.5g of dipotassium hydrogen phosphate, wherein 1000mL of purified water is utilized for preparation, and the pH value is adjusted to 7.2-7.4.
In some embodiments of the invention, the medium 1000mL system is as follows: 15g of peptone, 2.0g of soluble starch, 0.5g of fructose, 0.5g of sucrose, 0.2g of maltose, 0.2g of L-alanine, 0.3g of L-asparagine, 0.02g of bromocresol purple and 1.2g of dipotassium hydrogen phosphate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2-7.4.
In a fifth aspect, the invention provides the application of geobacillus stearothermophilus spores prepared by the preparation method in the first aspect in preparation of a sterilization biological indicator and/or in microorganism detection.
In some embodiments of the invention, the sterilization includes, but is not limited to, pressure steam sterilization, hydrogen peroxide low temperature plasma sterilization.
In some embodiments of the invention, the microbial detection includes, but is not limited to, an antibiotic residue test in fresh milk.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
1. the Geobacillus stearothermophilus spores prepared by the method have the spore inductivity of over 85 percent, the highest spore inductivity of 94.0 percent and high yield.
2. The Geobacillus stearothermophilus spores prepared by the method have short preparation period, and can be prepared in one batch in 1-3 days.
3. The Geobacillus stearothermophilus spore prepared by the method has large resistance regulation property and resistance D 121℃ Can be prepared in the range of about 1.23-2.65min according to requirements.
4. The Geobacillus stearothermophilus spores are prepared by liquid induction culture, the spores are easy to collect, and large-scale preparation can be realized.
5. The spore germination culture medium can ensure that Geobacillus stearothermophilus spores germinate rapidly, and is suitable for rapid detection.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clear, the present invention is further described in detail below with reference to the embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and materials cited therein are hereby incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 Geobacillus stearothermophilus spore preparation
1) Strain: geobacillus stearothermophilus purchased from Guangzhou culture CollectionG. stearothermophilusLyophilized spore powder of ATCC 7953;
2) activating strains: selecting lyophilized powder, placing in 0.3mol/L PBS buffer solution, shaking, mixing, and coating 100 μ L in TSA of 90mm dish, and culturing at 58 deg.C for 20-30 h; picking out the colonies, placing the colonies in a 2mL EP tube containing 1mL PBS buffer solution, fully shaking and uniformly mixing, taking 100 μ L of the colonies, coating the 100 μ L of the colonies in 90mm TSA culture medium, and carrying out 58 ℃ overnight culture (20-30 h) to form the colonies, and storing the colonies in a 4 ℃ refrigerator for later use;
3) preparing strains: inoculating activated strain (preserved TSA colony) in 500mL conical flask containing 200mL TSB culture medium, and culturing at 58 deg.C in 180rpm shake culture device for 10-20 h;
4) and (3) strain preservation: placing 40% glycerol and TSB bacterial suspension in a ratio of 1:1 in a refrigerator at-20 ℃ for storage for later use;
5) and (3) proliferation culture: the proliferation culture can adopt and is not limited to the following 2 methods: TSB proliferation method: inoculating a small amount of strain preserved by TSB into TSB for proliferation culture for 10-20 h; b. induction liquid proliferation method: inoculating a small amount of strain preserved by TSB into an induction culture medium for proliferation culture for 10-20 h;
6) induction (sporulation) culture: the induction culture may be performed by, and is not limited to, the following methods: a. inoculating 5-20% of the proliferation culture in inducing (spore-forming) culture medium, performing spore-forming culture at 55-65 deg.C and 180rpm for 1.5-3.5 days, sampling in the process, and observing spore generation; b. inoculating a small amount of TSB-preserved strain directly into inducing culture medium, and performing spore production at 55-65 deg.C and 180rpm for 1.5-3.5 days;
7) collecting and purifying: pouring the induction culture medium into a plurality of 50mL centrifuge tubes for differential centrifugation, discarding the supernatant, and reserving the bottom thalli and spores. Cracking the collected thalli and spores for 1-4h by using 0.2mg/mL egg albumen lysozyme, and then centrifuging and purifying to obtain high-purity spore suspension;
8) counting and storing: can be preserved at 4 deg.C with sterile water/PBS buffer solution; taking the stored spore suspension, performing gradient dilution, coating the spore suspension in a TSB, and counting to obtain the concentration (cfu/mL) of the stored spore suspension;
9) fast germination of spores (spore recovery): dropwise adding spore suspension with a certain concentration into a culture medium containing 1mL of rapidly recovered germination, and culturing in a dry constant-temperature culture at 58 +/-2 ℃ to observe the color change (purple-yellow) of the culture medium; meanwhile, the generation of thalli is observed under a sampling microscope every 15min, and the existence of the thalli indicates that spores are germinated.
