Imaging Flow Cytometry to Study Microbial Autoaggregation

Summary

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Abstract

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.

The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Introduction

Bacterial autoaggregation is considered a primary step in biofilm formation. In this process (sometimes also called autoagglutination or flocculation), bacteria of the same type form multicellular clumps that eventually settle at the bottom of culture tubes or attach to their target tissue or surface¹.

Autoaggregation is a widely observed phenomenon and has been shown so far in Gram-negative pathogens such as the opportunistic pathogen Acinetobacter baumannii², the dental pathogen Aggregobacter actinomycetemcomitans³, and the emerging pathogen Burk....

Protocol

The .ast file, with the template for Lacticaseibacillus rhamnosus GG (LGG) as an example, is provided in Supplementary Coding File 1.

1. Media preparation

1. Prepare Lactobacilli MRS broth following the manufacturer's instructions (55 g in 1 L of deionized water) and Lactobacilli MRS agar plates with 1.5% (w/v) agar (see Table of Materials). After autoclave sterilization, the medium is ready to use directly or can be stored at 4 °C.
2. Prepare 50% (15 g in 1 L of deionized water) Tryptic soy broth (TSB) (see Table of Materials), TSB supplem....

Results

The results demonstrate that this method can easily measure the differences in autoaggregation in response to dietary sugars in LAB bacteria. By separating individuals from aggregates, the method allows calculating the percentage of the population of the aggregation events out of all events in response to fermentable or non-fermentable sugars from the diet. Additionally, it was possible to measure if there are differences in the mean size of the aggregate's population between treatments.

The r.......

Discussion

Flow cytometry is a widely used method for quantifying fluorescence intensities in eukaryotic cells, but it may not provide accurate measurements for bacterial cells due to their larger size or small aggregates. These factors can significantly impact the precise quantification of autoaggregation and the basal level of aggregate formation in different conditions. To address this, imaging flow cytometry (IFC) was employed to gain a better resolution of how carbohydrates affect the aggregation of probiotic bacteria

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
14 mL culture tubes	Falcon	352051
15 mL centrifuge tube	Falcon	352096
Bacto Agar	Baeton,Dickinson and Company	214010
Bacto Typtic Soy Broth	Baeton,Dickinson and Company	211825
D-(+)-Glucose	Sigma	G7021-1KG
D-(+)-Raffinose pentahydrate	Sigma	83400-25G
Difco Lactobacilli MRS broth	Baeton,Dickinson and Company	288130
EASY-LOCK MICROPR. 1.5 mL (Eppendorf)	FL medical	23053
IDEAS Software	Amnis/EMD Millipore	N/A	Details available at: https://www.merckmillipore.com/INTL/en/20150212_144049?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1
ImageStream X Mark II	Amnis/EMD Millipore	N/A	Details available at: https://www.merckmillipore.com/INTL/en/20150121_205948?ReferrerURL=https%3A%2F%2Fwww.google.com%2F
MOPS, 3-(N-morpholino)propanesulfonic acid	Fisher bioreagents	BP308-500
Potassium phosphate dibasic	Fisher Scientific, 174.18 g/mol	BP363-1
Potassium phosphate monobasic	Sigma, 136.09 g/mol	P0662-500G