TSN Agar

Intended Use

TSN (Trypticase™ Sulfite Neomycin) Agar is used for the selective isolation of Clostridium perfringens.

Summary and Explanation

TSN Agar was developed by Marshall et al. as a medium that could achieve rapid enumeration of Clostridium perfringens.¹ The formulation is a modification of Mossel’s medium for the enumeration of sulfite-reducing clostridia in foods.² The 46°C temperature of incubation for TSN Agar permits specific and quantitative results.

Principles of the Procedure

Neomycin and polymyxin are inhibitory for gram-negative enteric bacilli. Neomycin at the concentration employed at least partially inhibits C. bifermentans. The relatively high incubation temperature of 46°C renders the medium highly specific for C. perfringens. The colonies are black due to the formation of ferric sulfide as a result of the reduction of the sulfite.

User Quality Control

Identity Specifications
TSN Agar
Dehydrated Appearance: Fine, homogeneous, may contain a large amount of minute
                                     to small dark particles.
Solution:                        4.0% solution, soluble in purified water upon boiling. Solution is
                                     light to medium, yellow to tan, clear to moderately hazy.      
Prepared Appearance:     Light to medium, yellow to tan, clear to moderately hazy.
Reaction of 4.0%
Solution at 25°C:            pH 7.2 ± 0.2
Cultural Response

TSN Agar
Prepare the medium per label directions. Inoculate and incubate at 46 ± 1°C anaerobically for 18-24 hours.

Formula

TSN Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................... 15.0 g
Sodium Sulfite.............................................................. 1.0 g
Neomycin Sulfate......................................................... 0.05 g
Polymyxin Sulfate........................................................ 0.02 g
Yeast Extract............................................................... 10.0 g
Ferric Citrate................................................................. 0.5 g
Agar........................................................................... 13.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 40 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Dispense and autoclave at 118°C for 12 minutes. Do not overheat.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use on the day of preparation. Inoculate tubes or plates of the medium by stabbing deep tubes or streaking plates with the test specimen. Incubate containers for 18-24 hours at 46 ± 0.1°C in an anaerobic atmosphere (BD GasPak™ EZ anaerobic system or equivalent).

Expected Results

C. perfringenes produces black colonies at 46°C. C. perfringens and C. bifermentans produce black colonies on TSN Agar at 37°C; however, C. bifermentans is inhibited at 46°C.¹

Limitations of the Procedure

*Store at 2-8° C.

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