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Resting cell preparation requires a centrifuge.
Resting cell preparation requires a centrifuge
.
Resting cell reaction, also known as resting cell, washes away various nutrients and growth factors in the culture medium and then suspends it in physiological saline for a period of time to consume endogenous nutrients, and cells that are in a starved state are used in the study of microbial growth.
It has a wide range of applications in physiology, biochemistry and metabolism
.
Its main feature is that although the cells are in a dormant state and do not grow and reproduce, they still contain various dangerous strains.
They have the ability to oxidize and ferment, and can resume growth under suitable conditions
.
In addition, the specificity of the resting cell reaction can improve the substrate conversion rate, and it is not easy to contaminate bacteria, and can reduce the inhibition of the product on bacterial growth and alcohol synthesis
.
Preparation and sterilization of culture medium: According to the type and quantity of culture medium in the experimental materials, configure the corresponding culture medium for sterilization
.
Strain activation and expanded culture Transfer the E.
coli cVcc249 preserved strains to the slant of the test tube complete solid medium, and cultivate at 37°C for 18-24h.
Select the activated strains with good growth and transfer them to the LB liquid medium, 37 Incubate with shaking at ℃ for 16-18h
.
Preparation of resting cells Pour the bacteria in the Erlenmeyer flask into a centrifuge tube, centrifuge at 4000 rpm for 15 min, and collect the precipitate
.
The precipitate was washed three times with 85% saline, and centrifuged at 4000 rpm for 15 min each time
.
Use phosphate buffered saline (PBS) 20mL of pH 7.
0 (the amount depends on the number of bacteria) to prepare bacterial suspension, put the prepared bacterial suspension into a 4°C refrigerator and cultivate for 72hr to reduce internal respiration and prepare resting cells
.
Preparation of substrates: Four substrates were prepared at a concentration of 0.
1 M: sodium acetate, sodium succinate, sodium α-ketoglutarate and sodium citrate, respectively
.
Dilutions of different concentration gradients were performed as needed
.
Enzyme and substrate reaction: In a 5mL centrifuge tube, add 0.
4ml of resting cells and 0.
4ml of substrate, keep the temperature at 37°C for 30min; add 40ul of thiazole salt, shake and mix, and react at 37°C for .
2hr; after the reaction , and then add 1.
5 mL of dimethyl sulfoxide to the centrifuge tube, mix and shake, and keep in a 37°C incubator for 30 min; add 1.
5 mL of distilled water
.
Determination of OD510 value: According to the instruction of spectrophotometer, determine the ODs1o value of each sample, and make a record of the experiment
.
Aseptic operation must be performed during the whole experiment, especially when the strain is activated, because the medium used is a complete medium, and general microorganisms can also grow.
In order to ensure that the measured dehydrogenase activity comes from the preserved strain , so the first step to activate the bacteria must ensure aseptic operation
.
Thiazole salts must be stored in the dark, because they are easily decomposed when exposed to light
.
In addition, since both thiazole salts and dimethyl sulfoxide are toxic, gloves should be worn when adding these two reagents
.
In the resting cell reaction experiment, when washing the cells, the suspension must be sufficient each time, otherwise it is easy to cause the washing to be unclean, resulting in residual nutrients and poor quality of the resting cells
.
When measuring its OD value with a spectrophotometer, since the suitable measurement range of the spectrophotometer used in this experiment is 0.
2~0.
7, it is necessary to check whether the OD value of the sample is within this range before measurement.
If it is too high, Then add an appropriate amount of PBS to dilute
.
In addition, before the measurement, the sample needs to be thoroughly mixed, and the action should be fast, so as to prevent the formazan from crystallizing again, which will affect the measurement result
.
The blank control was distilled water
.
Resting cell preparation requires a centrifuge
.
Resting cell reaction, also known as resting cell, washes away various nutrients and growth factors in the culture medium and then suspends it in physiological saline for a period of time to consume endogenous nutrients, and cells that are in a starved state are used in the study of microbial growth.
It has a wide range of applications in physiology, biochemistry and metabolism
.
Its main feature is that although the cells are in a dormant state and do not grow and reproduce, they still contain various dangerous strains.
They have the ability to oxidize and ferment, and can resume growth under suitable conditions
.
In addition, the specificity of the resting cell reaction can improve the substrate conversion rate, and it is not easy to contaminate bacteria, and can reduce the inhibition of the product on bacterial growth and alcohol synthesis
.
Preparation and sterilization of culture medium: According to the type and quantity of culture medium in the experimental materials, configure the corresponding culture medium for sterilization
.
Strain activation and expanded culture Transfer the E.
coli cVcc249 preserved strains to the slant of the test tube complete solid medium, and cultivate at 37°C for 18-24h.
Select the activated strains with good growth and transfer them to the LB liquid medium, 37 Incubate with shaking at ℃ for 16-18h
.
Preparation of resting cells Pour the bacteria in the Erlenmeyer flask into a centrifuge tube, centrifuge at 4000 rpm for 15 min, and collect the precipitate
.
The precipitate was washed three times with 85% saline, and centrifuged at 4000 rpm for 15 min each time
.
Use phosphate buffered saline (PBS) 20mL of pH 7.
0 (the amount depends on the number of bacteria) to prepare bacterial suspension, put the prepared bacterial suspension into a 4°C refrigerator and cultivate for 72hr to reduce internal respiration and prepare resting cells
.
Preparation of substrates: Four substrates were prepared at a concentration of 0.
1 M: sodium acetate, sodium succinate, sodium α-ketoglutarate and sodium citrate, respectively
.
Dilutions of different concentration gradients were performed as needed
.
Enzyme and substrate reaction: In a 5mL centrifuge tube, add 0.
4ml of resting cells and 0.
4ml of substrate, keep the temperature at 37°C for 30min; add 40ul of thiazole salt, shake and mix, and react at 37°C for .
2hr; after the reaction , and then add 1.
5 mL of dimethyl sulfoxide to the centrifuge tube, mix and shake, and keep in a 37°C incubator for 30 min; add 1.
5 mL of distilled water
.
Determination of OD510 value: According to the instruction of spectrophotometer, determine the ODs1o value of each sample, and make a record of the experiment
.
Aseptic operation must be performed during the whole experiment, especially when the strain is activated, because the medium used is a complete medium, and general microorganisms can also grow.
In order to ensure that the measured dehydrogenase activity comes from the preserved strain , so the first step to activate the bacteria must ensure aseptic operation
.
Thiazole salts must be stored in the dark, because they are easily decomposed when exposed to light
.
In addition, since both thiazole salts and dimethyl sulfoxide are toxic, gloves should be worn when adding these two reagents
.
In the resting cell reaction experiment, when washing the cells, the suspension must be sufficient each time, otherwise it is easy to cause the washing to be unclean, resulting in residual nutrients and poor quality of the resting cells
.
When measuring its OD value with a spectrophotometer, since the suitable measurement range of the spectrophotometer used in this experiment is 0.
2~0.
7, it is necessary to check whether the OD value of the sample is within this range before measurement.
If it is too high, Then add an appropriate amount of PBS to dilute
.
In addition, before the measurement, the sample needs to be thoroughly mixed, and the action should be fast, so as to prevent the formazan from crystallizing again, which will affect the measurement result
.
The blank control was distilled water
.