The culture medium and components involved in the preparation and recovery method are as follows:
TSA agar Medium
TSB Medium
PBS buffer
4. Induction medium
5. Culture medium for restoring germination of spores
EXAMPLE 2 growth culture
This example compares the growth culture conditions in step (5) of example 1.
1.2 mL EP tubes were used and 20% glycerol cultures were inoculated into 500mL Erlenmeyer flasks containing 200mL TSB and 200mL induction medium, respectively, and cultured in a constant temperature shaking incubator (ZQTY-70N) at 58 ℃ and 180rpm for 14h (overnight);
2. respectively sampling 1-2mL of TSB and induction culture medium, observing thallus growth condition under microscope (Nikon Ts 2) and testing OD in ultraviolet spectrophotometer 600 A value of (d);
as can be seen from the above table, neither the TSB proliferation method nor the induced medium proliferation method can obtain the thallus which is full and rod-shaped, and the TSB and the induced medium are utilized to proliferate for 14h and OD 600 To 1.425 and 1.232, respectively.
Example 3 Induction of sporulation
This example compares the conditions of the induced (sporulation) culture of step 6) of example 1.
Preparing an induction culture medium: 6.0g of tryptone; acid hydrolysis casein peptone 3.0 g; 3.2g of yeast extract powder; 2.5g of beef extract powder; 32mg of potassium chloride; 0.20g of calcium chloride; 10.0mg of manganese chloride; magnesium sulfate 0.41 g; 1000mL of purified water; the pH was adjusted to 7.2-7.4 using 2N sodium hydroxide solution for further use.
The induction inoculation method comprises the following steps:
a. inoculating 5-20% of the bacterial liquid after TSB proliferation into a 1000mL conical flask containing 300-500mL of induction liquid;
b. inoculating the bacterial liquid after proliferation of 5-20% of the induction liquid into a 1000mL conical flask containing 300-500mL of induction liquid;
c. directly inoculating 1-5mL of TSB preserved strain into a 1000mL conical flask containing 300-500mL of induction liquid; culturing at 58-60 deg.C under shaking at 180rpm for 2 d;
sampling and observing the sporulation state periodically after about 14 h; after 2d of culture, centrifugally collecting, cracking for 3h by using 0.2mg/mL lysozyme, purifying, collecting and counting; storing the purified collected spores in a refrigerator at 4 deg.C for further use
And (3) counting the sporulation condition:
example 4 spore resistance Regulation comparison (TSB proliferation)
This example uses TSB proliferation by adjusting the manganese ion content in the induction medium versus spore resistance at different induction times.
1. Media preparation
Preparing a TSB culture medium;
preparing an induction culture medium:
a. 6.0g of tryptone; acid hydrolysis casein peptone 3.0 g; 3.2g of yeast extract powder; 2.5g of beef extract powder; 32mg of potassium chloride; 0.20g of calcium chloride; 6.0mg of manganese chloride; magnesium sulfate 0.41 g; 1000mL of purified water;
b. 6.0g of tryptone; acid hydrolysis casein peptone 3.0 g; 3.2g of yeast extract powder; 2.5g of beef extract powder; 32mg of potassium chloride; 0.20g of calcium chloride; 18.0mg of manganese chloride; magnesium sulfate 0.41 g; 1000mL of purified water.
