Measuring the Migration and Biofilm Formation of Various Bacteria

Summary

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

Abstract

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923 and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Introduction

The mammalian host represented by the human body contains a large number of microorganisms. These microorganisms are widely distributed in the mouth, digestive tract, intestine, and blood of the host and have different effects on the host's health. The oral cavity is host to a plethora of microbes that can modulate the host's susceptibility to infections. Microbes such as Streptococci (e.g., S. mitis/oralis, S. pseudopneumoniae, and S. infantis) and Prevotella spp. colonize the oral cavity, forming a multispecies biofilm on the tongue surface causing bad breath and functioning as a microbial reservoir for microbial ....

Protocol

NOTE: See the Table of Materials for details about all the materials and equipment used in this protocol. Use aseptic techniques to avoid contamination.

1. Bacterial recovery to get a single colony

1. Prepare solid agar plates.
   1. Prepare Terrific Broth (TB) medium containing agar, with each liter of liquid containing 11.8 g of tryptone, 23.6 g of yeast extract, 9.4 g of K₂HPO₄, 2.2 g of KH₂PO₄, and.......

Discussion

We isolated and identified five species of bacteria by sequential methods. The growth of bacteria has minimal nutrient requirements: the minimal medium-a medium containing only inorganic salts, a carbon source, and water. Although the bacteria in the experimental group were found on MH solid plates, we used half-dose MH medium to verify the chemotaxis of the bacteria and achieved good results. However, we also performed control experiments using minimal medium. M9 basic medium was used in the experiment (see Tabl.......

Materials

Name	Company	Catalog Number	Comments
Chemical/Solution
1% crystal violet dye solution	Solarbio	G1062	100 mL
Agar	Sigma-Aldrich	V900500	Used to obtain semi-solid plates, 20 g
Centrifuge tube	Corning	430790	15 mL
Fluid thioglycollate medium	Kinghunt	K0001	29.3 g
Mueller Hinton II Broth medium	Solarbio	NO.11865	100 g
Petri dishes	Bkman	B-SLPYM90-15	Plastic Petri dishes, circular, 90 mm x 15 mm
Potassium Chloride	VETEC	WXBC4493V	0.2 g
Potassium Dihydrogen Phosphate	aladdin	04-11-7758	0.24 g
Sodium chloride	Macklin	S805275	8.0 g
Sodium phosphate dibasic	aladdin	7558-79-4	1.44 g
Terrific Broth medium	Solarbio	LA2520	200 g
Kits/ Equipment
Anaerobic incubator	Longyue
Biochemical incubator	Blue pard	LRH-70
Microplate reader	Spark
Tanon 5200multi imaging system	Tanon	5200CE
Thermostatic water bath	Jinghong	DK-S28