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 phenol-sulfuric acid⁶ methods can be employed. However, these methods detect any reducing sugar present in the sample, such as fructose and maltose, which can contribute to the signal and complicate the accurate quantification of a particular sugar.

In addition, they require strict temperature control and the handling of hazardous substances, which can complicate the process and affect the accuracy. These methods can also suffer interference from other compounds present in the sample, making accurate glucose quantification difficult. Conversely, high-performance liquid chromatography (HPLC) offers a more precise alternative, allowing for the discrimination between glucose and other reducing sugars, albeit with higher operational costs. These contrasting methods highlight the ongoing need for the development of accurate and cost-effective glucose quantification techniques⁷.

The glucose oxidase-peroxidase-sulfuric acid (GOX-H₂SO₄) method employs two enzymes, glucose oxidase (GOD) and peroxidase (POD). GOD is an oxidoreductase enzyme that is very selective for the oxidation of β-D-glucose⁸. This complex acts as a catalyst during the reaction that occurs when glucose is in the presence of oxygen, producing gluconic acid and hydrogen peroxide (H₂O₂). H₂O₂ is detected by means of a chromogenic oxygen acceptor, o-dianisidine, which, upon interaction with POD, causes its oxidation and H₂O₂ release, preventing gluconic acid from being converted back to glucose (see Figure 1). The color obtained is stabilized by the addition of sulfuric acid (H₂SO₄), which also stops the enzymatic reaction, thus avoiding possible interference that could occur if the reaction continues⁹.

Figure 1: Overview of the glucose quantification method using the glucose oxidase enzyme. which reacts with glucose to produce hydrogen peroxide (H₂O₂) and gluconic acid. Subsequently, the peroxidase enzyme reacts with H₂O₂ in the presence of O-dianisidine dihydrochloride. In the final step, sulfuric acid (H₂SO₄) is added to stop the enzymatic reaction, resulting in a pink color that is measured at 529 nm. Abbreviations: POD = peroxidase; GOD = glucose oxidase. Please click here to view a larger version of this figure.

The GOX-H₂SO₄ enzymatic method is a technique widely used to measure the concentration of glucose in biological samples, most of which are of clinical interest. However, few studies have investigated a technique that allows the quantification of the amount of glucose in a growth medium to evaluate its consumption. Therefore, in the present protocol, the methodology to quantify glucose by means of the enzymatic reaction of glucose oxidase, peroxidase, o-dianisidine dihydrochloride, and H₂SO₄ in microbiological liquid samples is presented, which arises as a modification of the work done by Bergmeyer⁹, by using 50% (v/v) H₂SO₄ and lower quantities of reagents.

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 to an amber container and store the solution at 4-8 °C for up to 3 months.
   CAUTION: Wear nitrile gloves throughout the preparation process to prevent skin contact and potential contamination, as o-dianisidine dihydrochloride is potentially carcinogenic.
2. Prepare a 1% w/v solution of o-dianisidine dihydrochloride. Weigh 0.01 g of o-dianisidine dihydrochloride and place it in a 2.0 mL centrifuge tube. Using a 1,000 mL micropipette, add 1.0 mL of H₂Odi to dissolve the compound, then mix slowly by pipetting up and down. Shield the solution from light by wrapping the container in aluminum foil and store it at -20 °C to maintain stability.
   NOTE: To facilitate support of the tube during measurement on the analytical balance, a 10 mL beaker can be used as additional support. This helps to stabilize the tube, ensuring accurate measurement by minimizing the risk of displacement or tilting that could alter the results obtained on the balance.
3. In a 100 mL volumetric flask, add 10 mL of phosphate buffer solution followed by 1 mL of the 1% v/v o-dianisidine dihydrochloride solution. Then, adjust the final volume to 100 mL with phosphate buffer to obtain a final concentration of 0.01% o-dianisidine-buffer mixture. Transfer the solution to an amber flask and wrap it with aluminum foil to protect it from light. Store the solution at 4-8 °C for up to 3-4 months.
   NOTE: Refrigeration is essential to prevent decomposition, which could compromise the integrity of experimental measurements. To avoid degradation of the o-dianisidine-buffer mixture, a small portion of 13 mL (sufficient for 12 samples) can be placed in a 14 mL plastic tube on ice until use.
4. Prepare 50% H₂SO₄ solution. Place a 100 mL volumetric flask with 30 mL of H₂Odi in an ice bath and allow it to cool for 10 min. Then, slowly pour 51 mL of H₂SO₄ (98% v/v) down the walls of the flask, shaking carefully to homogenize the solution. Wait for the mixture to cool down, as the reaction is exothermic (generates heat). Adjust the final volume to 100 mL with H₂Odi and transfer the solution to a glass amber flask.
   NOTE: Use a fume hood and follow all safety protocols, be sure to wear appropriate protective equipment. Prepare 50 mL of 40% calcium carbonate solution to neutralize any possible spills that may occur.
5. Prepare 50 mM sodium acetate solution by dissolving 0.04 g of sodium acetate in 5 mL of H₂Odi in a beaker. Adjust the pH to 5.1 using glacial acetic acid. Finally, adjust the volume to 10 mL with H₂Odi.

