Precise and High-Throughput Analysis of Bacterial Growth Using a Microplate Reader

Abstract

Source: Kurokawa, M., et al. Precise, High-throughput Analysis of Bacterial Growth. J. Vis. Exp. (2017)

This video demonstrates the high-throughput quantification of bacterial growth using a microplate reader. It outlines the steps involved in culture preparation, optical density measurement, and analysis of growth dynamics.

Protocol

1. Real-time recording of growth
   1. Draw a 96-well plate pictogram (8 × 12 table) to indicate the positions of the inoculated culture samples on the 96-well plate. Print out the table and use it as a reference for the experiment.
      NOTE: The wells located on the edges of the microplate should only contain blank medium because of evaporation.
   2. Place a sterilized 96-well flat-bottom microplate with lid, P-1000, 1000-µL pipette tips, P-200, 200-µL pipette tips, several 1.5-mL microtubes, an 8-multichannel pipette, a sterilized reagent reservoir, room temperature M63, and the Escherichia coli glycerol stocks on a clean bench.
   3. Add approximately 25 mL M63 to the reagent reservoir. Use this reservoir stock for all the following steps.
   4. Add 900 µL M63 to the microtubes in preparation for making serial dilutions.
   5. Thaw the glycerol stock at room temperature. Add 900 µL M63 to the thawed glycerol stock and vortex. This results in a 10-fold dilution of the original glycerol stock.
   6. Transfer 100 µL of the 10-fold dilution to another microtube containing 900 µL of M63 and vortex. This results in a 100-fold dilution.
   7. Repeat step 1.6 until the desired number of dilutions is achieved.
   8. Fill the wells at the edge of the microplate with 200 µL M63 using an 8-channel pipette (P-200).
      NOTE: These wells can be used as the blank.
   9. Load 200 µL of each diluted sample prepared in steps 1.4 - 1.7 to the microplate wells according to the reference table (step 1.1). Vortex the diluted samples prior to loading and load the same sample in multiple wells at varied locations on the plate.
   10. Place the 96-well microplate on the plate reader.
   11. Open "Read Now" in "Task Manager" and choose the program. Click "OK" to start measuring. Save the recording as a new experimental file for data analysis.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dipotassium phosphate	Wako	164-04295
Monopotassium phosphate	Wako	166-04255
Ammonium sulfate	Wako	019-03435
Magnesium sulfate heptahydrate	Wako	138-00415
Thiamine-HCl	Wako	201-00852
Glucose	Wako	049-31165
HCl	Wako	080-01066
Iron (II) sulfate heptahydrate	Wako	094-01082
KOH	Wako	168-21815
Glycerol	Wako	075-00611
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 meter	AS ONE	AS800 / 1-054-01
Pipetman P-200	GILSON	1-6855-05
Pipetman P-1000	GILSON	1-6855-06
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
EPOCH2	BioTek	2014-EP2-002 / EPOCH2T
BIO clean bench	Panasonic	MCV-B131F
Bacterial strains	Strain bank organization; National Bio Resource Project (NBRP) in Japan