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A kind of living bacteria enumeration method of bacterial cellulose production strain

A technology for producing bacterial cellulose and bacterial strains, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, and can solve the problems of single colonies, inability to effectively count, and inability to obtain

Inactive Publication Date: 2019-07-09
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when this method is applied to colony counting, it is still impossible to obtain a single colony, so it cannot be counted effectively

Method used

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  • A kind of living bacteria enumeration method of bacterial cellulose production strain
  • A kind of living bacteria enumeration method of bacterial cellulose production strain
  • A kind of living bacteria enumeration method of bacterial cellulose production strain

Examples

Experimental program
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Effect test

Embodiment 1

[0028] A method for counting live bacteria of bacterial cellulose production strains, the steps are as follows:

[0029] (1) Pick a ring of colonies and inoculate into 150mL fermentation medium. Medium formula: glucose 25g / L, yeast powder 7.5g / L, peptone 10g / L, Na 2 HPO 4 10 g / L, 3% (v / v) filter-sterilized cellulase (1000 U). Shake culture (180rpm) at 28°C for 48h.

[0030] (2) Inject 1mL of the bacterial solution into a test tube containing 8.9mL of sterile normal saline and 0.1mL of cellulase (1000U), shake the test tube to mix the solution in the tube, and make a 1:10 dilution.

[0031] (3) According to the above operation, do 10 times incremental dilution to 10 -6 , at 10 -4 、10 -5 and 10 -6 Take 100 μL of the dilution in the test tubes corresponding to the gradient, spread evenly on the solid plate, and incubate upside down at 28°C for 3-4 days, then count the colonies, see image 3 .

Embodiment 2

[0033] A method for counting live bacteria of bacterial cellulose production strains, the steps are as follows:

[0034] (1) Example Pick a ring of colony and inoculate into 50mL fermentation medium. Medium formula: glucose 25g / L, yeast powder 7.5g / L, peptone 10g / L, Na 2 HPO 4 10g / L. Incubate at 30°C for 72 hours.

[0035] (2) Pour all the fermentation broth and the produced BC into a 50mL centrifuge tube, and centrifuge at 5000rpm for 5min. Discard the supernatant, add 5 mL membrane-filtered cellulase (1000 U) to the precipitate, and let stand at 30°C for 30 min until the cellulose is completely degraded. Centrifuge at 5000rpm for 5min and discard the supernatant.

[0036] (3) Resuspend the precipitate with 50 mL of sterile normal saline, mix thoroughly, and inject 1 mL of bacterial solution into a test tube containing 8.8 mL of sterile phosphate buffer and 0.2 mL of cellulase (1000 U). Shake the test tube to mix the solution in the tube to make a 1:10 dilution. Accord...

Embodiment 3

[0039] A method for counting live bacteria of bacterial cellulose production strains, the steps are as follows:

[0040] (1) Pick a ring of colonies and inoculate into 150mL fermentation medium. The formula of the medium is the same as that of case 2. After 20 hours of shaking culture (160 rpm) at 30°C, 5 mL of cellulase (1000 U) was added, and shaking culture was continued for 4 hours.

[0041] (2) Gradually dilute according to the method of Case 1, and take 150 μL of the diluted solution and spread it evenly on the solid plate. After inverting culture at 30°C for 3-4 days, count the colonies.

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Abstract

The invention relates to a viable bacterium counting method for bacterial cellulose production strains. The method comprises the steps of 1, preparing bacterial crude suspension; 2, after the crude suspension obtained in step 1 is subjected to centrifugation, obtaining bacteria, and weighing the bacteria with normal saline or a phosphate buffer solution containing a certain amount of cellulose till the initial volume is reached; 3, conducting gradient dilution, wherein firstly, a certain amount of cellulose is added to a gradient dilution solution, and then gradient dilution experiments are conducted; 4, after the materials are fully and uniformly mixed, conducting coating counting or pouring counting. According to the method, the bacteria are released from bacterial cellulose through cellulose, and the uniform bacterium suspension is obtained. Cellulose is added to the gradient dilution solution, so that the purpose of fully degrading cellulose fibers connected among the bacteria is achieved, the situation that in the counting process, due to existence of cellulose, the bacteria cannot be differentiated completely, so that bacterial colonies gather or cannot be counted is avoided, and when the method is applied to coating counting and pouring counting methods, the bacterium separation effect is extremely good.

Description

technical field [0001] The invention relates to microorganism culture technology, in particular to a method for counting live bacteria of bacterial cellulose production strains. Background technique [0002] Bacterial Cellulose (BC) is synthesized by microorganisms such as Acetobacter, Rhizobium, Agrobacterium and Sarcina, and is different from plant fiber. , named "bacterial cellulose" or "microbial cellulose". At present, Acetobacter is the genus with the widest research scope and relatively good properties of the bacterial cellulose produced. [0003] Studies by Czaja W et al. and Tang WH et al. have shown that during the static culture and shaking culture of Acetobacter to produce bacterial cellulose, most of the bacteria are wrapped in membranous and spherical bacterial cellulose. Brown et al. reported that dense cellulose secretion sites are distributed on the surface of the cell membrane of Acetobacter xylinum. The filaments are continuously extended at a speed of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/06
CPCC12Q1/06
Inventor 钟成刘淼邓婷月贾士儒郑鑫杨啸天谭之磊韩培培
Owner TIANJIN UNIV OF SCI & TECH