CTA Agar

Intended Use

CTA Agar is primarily used for carbohydrate fermentation tests with corynebacteria and especially for differentiation of C. diphtheriae from related species.
Cystine Tryptic Agar and CTA Medium (Cystine Trypticase™ Agar Medium) are for the maintenance of microorganisms, as well as for the detection of bacterial motility and, with added carbohydrate, for fermentation reactions of fastidious microorganisms; i.e., Neisseria, pneumococci, streptococci and nonsporeforming anaerobes.

Summary and Explanation

CTA Agar
CTA Medium™, a semi-solid formulation, was developed by Vera and is widely used for fermentation and motility determinations by a wide variety of microorganisms.¹ CTA Agar is the solid form of CTA Medium and, when employed as a plated medium and used in conjunction with BBL™ Taxo™ carbohydrate discs, is useful in the speciation of Corynebacterium isolates of medical importance.² Supplemented with carbohydrates and prepared as slants, it is used for the differentiation of Neisseria species.³
CTA Medium
This formulation was developed by Vera as a simple semi-solid medium for the identification and maintenance of the gonococcus and other bacteria.¹ Without carbohydrates, it can be used for maintenance of cultures, including fastidious organisms, for extended periods when stored at appropriate temperatures. With the appropriate carbohydrate, it is recommended for the differentiation of fastidious organisms by means of fermentation reactions. In the semisolid agar, acid reactions are easily detected because the acid formed is not immediately diffused throughout the entire culture as in a broth. When no fermentable carbohydrate is present, most cultures show an alkaline shift. Motility can be readily detected in the semisolid medium.² Stab cultures show growth out from the line of inoculation. Nonmotile organisms grow in the inoculated area, while the surrounding area remains clear. BBL™ Taxo™ carbohydrate discs can be selected and added, as needed, to tubes of plain CTA Medium when fermentation reactions are to be determined. For clostridia, bacilli, common micrococci, enteric bacilli and other organisms not generally considered to be nutritionally fastidious, the use of Trypticase Agar Base is recommended instead of this formulation.

Principles of the Procedure

CTA Agar
CTA Agar utilizes peptone as a carbohydrate-free source of nutrients. Inorganic salts are included in order to supply essential ions. Phenol red is an indicator of pH changes in the medium surrounding the Taxo carbohydrates discs, which are applied to the surface of inoculated plates.
CTA Medium
The medium contains cystine and peptone to supply the nutrients necessary to support the growth of fastidious microorganisms. Carbohydrate fermentation is detected by a visible color change of the medium due to the incorporation of the pH indicator dye, phenol red. When the carbohydrate present is metabolized by the organism, organic acids are produced and the medium becomes acidified. However, the peptone present in the medium is also degraded by the bacteria present and yields substances that are alkaline in pH.
The phenol red indicator changes from reddish-orange to yellow when the amount of acid produced by carbohydrate fermentation is greater than the alkaline end products of peptone degradation. The color change with phenol red occurs around pH 6.8, near the original pH of the medium.

User Quality Control

Identity Specifications
BBL™ CTA Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       4.0% solution, soluble in purified water upon boiling.
                                   Solution is medium, orange-red to red-rose,
                                   clear to slightly hazy.
Prepared Appearance:    Orange-red to red-rose, slightly hazy.
Reaction of 4.0%
Solution at 25°C:           pH 7.3 ± 0.2
Cultural Response
BBL™ CTA Agar
Prepare the medium per label directions. Inoculate with fresh cultures and incubate at 35 ± 2°C for 18-48 hours.

Identity Specifications
Difco™ Cystine Tryptic Agar
Dehydrated Appearance: Pink, free-flowing, homogeneous.
Solution:                       2.85% solution, soluble in purified water upon
                                    boiling. Solution is red, very slightly opalescent.
Prepared Appearance:     Red, very slightly opalescent.
Reaction of 2.85%
Solution at 25°C:            pH 7.3 ± 0.2
Cultural Response
Difco™ Cystine Tryptic Agar
Prepare the medium per label directions without and with 0.5% dextrose. Inoculate tubes with fresh broth cultures (Neisseria from chocolate agar) by straight stab and incubate with caps tightened at 35 ± 2°C for 18-48 hours (up to 72 hours if necessary).

Identity Specifications
BBL™ CTA Medium™
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       2.85% solution, soluble in purified water upon
                                    boiling. Solution is light to medium, red-orange
                                   to orange-red to red-rose, clear to slightly hazy.
Prepared Appearance:    Red-orange to red-rose, slightly hazy.
Reaction of 2.85%
Solution at 25°C:           pH 7.3 ± 0.2
Cultural Response
BBL™ CTA Medium™
Prepare the medium per label directions (without added carbohydrate). Inoculate tubes with fresh broth cultures (Neisseria from chocolate agar) by straight stab and incubate at 35 ± 2°C under appropriate atmospheric conditions (Neisseria with tightened caps) for 72 hours.

