Hardy Diagnostics HUGO

MYCOPLASMA MEDIA

Cat. no. G102 Mycoplasma Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. G212 Mycoplasma Agar with Cefoperazone, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. R102 Mycoplasma Broth, 13x100mm Tube, 4ml 20 tubes/box
Cat. no. U187 Mycoplasma Broth, 1L Polycarbonate Bottle, 1000ml 1 each

INTENDED USE

Hardy Diagnostics Mycoplasma Media is recommended for use in isolation of Mycoplasma species associated with bovine mastitis.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Mastitis, or inflammation of the mammary gland, is the most common and most expensive disease of dairy cattle throughout most of the world. Mycoplasmal mastitis was first reported in the U.S. in 1961. (4)

Several species of Mycoplasma can cause mastitis in dairy cows ( M. bovis, M. californium, M. canadense, M. arginini, etc.), with M. bovis being the most frequent and pathogenic bovine mycoplasma in the U.S. (4,8) Mycoplasma spp. are highly contagious and can be spread from cow-to-cow during milking by means of fomites such as milking machines and teat cups, contaminated hands and airborne transmission in poorly ventilated barns. (5,8) Mastitis-causing mycoplasma, M. bovis in particular, are commonly found in the mucous membranes of the respiratory and urogenital tracts of healthy cows, and transfer of the microorganisms from the lungs to the mammary gland can occur. (6-8)
M. bovis is a frequent cause of mastitis, arthritis and, less often, of genital infections. (6)

There is no effective treatment for mycoplasmal mastitis. Control of the disease relies on identification of infected cows through culturing milk samples from all cows in the herd and through segregation of infected animals. (7,8) Although mycoplasmal masitis can be clinically severe, there is rarely any systemic involvement. However, a mastitis problem within a dairy herd can cause major economic loss: deaths due to peracute forms of mastitis, loss of cows through premature culling, and loss of milk production. (4)

Most species of Mycoplasma use either glucose or arginine as their major source of energy and require cholesterol or related sterols for growth. Therefore, special or complex media are required for successful isolation of Mycoplasma spp. (4,6) Hardy Diagnostics' Mycoplasma Media contain inactivated horse serum to provide cholesterol and a source of protein. Thallium acetate and ampicillin, or cefoperazone are also added to inhibit bacterial growth. Mycoplasma Agar is used to detect mycoplasma upon direct culture method, whereas, Mycoplasma Broth is used as an enrichment before subculturing onto Mycoplasma Agar.

FORMULA

Ingredients per liter of deionized water:*

Mycoplasma Broth:
Pancreatic Digest of Casein 7.0gm
Sodium Chloride 5.0gm
Beef Extract 3.0gm
Yeast Extract 3.0gm
Beef Heart Infusion 2.0gm
Thallium Acetate, 10% Solution 5.0ml
Ampicillin 0.25gm
DNA, 0.2% Solution 10.0ml
Horse Serum, Heat Inactivated 150.0ml

In addition,

Mycoplasma Agar contains:
Noble Agar 15.0gm
Cefoperazone (replaces Ampicillin) - G212 0.07gm

Final pH 7.8 +/- 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., media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Sample Collection: All Mycoplasma are sensitive to pH changes in milk. The best recovery rates are achieved when fresh milk samples are plated soon after collection and delivery to the laboratory. Samples can by kept refrigerated for three days or frozen for longer periods before culturing onto Mycoplasma Medium. (8)

Mycoplasma Agar: For direct culture, composite or quarter milk samples are usually streaked over one-half of the media surface, while bulk tank milk samples are streaked over the entire plate surface. (8) Mycoplasma Agar is then incubated in 5-10% CO 2 , at 35-37ºC., for at least 24-36 hours. Plates are held for up to seven days before being reported as negative.

Mycoplasma Broth: For the enrichment broth method, milk samples are directly inoculated into the broth and incubated aerobically with tight caps, at 35-37ºC., for 48 hours. After incubation, the broth is subcultured onto Mycoplasma Agar (Cat. no. G102 or G212) as described above. (5)

INTERPRETATION OF RESULTS

After incubation, Mycoplasma Agar are examined for colonies under low power on a standard microscope or, more effectively, under a stereomicroscope or dissecting microscope. Growth may be visible after three days of incubation, however, 5 to 7 days of incubation are needed for the full development of mycoplasma colonies. Colonies appear transparent, flat, and often resemble a fried egg. Plates should be incubated for 7 to 10 days before negative results are reported. (5,6,8)

LIMITATIONS

Most Mycoplasma isolated from bulk tank milk and cow milk samples are pathogenic, but some milk samples may contain Acholesplasma laidlawii , a common non-pathogenic saprophytic contaminant often found in the dairy environment and on teat skin. Therefore, species identification (accomplished by immunofluorescence or an indirect immunoperoxidase test) of Mycoplasma -like colonies is recommended. (8)

There is no published research for the comparison between direct culture and enrichment culture for bulk tank milk. It can only be assumed that enrichment culture will detect Mycoplasma in small numbers more effectively than direct culture. However, enrichment culture of bulk tank milk may also detect nonpathogenic Mycoplasma in small numbers, especially during rainy weather. (5)

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 Results
Mycoplasma bovis
ATCC ® 25025*
Growth
Staphylococcus aureus
ATCC ® 25923
Partial to complete inhibition
Escherichia coli
ATCC ® 25922*
Partial to complete inhibition

* Recommended QC strains for User Quality Control according to the CLSI document M22, when applicable.

User Quality Control

PHYSICAL APPEARANCE

Mycoplasma Agar and Mycoplasma Broth should appear clear, and pale amber in color. Mycoplasma Agar and Mycoplasma Broth may form a slight precipitate at refrigerated storage that clears upon incubation.

M. bovis growing on Mycoplasma Agar
Microscopic image of Mycoplasma bovis (ATCC ® 25205)colonies growing on Mycoplasma Agar (Cat. no. G102). Incubated in CO 2 for 72 hours at 35ºC.


REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

2. Quinn, P.J., et al. 1994. Clinical Veterinary Microbiology, Wolfe Publishing, London, England.

3. National Mastitis Council, Laboratory Handbook on Bovine Mastitis . 1999. NMC Inc., Madison, WI.

4. Carter, G.R., et al. 1995. Essentials of Veterinary Microbiology , 5th ed. Williams & Wilkins, Philadelphia, PA.

5. UC Davis Veterinary Medicine Extension, Culturing for Mycoplasma .

6. Thurmond M.C., Tyler J.W., Luiz D.M., Holmberg C.A. and Picanso J.P. 1989. The effect of pre-enrichment on recovery of Streptococcus agalactiae , Staphylococcus aureus and Mycoplasma from bovine milk. Epidem Inf. 103:465-474.

7. A Practical Look at Contagious Mastitis . Internet: www.nmconline.org /contmast, 04/18/02.

8. Gonzalez, R.N. Mycoplasma Mastitis in Dairy Cattle: If Ignored, it Can Be a Costly Drain on the Milk Producer . Internet: www.nmconline.org /articles/mycoplasma. Quality Milk Promotion Services, College of Veterinary Medicine, Cornell University, Ithaca, New York.


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

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