The research evaluates glucose concentration in microbial samples using quantification techniques from bacterial and yeast filters. The objective is to improve the accuracy of glucose mass gram in microbiological studies. Challenges include accurately quantifying low glucose concentration in complex microbial samples and distinguishing between microbial and environmental sources of glucose in mixed cultures.
The research has shown a clear correlation between specific microbial strains and their glucose consumption rates, revealing how environmental factors influence metabolic pathways. Our protocol enhances glucose detection by improving specificity and sensitivity while reducing processing time. Using sulfuric acid in a controlled way minimizes interference.
Future research will focus on optimizing glucose detection methods for industrial microbiology applications, and investigating the role of a specific genes in glucose metabolism under varying environmental conditions. To begin, weigh 1.28 grams of potassium dihydrogen phosphate and 0.1304 grams of dipotassium phosphate in a glass beaker. Add 70 milliliters of deionized water, and adjust the pH to 5.5 using 1 molar hydrochloric acid or potassium hydroxide.
Then transfer the solution to a 100-milliliter volumetric flask, and adjust the volume to 100 milliliters with deionized water. Store the prepared solution in an amber container at 4-8 degrees Celsius for up to three months. Next, weigh 0.01 grams of o-Dianisidine dihydrochloride in a 2-milliliter centrifuge tube.
Using a 1, 000-microliter pipette, add 1 milliliter of deionized water to dissolve the compound, and mix the solution slowly. Wrap the centrifuge tube in aluminum foil to shield it from light, and store it at 20 degrees Celsius. Then add o-Dianisidine dihydrochloride solution to 10 milliliters of phosphate buffer taken in a 100-milliliter volumetric flask, and adjust the final volume to 100 milliliters with the phosphate buffer.
After transferring the solution to an amber flask, store it at 4-8 degrees Celsius for 3-4 months. Now place a 100-milliliter volumetric flask with 30 milliliters of deionized water in an ice bath. After 10 minutes, slowly pour 51 milliliters of 98%sulfuric acid down the walls of the flask while gently shaking it.
Later, adjust the final volume to 100 milliliters with deionized water, and transfer the solution to a glass amber flask for storage. For enzyme preparation, weigh 0.01 grams of glucose oxidase in a 2-milliliter sterile microtube. Add 1 milliliter of 50-millimolar sodium acetate buffer at pH 5 to the microtube and mix gently.
Then in a new 2-milliliter microtube, weigh 0.0031 grams of peroxidase enzyme, and dissolve it in 1 milliliter of phosphate buffer set to pH 5.5. Cover both the microtubes containing enzymes with aluminum foil, and store them at 20 degrees Celsius. Then prepare 1 gram per liter D-Glucose standard solution using deionized water.
To begin, prepare the glucose standard and all other required solutions for the assay. Add the appropriate volume of glucose to fresh 2-milliliter tubes. Set a dry thermobath to 37 degrees Celsius, and allow it to stabilize.
Add the appropriate volumes of buffer to each microtube, followed by 3.3 microliters of the glucose oxidase and 1.2 microliters of peroxidase. Incubate the tubes at 37 degrees Celsius, and set the timer for 20 minutes. Immediately after incubation, add 750 microliters of 50%sulfuric acid to each microtube, and place them in an ice bath for 2 minutes to cool.
Transfer the cooled 1, 500 microliter mixture to a plastic cuvette, and measure the absorbance at 529 nanometers. Then clean the work area with a 70%alcohol solution and light a burner to ensure asepsis. Obtain bacteria or yeast in a liquid culture medium.
Transfer 100 microliters of the culture into 1.5-milliliter microtubes using sterile tips. Centrifuge the samples at 7, 500 G for 7 minutes at 4 degrees Celsius. After centrifugation, carefully remove the supernatant, which contains glucose in solution.
Mix 0.5 microliters of the supernatant with all the required components to prepare the reaction mixture. Incubate the microtubes for 20 minutes at 37 degrees Celsius, and immediately add 750 microliters of 50%sulfuric acid. Let the mixture cool in an ice bath for 2 minutes.
Finally, measure the absorbance at 529 nanometers. The spectrophotometric analysis indicated a maximum absorbance peak at 529 nanometers. The analysis of glucose consumption by Debaryomyces hansenii demonstrated consistent results between glucose oxidase and DNS methods until significant differences emerged at 6, 8, and 12 hours.
The application of the glucose oxidase sulfuric acid method facilitated analysis of high glucose concentrations up to 100 grams per liter, with reliable results following dilution.
