Method for determining total number of plate bacterial colonies and application of method

Abstract

The invention discloses a method for measuring the total number of plate bacterial colonies and application of the method. The determination method comprises the following steps: preparing a plate counting culture medium, and carrying out water bath heat preservation on the plate counting culture medium; the plate counting culture medium contains peptone, yeast extract powder, glucose, agar, sodium pyruvate, L-cysteine hydrochloride, magnesium sulfate and hemin; performing gradient dilution on the target bacteria; fully and uniformly mixing the sample homogenate with a plate counting culture medium; covering the surface of the solidified plate counting culture medium with a layer of same plate counting culture medium to form a covering layer; carrying out constant-temperature culture on the solidified flat plates with various dilution degrees; when macroscopic bacterial colonies are formed in the culture medium plate, selecting a plate with 30-300 bacterial colonies on each plate for counting. The method does not need coating operation, can effectively reduce the pollution risk, shortens the detection time, has clear colonial morphology and reliable data, and is suitable for rapid determination of the total number of colonies in various samples.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for determining the total number of bacterial colonies on a plate and its application. Background Technology

[0002] Plate count is an effective method for determining the bacterial count in a sample. The method is as follows: After appropriate dilution of the sample, the microorganisms are fully dispersed into single cells. A certain amount of the diluted sample is spread onto a plate and incubated. Each single cell grows and multiplies to form a visible colony; a single colony represents one single cell from the original sample. The number of colonies is counted, and the bacterial count in the sample can be calculated based on the dilution factor and the inoculum size. However, since the sample is often not completely dispersed into single cells, a single colony may originate from 2-3 or more cells in the sample. Therefore, the accuracy of plate count results is often low. To clearly illustrate the results of plate count, colony-forming units (CFUs) are preferred over absolute colony counts to represent the viable bacterial content of the sample.

[0003] Currently, in laboratories, plating apparatus is often used to spread bacterial solutions on plates for cultivation and counting. However, plating apparatus needs to be heated at high temperatures to remove any residual bacterial solution from the previous operation before it can be used, and it also needs to be cooled to room temperature before it can be used. This wastes a lot of time and is also prone to contamination, affecting the accuracy of experimental results.

[0004] Therefore, there is an urgent need for a simple method to determine the total number of bacterial colonies on plates that does not require the use of a coating stick and can reduce contamination. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a method for determining the total bacterial count on a plate and its application. This method eliminates the need for plating, effectively reduces the risk of contamination, shortens the detection time, and provides clear colony morphology and reliable data. It is suitable for the rapid determination of the total bacterial count in various samples.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for determining the total number of bacterial colonies on a plate, the method comprising the following steps:

[0008] S1. Prepare plate counting medium and incubate the plate counting medium in a water bath; the plate counting medium contains peptone, yeast extract, glucose, agar, sodium pyruvate, L-cysteine ​​hydrochloride, magnesium sulfate, and heme chloride;

[0009] S2. The target bacteria are serially diluted to obtain serially diluted sample homogenates, and the dilutions are selected from high to low for detection.

[0010] S3. First, inject the sample homogenate into a sterile plate, then inject the plate counting medium kept in a water bath into the same plate. Immediately rotate the plate clockwise to ensure that the sample homogenate and the plate counting medium are thoroughly mixed.

[0011] S4. After the plate counting medium in the plate has solidified, cover the surface of the solidified plate counting medium with another layer of the same plate counting medium to form a covering layer, and then place the plate on a horizontal platform to allow it to solidify.

[0012] S5. Incubate the solidified plates of each dilution at a constant temperature.

[0013] S6. Once visible colonies have formed on the culture medium plates, select plates with 30-300 colonies per plate for counting.

[0014] The method for determining the total number of colonies on a plate according to the present invention is simple to operate, does not require the use of a plating stick for plating, saves time, reduces contamination caused by the use of external objects, and the test data is stable and consistent with the results of plating the target bacteria with a plating stick. It has a wide range of applications and can be used to detect a variety of target bacteria that require plate colony counting.

