Generating Pure Bacterial Cultures in a Liquid Medium

Abstract

Source: Brumfield, K. D. et. al., Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (2017)

This video demonstrates the growth of bacterial cultures in a liquid medium. Shaking promotes aeration and distributes nutrients evenly, supporting exponential growth and resulting in a uniform, metabolically active suspension suitable for downstream applications.

Protocol

1. Maintenance and Growth of Vibrio cholerae Strains

1. Preparation and Use of Frozen Stock Cultures
   1. Pellet 1.8 mL of liquid overnight culture for 2 min by centrifugation (≥8,600 x g) in a sterile 2 mL microcentrifuge tube, remove supernatant, and resuspend the cell pellet in 900 µL of fresh Luria-Bertani (LB) broth by pipetting.
   2. Add 900 µL of sterile 60% (v/v) glycerol to the culture and mix by vortexing.
   3. Transfer the mixture to a sterile 2 mL cryogenic tube and store indefinitely at -80 °C.
   4. To use, remove a small amount of frozen stock using a sterile inoculating loop and streak for single colonies on LB agar plates. Immediately return frozen stock to -80 °C after use to prevent cultures from completely thawing. Incubate plates lid-side down for 12 to 16 h at 37 °C.
2. Growth of Overnight Cultures in Liquid Medium
   1. Streak for single colonies (as per section 1.1.4) from frozen stock onto LB agar plates. Incubate plates lid-side down for 12 to 16 h at 37 °C.
   2. Inoculate 4 mL of liquid LB broth in a sterile 10 mL culture tube with a single colony by touching the surface of the single colony with a sterile applicator stick and transferring the colony into the liquid broth.
   3. Incubate with aeration in a shaker incubator at 225 revolutions per minute (rpm) for 12 to 16 h at 37 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
60% Glycerol	Calbiochem	356352	http://www.emdmillipore.com/US/en/product/Glycerol%2C-Molecular-Biology-Grade---CAS-56-81-5---Calbiochem,EMD_BIO-356352
Agar	Becto, Dickinson and Co.	214030	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch
HCl	Fisher Scientific	A144-212	Corrosive; https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&searchTerm=A144212&searchType=RAPID
Le loop	Decon Labs Inc.	MP190-25	http://deconlabs.com/products/leloop/
Le stab	Decon Labs Inc.	MP186-5	http://deconlabs.com/products/lestab/
NaCl	Fisher Scientific	S271-10	https://www.fishersci.com/shop/products/sodium-chloride-crystalline-certified-acs-fisher-chemical-6/s27110?searchHijack=true&searchTerm=S27110&searchType=RAPID
Petri dishes (100 mm x 15 mm)	Fisher Scientific	FB0875712	https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875712#?keyword=FB0875712
Tryptone	Becto, Dickinson and Co.	211705	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch
Yeast extract	Becto, Dickinson and Co.	212750	http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch