Method for rapidly and accurately preparing quantitative bacteria

Abstract

The method for rapidly and accurately preparing the quantitative bacteria not only can improve the accuracy of a microbiological experiment, but also can reduce the experiment cost and time. Meanwhile, the method also has good repeatability and applicability, can stably work under different experimental conditions, and is particularly suitable for microbiological experiments with high requirements on bacterial growth state, bacterial quantity and batch repeatability. According to the method, after a standardized method is established, quantitative bacteria in the logarithmic phase can be accurately obtained only in two days each time, the process is simple and reliable, quantification is accurate, and more time and energy are saved compared with a traditional mode.

Description

Technical Field

[0001] The present invention relates to a bacteria cultivation method, in particular to a standardized method for rapidly and accurately preparing logarithmic phase bacteria quantitatively. Background Art

[0002] The bacterial growth process is divided into four phases according to the growth curve: lag phase, logarithmic phase, stationary phase, and decay phase. Bacteria in the logarithmic phase have the fastest growth rate, the most active metabolism, a similar number of live bacteria to the total bacterial count, and generally consistent physical and chemical properties. To achieve stable and accurate experimental results, most microbial experiments tend to use bacteria in the logarithmic phase as the primary experimental subject.

[0003] Conventional preparation of logarithmic-phase bacteria typically involves dilution and subculture. An overnight bacterial seed culture is diluted into fresh culture medium, followed by intermittent monitoring of bacterial growth using a spectrophotometer or microplate reader until the bacterial growth reaches the logarithmic phase. The bacterial concentration is then estimated empirically, resulting in low accuracy and large errors. For example, patent CN201510272317.5 discloses a method utilizing a fibrous microbacterial bioflocculant, which includes the following steps: A. Inoculate a slant culture medium with fibrous microbacteria and store at 4°C; B. Inoculate the seed culture medium with the culture medium and culture it in a shaker at 37°C to prepare a seed solution; C. Transfer the seed solution to a fermentation medium containing corn straw as the sole carbon source at a ratio of 1% and culture it in a shaker at 37°C at 180 rpm for 48 hours. To obtain an accurate bacterial concentration, a commonly used traditional method involves simultaneous bacterial sampling, serial dilution, overnight culture, and plate counting. The logarithmic-phase bacterial stock solution is then refrigerated to 4°C, and the bacterial concentration is estimated the next day based on the plate count results. However, the logarithmic phase bacteria that have been stored overnight still have a small amount of proliferation at 4°C, and there is a certain error between the actual bacterial concentration and the bacterial concentration obtained by plate counting. The operation is cumbersome and time-consuming, and has high requirements for the experimental environment and operators. Summary of the Invention

[0004] This invention addresses the shortcomings of existing bacterial cultivation methods and proposes a rapid, accurate, and simple method for preparing quantitative logarithmic-phase bacteria. This method improves the accuracy of microbiological experiments while reducing experimental costs and time. Furthermore, the method should exhibit good repeatability and applicability, operating stably under diverse experimental conditions. It is particularly suitable for microbiological experiments that place high demands on bacterial growth status, bacterial count, and batch reproducibility.

[0005] The present invention relates to a method for rapidly and accurately preparing a quantitative amount of bacteria, characterized in that the method comprises the following steps: S1, Day 1: Take a single colony D1 and inoculate it into fresh liquid culture medium. Incubate it in a biological incubator at a temperature of 24°C to 37°C with shaking or standing overnight. The overnight time ranges from 15 to 18 hours to obtain seed liquid D2. S2, Day 2: Dilute the seed solution D2 described in step 1 into fresh liquid culture medium at a ratio of 1:5-1:10 to obtain the passaged colony solution D3; S3. Place the subcultured bacterial liquid D3 in a biological incubator controlled at a temperature of 24°C to 37°C and culture it in the incubator for 1 to 3 hours until the logarithmic phase bacterial liquid D4 is obtained; S4. Using a spectrophotometer, measure the optical density of the logarithmic phase bacterial solution D4 and normalize the optical density to a fixed value X, wherein the fixed value X is set in a range of 0.5 to 1.0; S5. Perform a gradient dilution of the normalized bacteria and count the bacteria on the plate. On the third day, obtain the corresponding bacterial concentration value F. S6. Take another batch of bacteria that need to be quantitatively cultured to the logarithmic phase and process them according to steps S1 to S4 above. On Day 2, after the bacteria are cultured to the logarithmic phase bacterial solution D4, the optical density at the same wavelength is measured and normalized to the fixed value X determined in step S5. The optical density is then converted according to the known bacterial concentration F. A quantitative count of logarithmic phase bacteria can be quickly and accurately obtained on the next day.

