Bergmeyer Glucose Quantification for Microbiological Samples

Summary

Bergmeyer glucose quantification is a spectrophotometric enzymatic technique mainly used for clinical tests that accurately and sensitively measure the amount of glucose. Herein, we present a protocol for using this method for microbiological samples.

Abstract

The glucose concentration is a key indicator of cellular metabolism and could indicate the total metabolism rate, an aberration in glucose metabolism, and, in some cases, how cells couple glucose metabolism to energetic metabolism. In addition, intracellular and extracellular glucose levels are indicative of the cellular metabolic status. Enzymatic techniques, such as Bergmeyer glucose quantification, are more accurate and sensitive than other techniques, such as dinitrosalicylic acid or fluorescence methods, which are usually utilized in microbiology. Although mainly used in the clinical area, Bergmeyer glucose quantification can also be applied to any cell but has not been reported in detail for bacteria, fungi, yeasts, or other microorganisms.

Herein, we present a methodology to quantify glucose from bacteria and yeast samples using the enzymatic Bergmeyer glucose quantification method. The procedure involved the enzymes glucose oxidase, peroxidase, and o-dianisidine dihydrochloride incubated at 37 °C for 20 min, followed by the addition of sulfuric acid. The absorbance is then measured at 545 nm. It is important to highlight that although this technique presents difficulties in measuring high concentrations of glucose (above 60 g/L), it is possible to measure concentrations below 50 g/L using dilution factors. This enzymatic approach is valuable for research and analysis in microbiology and other scientific areas. The precision and sensitivity of the method make it helpful for detecting even low concentrations of glucose in microbiological samples.

Introduction

Glucose serves as the primary energy and carbon source for numerous microorganisms, including bacteria, yeasts, and fungi. These microorganisms take up glucose through transporters located on the extracellular membrane, where it undergoes a series of biochemical reactions within the glycolysis metabolic pathway to be converted into pyruvate¹. There are various techniques for the quantification of reducing sugars such as glucose. For example, Benedict's reagent², the use of 3,5-dinitrosalicylic acid (DNS)^3,4, the anthrona method⁵, or ph....

Protocol

1. Solution preparation

1. Prepare 100 mL of 0.1 M phosphate buffer. Weigh 1.28 g of potassium dihydrogen phosphate (KH₂PO₄) and 0.1304 g of dipotassium phosphate (K₂HPO₄) and add them to a glass beaker. Now, add 70 mL of deionized water (H₂Odi) and adjust the pH to 5.5 using 1 M hydrochloric acid (HCl) or potassium hydroxide (KOH). Transfer the solution to a volumetric flask and adjust the final volume to 100 mL. Then, transfer the solution.......

Discussion

Quantifying glucose in culture media is essential for understanding microbial growth and metabolic activity, as glucose serves as a primary energy source for many microorganisms. In this study, we employed the GOX-H₂SO₄ method to accurately measure glucose levels in culture media, aiming to optimize microbial processes in bacteria or yeast fermentations. Our findings are consistent with those of Yuen and McNeill¹¹; however, unlike them, our findings indicate that the readings.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL microtube	Eppendorf	-	-
2.0 mL microtube	Eppendorf	-	-
CaCO3	Meyer	471-34-1	Calcium Carbonate
D-Glucose	Meyer	50-99-7	D-Glucose
Drybath	FisherScientific	11-718-4	Serial 911NO251. Block WxDxH mm/in: 124 x 76 x 39 / 4.9 x 3.0 x 1.5
Glucose oxidase	Sigma Aldrich	G6125-50KU	Enzyme powder
H2O	-	-	Deionized water
H2SO4	Meyer	7664-93-9	Sulfuric acid
HCl	Meyer	7647-01-0	Hydrochloridric acid
K2HPO4	Meyer	7758-11-4	Dipotassium phosphate
KH2PO4	Meyer	7778-77-0	Monopotassium phosphate
KOH	Meyer	1310-58-3	Potassium hydroxide
Lambda 35	PerkinElmer	-	Spectrophotometer
Microtube centrifuge	-	-	-
o-Dianisidine dihydrochloride	Sigma Aldrich	D3252	Chromogenic
Peroxidase	Sigma Aldrich	P8125-50KU	Enzyme powder
pH-meter	Hanna	1131	Hanna 1170
Sodium acetate	Meyer	127-09-3	Meyer
Weighing spatulas	-	-	-