Formula

BBL™ CTA Agar
Approximate Formula* Per Liter
L-Cystine...................................................................... 0.5 g
Pancreatic Digest of Casein........................................... 20.0 g
Agar........................................................................... 14.0 g
Sodium Chloride............................................................ 5.0 g
Sodium Sulfite............................................................... 0.5 g
Phenol Red................................................................. 17.0 mg
Difco™ Cystine Tryptic Agar
Approximate Formula* Per Liter
Tryptose..................................................................... 20.0 g
L-Cystine...................................................................... 0.5 g
Sodium Chloride............................................................ 5.0 g
Sodium Sulfite.............................................................. 0.5 g
Agar............................................................................ 2.5 g
Phenol Red................................................................ 17.0 mg
BBL™ CTA Medium™
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................... 20.0 g
L-Cystine...................................................................... 0.5 g
Sodium Chloride............................................................ 5.0 g
Sodium Sulfite.............................................................. 0.5 g
Agar............................................................................ 2.5 g
Phenol Red................................................................ 17.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

CTA Agar
1. Suspend 40 g of the powder in 1 L purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 118°C for 15 minutes.
4. If desired, add 2 drops of sterile rabbit serum per tube prior to solidification in order to enhance the recovery of C. diphtheriae.
5. Test samples of the finished product for performance using stable, typical control cultures.
CTA Medium
Difco™ Cystine Tryptic Agar 1. Suspend 28.5 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 not over 118°C for 15 minutes.
4. To prepare fermentation medium, add 5-10 g of carbohydrate before autoclaving or dissolve medium in 900 mL water, autoclave, and aseptically add 100 mL sterile 5-10% carbohydrate solution.
5. Test samples of the finished product for performance using stable, typical control cultures.
BBL™ CTA Medium™
1. Suspend 28.5 g of the powder in 1 L of purified water. Add carbohydrate (0.5 to 1.0%) if desired, and adjust the pH if necessary. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute or until solution is complete.
3. Tube and autoclave at not over 118°C for 15 minutes. Cool in the upright position.
4. Store at room temperature. Do not refrigerate unless in tightly closed, screw-capped tubes.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

CTA Agar
Inoculate a pure culture of the organism onto the surface of the plated medium using a swab technique to inoculate the entire surface. Taxo carbohydrate discs are then applied to the agar surface using no more than four discs per plate.>
Incubate plates for 18-48 hours at 35 ± 2°C in an aerobic atmosphere.
CTA Medium
1. Loosen caps, boil, tighten caps and cool before use.
2. Remove fresh colony growth from the surface of a suitable culture medium; e.g., Chocolate Agar, not from a selective, primary isolation plate.3
3. For fermentation tests with members of the genus Neisseria, only the surface of the tubed medium is inoculated.
For facultative organisms, such as streptococci and strictly anaerobic organisms, inoculate by stabbing the center of the medium with an inoculating needle to about 1/2 the depth of the medium.
4. Repeat for each tube to be inoculated.
5. Incubate at 35 ± 2°C with loosened caps aerobically or anaerobically depending upon the organisms being tested; Neisseria should be incubated with tight caps⁴ especially if tubes must be incubated in a CO₂ incubator,^5-6 or with loose caps in a non-CO₂ incubator.^7-8 Examine periodically up to 24 hours for growth (turbidity), evidence of motility, and acid production in carbohydrate-containing medium (yellow color in upper layer of medium). A few strains may require incubation for up to 48-72 hours.⁹
6. Many fastidious organisms, including Neisseria, Pasteurella, streptococci, Brucella, corynebacteria and vibrios, may be readily cultivated in this medium, no added carbon dioxide, serum or other enrichments being required.
7. For more rapid growth and also for more rapid fermentation reactions, anaerobic cultures preferably should be incubated in the presence of carbon dioxide as well as hydrogen or nitrogen. Some strict anaerobes fail to grow or grow poorly in the absence of carbon dioxide.

Expected Results

CTA Agar
Typical diphtheria bacilli ferment dextrose and maltose, but not sucrose.
Typical carbohydrate reactions for selected corynebacteria on CTA Agar plates containing Taxo carbohydrate discs are as follows:
Current schemes recommended for the identification of medically significant corynebacteria include carbohydrate utilization as part of the testing regimen. Appropriate references should be consulted for a discussion of the other tests, which enable a definitive identification of the above-named organisms as well as other clinically important species of corynebacteria.^4,5
CTA Medium
A yellow color either in the upper one-third or throughout the medium indicates acid production; i.e., fermentation of the carbohydrate. A red (alkaline) to orange (neutral) color indicates that the carbohydrate has not been degraded and that only the peptone has been utilized. Inoculated medium (without carbohydrate) also exhibits a red to orange color.
Motile organisms show growth out from the line of stabinoculation. Nonmotile organisms only grow along the stab line with the surrounding agar remaining clear.

Limitation of the Procedure

1. CTA requires a heavy inoculum.¹⁰
2. Prolonged incubation may lead to changes in pH indicator or abnormal lactose/sucrose reactions with Neisseria pathogens.^11-12
3. Neisseria species usually produce acid only in the area of stabs (upper third). If there is a strong acid (yellow color) throughout the medium, a contaminating organism may be present. If in doubt about a tube containing a Neisseria species, a Gram stain and oxidase test should be performed on the growth.¹⁰
*Store at 2-8° C. †QC testing performed according to USP/EP/JP performance specifications.

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