Adjusting pH to 7.2-7.4 with 2N sodium hydroxide, and packaging into 1000mL conical flask, and steam sterilizing at 121 deg.C for 20 min.
Inoculating the strain into TSB according to the ratio of the strain to the TSB proliferation liquid of 1-5% and proliferating for 12-14h at the same time to obtain the TSB proliferation liquid.
Inoculating the TSB proliferation solution into an induction culture medium according to the proportion of the TSB proliferation solution to the induction culture medium of 10-20%, culturing at 58 ℃, collecting spores at 180rpm for different periods of time, and testing spore resistance.
The results are shown in the following table:
as can be seen from the above table, the manganese ions have a relatively obvious enhancing effect on spore resistance; the induction rate and yield increase with the induction time over a period of time; resistance shows a tendency to increase and then decrease with increasing induction time.
Example 5 comparison of spore resistance regulation (Induction liquid proliferation protocol)
In this example, the inducing solution was used as a proliferation medium, and spore resistance was compared by adjusting the content of calcium ions in the inducing medium for different inducing times.
Preparing an induction culture medium:
a. 6.0g of tryptone; acid hydrolysis casein peptone 3.0 g; 3.2g of yeast extract powder; 2.5g of beef extract powder; 32mg of potassium chloride; 0.10g of calcium chloride; 10.0mg of manganese chloride; magnesium sulfate 0.41 g; 1000mL of purified water;
b. 6.0g of tryptone; acid hydrolysis casein peptone 3.0 g; 3.2g of yeast extract powder; 2.5g of beef extract powder; 32mg of potassium chloride; 0.30g of calcium chloride; 10.0mg of manganese chloride; magnesium sulfate 0.41 g; 1000mL of purified water.
Adjusting pH to 7.2-7.4 with 2N sodium hydroxide, and packaging into 1000mL conical flask, sterilizing at 121 deg.C for 20min under pressure.
Inoculating the strain into the induction culture solution according to the ratio of strain to induction culture solution of 1-5%, and proliferating overnight for 12-14 hr to obtain induced proliferation solution.
Inoculating the induced culture solution obtained in the step (2) into an induced culture medium according to the proportion of the induced proliferation solution to the induced culture medium being 10-20%, culturing at 58 ℃ and 180rpm for different periods of time, and testing spore resistance.
The results are shown in the following table:
as can be seen from the above table, calcium ions have a more obvious enhancing effect on spore resistance; the induction rate and yield increase with the induction time over a period of time; resistance shows a tendency to increase and then decrease with increasing induction time.
EXAMPLE 6 spore resistance modulation (direct Induction)
This example regulates resistance of spores generated by non-proliferating Geobacillus stearothermophilus directly using the induction medium by adjusting the induction time.
1. Preparing an induction culture medium:
6.0g of tryptone; acid hydrolysis casein peptone 3.0 g; 3.2g of yeast extract powder; 2.5g of beef extract powder; 32mg of potassium chloride; 0.25g of calcium chloride; 10.0mg of manganese chloride; magnesium sulfate 0.41 g; 1000mL of purified water;
adjusting pH to 7.2-7.4 with 2N sodium hydroxide, and packaging into 1000mL conical flask, and steam sterilizing at 121 deg.C for 20 min.
2. 1-5mL of strains are directly inoculated to an induction culture medium for induction culture, spores are collected at 58 ℃ and 180rpm for different periods of time, and spore resistance is tested.
The results are shown in the following table:
as can be seen from the above table, the induction rate and yield increased with the increase of the induction time over a period of time; resistance shows a tendency to increase and then decrease with increasing induction time.
