Preparation of Streptococcus mitis Culture for Host-Pathogen Interaction Studies

Abstract

Source: Naji, A., et al. Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans. J. Vis. Exp. (2019).

This video demonstrates the method for culturing Streptococcus mitis under microaerophilic conditions using Todd-Hewitt Yeast Extract, or THY agar medium, to prepare bacterial cultures for host-pathogen interaction studies.

Protocol

1. Preparation of Mitis Group Streptococci for Infection

1. Streak out desired strains of streptococci on the 100 mm x 15 mm THY (Todd-Hewitt Yeast Extract) agar (if plates were stored at 4 °C, pre-warm the plates to 37 °C before streaking the respective strains), then incubate the plates at 37 °C overnight (~18 h) in a candle jar providing a microaerophilic environment for the growth of the streptococci (the streaked plates can be stored for a week at 4 °C).
2. To propagate clinical isolates of streptococci, use tryptic soy blood agar. Incubate the plates at 37 °C in a candle jar overnight (~18 h).
3. The next day, remove the plates from the candle jar and pick isolated colonies using a sterile loop. Inoculate 15 mL sterile conical tubes containing 2 mL of THY broth. To propagate clinical isolates of streptococci, supplement the THY broth with 5% v/v of sheep blood. Close the caps tightly and incubate the tubes at 37 °C under static conditions.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD Bacto Todd Hewitt Broth	Fisher Scientific	DF0492-17-6
Yeast extract	Fisher Scientific	BP1422-500
Disposable Polystyrene Serological Pipettes 10 mL	Fisher Scientific	07-200-574
Disposable Polystyrene Serological Pipettes 25 mL	Fisher Scientific	07-200-575
Falcon Bacteriological Petri Dishes with Lid (35 mm x 10 mm)	Fisher Scientific	08-757-100A
Petri Dish with Clear Lid (60 mm x 15 mm)	Fisher Scientific	FB0875713A
Petri Dishes with Clear Lid (100 mm x 15 mm)	Fisher Scientific	FB0875712
S. mitis ATCC 49456
Agar