[0006] The so-called single colony in step one is a single type of bacteria or a single type of fungi.

[0007] The normalized optical density value of the colony is performed by concentration or dilution.

[0008] The methods involved in the present invention are all based on the establishment of standardized methods. After that, it only takes two days each time to accurately obtain quantitative logarithmic phase bacteria. The process is simple, reliable and quantitatively accurate, which saves more time and energy than traditional methods. DETAILED DESCRIPTION

[0009] The present invention will be described in detail below with reference to the embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0010] In the description of the present invention, it should be understood that the terms "center", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating orientations or positional relationships, are positional relationships, which are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. Example 1:

[0011] Test goal: Multiple times, the bacterial concentration in 100ml is 2*10 5 CFU / ml of logarithmic phase Escherichia coli, The cultivation process of traditional experiments is: S1, Day 1: Take a single E. coli colony and inoculate it into fresh liquid culture medium. Incubate it in a 37°C incubator with shaking overnight for 16 hours to obtain seed solution. S2. The next day: dilute the seed solution into fresh liquid culture medium at a ratio of 1:100 to obtain a passaged bacterial solution; S3. Place the subcultured bacterial solution in a 37°C incubator for shaking culture. S4. Take samples regularly and measure the optical density at 600 nm every 15 minutes using a spectrophotometer. Plot the OD 600nm Growth curve corresponding to subculture time.

[0012] S5. Collect OD600 nm For bacteria with a value of 0.2-0.8, the estimated bacterial concentration is 2*10 8 CFU / ml, use directly or store at 4℃.

[0013] S6. Take 1 ml of the bacteria stored at 4°C and perform gradient dilutions and plate counts. On the third day, the actual bacterial concentration is 3*10 8 CFU / ml, and then take out the bacteria stored overnight (a small amount of proliferation) and dilute them in the corresponding proportion. That is, on the third day of the experiment, the bacterial concentration in 100ml can be 2*10 5 Logarithmic phase Escherichia coli in CFU / ml (small deviation in concentration).

[0014] To achieve the above goals, the traditional test method requires three days each time, which is time-consuming and the final bacterial concentration obtained has certain deviations.

[0015] The specific implementation process of the method adopted by the present invention is: S1. Preliminary experiment Day 1: Take a single Escherichia coli bacterial colony D1 and inoculate it into fresh liquid culture medium. Incubate it in a biological incubator at 37°C with shaking overnight for 18 hours to obtain seed liquid D2. S2, Day 2: dilute the seed solution D2 into fresh liquid culture medium at a ratio of 1:10 to obtain the subcultured colony solution D3; S3. Place the subcultured bacterial solution D3 in a 37°C biological incubator and culture for 1.5 hours until the logarithmic phase bacterial solution D4 is obtained; S4. Use a spectrophotometer to measure the optical density (OD) of the logarithmic phase bacterial solution D4 at a wavelength of 600 nm, that is, the OD 600nm The normalized value is 0.6 / 0.5 = 1.2 times the OD 600nm =0.5; S5. Perform gradient dilutions on the normalized bacteria and count the bacteria on the plate. On the third day, the corresponding bacterial concentration is 2*10 8 CFU / ml.

[0016] Standardized method: All subsequent bacterial concentrations in 100 ml are required to be 2*10 8 The experiment of CFU / ml logarithmic phase Escherichia coli can be carried out according to the above method. After the bacteria are cultured to the logarithmic phase, the optical density (OD) is normalized (diluted or concentrated). 600nm is 0.5, and then according to the above known bacterial concentration 2*10 8 CFU / ml is diluted in the corresponding ratio, and the quantitative logarithmic phase bacteria can be quickly and accurately obtained on the second day of the experiment.