[0015] Preferably, in step S1, the concentration of peptone in the plate counting medium is 5-10 g / L (e.g., 5 g / L, 8 g / L, or 10 g / L), the concentration of yeast extract is 2.5-10 g / L (e.g., 2.5 g / L, 8 g / L, or 10 g / L), the concentration of glucose is 1-10 g / L (e.g., 2.5 g / L, 8 g / L, or 10 g / L), the concentration of agar is 15-20 g / L (e.g., 15 g / L, 18 g / L, or 20 g / L), the concentration of sodium pyruvate is 1-10 g / L (e.g., 2.5 g / L, 8 g / L, or 10 g / L), the concentration of L-cysteine ​​hydrochloride is 0.2-1 g / L (e.g., 0.2 g / L, 0.8 g / L, or 1 g / L), and the concentration of magnesium sulfate is 0.5-1 g / L (e.g., 0.5 g / L, 0.8 g / L, or 1 g / L). The concentration of heme chloride is 10-20 mg / L (e.g., 10 mg / L, 15 mg / L or 20 mg / L).

[0016] Preferably, the water bath temperature in step S1 is 45-50℃ (e.g., 45℃, 48℃ or 50℃).

[0017] Preferably, the target bacteria in step S2 includes any one of microaerophilic bacteria, microaerophilic bacteria, or anaerobic bacteria.

[0018] Preferably, the step S2 of performing gradient dilution of the target bacteria includes adding the target bacterial solution to physiological saline and performing serial dilutions at a 1:10 gradient to obtain 1:1×10⁻⁶ solutions. -3 1:1×10 -4 1:1×10 -5 1:1×10 -6 ...1:1×10 -n A diluted suspension of the target bacteria, where n ≥ 3.

[0019] Preferably, the gradient dilution includes selecting 3 dilutions for each target bacterium, with each dilution performed in 2-3 replicates; the 3 dilutions include 10... -7 Dilution, 10 -6 Dilution and 10 -5 Dilution rate.

[0020] Preferably, the volume of sample homogenate injected in step S3 is 1-2 mL (e.g., 1 mL or 2 mL), and the volume of plate counting medium injected is 15-20 mL (e.g., 15 mL or 20 mL).

[0021] Preferably, the amount of culture medium poured into the covering layer in step S4 is 4-6 mL (e.g., 5 mL or 6 mL).

[0022] Preferably, the isothermal incubation in step S5 is carried out at a temperature of 28°C-35°C (e.g., 28°C, 30°C or 50°C) for a time of 24-48 h (e.g., 24 h or 48 h).

[0023] Secondly, the present invention provides the application of the plate total colony count determination method described in the first aspect in the cultivation of microaerophilic bacteria, microaerophilic bacteria, or anaerobic bacteria.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The method for determining the total number of colonies on a plate according to the present invention is simple to operate, does not require the use of a plating stick for plating, saves time, reduces contamination caused by the use of external objects, and the detection data is stable and consistent with the results of the target bacteria when the plating stick is used to spread the bacteria. It has a wide range of applications and can be applied to the detection of a variety of target bacteria that require the plate colony counting method.

[0026] (2) In this invention, peptone provides basic nitrogen source and amino acids, yeast extract supplements vitamins and growth factors, glucose provides energy as a priority energy source, sodium pyruvate can replace carbon source and participate in energy metabolism, L-cysteine ​​hydrochloride, in addition to being a source of sulfur-containing amino acids, can also create a microaerobic environment, which is conducive to the recovery of microaerobic and anaerobic bacteria; magnesium sulfate provides magnesium ions, which serve as cofactors for various enzymes (such as kinases and synthases), and heme chloride is a precursor for heme synthesis, which can promote the early colonization and energy metabolism of aerobic bacteria;

