Subculturing Sulfite-Reducing Anaerobic Bacteria from an Agar Culture

Samenvatting

Source: Anwar, Z., et al. Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples. J. Vis. Exp. (2019).

In this video, sulfite-reducing bacteria are cultured in a sulfite-containing agar medium, where they grow anaerobically and reduce sulfite to hydrogen sulfide, forming black precipitates with iron ions. Black colonies are isolated from the agar depth using aseptic techniques and transferred to fresh medium for continued anaerobic incubation.

Protocol

NOTE: Clostridium perfringens is considered a biosafety hazard level 2 (BSL2) organism. Although not all food samples will contain C. perfringens, all cultured samples should be treated as such. All proper precautions and personnel protective equipment (PPE) should be worn at all times. Decontaminate all material prior to disposal.

1. Prepare modified RPM (Rapid perfringens medium)

1. Combine 30 g/L fluid thioglycolate medium, 60 g/L gelatin, 5 g/L peptone, 5 g/L glucose, 5 g/L potassium phosphate dibasic, 3 g/L yeast extract, 1.5 g/L sodium chloride, 0.5 g/L ferrous sulfate in deionized water until evenly dispersed. We typically make 1 L batch....

Resultaten

Figure 1: Schematic of TSC agar sandwich technique.

RPM media containing the C. perfring.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
D-Cycloserine	Sigma-Aldrich	C6880
Dextrose	Sigma Life Science	D9434-250G
Disposable Transfer Pipets	any brand		Select one with slim tip like Thermo Scientific Disposable Transfer Pipets 137116M/EMD
DNeasy Blood & Tissue Kits	Qiagen	69504	Note: numerous DNA and plasmid extractions kits were evaluated, this kit gave the most desirable results.
Dry Incubator	any brand
Fluid thioglycolate medium	Remel	R453452
Gelatin from porcine skin	Sigma Life Science	G1890-500G
Iron (II) sulfate heptahydrate	Sigma Life Science	F8633-250 G
Lysozyme from chicken egg white	Sigma-Aldrich	L6876	Needed for DNA extraction, not provided in kit
Parafilm or plastic wrap	any brand
Peptone from casein and other animal proteins	Sigma-Aldrich	70173-100G
Perfringens Agar Base (TSC + SFP)	Oxoid	CM0587	Make TSC agar according to instructions
Potassium phosphate dibasic	Sigma-Aldrich	P2222-100G
Sodium chloride	Sigma-Aldrich	S-7653
Sterile Cell Scraper	any brand
Sterile cell Spreader	any brand
Sterile petri dishes	any brand
Yeast extract	Sigma-Aldrich	70161-100G