SP4 MEDIA

Cat. no. G21 SP4 Agar with Glucose, 15x60mm Plate, 11ml 10 plates/bag
Cat. no. G32 SP4 Agar with Arginine, 15x60mm Plate, 11ml 10 plates/bag
Cat. no. R85 SP4 Broth with Arginine, 13x100mm Tube, 2ml 20 tubes/box
Cat. no. R86 SP4 Broth with Glucose, 13x100mm Tube, 2ml 20 tubes/box
Cat. no. R87 SP4 Broth with Urea, 13x100mm Tube, 2ml 20 tubes/box
Cat. no. U86* SP4 Broth with Glucose, 125ml Polypropylene Bottle, 90ml 1 each
Cat. no. U191* SP4 Broth with Glucose, 4oz. Boston Round, 100ml 1 each
Cat. no. U513* SP4 Broth with Urea, 500ml Polycarbonate Bottle, 500ml 1 each

INTENDED USE

Hardy Diagnostics SP4 Media are recommended for the isolation, differentiation and maintenance of mycoplasma, including M. hominis, M. pneumoniae, and Ureaplasma urealyticum.

*Bottle format not useful for clinical procedures.

SUMMARY

SP4 Media are highly nutritious due to the addition of beef heart infusion, peptone supplemented with yeast extract, CMRL 1066 Medium, and fetal bovine serum. Yeast extract provides diphosphopyridine nucleotides and serum provides cholesterol and an additional source of protein. Specific substrates, such as glucose, arginine, or urea, may be added and used in conjunction with pH to differentiate and select for certain mycoplasma. Amphotericin B, polymyxin B, and penicillin are added to inhibit faster growing contaminants. Phenol Red is added to broth media as a pH indicator.

FORMULA

Ingredients per 690ml of deionized water:*

SP4 Broth, Base**:
Pancreatic Digest of Casein 10.0gm
Pancreatic Digest of Gelatin 5.0gm
PPLO Broth without CV 3.5gm
Polymyxin B 50.0mg
Amphotericin B 5.0mg
Fetal Bovine Serum 170.0ml
CMRL 1066 Medium (10X) 50.0ml
Yeast Extract 35.0ml
Yeastolate 10% 20.0ml
Penicillin 1,000,000U

Note: In addition to the above ingredients;


SP4 Broth with Glucose** contains:
Glucose 5.0gm/L

SP4 Broth with Arginine** contains:
Arginine 5.0gm/L

SP4 Broth with Urea** contains:
Urea 1.0gm/L

SP4 Agar Media contains:
Agar 9.0gm/L

**All broth media contain:
Phenol Red 18.0mg/L

Final pH at 25°C:

SP4 Broth with Arginine
7.0 +/- 0.2

SP4 Agar with Glucose
SP4 Broth with Glucose
7.4 +/- 0.2

SP4 Broth with Urea
6.0 +/- 0.2

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8°C 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: Specimen/swab should be placed in a tightly sealed transport container with sufficient transport medium to prevent drying. Samples should be taken directly to the laboratory and processed as soon as possible. If there is to be a delay in culturing, specimens should be refrigerated. For long-term storage, or if cultures cannot be set up within 24 hours of collection, freeze specimens at -70°C in a cryopreservative such as Brucella with Glycerol (Cat. no. D04). Do not freeze at temperatures warmer than -70°C Consult listed references for more information regarding cultivation and isolation of mycoplasmas.

SP4 Agar Media

1. If using a liquid inoculum, add 0.1ml of liquid to the agar surface and distribute evenly by rocking the plate back and forth or spreading the inoculum with a sterile bent glass rod. If culturing directly from a swab, roll the swab over the agar surface and streak for isolation. Increased recovery may be enhanced by diluting and plating the specimen serially up to 10-3. Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma.(1)

2. Apply tape or similar sealant over agar plates to restrict dehydration.

3. Incubate plates in 5-10% CO2 at 35°C for up to 30 days.

4. Invert plates and examine microscopically at 100x magnification.

5. Observe for typical tiny "fried-egg" colonies or finely granular colonies with a berry-like appearance that penetrate the agar surface. Colonies range from 20-300µm in diameter.