[0027] (3) In this invention, the four additive factors, sodium pyruvate, heme chloride, L-cysteine ​​hydrochloride and magnesium sulfate, work synergistically: sodium pyruvate and heme chloride together enhance energy metabolism efficiency, and the reducing environment created by L-cysteine ​​hydrochloride and the enzyme system activated by magnesium ions work together to promote the rapid recovery of bacteria from the sub-damaged state and start division, thereby significantly increasing the number of colony forming units (CFU) and the size of the colony, shortening the lag period, and with appropriate water bath temperature control, can further promote the rapid and uniform growth of the colony and effectively inhibit its spread. Attached Figure Description

[0028] Figure 1 This is a flowchart of the method for determining the total number of bacterial colonies on a plate according to the present invention. Detailed Implementation

[0029] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0030] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Example 1

[0032] This embodiment provides a method for determining the total number of bacterial colonies on a plate, as detailed below:

[0033] S1. Preparation of culture medium: Prepare plate counting medium containing the following components and concentrations: peptone 8 g / L, yeast extract 5 g / L, glucose 5 g / L, agar 18 g / L, sodium pyruvate 5 g / L, L-cysteine ​​hydrochloride 0.5 g / L, magnesium sulfate 0.8 g / L, and heme chloride 15 mg / L. Incubate the prepared culture medium in a 46°C water bath. At the same time, prepare PBS solution and 0.9% physiological saline for later use.

[0034] S2. Gradual dilution: The target bacteria are serially diluted to obtain serially diluted sample homogenates. Appropriate dilutions are selected from high to low for detection.

[0035] The serial dilution of the target bacteria includes adding the target bacteria to sterile water and performing serial dilutions at a ratio of 1:10 to obtain 1:1×10⁻⁶ samples. -7 1:1×10 -6 1:1×10 -5 For each diluted target bacterial suspension, replace the sterile pipette.

[0036] S3. Mixing and pouring: Use a pipette to first draw a certain amount of sample homogenate into a sterile plate, then pour the water-bath-insulated culture medium into the same plate, and immediately rotate the plate clockwise to make the sample homogenate and culture medium fully mixed.

[0037] Among them, a certain amount of sample homogenate was taken with a pipette and the volume was 1 mL, the volume of culture medium was 15 mL, and the mixing direction was clockwise.

[0038] S4. Prevention of spread: After the culture medium in the plate has solidified, cover its surface with a thin layer of the same culture medium to form a covering layer, and place the plate on a horizontal platform to allow it to solidify.

[0039] The culture medium coverage volume is 5 mL.

[0040] S5. Constant temperature incubation: Place the solidified plates of each dilution in a constant temperature incubator for incubation.

[0041] The culture temperature was 30℃ and the culture time was 48 h.

[0042] Observation and counting: Once visible colonies have formed on the culture medium plates, select plates with 30-300 colonies per plate for counting;

[0043] Among them, culture plates with a target colony count of 30-300 were all included in the statistical analysis, which is more accurate and clear.

[0044] The flowchart of the detection method described in Embodiment 1 of the present invention is as follows: Figure 1 As shown.

[0045] Example 2

[0046] This embodiment provides a method for determining the total number of bacterial colonies on a plate, as detailed below:

[0047] S1. Preparation of culture medium: Prepare plate counting medium containing the following components and concentrations: peptone 5 g / L, yeast extract 2.5 g / L, glucose 1 g / L, agar 15 g / L, sodium pyruvate 1 g / L, L-cysteine ​​hydrochloride 0.2 g / L, magnesium sulfate 0.5 g / L, and heme chloride 10 mg / L. Incubate the prepared culture medium in a 45°C water bath. At the same time, prepare PBS solution and 0.9% physiological saline for later use.

[0048] S2. Gradual dilution: The target bacteria are serially diluted to obtain serially diluted sample homogenates. Appropriate dilutions are selected from high to low for detection.

