LYSOSTAPHIN DIFFERENTIATION DISKS

Cat. no. Z112 Lysostaphin Differentiation Disks 50 disks/vial

INTENDED USE

HardyDisk™ Lysostaphin Differentiation Disks are used to rapidly differentiate Staphylococcus spp. and Micrococcus spp. based on lysostaphin resistance.

SUMMARY

Susceptibility to lysostaphin is a rapid test method in differentiating staphylococci from micrococci. Other differentiation tests, such as glucose fermentation, bacitracin or furazolidone susceptibility tests require at least 24 hours incubation. In contrast, the lysostaphin disk test is a 2.5 hour test.

The peptidoglycan in the cell walls of most Staphylococcus contain an interpeptide bridge consisting of glycine-rich peptides. Lysostaphin is an endopeptidase that cleaves these peptide linkages, rendering the cells susceptible to osmotic lysis (lysostaphin susceptible) (2,4) . The interpeptide bridge of Micrococcus does not contain glycine. Since the presence of glycine is essential for the action of lysostaphin, the Micrococcus cells are not affected and are lysostaphin resistant. (2,4)

FORMULA

Each HardyDisk™ Lysostaphin Differentiation Disk is a filter paper disk (0.25 inch in diameter) containing 20ug of lysostaphin.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. Product should not be used if there are any signs of deterioration, discoloration, or if the expiration date has passed. Protect from light, excessive heat, and moisture.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for the primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

1. Allow disks to equilibrate to room temperature before opening vial.

2. Using a pure 18-24 hour culture, prepare a heavy suspension (equivalent in density to a McFarland 2.0 standard) in 2ml of sterile saline.

3. Transfer 1ml of the bacterial suspension into a second test tube.

4. Add one lysostaphin disk to one of the bacterial suspensions and shake vigorously. Use the second suspension as a negative control.

5. Incubate both suspensions aerobically at 35-37ºC. for 2.5 hours in an incubator, or for 2 hours in a 35-37ºC. waterbath. Do not disturb suspension before 2 hours.

6. After incubation, compare the turbidity of the bacterial suspension with the lysostaphin disk against the control suspension.

INTERPRETATION OF RESULTS

Positive Reaction: Complete clearing of the bacterial suspension or marked decrease in its turbidity as compared with the negative control suspension, indicates susceptibility to lysostaphin ( Staphylococcus spp.).

Negative Reaction: The suspension remains turbid and does not clear, indicates resistance to lysostaphin ( Micrococcus spp.).

LIMITATIONS

Certain Staphylococcus species (i.e., S. aureus, S. simulans, S. cohnii, and S. xylosus ) are more susceptible to lysostaphin than others (e.g., S. hominis, S. saprophyticus , and S. haemolyticus ); making standardization and interpretation of this test sometimes difficult. (4)

Glycine is an important part of the staphylococcal cell wall and is essential for the action of lysostaphin. (2,4,5) For optimal results with the lysostaphin test, organisms should be grown on a beef peptone-based medium (which are high in glycine) such as Tryptic Soy Agar, Nutrient Agar or Chocolate Agar. (4,5)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, culture media, test tubes, McFarland 2.0 standard, 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
Staphylococcus aureus
ATCC ® 25923
D 2.5hr 35ºC Aerobic Positive (lysostaphin susceptible); complete clearing of bacterial suspension or marked decrease in its turbidity as compared with the negative control
Staphylococcus epidermidis
ATCC ® 12228
D 2.5hr 35ºC Aerobic Positive (lysostaphin susceptible); complete clearing of bacterial suspension or marked decrease in its turbidity as compared with the negative control
Micrococcus luteus
ATCC ® 49732
D 2.5hr 35ºC Aerobic Negative (lysostaphin resistant); bacterial suspension remains turbid

User Quality Control

PHYSICAL APPEARANCE

HardyDisk™ Lysostaphin Differentiation Disks are 0.25 inch (in diameter) filter paper disks with the letters LYS ID printed on both sides, and should appear white in color.

Lysostaphin Susceptible

Showing positive reaction (lysostaphin susceptible).
A 1mL aliquot of a heavy Staphylococcus aureus (ATCC ® 25923) suspension (McFarland 2.0) in saline was incubated aerobically for 2.5 hours at 35ºC. with a Lysostaphin Differentiation Disk (Cat. no. Z112). The loss of turbidity was indicative of a positive reaction (lysostaphin susceptible), as compared to the negative control (LEFT).

Lysostaphin Resistant

Showing negative reaction (lysostaphin resistant).
A 1mL aliquot of a heavy Micrococcus luteus (ATCC ® 49732) suspension (McFarland 2.0) in saline was incubated aerobically for 2.5 hours at 35ºC. with a Lysostaphin Differentiation Disk (Cat. no. Z112). No loss of turbidity was indicative of a negative reaction (lysostaphin resistant) as compared to the negative control (LEFT).

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

5. Gunn, B.A. 1981. Journal of Clinical Microbiology, Vol.14; 195-200, no.2.

6. Baker, John S. 1984. Journal of Clinical Microbiology, Vol.19; 875-879, no.6.


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

031016gr