2. Preparation of enzymes

1. In a 2.0 mL sterile microtube, weigh 0.01 g of glucose oxidase and mix with 1 mL of 50 mM sodium acetate, pH 5.0, to obtain a final concentration of 10 mg/mL. Cover the tube with aluminum foil and store at -20 °C.
   NOTE: The solution can be stored for up to 2 months at -20 °C.
2. Calculate the volume of enzyme solution V₂ needed to achieve the desired enzyme activity in each plastic cuvette (with a total volume of 1.5 mL) using Eq (1): V₁C₁ = V₂C₂, where V₁ is 1,500 µL, C₁ is 28.5 U of enzyme activity, and C₂ (the concentration of the enzyme solution) is 12,970 U/mL (10 mg of the enzyme [129,000 units/g] in 1 mL of H₂Odi).
   V₂ = (1,500 µL × 28.5 U)/12,970 U = 3.29 µL    (1)
   NOTE: For more accurate pipetting, the value can be rounded to 3.3 µL.
3. In a new 2 mL microtube, weigh 0.0031 g of peroxidase enzyme and add 1 mL of phosphate buffer, pH 5.5, to achieve a final concentration of 3.1 mg/mL. Cover the microtube with aluminum foil and store at -20 °C.
4. Calculate the volume of the peroxidase solution required to achieve the desired enzyme activity per cuvette using Eq (1). Start with a total cuvette volume of 1,500 µL and a target enzyme activity of 1.25 U. Then, determine the necessary volume of the enzyme solution, given its concentration of 1,551 U.
   V₂ = (1,500 µL × 1.25 U)/1,551 U = 1.208 µL
   NOTE: For more accurate pipetting, the value was rounded to 1.20 μL.

3. Glucose standard

1. Prepare a 1 g/L D-glucose standard by adding 0.01 g of D-glucose to 10 mL of H₂Odi in a volumetric flask. To dilute the concentration to 0.5 g/L, take 500 µL of the stock solution and mix it with 500 µL of H₂Odi to achieve a final volume of 1 mL.
   NOTE: This dilution process is essential for creating accurate standard solutions.

4. Standard curve preparation

1. Using a new 2 mL microtube, add the buffer and glucose volumes indicated in Table 1.
2. Set the dry thermobath to 37 °C. Once the temperature is stable, add 3.3 µL of glucose oxidase to all the tubes and then add 1.2 µL of peroxidase to each tube. Incubate the tubes at 37 °C for 20 min.
3. After incubation, immediately add 750 µL of 50% H₂SO₄ (v/v) to each microtube and let the mixture cool in an ice bath for 2 min. This results in a final volume of 1,500 µL. Finally, transfer the solution to a plastic cuvette and measure the absorbance at 529 nm.

Table 1: Glucose calibration curve for the GOX-H₂SO₄ method. Abbreviations: GOD = glucose oxidase; POD = peroxidase; H₂SO₄ = sulfuric acid. Please click here to download this Table.