[0049] The serial dilution of the target bacteria includes adding the target bacteria to sterile water and performing serial dilutions at a ratio of 1:10 to obtain 1:1×10⁻⁶ samples. -7 1:1×10 -6 1:1×10 -5 For each diluted target bacterial suspension, replace the sterile pipette.

[0050] S3. Mixing and pouring: Use a pipette to first draw a certain amount of sample homogenate into a sterile plate, then pour the water-bath-insulated culture medium into the same plate, and immediately rotate the plate clockwise to make the sample homogenate and culture medium fully mixed.

[0051] Among them, a certain amount of sample homogenate was taken with a pipette and the volume was 1 mL, the volume of culture medium was 15 mL, and the mixing direction was clockwise.

[0052] S4. Prevention of spread: After the culture medium in the plate has solidified, cover its surface with a thin layer of the same culture medium to form a covering layer, and place the plate on a horizontal platform to allow it to solidify.

[0053] The culture medium coverage volume is 5 mL.

[0054] S5. Constant temperature incubation: Place the solidified plates of each dilution in a constant temperature incubator for incubation.

[0055] The culture temperature was 28℃ and the culture time was 24 h.

[0056] Observation and counting: Once visible colonies have formed on the culture medium plates, select plates with 30-300 colonies per plate for counting;

[0057] Among them, culture plates with a target colony count of 30-300 were all included in the statistical analysis, which is more accurate and clear.

[0058] Example 3

[0059] This embodiment provides a method for determining the total number of bacterial colonies on a plate, as detailed below:

[0060] S1. Preparation of culture medium: Prepare plate counting medium containing the following components and concentrations: peptone 10 g / L, yeast extract 10 g / L, glucose 10 g / L, agar 20 g / L, sodium pyruvate 10 g / L, L-cysteine ​​hydrochloride 1 g / L, magnesium sulfate 1 g / L, and heme chloride 20 mg / L. Place the prepared culture medium in a 50°C water bath for incubation. At the same time, prepare PBS solution and 0.9% physiological saline for later use.

[0061] S2. Gradual dilution: The target bacteria are serially diluted to obtain serially diluted sample homogenates. Appropriate dilutions are selected from high to low for detection.

[0062] The serial dilution of the target bacteria includes adding the target bacteria to sterile water and performing serial dilutions at a ratio of 1:10 to obtain 1:1×10⁻⁶ samples. -7 1:1×10 -6 1:1×10 -5 For each diluted target bacterial suspension, replace the sterile pipette.

[0063] S3. Mixing and pouring: Use a pipette to first draw a certain amount of sample homogenate into a sterile plate, then pour the water-bath-insulated culture medium into the same plate, and immediately rotate the plate clockwise to make the sample homogenate and culture medium fully mixed.

[0064] Among them, a certain amount of sample homogenate was taken with a pipette and the volume was 1 mL, the volume of culture medium was 15 mL, and the mixing direction was clockwise.

[0065] S4. Prevention of spread: After the culture medium in the plate has solidified, cover its surface with a thin layer of the same culture medium to form a covering layer, and place the plate on a horizontal platform to allow it to solidify.

[0066] The culture medium coverage volume is 5 mL.

[0067] S5. Constant temperature incubation: Place the solidified plates of each dilution in a constant temperature incubator for incubation.

[0068] The culture temperature was 35℃ and the culture time was 48 h.

[0069] Observation and counting: Once visible colonies have formed on the culture medium plates, select plates with 30-300 colonies per plate for counting;

[0070] Among them, culture plates with a target colony count of 30-300 were all included in the statistical analysis, which is more accurate and clear.

[0071] Comparative Example 1

[0072] The only difference between this comparative example and Example 1 is that sodium pyruvate in the plate counting medium is replaced with an equal amount of α-ketoglutarate.

