Precise, High-throughput Analysis of Bacterial Growth

Summary

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Abstract

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Introduction

Microbiological studies often start with the culture of bacterial cells and the assessment of the bacterial growth curves, which represent a fundamental phenomenon of bacterial physiology^1,2,3. Basic culture principles are widely available in the published research literature and textbooks because bacterial culture is a fundamental methodology. At the bench level, substantial attention has traditionally been focused on optimizing growth media and culturing conditions, but controlling the growth rate, which would likely provide even greater understanding of microbial physiolog....

Protocol

1. Preparing the Growth Medium

NOTE: The chemical composition of minimal medium M63 is as follows: 62 mM K₂HPO₄, 39 mM KH₂PO₄, 15 mM (NH₄)₂SO₄, 1.8 µM FeSO₄, 15 µM thiamine-HCl, 0.2 mM MgSO₄, and 22 mM glucose. M63 is made by mixing three stock solutions: Five X solution, 20% glucose and MgSO₄ thiamine solution. Store all solutions at 4 °C.

1. Preparing the Five X solution
   1. To prepare FeSO₄ solution, use an electrical pipette and a disposable serological pipette to add ddH₂O to a 50-mL centr....

Results

The described method provides a means to capture dynamic bacterial growth in a continuous, high-throughput manner by utilizing a 96-well format reader that takes multiple optical density measurements at various time intervals (from minutes to hours to days). The growth curves of an assortment of E. coli strains expressing various genomes can be precisely acquired in a single experiment (Figure 1A). In comparison to the described method, the tradition.......

Discussion

Critical steps in the protocol include the preparation of a common stock of exponentially growing cells and the replication of the same samples in multiple wells at various positions on the microplate. Previously, microbiologists started the culture from an overnight pre-culture. While this method may reduce the lag time of bacterial growth, it is difficult to achieve reproducible growth curves. As shown in Figure 2, the independent measurements using the common glycerol stocks resulted in n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
K2HPO4	Wako	164-04295
KH2PO4	Wako	166-04255
(NH4)2SO4	Wako	019-03435
MgSO4-7H2O	Wako	138-00415
Thiamine-HCl	Wako	201-00852
glucose	Wako	049-31165
HCl	Wako	080-01066
Iron (II) sulfate heptahydrate (FeSO4-7H2O)	Wako	094-01082
KOH	Wako	168-21815
Glycerol	Wako	075-00611
Centrifuge tube (50 mL, sterilized)	WATSON	1342-050S
Pipette Tips, 200 µL	WATSON	110-705Y
Pipette Tips, 1,000 µL	WATSON	110-8040
Microtube (1.5 mL)	WATSON	131-715C
8 multichannel-pipette	WATSON	NT-8200
PASORINA STIRRER	AS ONE	2-4990-02
Glass cylinder (200 mL)	AS ONE	1-8562-07
Precision pH mater	AS ONE	AS800 / 1-054-01
Pipetman P-200	GILSON	1-6855-05
Pipetman P-1000	GILSON	1-6855-06
Disposable Serolocical Pipettes (10 mL)	SANPLATEC	SAN27014
Disposable Serolocical Pipettes (25 mL)	SANPLATEC	SAN27015
Microtube stand	BM Bio	801-02Y
Vortex	BM Bio	BM-V1
Corning Costar 96-well microplate with lid (Flat bottom, Clear)	Sigma-Aldrich	Corning, 3370
Corning Costar reagent reservoir (50 mL)	Sigma-Aldrich	Corning, 4870
Stericup GV PVDF (250 mL, 0.22 µM)	Merck Millipore	SCGVU02RE
Pipet-Aid XP	DRUMMOND	4-000-101
Bioshaker (BR-23UM MR)	TAITEC	0053778-000
Disposal cell (1.5 mL)	Kartell	1938 / 2-478-02
DU 730 Life Science UV/Vis Spectrophotometer	Beckman Coulter	A23616
EPOCH2	BioTek	2014-EP2-002 / EPOCH2T
Beaker (500 mL)	IWAKI	82-0008
BIO clean bench	Panasonic	MCV-B131F
Glass tubes	NICHIDEN RIKA GLASS	P-10M~P-30 /101019
Silicone rubber stoppers	ShinEtsu Polymer	T-19
Bacterial strains	Strain bank organization; National Bio Resource Project (NBRP) in Japan