Method for evaluating formation amount of streptococcus mutans biological membrane and application

A Streptococcus mutans and biofilm technology, applied in the field of microorganisms, can solve the problems of inability to stain biofilms, poor data reproducibility and low sensitivity of parallel samples, and achieve the effects of good application prospects, good data reproducibility and high sensitivity.

Inactive Publication Date: 2019-07-09
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The measurement of biofilm formation is an important means to carry out research work and evaluate the treatment effect, but the current methods for measuring biofilm have the following disadvantages: low sensitivity, high cost, complicated operation, and poor reproducibility of data in parallel samples
However, in this method, the dye cannot stain the biofilm at the bottom of the plate, and the results obtained have poor stability and low sensitivity

Method used

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  • Method for evaluating formation amount of streptococcus mutans biological membrane and application
  • Method for evaluating formation amount of streptococcus mutans biological membrane and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Materials and methods

[0050] (a) Preparation of bacterial suspension:

[0051] Preparation of Streptococcus mutans bacteria suspension: Dissolve the freeze-dried powder of Streptococcus mutans CGMCC No.1.2499 (purchased from CGMCC, China) with a small amount of sterile distilled water, and use an inoculation loop to get a ring and streak it on the BHI solid medium (purchased From OXOID Co., UK), anaerobic culture at 37°C for 24 h, pick a single colony with an inoculation loop and put it into 10 mL of BHI liquid medium (purchased from OXOID Co., UK), use a vortex shaker to evenly disperse the colony in In the liquid medium, take it out after anaerobic culture at 37°C for 24 hours, and inoculate it in BHI liquid medium with 2% (v / v) inoculum. After washing twice with sterile distilled water, resuspend in BHI liquid medium to the desired concentration.

[0052] Preparation of Streptococcus salivarius bacterial suspension: Dissolve the freeze-dried powder of Streptoc...

Embodiment 2

[0070] 1. Materials and methods

[0071] (a) Preparation of bacterial suspension: same as in Example 1.

[0072] (b) a method for evaluating the amount of Streptococcus mutans biofilm formation, specifically comprising the following steps:

[0073] (1) Streptococcus mutans biofilm formation:

[0074] (11) Submerge sterile stainless steel beads (5mm in diameter) in sterile saliva and incubate at 25°C for 6h.

[0075] (12) Pick up the stainless steel beads with a sterile magnetic stirrer (diameter length: 10×50 mm), rinse with 10 mM, pH 7.0 PBS buffer, remove the stainless steel beads, and place them in the culture wells of a 24-well plate .

[0076] (13) Add 1.5mL BHI liquid medium and 300μL mutans Streptococcus suspension (10 4 CFU / mL) and 300 μL of the sample solution to be tested (or blank control solution), and then incubated anaerobically at 25°C for 36h.

[0077] (14) Pick up the stainless steel beads with a magnetic stirring bar, rinse with PBS buffer, remove the s...

Embodiment 3

[0088] 1. Materials and methods

[0089] (a) Preparation of bacterial suspension: same as in Example 1.

[0090] (b) a method for evaluating the amount of Streptococcus mutans biofilm formation, specifically comprising the following steps:

[0091] (1) Streptococcus mutans biofilm formation:

[0092] (11) Submerge sterile stainless steel beads (5mm in diameter) in sterile saliva and incubate at 45°C for 2h.

[0093] (12) Pick up the stainless steel beads with a sterile magnetic stirrer (diameter length: 10×50 mm), rinse with 10 mM, pH 7.0 PBS buffer, remove the stainless steel beads, and place them in the culture wells of a 24-well plate .

[0094] (13) Add 1.8 mL of BHI liquid medium and 100 μL of Streptococcus mutans suspension (10 8 CFU / mL) and 500 μL of the sample solution to be tested (or blank control solution), and incubated anaerobically at 45°C for 12h again.

[0095] (14) Pick up the stainless steel beads with a magnetic stirring bar, rinse with PBS buffer, rem...

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Abstract

The invention discloses a method for evaluating the formation amount of a streptococcus mutans biological membrane. The method comprises the following steps that 11, stainless steel beads are immersedin saliva for incubation; 12, the stainless steel beads are taken down after being sucked and washed, and are placed in culture holes of a 24-hole plate; 13, a BHI liquid culture medium, a streptococcus mutans strain suspension and a to-be-detected sample solution are added, and incubation is carried out again; 14, the stainless steel beads are sucked, washed, taken down and put into the cultureholes of the 24-hole plate to be naturally dried; 21, a crystal violet solution is added for dyeing, the stainless steel beads are sucked, washed, taken down and put into the culture holes of the 24-hole plate to be naturally dried; 22, absolute ethyl alcohol is added for decolorizing, then the stainless steel beads are transferred to a 96-hole plate, and OD is measured to be 600 nm through a microplate reader. The invention also discloses application of the method in screening a streptococcus mutans biological membrane inhibitor. Compared with a traditional method, the method is easy to operate, low in cost, high in data stability and sensitivity and good in application prospect.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a method and application for evaluating the biofilm formation amount of Streptococcus mutans. Background technique [0002] Dental caries, commonly known as dental caries and tooth decay, is a major common oral disease originating from bacterial infection. In severe cases, it can be followed by pulpitis and periapical periodontitis, and even cause alveolar bone and jaw inflammation. Chronic oral infectious diseases prevalent in children and adults are closely related to the colonization of Streptococcus mutans on the tooth surface. Modern medical research believes that the plaque formed by Streptococcus mutans biofilm adsorbing other bacteria on the surface of tooth enamel is also one of the key factors for caries, and reducing or inhibiting the formation of biofilm can also achieve the purpose of preventing dental caries. The measurement of biofilm formation is an ...

Claims

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Application Information

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IPC IPC(8): C12Q1/06C12Q1/18C12R1/46
CPCC12Q1/06C12Q1/18G01N2333/315
Inventor 吴正钧冯华峰韩瑨赵磊乔祯逸
Owner BRIGHT DAIRY & FOOD
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