[0073] Comparative Example 2

[0074] The only difference between this comparative example and Example 1 is that the heme chloride in the plate counting medium is replaced with an equal amount of heme iron.

[0075] Comparative Example 3

[0076] The only difference between this comparative example and Example 1 is that the L-cysteine ​​hydrochloride in the plate counting medium is replaced with an equal amount of glutathione.

[0077] Comparative Example 4

[0078] The only difference between this comparative example and Example 1 is that the plate counting medium does not contain sodium pyruvate, which is added in proportion to heme chloride, L-cysteine ​​hydrochloride and magnesium sulfate.

[0079] Comparative Example 5

[0080] The only difference between this comparative example and Example 1 is that the plate counting medium does not contain heme chloride, which is added in proportion to sodium pyruvate, L-cysteine ​​hydrochloride and magnesium sulfate.

[0081] Comparative Example 6

[0082] The only difference between this comparative example and Example 1 is that the plate counting medium does not contain L-cysteine ​​hydrochloride, which is added to magnesium sulfate, sodium pyruvate and heme chloride in proportion by mass.

[0083] Comparative Example 7

[0084] The only difference between this comparative example and Example 1 is that the plate counting medium does not contain magnesium sulfate, which is added in proportion to L-cysteine ​​hydrochloride, sodium pyruvate and heme chloride.

[0085] Comparative Example 8

[0086] The only difference between this comparative example and Example 1 is that the plate counting medium does not contain sodium pyruvate, heme chloride, L-cysteine, and magnesium sulfate, which are added to all other components of the medium in proportion to their mass.

[0087] Comparative Example 9

[0088] The only difference between this comparative example and Example 1 is the plate counting medium used.

[0089] Test Example 1

[0090] Detection of total bacterial count in soy milk.

[0091] The total bacterial count in soy milk was determined according to the methods of each embodiment and comparative example: 10 μL of the serially diluted soy milk was selected. -7 10 -6 10 -5 These three dilutions were tested. First, 1 mL of diluted soy milk was pipetted into a plate. Then, the pre-prepared culture medium, which had been prepared in a water bath, was poured into the plate and thoroughly mixed with the soy milk. The mixture was then tested at 10... -7 10 -6 10 -5 Each of the three dilutions was performed in triplicate. The treated culture medium plates were then anaerobically incubated at 30°C for 48 h, and the samples were counted. The results are shown in Table 1.

[0092] Table 1

[0093]

[0094] As shown in Table 1, the detection methods described in Examples 1-3 of this invention can effectively and accurately detect the total bacterial count in soy milk without the need for a coating stick, thus reducing contamination from external sources. Comparative Examples 1-9 illustrate that the four additives in the plate counting culture medium of this invention—sodium pyruvate, heme chloride, L-cysteine ​​hydrochloride, and magnesium sulfate—work synergistically to significantly increase the number and size of colony-forming units (CFUs), shorten the lag phase, and, with appropriate water bath temperature control, further promote rapid and uniform colony growth while effectively inhibiting spread.

[0095] Test Example 2

[0096] Detection of Portuguese yeast.

[0097] The total colony count of *Corynebacterium lucida* was determined according to the methods of each embodiment and comparative example: 10- colonies of serially diluted *Corynebacterium lucida* were selected. -7 10 -6 10 -5 These three dilutions were tested by first pipetting 1 mL of the diluted bacterial suspension into a plate, then pouring the pre-prepared culture medium from the water bath into the plate, mixing thoroughly with the bacterial suspension, and so on, for 10... -7 10 -6 10 -5 Each of the three dilutions was performed in triplicate. The treated culture medium plates were then anaerobically incubated at 30°C for 48 h, and the samples were counted. The results are shown in Table 2.