Example 7 Geobacillus stearothermophilus spores Rapid Geobacillus germination
1. Preparing a rapid germination culture medium:
15g of peptone; 2.0g of soluble starch; 0.5g of fructose; 0.5g of cane sugar; maltose 0.2 g; 0.2g of L-alanine; 0.3g of L-asparagine; bromcresol purple (ph indicator) 0.02 g; dipotassium phosphate 1.2 g; 1000mL of purified water. Adjusting pH to 7.2-7.4 with 2N sodium hydroxide, and steam sterilizing at 121 deg.C for 20 min.
2. Get about 10 7 The spore amount of (A) was added dropwise to a 2mL EP tube containing 1mL of a fast germinant, and the medium was incubated in a dry thermostat at 58 ℃.
3. Sampling every 15 minutes and performing microscopic examination by using an inverted phase contrast microscope to observe whether thalli are formed or not; the color change in 2mL EP tubes was also recorded.
Microscopic examination and color change in the culture broth were as follows:
thus, when Geobacillus stearothermophilus spores are cultured by using the germination medium, thallus production can be observed under a microscope within 60min, and the color of the medium can be observed to start changing within 75 min. After 135min, the number of thallus reaches the peak value, and the color of the culture medium turns yellow completely at 195 min. The germination medium can rapidly promote germination of Geobacillus stearothermophilus spores and is suitable for rapid detection of microorganisms.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (2)
1. A germination method of Geobacillus stearothermophilus spores, which is characterized by comprising the step of adding the spores to a germination medium, wherein a 1000mL system of the germination medium is as follows: 5-20g of peptone, 0.5-7.0g of soluble starch, 0.2-5.0g of fructose, 0.2-5.0g of sucrose, 0.2-5.0g of maltose, 0.1-3.0g of L-alanine, 0.05-0.30g of L-asparagine, 0.01-0.10g of bromocresol purple and 0.25-2.5g of dipotassium phosphate, wherein the preparation method comprises the steps of preparing 1000mL of purified water and adjusting the pH value to 7.2-7.4, wherein the preparation method of Geobacillus stearothermophilus spores comprises the following steps:
(1) activating strains: preparing Geobacillus Stearothermophilus strain ATCC 7953 freeze-dried powder into a bacterial suspension by using PBS buffer solution, and coating the bacterial suspension on a TSA culture medium for activation;
(2) preparing strains: inoculating the activated strain to a TSB culture medium, and performing shaking culture for 10-20 h;
(3) carrying out secondary proliferation on the strains in an induction culture medium;
(4) spore induction: inoculating the strain into an induction culture medium for induction culture, collecting the thalli and spores of Geobacillus stearothermophilus, removing the thalli to obtain Geobacillus stearothermophilus spores,
wherein the content of the first and second substances,
the TSA culture medium 1000mL system is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride and 16g of agar, preparing 1000mL of purified water, and adjusting the pH value to 7.2 +/-0.2;
the TSB culture medium 1000mL system is as follows: 17g of tryptone, 3g of soybean peptone, 5g of sodium chloride, 2.5g of glucose and 2.5g of anhydrous dipotassium hydrogen phosphate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2 +/-0.2;
the induction medium 1000mL system is as follows: 3.0-12g of tryptone, 1.0-6.0g of acid hydrolyzed casein, 1.0-6.0g of yeast extract powder, 1.0-6.0g of beef extract powder, 10.0-60.0mg of potassium chloride, 0.1-0.6g of calcium chloride, 2.0-20.0mg of manganese chloride and 0.2-0.8mg of magnesium sulfate, wherein 1000mL of purified water is used for preparation, and the pH value is adjusted to 7.2 +/-0.2.
2. Germination method according to claim 1, characterised in that the step of re-propagation is:
inoculating the strain obtained in the step (2) into an induction culture medium for proliferation culture for 10-20 h.
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