[0017] That is, to achieve the above-mentioned goal, the test method of the present invention only takes 2 days each time after the standardized method is established, which is relatively short and can quickly and accurately obtain quantitative logarithmic phase bacteria. Example 2:

[0018] Test goal: Multiple times, 100ml of bacteria concentration is required to be 1*10 6 CFU logarithmic phase Staphylococcus aureus Traditional test: S1, Day 1: Take a single Staphylococcus aureus colony and inoculate it into fresh liquid culture medium. Incubate it in a 37°C incubator with shaking overnight for 16 hours to obtain seed solution. S2. The next day: dilute the seed solution into fresh liquid culture medium at a ratio of 1:100 to obtain a passaged bacterial solution; S3. Place the subcultured bacterial solution in a 37°C incubator for shaking culture. S4. Take samples regularly and measure the optical density at 600 nm every 15 minutes using a spectrophotometer. Plot the OD 600nm Growth curve corresponding to subculture time.

[0019] S5. Collect OD600 nm For bacteria with a value of 0.2-0.8, the estimated bacterial concentration is 3*10 8 CFU / ml, use directly or store at 4℃.

[0020] S6, the bacteria stored at 4℃ above are sampled 1ml, gradient dilution is carried out, and plate counting is carried out, and the actual bacterial concentration of 1.5*10 8 CFU / ml is obtained on the third day, and the bacteria (small amount of proliferation) stored overnight are taken out again, and corresponding proportional dilution is carried out, that is, 100ml of bacteria with a concentration of 1*10 6 CFU / ml (slightly deviated concentration) of logarithmic phase Staphylococcus aureus can be obtained on the second day of the experiment.

[0021] In order to achieve the above-mentioned goal, the traditional test method needs 3 days each time, which is time-consuming and the final obtained bacterial concentration has a certain deviation.

[0022] The specific implementation process of the method adopted by the present application is as follows: S1, pre-experiment Day1: a single Staphylococcus aureus bacterial colony D1 is inoculated into fresh liquid medium, and is cultured in a 37℃ biological incubator for 15h overnight to obtain seed liquid D2; S2, Day2: the seed liquid D2 is diluted by 1:5 into fresh liquid medium to obtain the colony liquid D3 after subculture; S3, the colony liquid D3 after subculture is placed in a 37℃ biological incubator for 2h to obtain the logarithmic phase colony liquid D4; S4, the optical density value (optical density OD) of the logarithmic phase bacteria liquid D4 is detected at 600nm wavelength by using a spectrophotometer, that is, the OD 600nm is measured to be 0.45, and is normalized, that is, it is concentrated by 0.6 / 0.45=1.33 times to OD 600nm equals 0.6; S5, the normalized bacteria are gradient diluted and plate counted, and the corresponding bacterial concentration of 1.5*10 6 CFU / ml is obtained on the third day.

[0023] Standardized method: all experiments requiring 100ml of Staphylococcus aureus bacteria with a concentration of 1*10 6 CFU / ml can be carried out according to the above method, the logarithmic phase bacteria are cultured, the optical density OD 600nm is normalized (diluted or concentrated) to 0.6, and corresponding proportional dilution is carried out according to the known bacterial concentration 1.5*10 6 CFU / ml, and quantitative logarithmic phase bacteria can be quickly and accurately obtained on the second day of the experiment.

[0024] That is, in order to achieve the above-mentioned goal, after the establishment of the standardized method, the test method of the present application only needs 2 days each time, which is time-saving, and quantitative logarithmic phase bacteria can be quickly and accurately obtained. Example 3:

[0025] Test goal: Multiple times, 300ml of bacteria concentration is required to be 3*10 6 CFU / ml of logarithmic phase Enterococcus faecalis.

[0026] Traditional test: S1, Day 1: Take a single Enterococcus faecalis colony and inoculate it into fresh liquid culture medium. Incubate it in a 37°C incubator with shaking overnight for 16 hours to obtain seed solution. S2. The next day: dilute the seed solution into fresh liquid culture medium at a ratio of 1:100 to obtain a passaged bacterial solution; S3. Place the subcultured bacterial solution in a 37°C incubator for shaking culture. S4. Take samples regularly and measure the optical density at 600 nm every 15 minutes using a spectrophotometer. Plot the OD 600nm Growth curve corresponding to subculture time.

[0027] S5. Collect OD 600nm For bacteria with a value of 0.2-0.8, the estimated bacterial concentration is 3*10 8 CFU / ml, use directly or store at 4℃.

