Cystine Tryptic Agar Composition, Preparation, Principle, Uses

Cystine tryptic agar (CTA) which is also referred to as cystine trypticase agar was created by Vera as an semi-solid growth medium to identify and maintain the health of microorganisms for long time period of time when stored at suitable temperatures. It’s used for determination of the motility of bacteria and, when combined with carbohydrate, is involved in the fermentation process and the differentiation of microorganisms that are fastidious, such as Neisseria and streptococci, as well as pneumococci and non-spore-forming anaerobes.

Composition of Cystine Tryptic Agar

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

Principle of Cystine Tryptic Agar

The medium is made up of peptone and cystine to provide the essential nutrients needed to promote the growth of microorganisms that are fastidious. CTA Media supplemented with a one percent concentration of a specific carbohydrate is utilized to identify the fermentation processes. Carbohydrate fermentation can be detected through the visible change in color of the medium as a result of the inclusion in the indicator color pH the phenol red. As the carbohydrate is broken down by the organism organic acids are created and the medium is acidified. 

The acid created through carbohydrate fermentation results in an increase in pH which causes a change in color within the medium that ranges from pink-red to yellow. But, the peptone in the medium gets destroyed by the bacteria in the medium and produces products that are alkaline in the pH range. The indicator for phenol red change from orange to red in the event that the acid created by the carbohydrate fermentation process is greater than the alkaline products of peptone degrading. 

The change in color with the red phenol occurs at pH 6.8 which is close to the original level of pH in the medium. In addition, adding agar the medium permits observation of the motility along the stabbing line of inoculation. The motile organisms extend out from the stab line, and cause clouds or turbidity throughout the medium, whereas non-motile organisms only grow within the stab line and leave the medium unclouded.

Intended Use

Cystine Tryptic Agar and CTA Medium (Cystine Trypticase(tm) Agar Medium) serve for the care of microorganisms and also for determination of motility in bacterial cells and, when combined with carbohydrate, to aid in the fermentation process of fastidious microorganisms, i.e., Neisseria, streptococci, pneumococci as well as nonsporeforming anaerobes.

Preparation of Cystine Tryptic Agar

Difco™ Cystine Tryptic Agar

1. Suspend 28.5 grams from the mixture in 1 liter of water purified. Mix thoroughly.
2. Then, with constant stirring, simmer for one minute, allowing the powder.
3. Autoclave at a temperature not exceeding 118degC for 15 minutes.
4. For the preparation of fermentation media, mix 5-10 grams of carbohydrate prior to autoclaving. dissolve the medium in 900 mL water, autoclave, then aseptically add 100mL of 5–10% solution of carbohydrate that is sterile.
5. Samples of the final product to determine its effectiveness using stable, typical control cultivars.

BBL™ CTA Medium™

1. Suspend 28.5 grams of powder into 1L of water purified. Incorporate the carbohydrate (0.5 to 1.0 percent) as desired. Adjust the pH as needed. Mix thoroughly.
2. Stir frequently and boil for one minute or until the solution is fully dissolved.
3. Tube and autoclave at no more than 118degC for fifteen minutes. Then cool in an upright position.
4. Keep at the room temperatures. Avoid refrigerating unless you are inside screw-capped, tightly sealed tubes.
5. Samples of the final product for their performance by using stable, common control culture.

Plating Procedure of Cystine Tryptic Agar

1. Lock caps loosen caps, boil, tighten caps, and let cool before using.
2. Remove new colonies off the top of a suitable medium, e.g., Chocolate Agar but not from a selective primarily isolated plate.
3. In the case of tests on fermentation with members of the Genus Neisseria only the the medium tube is inoculated.
4. For facultative organisms like streptococci or strictly anaerobic organisms, you can inoculate by stabbing the middle of the medium using an inoculating needle that is approximately 1/2 of the depth of medium.
5. Repeat this for each tube to be inoculated.
6. Incubate at 35 + 2 degC, using loose caps either aerobically or anaerobically based on the organisms that are being studied; Neisseria should be incubated with tightly fitting caps, particularly in the case of tubes that must be kept inside a CO2 incubator as well as with caps that are loose in an incubator that is not CO2. 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). Some strains might require an incubation period of up to 48-72 hours.
7. Many of the most snobbly organisms, like Neisseria, Pasteurella, streptococci, Brucella, corynebacteria and vibrios can be easily cultivated in the medium, with no additional serum, carbon dioxide or other enrichments are required.
8. To speed up growth, and also for fast fermentation, anaerobic culture prefer to be grown within the presence of carbon dioxide, as well as nitrogen or hydrogen. Certain strict anaerobes do not develop or are not able to grow when there is no carbon dioxide.

Result Interpretation of Cystine Tryptic Agar

Fermentation

• Positive reaction: the development of the color of yellow in the inoculated portion (stab lines) in the media.
• Negative reaction: deep red or red-pink color within the medium. The development of a deep red-orange hue in the media means that the carbohydrate is not been utilized and the degradation of peptone has occurred.

Motility

• Positive test: Cloudy or turbid growth is seen beyond the line of vaccination.
• Negative test: Growth is just along the stagger lines, leaving the area clear.

Storage of Cystine Tryptic Agar

This product should be stored between 2 and 8 degrees Celsius. A proper storage environment prolongs the shelf life and quality of the product. Overheating and freezing can cause extreme degradation of the media. The media must not be used past the expiration dates. The expiration dates are for not-opened tubes that are properly maintained.

Uses of Cystine Tryptic Agar

• It is used to aid in the monitoring and identification of gonococcus as well as other bacteria.
• It is utilized for the analysis of the carbohydrate fermentation process by microorganisms that are fastidious.
• It can also be utilized to detect bacteria’s motility. Additionally, the base is an anchoring medium for maintaining of fastidious microorganisms.

Limitations of Cystine Tryptic Agar

• It is suggested that biochemical, immunological mass spectrometry or molecular tests be conducted on pure-cultured colonies to ensure complete identification.
• Small amounts of acid are produced by Neisseria because the organisms use carbohydrates for oxidation.
• Incubation for a long time can cause changes in pH indicators or abnormal lactose/sucrose reactions Neisseria pathogens.
• Aerobic incubation is required since incubation in CO 2 could result in incorrect results.
• CTA requires a large inoculum. Insufficient inoculum could result in incorrect results.
• Inoculating the bottom of the tube inadequate inoculation can result in low acidity, making it difficult to interpret test results.
• Utilizing peptone results in the creation of alkaline by-products. Longer incubation can lead to an inverse reaction in which alkaline by-products disguise acid by-products resulting through the utilization of carbohydrate.
• Because some strains of meningococci, primarily sulfonamide-resistant strains, do not produce acid from maltose, repeated subcultures to non-inhibitory media may be required to restore their maltose-utilizing capability.
• Certain strains of gonococci require extra compounds not offered by CTA Media formulations and will consequently not develop with CTA Media.