[0098] Table 2

[0099]

[0100] As shown in Table 2, the detection methods described in Examples 1-3 of this invention can effectively and accurately detect the total colony count of *Corynebacterium lucida* without the need for a plating stick, thus reducing contamination from external sources. Comparative Examples 1-9 illustrate that the four additives in the plate counting medium of this invention—sodium pyruvate, heme chloride, L-cysteine ​​hydrochloride, and magnesium sulfate—work synergistically to significantly increase the number and size of colony-forming units (CFUs), shorten the lag phase, and, with appropriate water bath temperature control, further promote rapid and uniform colony growth while effectively inhibiting spread.

[0101] In summary, the plate colony counting method provided by this invention is more accurate than the plating method, and the method used in this invention is simple to operate, saves time, and reduces contamination caused by external objects.

[0102] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for measuring total plate count, characterized by, The determination method comprises the following steps: S1, preparing a plate count medium, and water-bath incubating the plate count medium; the plate count medium contains peptone, yeast extract powder, glucose, agar, sodium pyruvate, L-cysteine hydrochloride, magnesium sulfate, and hemin chloride; S2, gradient diluting the target bacteria to obtain a gradient-diluted sample homogenate, and selecting dilution degrees from high to low for detection; S3, first injecting the sample homogenate into a sterile plate, then injecting the water-bath incubated plate count medium into the same plate, and immediately rotating the plate in a clockwise direction to fully mix and evenly distribute the sample homogenate and the plate count medium; S4, after the plate count medium in the plate is solidified, covering a layer of the same plate count medium on the surface of the solidified plate count medium to form a cover layer, and solidifying the plate on a water platform; S5, incubating the solidified plates of different dilution degrees at a constant temperature; S6, after visible colonies are formed in the medium plate, selecting plates with 30-300 colonies per plate for counting.

2. The method of claim 1, wherein: In step S1, the concentration of peptone in the plate count medium is 5-10 g / L, the concentration of yeast extract powder is 2.5-10 g / L, the concentration of glucose is 1-10 g / L, the concentration of agar is 15-20 g / L, the concentration of sodium pyruvate is 1-10 g / L, the concentration of L-cysteine hydrochloride is 0.2-1 g / L, the concentration of magnesium sulfate is 0.5-1 g / L, and the concentration of hemin chloride is 10-20 mg / L.

3. The method for measuring the total number of colonies of flat plates according to claim 1 or 2, characterized by, In step S1, the temperature of water-bath incubation is 45-50℃.

4. The plate count method according to any one of claims 1 to 3, characterized in that, In step S2, the target bacteria include any one of microaerophilic bacteria, microaerophilic bacteria, or anaerobic bacteria.

5. The plate count method according to any one of claims 1 to 4, characterized in that: The gradient dilution of the target bacteria in step S2 includes adding the target bacteria liquid into physiological saline, performing serial dilution with a gradient of 1:10 to obtain 1:1×10 -3 , 1:1×10 -4 , 1:1×10 -5 , 1:1×10 -6 ……1:1×10 -n diluted target bacteria suspensions, wherein n≥3.

6. The plate count method according to claim 5, wherein: The gradient dilution includes 3 dilution degrees for each target bacteria, and 2-3 parallels are made for each dilution degree; the 3 dilution degrees include 1:1×10 7 Dilution, 1:1×10 6 Dilution, and 1:1×10 5 Dilution.

7. The plate count method according to any one of claims 1 to 6, characterized in that: In step S3, the injection amount of the sample homogenate is 1-2 mL, and the injection amount of the plate count medium is 15-20 mL.

8. The plate count method according to any one of claims 1 to 7, characterized in that: In step S4, the pouring amount of the medium of the cover layer is 4-6 mL.

9. The plate count method according to any one of claims 1 to 8, characterized in that: In step S5, the temperature of constant-temperature incubation is 28℃-35℃, and the time is 24-48 h.

10. The plate colony total number determination method according to any one of claims 1-9 is applied to culturing microaerophilic bacteria, microaerophilic bacteria, or anaerobic bacteria.

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