[0028] S6. Take 1 ml of the bacteria stored at 4°C and perform gradient dilutions and plate counts. On the third day, the actual bacterial concentration is 2*10 8 CFU / ml, and then take out the bacteria stored overnight (a small amount of proliferation) and dilute them in the corresponding proportion. That is, on the third day of the experiment, the bacterial concentration of 300ml can be 3*10 6 Logarithmic phase Enterococcus faecalis in CFU / ml (small deviation in concentration).

[0029] To achieve the above goals, the traditional test method requires three days each time, which is time-consuming and the final bacterial concentration obtained has certain deviations.

[0030] The specific implementation process of the method adopted by the present invention is: S1. Preliminary experiment Day 1: Take a single Enterococcus faecalis colony D1 and inoculate it into fresh liquid culture medium. Incubate it in a biological incubator at 37°C overnight for 18 hours to obtain seed liquid D2. S2, diluting the seed solution D2 into fresh liquid culture medium at a ratio of 1:10 to obtain a passaged colony solution D3; S3. Place the subcultured colony solution D3 in a 37°C biological incubator and culture for 2 hours until the logarithmic phase colony solution D4 is obtained; S4. Use a spectrophotometer to measure the optical density (OD) of the above-mentioned logarithmic phase bacteria at a wavelength of 650nm. 650nmThe normalized value is 0.6 / 0.5 = 1.2 times the OD 650nm =0.5; S5. Perform gradient dilution of the normalized bacteria and count the bacteria on the plate to obtain the corresponding bacterial concentration 1*10 8 CFU / ml.

[0031] Standardization method: All subsequent bacterial concentrations in 100 ml are 3*10 8 The experiments on CFU / ml logarithmic phase Enterococcus faecalis can be carried out according to the above method. After the bacteria are cultured to the logarithmic phase, the optical density (OD) is normalized (diluted or concentrated). 650nm is 0.5, and then according to the above known bacterial concentration 1*10 8 CFU / ml is diluted in the corresponding ratio, and the quantitative logarithmic phase bacteria can be quickly and accurately obtained on the second day of the experiment.

[0032] That is, to achieve the above-mentioned goal, the test method of the present invention only takes 2 days each time after the standardized method is established, which is relatively short and can quickly and accurately obtain quantitative logarithmic phase bacteria.

[0033] The methods involved in the present invention are all based on the establishment of standardized methods. After that, it only takes two days each time to accurately obtain quantitative logarithmic phase bacteria. The process is simple, reliable and quantitatively accurate, which saves more time and energy than traditional methods.

[0034] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention is disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the technical content disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technology of the present invention, which do not depart from the content of the technical solution of the present invention, are within the scope of the technical solution of the present invention.

Claims

1. A method for rapidly and accurately preparing quantitative bacteria, characterized in that: The method comprises the following steps: S1, Day 1: Take a single colony D1 and inoculate it into fresh liquid culture medium. Incubate it in a biological incubator at a temperature of 24°C to 37°C with shaking or standing overnight. The overnight time ranges from 15 to 18 hours to obtain seed liquid D2. S2, Day 2: Dilute the seed solution D2 described in step 1 into fresh liquid culture medium at a ratio of 1:5-1:10 to obtain the passaged colony solution D3; S3. Place the subcultured bacterial liquid D3 in a biological incubator controlled at a temperature of 24°C to 37°C and culture it in the incubator for 1 to 3 hours until the logarithmic phase bacterial liquid D4 is obtained; S4. Using a spectrophotometer, measure the optical density of the logarithmic phase bacterial solution D4 and normalize the optical density to a fixed value X, wherein the fixed value X is set in a range of 0.5 to 1.0; S5. Perform a gradient dilution of the normalized bacteria and count the bacteria on the plate. On the third day, obtain the corresponding bacterial concentration value F. S6. Take another batch of bacteria that need to be quantitatively cultured to the logarithmic phase and process them according to steps S1 to S4 above. On Day 2, after the bacteria are cultured to the logarithmic phase bacterial solution D4, the optical density at the same wavelength is measured and normalized to the fixed value X determined in step S5. The optical density is then converted according to the known bacterial concentration F. A quantitative count of logarithmic phase bacteria can be quickly and accurately obtained on the next day.

2. The method for rapid and accurate preparation of quantitative bacteria according to claim 1, characterized in that: The so-called single colony in step 1 is a single type of bacteria or a single type of fungi.

3. The method for rapid and accurate preparation of quantitative bacteria according to claim 1, characterized in that: The normalized optical density value of the colony is performed by concentration or dilution.