RTF AGAR (MODIFIED CASMAN)

Cat. no. A68 RTF Agar (Modified Casman), 15x100mm Plate, 17ml 10 plates/bag

INTENDED USE

Hardy Diagnostics RTF Agar is recommended for the cultivation and differentiation of fastidious microorganisms, especially Haemophilus spp. by hemolytic reaction.

SUMMARY

Casman, in 1947, developed a medium which allowed for the cultivation of fastidious microorganisms but did not require the use of fresh meat infusion in the basal medium. (6)

The basal medium is composed of beef and yeast extracts and peptones. Beef extract replaces the infusion from fresh meat which enhances pathogenic cocci development. (7,8) Along with peptones, beef extract also provides amino acids and other complex nitrogenous nutrients. Yeast extract serves as a source of the B-complex vitamins. Dextrose, corn starch and purified agar are incorporated into the medium. Dextrose enhances the development of pathogenic cocci. Corn starch and purified agar allow the growth of Neisseria gonorrhoeae without interfering with hemolytic reactions.

RTF Casman is an excellent substitute for Rabbit and/or Horse Blood Agar. In comparison, the sheep blood in RTF has a longer shelf life and is less prone to spontaneous hemolysis and contamination.

The medium is supplemented with sheep blood in order to supply hemin (X-factor), and nicotinamide adenine dinucleotide (NAD or V-factor) which are growth factors required by Haemophilus influenzae . Nicotinamide (also known as niacinamide or nicotinic acid amide) is added to retard the nucleotidase of sheep blood erythrocytes that destroys the V-factor. (8)

RTF Casman Agar is also useful in identifying the various species of Haemophilus by pattern of hemolysis. H. influenzae and H. parainfluenzae will grow but will not exhibit beta-hemolysis. H. hemolyticus and H. parahemolyticus will also grow and will show a zone of beta-hemolysis.

FORMULA

Ingredients per liter of deionized water:*

Yeast Extract 10.0gm
Casein Peptone 5.0gm
Meat Peptone 5.0gm
Sodium Chloride 5.0gm
Beef Extract 3.0gm
Corn Starch 1.0gm
Niacinamide 0.5gm
Dextrose 0.5gm
Sheep Blood 50.0ml
Agar 14.0gm

Final pH 7.3 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.

Method of Use: Prior to inoculation, the medium should be brought to room temperature. Using aseptic technique, inoculate medium then streak to obtain isolated colonies. Incubate the plate in 5-10% CO 2 at 35-37ºC. for 18-24 hours. Examine plates for typical colonial growth and hemolytic reactions.

INTERPRETATION OF RESULTS

Observe for typical colonial growth and morphology. Consult listed references for the identification of colony morphology and further biochemical tests required for identification. (1-4)

Colonies of Haemophilus influenzae appear colorless to gray, are transparent and moist, and produce a characteristic "mousy" odor. Haemophilus haemolyticus and Haemophilus parahaemolyticus are similar in appearance to H. influenzae except that the colonies are surrounded by a zone of beta-hemolysis. Colonies of Neisseria gonorrhoeae appear colorless to grayish-white, are small, translucent, raised, and moist.

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
RTF Agar:
Haemophilus influenzae
ATCC ® 10211
A 24-48hr 35°C CO 2 ** Growth with no hemolysis
Haemophilus parahaemolyticus
ATCC ® 10014
A 24-48hr 35°C CO 2 ** Growth with beta-hemolysis

** Atmosphere of incubation is enriched with 5-10% CO 2 .

USER QUALITY CONTROL

PHYSICAL APPEARANCE

RTF Casman (Modified) should appear opaque, and cherry red in color.

H. influenzae growing on RTF Agar

Haemophilus influenzae (ATCC ® 10211) colonies growing on RTF Agar (Cat. no. A68). Incubated in CO 2 for 48 hours at 35ºC.

H. parahaemolyticus growing on RTF Agar

Haemophilus parahaemolyticus (ATCC ® 10014) colonies growing on RTF Agar (Cat. no. A68). Incubated in CO 2 for 48 hours at 35ºC.

H. influenzae growing on RTF Agar

Haemophilus influenzae (ATCC ® 10211) colonies growing on RTF Agar (Cat. no. A68). Plate shown against backlight to demonstrate lack of beta-hemolysis. Incubated in CO 2 for 48 hours at 35ºC.

H. parahaemolyticus growing on RTF Agar

Haemophilus parahaemolyticus (ATCC ® 10014) colonies growing on RTF Agar (Cat. no. A68). Plate shown against backlight to demonstrate beta-hemolysis. Incubated in CO 2 for 48 hours at 35ºC.

RTF Agar

Uninoculated plate of RTF Agar (Cat. no. A68).

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

6. Casman. 1947. Am. J. Clin. Pathol.; 17:281.

7. Casman. 1942. J. Bacteriol.; 43:33.

8. Casman. 1947. J. Bacteriol.; 53:561.


ATCC is a registered trademark of the American Type Culture Collection.

041116gr