We are grateful for the partial donations from the Tecnol&#243;gico Nacional de M&#233;xico in the 2023 and 2024 Call for Proposals for Scientific Research, Technological Development (16432.23-P y 19545.24-P). We would like to thank the National Council for Humanities, Sciences, and Technologies (CONAHCyT) for the scholarship received (No. 832315) during the doctoral studies (IHRH), and the participation and collaboration of Wendolyne Monroy-Mart&#237;nez is gratefully acknowledged. Figure 1 was created with BioRender.com.

1. Solution preparation
<ol>
	<li>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 &#176;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.</li>
	<li>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 &#176;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.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Preparation of enzymes
<ol>
	<li>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 &#176;C. 
	NOTE: The solution can be stored for up to 2 months at -20 &#176;C.</li>
	<li>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 &#181;L, 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 &#181;L &#215; 28.5 U)/12,970 U = 3.29 &#181;L&#160; &#160; (1) 
	NOTE: For more accurate pipetting, the value can be rounded to 3.3 &#181;L.</li>
	<li>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 &#176;C.</li>
	<li>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 &#181;L 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 &#181;L &#215; 1.25 U)/1,551 U = 1.208 &#181;L 
	NOTE: For more accurate pipetting, the value was rounded to 1.20 &#956;L.</li>
</ol>
3. Glucose standard
<ol>
	<li>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 &#181;L of the stock solution and mix it with 500 &#181;L of H2Odi to achieve a final volume of 1 mL. 
	NOTE: This dilution process is essential for creating accurate standard solutions.</li>
</ol>
4. Standard curve preparation
<ol>
	<li>Using a new 2 mL microtube, add the buffer and glucose volumes indicated in Table 1.</li>
	<li>Set the dry thermobath to 37 &#176;C. Once the temperature is stable, add 3.3 &#181;L of glucose oxidase to all the tubes and then add 1.2 &#181;L of peroxidase to each tube. Incubate the tubes at 37 &#176;C for 20 min.</li>
	<li>After incubation, immediately add 750 &#181;L 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 &#181;L. Finally, transfer the solution to a plastic cuvette and measure the absorbance at 529 nm.</li>
</ol>
Table 1: Glucose calibration curve for the GOX-H2SO4 method. Abbreviations: GOD = glucose oxidase; POD = peroxidase; H2SO4 = sulfuric acid. <a href="https://www.jove.com/files/ftp_upload/67126/67126_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
5. Testing microbiological samples
<ol>
	<li>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 &#176;C, while Debaryomyces hansenii was cultured with an agitation speed of 200 rpm at 30 &#176;C in an orbital shaker. In the specific case of D. hansenii, edible canola oil was also used as an inducer for lipase production.</li>
	<li>Clean the work area with a 70% alcohol solution or a suitable cleaning agent according to biosafety protocols. Light a burner to ensure asepsis.</li>
	<li>Transfer 100 &#181;L of the culture media into a 1.5 mL or 2 mL micro tube using sterile tips.</li>
	<li>Centrifuge the samples at 7,500 &#215; g for 7 min at 4 &#176;C, or at 10,000 &#215; g for 7 min without temperature control. After centrifugation, carefully remove the supernatant, which contains glucose in solution, and discard the pellet.</li>
	<li>Use 0.5 &#181;L of the supernatant for glucose concentrations ranging from 1 to 20 g/L, then mix with 749.5 &#181;L of o-dianisidine-buffer, 3.3 &#181;L of GOD, and then 1.2 &#181;L of POD. Incubate the microtubes for 20 min at 37 &#176;C, as detailed in step 4.2 and immediately add 750 &#181;L of 50% H2SO4 and let it cool in an ice bath for 2 min.
	<ol>
		<li>For concentrations above 20 g/L, perform a 1:1 dilution with sterile H2Odi before proceeding.</li>
		<li>If the supernatant samples were previously frozen at -80 &#176;C or -20 &#176;C, thaw them at room temperature and vortex for 30 s before use. If dilution is necessary, use sterile H2Odi.</li>
		<li>For samples with glucose concentrations above 30 g/L, perform a 1:1 dilution with sterile H2Odi, resulting in a dilution factor of 2.</li>
	</ol>
	</li>
	<li>Calculate the total glucose concentration in each sample using Eq (2) in a spreadsheet program. 
	<img alt="Equation 11" src="/files/ftp_upload/67126/67126eq11.jpg" style="margin: auto;" />&#160; &#160; &#160;&#8203;(2) 
	Where: 
	x = concentration of glucose (g/L)&#160; &#160; &#160;&#948; = Final reaction volume (0.0015 L) 
	y = absorbance&#160; &#160; &#160; &#160;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 &#181;L of the supernatant, M.S. = 0.0000005 L = 0.5 &#215; 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.</li>
</ol>
6. Analytical validation
<ol>
	<li>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).
	<ol>
		<li>Calculate precision as a percentage of the coefficient of variation or %CV = (S/x) &#215; 100, where x is the sample group mean and S is the standard deviation with an acceptance value of &#8804;10%.</li>
		<li>Determine linearity by fitting the standard curve to linear model R2 above 0.995.</li>
		<li>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</li>
		<li>Calculate the systematic error using this equation: Slant = x - m, where x = average of the experimental concentration and m is the theoretical concentration.</li>
		<li>Determine accuracy as the degree of agreement between a real value and a theoretical value. 
		Recovery% = (CR/CV) &#215; 100 
		Where CR is the average of the experimental concentration and CV is the coefficient of variation of the theoretical concentration.</li>
	</ol>
	</li>
</ol>
7. Interference by other reducing sugars
<ol>
	<li>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.</li>
</ol>

