Cystine Heart Agar

Intended Use

Cystine Heart Agar is used with hemoglobin for cultivating Francisella tularensis and without enrichment for cultivating gram-negative cocci and other microorganisms.

Summary and Explanation

Francisella tularensis was first described in humans in 1907.¹ Several media formulations were employed to isolate this microorganism. Initial formulations contained egg or serum
and were difficult to prepare. Edward Francis,² who dedicated his career to the study of this organism, reported that blood dextrose cystine agar was a satisfactory medium for cultivating this fastidious pathogen. Shaw3 added 0.05% cystine and 1% dextrose to Heart Infusion Agar for the cultivation of F. tularensis.
While experimenting with Francis’ blood dextrose cystine agar, Rhamy4 added hemoglobin to Cystine Heart Agar to develop a satisfactory medium for growth of F. tularensis.
Cystine Heart Agar, also known as Cystine Glucose Blood agar, is the historical medium of choice for isolating F. tularensis.²

Principles of the Procedure

Infusions from beef heart, peptone and L-cystine provide nitrogen, vitamins and amino acids in Cystine Heart Agar. Dextrose is a carbon source. Sodium chloride maintains the osmotic balance and agar is the solidifying agent. Enrichment with 2% hemoglobin provides additional growth factors. Without enrichment, Cystine Heart Agar supports excellent growth of gram-negative cocci and other pathogenic microorganisms. Rabbit blood and antimicrobial agents can be added to this medium.⁵

User Quality Control

Identity Specifications
Cystine Heart Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                       5.1% solution, soluble in purified water upon
                                    boiling. Solution is light to medium amber, very
                                    slightly to slightly opalescent, may have fine precipitate.
Prepared Appearance:     Plain – Light to medium amber, slightly opalescent,
                                    may have a fine precipitate.
                                    With Hemoglobin – Chocolate, opaque.
Reaction of 5.1%
Solution at 25°C:            pH 6.8 ± 0.2
Cultural Response
Cystine Heart Agar
Prepare the medium per label directions without and with hemoglobin. Incubate inoculated medium at 35 ± 2oC aerobically for 66-72 hours. Incubate Neisseria meningitidis under increased CO₂.

*Minimally, one strain of F. tularensis should be used for performance testing. F. tularensis ATCC 29684 can be
substituted for BD Diagnostics strain 16223.

Formula

Cystine Heart Agar
Approximate Formula* Per Liter
Beef Heart, Infusion from 500 g................................. 10.0 g
Proteose Peptone..................................................... 10.0 g
Dextrose................................................................. 10.0 g
Sodium Chloride........................................................ 5.0 g
L-Cystine.................................................................. 1.0 g
Agar....................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Precautions

Francisella tularensis is a Biosafety Level 2 pathogen that can be transmitted by aerosols or by penetration of unbroken skin.⁵ Wearing of gowns, gloves and masks is advocated for laboratory staff handling suspected infectious material.⁶

Directions for Preparation from Dehydrated Product

Enriched Medium
1. Suspend 10.2 g of the powder in 100 mL of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes. Cool to 50-60°C.
4. Add 100 mL sterile 2% hemoglobin solution and mix well.
Use:
• Hemoglobin Solution 2%; or,
• Prepare a 2% hemoglobin solution as follows: Place 2 g of hemoglobin powder in a dry flask. Add 100 mL of cold purified water while agitating vigorously. Continue intermittent agitation for 10-15 minutes until solution is complete. Autoclave at 121°C for 15 minutes. Cool to 50-60°C.
5. Dispense into sterile Petri dishes or tubes.
6. Test samples of the finished product for performance using
stable, typical control cultures.
Unenriched Medium
1. Suspend 51 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. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

1. Inoculate and streak specimens as soon as possible. For a complete discussion on the inoculation and identification of Francisella, consult appropriate references.
2. Overgrowth by contaminating organisms can be reduced by incorporating 100-500 units penicillin per mL into the medium.¹

Expected Results

Refer to appropriate references and procedures for results.

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