SP4 Broth Media

1. Inoculate broth with 0.1ml of transport media containing swab. Alternatively, broth may be inoculated at a 1:10 ratio with blood or CSF. Tissue specimens may be inoculated by placing several minced fragments directly into the broth. Increased recovery may be enhanced by diluting and plating the specimen serially up to 10-3. Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma.(1)

2. Tighten cap and incubate inoculated broth aerobically at 35°C for up to 30 days.

3. Examine tubes daily for a change in the pH, detectable by a color change in the medium.

4. As soon as a pH shift is noted, subculture the broth to an appropriate agar medium. Consult listed references for more information regarding cultivation and isolation of mycoplasma.

INTERPRETATION OF RESULTS

SP4 Agar Media

Mycoplasma appear as tiny "fried-egg" colonies or as finely granular colonies with a berry-like appearance that penetrate the agar surface. Colonies range from 20-300µm in diameter.

SP4 Broth Media

Growth of glucose fermenting mycoplasma will cause SP4 with Glucose to turn from its original red color to yellow.

Ureaplasmal growth in SP4 with Urea will turn the medium from yellow-orange to red.

Growth of arginine using mycoplasma in SP4 with Arginine results in the medium changing from orange to red.

LIMITATIONS

Occasional breakthrough of bacterial growth may occur on these media. Similarities of L-form bacteria and mycoplasma organisms on the agar medium may cause some confusion because they both exhibit "fried egg" colonies that penetrate the agar surface. L-form colonies tend to be larger and demonstrate a rougher surface. Many L-forms will revert back to the bacterial form if passed to a penicillin-free media.

Increased recovery may be enhanced by diluting and plating the specimen serially up to 10-3. Diluting the specimen minimizes the effect of bacterial inhibitors on the growing mycoplasma.(1)

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
The following organisms are inoculated to all media listed below:
Escherichia coli
ATCC® 25922
B 24hr 35°C Aerobic Inhibited
Staphylococcus aureus
ATCC® 25923
B 24hr 35°C Aerobic Inhibited
Candida albicans
ATCC® 10231
B 24hr 35°C Aerobic Inhibited
SP4 Agar with Glucose:
Mycoplasma pneumoniae
ATCC® 15531
K 3-20 days 35°C CO2** Growth; appears as tiny "fried-egg" colonies or finely granular berry-like colonies that penetrate the agar surface, media changes from its original salmon to yellow
Mycoplasma arginini
ATCC® 23838
K 3-20 days 35°C CO2** Growth; appears as tiny "fried-egg" colonies or finely granular berry-like colonies that penetrate the agar surface, media will not have a color change
SP4 Agar with Arginine:
Mycoplasma hominis
ATCC® 23114
K 1-4 days 35°C CO2** Growth; appears as tiny "fried-egg" colonies or finely granular berry-like colonies that penetrate the agar surface, media changes from its original red to pink
SP4 Broth with Arginine:
Mycoplasma hominis
ATCC® 23114
K 1-4 days 35°C Aerobic Growth; color change in medium from its original orange to red, subculture to appropriate agar medium
SP4 Broth with Urea:
Ureaplasma urealyticum
ATCC® 27618
K 1-4 days 35°C Aerobic Growth; color change in medium from its original yellow-orange to red, subculture to appropriate agar medium
SP4 Broth with Glucose:
Mycoplasma pneumoniae
ATCC® 29085
K 3-20 days 35°C Aerobic Growth; color change in medium from its original red to yellow, subculture to appropriate agar medium
Mycoplasma arginini
ATCC® 23838
K 3-20 days 35°C Aerobic Growth, slight turbidity; no color change; subculture to appropriate agar medium

** Atmosphere of incubation is enriched with 5-10% CO2

User Quality Control

Physical Appearance

M. hominis and U. urealyticum growing on SP4 Agar

Microscopic image of Mycoplasma hominis (ATCC® 23114) and Ureaplasma urealyticum (ATCC® 27618) colonies growing on SP4 Agar with Glucose (Cat. no. G21). Incubated in CO2 for 72 hours at 35°C. All colonies are mycoplasma except the dark one, which is ureaplasma.

REFERENCES

1. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

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

3. 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.

4. Cumitech 19; Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections. American Society for Microbiology, Washington D.C., August, 1984.

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

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

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


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

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