Enzymatic Bergmeyer Glucose Quantification for Microbial and Yeast Analysis

<ol><li>Huang, W. C., Tang, I. C. Bacterial and yeast cultures: process characteristics, products, and applications. Bioprocessing for value-added products from renewable resources. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHuang%2C+WC+%22New+Technologies+and+Applications%22">New Technologies and Applications</a>. , Elsevier. 185-223 (2007).</li>
<li>Hernandez-Lopez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+reducing+sugars+based+on+the+qualitative+technique+of+Benedict%5BTitle%5D)+AND+%22ACS+Omega%22%5BJournal%5D)">Quantification of reducing sugars based on the qualitative technique of Benedict.</a> ACS Omega. 5 (50), 32403-32410 (2020).</li>
<li>Miller, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+dinitrosalicylic+acid+reagent+for+determination+of+reducing+sugar%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Use of dinitrosalicylic acid reagent for determination of reducing sugar.</a> Anal Chem. 31 (3), 426-428 (1959).</li>
<li>Deshavath, N. N., Mukherjee, G., Goud, V. V., Veeranki, V. D., Sastri, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pitfalls+in+the+3%2C5-dinitrosalicylic+acid+%28DNS%29+assay+for+the+reducing+sugars%3A+interference+of+furfural+and+5-hydroxymethylfurfural%5BTitle%5D)+AND+%22Int+J+Biol+Macromol%22%5BJournal%5D)">Pitfalls in the 3,5-dinitrosalicylic acid (DNS) assay for the reducing sugars: interference of furfural and 5-hydroxymethylfurfural.</a> Int J Biol Macromol. 156, 180-185 (2020).</li>
<li>De Abreu, J. R., Santos, C. D. D., De Abreu, C. M. P., Corrêa, A. D., De Oliveira Lima, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sugar+fractionation+and+pectin+content+during+the+ripening+of+guava+cv.+Pedro+Sato%5BTitle%5D)+AND+%22Food+Sci+Technol%22%5BJournal%5D)">Sugar fractionation and pectin content during the ripening of guava cv. Pedro Sato.</a> Food Sci Technol. 32 (1), 156-162 (2020).</li>
<li>Dubois, M., Gilles, K. -A., Hamilton, J. K., Roberts, P. A., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+colorimetric+method+for+the+determination+of+sugars%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A colorimetric method for the determination of sugars.</a> Nature. 168 (4265), 167(1951).</li>
<li>Gonzalez, N. M., Fitch, A., Al-Bazi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+RP-HPLC+method+for+determination+of+glucose+in+Shewanella+oneidensis+cultures+utilizing+1-phenyl-3-methyl-5-pyrazolone+derivatization%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Development of a RP-HPLC method for determination of glucose in Shewanella oneidensis cultures utilizing 1-phenyl-3-methyl-5-pyrazolone derivatization.</a> PLoS ONE. 15 (3), e0229990(2020).</li>
<li>Galant, A. L., Kaufman, R. C., Wilson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose%3A+Detection+and+analysis%5BTitle%5D)+AND+%22Food+Chem%22%5BJournal%5D)">Glucose: Detection and analysis.</a> Food Chem. 188, 149-160 (2015).</li>
<li>Bergmeyer, H. U. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBergmeyer%2C+HU+%22Methods+of+enzymatic+analysis%22">Methods of enzymatic analysis</a>. 2, Elsevier Inc. (2012).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Gu%C3%ADa+t%C3%A9cnica+sobre+trazabilidad+e+incertidumbre+en+las+mediciones+anal%C3%ADticas+que+emplea+la+t%C3%A9cnica+de+espectrofotometr%C3%ADa+de+ultravioleta-visible%22">Guía técnica sobre trazabilidad e incertidumbre en las mediciones analíticas que emplea la técnica de espectrofotometría de ultravioleta-visible</a>. , CENAM-EMA. (2008).</li>
<li>Yuen, V. G., McNeill, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+the+glucose+oxidase+method+for+glucose+determination+by+manual+assay+and+automated+analyzer%5BTitle%5D)+AND+%22J+Pharmacol+Toxicol+Methods%22%5BJournal%5D)">Comparison of the glucose oxidase method for glucose determination by manual assay and automated analyzer.</a> J Pharmacol Toxicol Methods. 44 (3), 543-546 (2000).</li>
<li>Hansen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specificity+of+glucose+oxidase+reaction+and+interference+with+quantitative+glucose+oxidase-peroxidase-O-dianisidine+method%5BTitle%5D)+AND+%22Scand+J+Clin+Lab+Investig%22%5BJournal%5D)">Specificity of glucose oxidase reaction and interference with quantitative glucose oxidase-peroxidase-O-dianisidine method.</a> Scand J Clin Lab Investig. 14 (6), 651-655 (1962).</li>
<li>Kumar, V., Gill, K. D. Estimation of blood glucose levels by glucose oxidase method. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKumar%2C+V+%22Basic+concepts+in+clinical+biochemistry%3A+A+practical+guide%22">Basic concepts in clinical biochemistry: A practical guide</a>. , Springer eBooks. 57-60 (2018).</li>
</ol>