5. Testing microbiological samples

1. Obtain the sample by growing bacteria or yeast in a liquid culture medium under specific conditions, including temperature, agitation speed, and incubation time. Once the culture reaches the desired optical density (OD) or cell concentration, collect the culture for further processing.
   NOTE: Pseudomonas reptilivora was cultured at a constant agitation speed of 300 rpm and 28 °C, while Debaryomyces hansenii was cultured with an agitation speed of 200 rpm at 30 °C in an orbital shaker. In the specific case of D. hansenii, edible canola oil was also used as an inducer for lipase production.
2. Clean the work area with a 70% alcohol solution or a suitable cleaning agent according to biosafety protocols. Light a burner to ensure asepsis.
3. Transfer 100 µL of the culture media into a 1.5 mL or 2 mL micro tube using sterile tips.
4. Centrifuge the samples at 7,500 × g for 7 min at 4 °C, or at 10,000 × g for 7 min without temperature control. After centrifugation, carefully remove the supernatant, which contains glucose in solution, and discard the pellet.
5. Use 0.5 µL of the supernatant for glucose concentrations ranging from 1 to 20 g/L, then mix with 749.5 µL of o-dianisidine-buffer, 3.3 µL of GOD, and then 1.2 µL of POD. Incubate the microtubes for 20 min at 37 °C, as detailed in step 4.2 and immediately add 750 µL of 50% H₂SO₄ and let it cool in an ice bath for 2 min.
   1. For concentrations above 20 g/L, perform a 1:1 dilution with sterile H₂Odi before proceeding.
   2. If the supernatant samples were previously frozen at -80 °C or -20 °C, thaw them at room temperature and vortex for 30 s before use. If dilution is necessary, use sterile H₂Odi.
   3. For samples with glucose concentrations above 30 g/L, perform a 1:1 dilution with sterile H₂Odi, resulting in a dilution factor of 2.
6. Calculate the total glucose concentration in each sample using Eq (2) in a spreadsheet program.
        ​(2)
   Where:
   x = concentration of glucose (g/L)     δ = Final reaction volume (0.0015 L)
   y = absorbance       M.S. = amount of sample used*
   NOTE: The b and m values are from the equation that models the trend line of the calibration curve for the method (y = mx + b). *The M.S. value must be based on the amount of sample used to obtain the ratio at the corresponding dilution (i.e., if you take 0.5 µL of the supernatant, M.S. = 0.0000005 L = 0.5 × 10^-6 L); if a dilution factor (DF) is used, multiply the obtained absorbance by the DF.
   For example, if an absorbance of 0.5 is obtained and a DF of 2 was used, the result would be 1.0. Therefore, y = 1.0, and this value should be entered into Eq (2), as previously described.

6. Analytical validation

1. Calculate the analytical validation parameters of the GOX-H₂SO₄ method according to what is established by the National Metrology Center and the Mexican Accreditation Entity¹⁰ (CENAM-ema).
   1. Calculate precision as a percentage of the coefficient of variation or %CV = (S/x) × 100, where x is the sample group mean and S is the standard deviation with an acceptance value of ≤10%.
   2. Determine linearity by fitting the standard curve to linear model R² above 0.995.
   3. Calculate the limit of quantification (LOQ) using the equation: LOQ = yB + 10sB, where yB represents the analyte concentration that produces a signal equivalent to the target signal, and 10sB denotes 10x the standard deviation of the blank
   4. Calculate the systematic error using this equation: Slant = x - m, where x = average of the experimental concentration and m is the theoretical concentration.
   5. Determine accuracy as the degree of agreement between a real value and a theoretical value.
      Recovery% = (CR/CV) × 100
      Where CR is the average of the experimental concentration and CV is the coefficient of variation of the theoretical concentration.

7. Interference by other reducing sugars

1. Evaluate four reducing sugars separately: glucose, fructose, galactose, and xylose from a 0.5 g/L stock solution. Additionally, use trehalose as a negative control. Follow the protocol for all samples and measure the absorbance at 545 and 529 nm.

Results

The spectrophotometric analysis of GOX-H₂SO₄ revealed a single maximum absorbance at 529 nm (λmax) (Figure 2A), with additional absorbance values observed at 545 nm. By analyzing the absorbance values at different glucose concentrations, we obtained a linearity (R²) of 0.9977 for λ529 nm and R² of 0.9967 for λ545 nm (Figure 2B). Linearity, within a given range, refers to the ability to provide results directly proportional to the glucos...

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

List of materials used in this article

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	-	-	-