Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies

Human gut microbiota composition, structure, and density varies among individuals mainly due to age, eating habits, gender, and other factors. Although human microbial biodiversity varies between individuals with respect to species composition, variable fecal bacterial load may introduce an artifact...

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Bibliographic Details
Main Author:	Troncoso S., Ana V.
Other Authors:	Gonzales, Miguel
Format:	Tesis
Language:	Inglés
Published:	Zamorano: Escuela Agrícola Panamericana 2024
Subjects:	bacterial density fluorophore normalization microbiota
Online Access:	https://bdigital.zamorano.edu/handle/11036/7767

id	ZAMORANO7767
record_format	dspace
spelling	ZAMORANO77672024-01-11T10:58:52Z Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies Troncoso S., Ana V. Gonzales, Miguel Lindemann, Stephen Luna, Ligia bacterial density fluorophore normalization microbiota Human gut microbiota composition, structure, and density varies among individuals mainly due to age, eating habits, gender, and other factors. Although human microbial biodiversity varies between individuals with respect to species composition, variable fecal bacterial load may introduce an artifact during in vitro fermentations, resulting in variable fermentation outcomes in terms of metabolite production and composition and structure of the microbiota. Multiple methods have been developed for the estimation of bacterial density; however, these tend to require considerable time to obtain results and/or expensive equipment, making their use difficult for measurement of bacterial density in inoculum before in vitro experiments. Here, we describe a convenient and cost-effective method to estimate bacterial density from diluted stool by staining bacteria with a fluorophore and measuring its fluorescence intensity. For our preliminary test, Escherichia coli cells were stained with SYBR Gold, SYBR Green, and DAPI to identify the fluorophore with the highest fluorescence intensity. SYBR Gold was selected as the best fluorophore and the method was optimized by maintaining the sample wash step and fluorophore concentration. Fluorescence intensity measurements were collected on a microplate reader to assess staining efficiency and cell numbers were estimated using a hemacytometer. Finally, the method was used to estimate the bacterial density in diluted feces obtained from two donors to later use this method to test the effect of normalizing the fecal bacterial load before in vitro fermentation. 2024-01-11T16:55:47Z 2024-01-11T16:55:47Z 2023 Thesis https://hdl.handle.net/11036/7767 eng Copyright Escuela Agrícola Panamericana, Zamorano https://creativecommons.org/licenses/by-nc-nd/3.0/es/ application/pdf Zamorano: Escuela Agrícola Panamericana
institution	Universidad Zamorano
collection	Biblioteca Digital Zamorano
language	Inglés
topic	bacterial density fluorophore normalization microbiota
spellingShingle	bacterial density fluorophore normalization microbiota Troncoso S., Ana V. Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies
description	Human gut microbiota composition, structure, and density varies among individuals mainly due to age, eating habits, gender, and other factors. Although human microbial biodiversity varies between individuals with respect to species composition, variable fecal bacterial load may introduce an artifact during in vitro fermentations, resulting in variable fermentation outcomes in terms of metabolite production and composition and structure of the microbiota. Multiple methods have been developed for the estimation of bacterial density; however, these tend to require considerable time to obtain results and/or expensive equipment, making their use difficult for measurement of bacterial density in inoculum before in vitro experiments. Here, we describe a convenient and cost-effective method to estimate bacterial density from diluted stool by staining bacteria with a fluorophore and measuring its fluorescence intensity. For our preliminary test, Escherichia coli cells were stained with SYBR Gold, SYBR Green, and DAPI to identify the fluorophore with the highest fluorescence intensity. SYBR Gold was selected as the best fluorophore and the method was optimized by maintaining the sample wash step and fluorophore concentration. Fluorescence intensity measurements were collected on a microplate reader to assess staining efficiency and cell numbers were estimated using a hemacytometer. Finally, the method was used to estimate the bacterial density in diluted feces obtained from two donors to later use this method to test the effect of normalizing the fecal bacterial load before in vitro fermentation.
author2	Gonzales, Miguel
author_facet	Gonzales, Miguel Troncoso S., Ana V.
format	Tesis
author	Troncoso S., Ana V.
author_sort	Troncoso S., Ana V.
title	Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies
title_short	Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies
title_full	Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies
title_fullStr	Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies
title_full_unstemmed	Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies
title_sort	development of a fluorescence-based method to estimate bacterial density in in vitro fermentation studies
publisher	Zamorano: Escuela Agrícola Panamericana
publishDate	2024
url	https://bdigital.zamorano.edu/handle/11036/7767
work_keys_str_mv	AT troncososanav developmentofafluorescencebasedmethodtoestimatebacterialdensityininvitrofermentationstudies
_version_	1808119450664173568

Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies

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