The authors declare no conflicts of interest

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...

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&#39;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 &#946;-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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67126/67126fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/67126/67126fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.

<table><tbody><tr><td>1.5 mL microtube</td><td>Eppendorf</td><td>-</td><td>-</td></tr><tr><td>2.0 mL microtube</td><td>Eppendorf</td><td>-</td><td>-</td></tr><tr><td>CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Meyer</td><td>471-34-1</td><td>Calcium Carbonate</td></tr><tr><td>D-Glucose</td><td>Meyer</td><td>50-99-7</td><td>D-Glucose&amp;nbsp;</td></tr><tr><td>Drybath</td><td>FisherScientific</td><td>11-718-4</td><td>Serial 911NO251. Block WxDxH mm/in: 124 x 76 x 39 / 4.9 x 3.0 x 1.5</td></tr><tr><td>Glucose oxidase&amp;nbsp;</td><td>Sigma Aldrich</td><td>G6125-50KU</td><td>Enzyme powder</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O</td><td>-</td><td>-</td><td>Deionized water</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Meyer</td><td>7664-93-9</td><td>Sulfuric acid</td></tr><tr><td>HCl</td><td>Meyer</td><td>7647-01-0</td><td>Hydrochloridric acid&amp;nbsp;</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Meyer</td><td>7758-11-4</td><td>Dipotassium phosphate</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Meyer</td><td>7778-77-0</td><td>Monopotassium phosphate</td></tr><tr><td>KOH</td><td>Meyer</td><td>1310-58-3</td><td>Potassium hydroxide</td></tr><tr><td>Lambda 35</td><td>PerkinElmer</td><td>-</td><td>Spectrophotometer</td></tr><tr><td>Microtube centrifuge</td><td>-</td><td>-</td><td>-</td></tr><tr><td>o-Dianisidine dihydrochloride</td><td>Sigma Aldrich</td><td>D3252</td><td>Chromogenic</td></tr><tr><td>Peroxidase</td><td>Sigma Aldrich</td><td>P8125-50KU</td><td>Enzyme powder</td></tr><tr><td>pH-meter</td><td>Hanna&amp;nbsp;</td><td>1131</td><td>Hanna 1170</td></tr><tr><td>Sodium acetate</td><td>Meyer</td><td>127-09-3</td><td>Meyer</td></tr><tr><td>Weighing spatulas&amp;nbsp;</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

bergmeyer glucose quantification for microbiological samples

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 &#176;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.

The spectrophotometric analysis of GOX-H2SO4 revealed a single maximum absorbance at 529 nm (&#955;max) (Figure&#160;2A), with additional absorbance values observed at 545 nm. By analyzing the absorbance values at different glucose concentrations, we obtained a linearity (R&#178;) of 0.9977 for &#955;529 nm and R&#178; of 0.9967 for &#955;545 nm (Figure 2B). Linearity, within a given range, refers to the ability to provide results direc...

Bergmeyer Glucose Quantification for Microbiological Samples

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.

The glucose concentration is a key indicator of cellular metabolism and could indicate the total metabolism rate, an aberration in glucose metabolism, ...

bergmeyer-glucose-quantification-for-microbiological-samples

Research

JoVE Journal

Biochemistry

409 Views.  Tecnológico Nacional de México/Instituto Tecnológico de Morelia. Het onderzoek evalueert de glucoseconcentratie in microbiële monsters met behulp van kwantificeringstechnieken van bacterie- en gistfilters. Het doel is om de nauwkeurigheid van glucosemassagram in microbiologische onderzoeken te verbeteren. Uitdagingen zijn onder meer het nauwkeurig kwantificeren van lage glucoseconcentraties in complexe microbiële monsters en het onderscheid maken tussen microbiële en omgevingsbronnen van glucose in gemengde culturen.

Video: Bergmeyer glucosekwantificering voor microbiologische monsters

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Bioengineering

Enhanced Reproducibility and Precision of High-Throughput Quantification of Bacterial Growth Data Using a Microplate Reader

This study aimed to develop a repeatable, reliable, high-throughput protocol to monitor bacterial growth in 96-well plates and analyze the maximum growth rate. The growth curves and maximum growth rates of two bacterial species were determined. Issues including (i) lid condensation, (ii) pathlength correction, (iii) inoculation size, (iv) sampling time interval, and (v) spatial bias were investigated. The repeatability of the protocol was assessed with three independent technical replications, with a standard deviation of 0.03 between the runs. The maximum growth rates of Bacillus mycoides and Paenibacillus tundrae were determined to be&#160;(mean&#160;&#177; SD) 0.99 h&#8722;1 &#177;&#160; 0.03 h&#8722;1 and 0.85 h&#8722;1 &#177; 0.025 h&#8722;1, respectively. These bacteria are more challenging to monitor optically due to their affinity to clump together. This study demonstrates the critical importance of inoculation size, path length correction, lid warming, sampling time intervals, and well-plate spatial bias to obtain reliable, accurate, and reproducible data on microplate readers. The developed protocol and its verification steps can be expanded to other methods using microplate readers and high-throughput protocols, reducing the researchers&#39; innate errors and material costs.

Here, a high-throughput protocol is presented to measure growth data, including growth curves, growth rate, and maximum growth rate. The protocol was verified and validated using two biofilm-producing bacteria. The results and approach applied in this study can be expanded to other high-throughput protocols using microplate readers.

This study aimed to develop a repeatable, reliable, high-throughput protocol to monitor bacterial growth in 96-well plates and analyze the maximum growth ...

Environment

Modeling Methanotroph Niches – The Gradient Syringe Model Ecosystem

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists' understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs' natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.

Author Spotlight: Designing Simple and Inexpensive Techniques to Grow Methane-Oxidizing Bacteria in the Laboratory

A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Immunology and Infection

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Biology

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Assays for Qualitative and Quantitative Assessment of Mouse Urine Glucose Concentrations

This video demonstrates a procedure for collecting mouse urine and assessing its glucose levels. To evaluate the glucose level, immerse a test strip coated with enzymes and a chromogenic dye into the sample, initiating a color change that indicates the glucose levels. For quantifying urine glucose levels, introduce a urine sample to a multiwell plate with escalating glucose concentrations, add the reaction mixture, and incubate. Measure the absorbance to create a standard curve and compute the glucose levels based on the sample absorbance.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Spot ...

Bergmeyer Glucose Quantification for Microbiological Samples

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