UriStar-AST™

Cat. no. US50

UriStar-AST™, 6oz. Plastic Screw-cap Jar, Five-tip Ring

50 rings/jar

INTENDED USE

Hardy Diagnostics UriStar-AST™ is a ring of susceptibility disks that is recommended for semi-quantitative in vitro susceptibility testing by the agar diffusion test procedure (Kirby-Bauer) for rapidly growing bacterial pathogens isolated from urine cultures.

SUMMARY

Agar diffusion methods employing dried filter paper disks impregnated with specific concentrations of antimicrobial agents were developed in the 1940s. In order to eliminate or minimize variability in the testing, Bauer, et al., developed a standardized procedure in which Mueller Hinton Agar was selected as the test medium.^(1,2)

Various regulatory agencies and standards-writing organizations subsequently published standardized reference procedures based on the Kirby-Bauer method. Early procedures were published by the U.S. Food and Drug Administration (FDA) and the World Health Organization (WHO) and were later adopted as a consensus standard by the Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS).^(3-7) These standards are updated frequently and the latest CLSI document revisions should be consulted for current recommendations.^(6-9)

Hardy Diagnostics UriStar-AST™ contains five of the most commonly used antibiotic disks used for antimicrobial susceptibility testing on urine isolates. The 6mm disks are linked by a ring structure which enables easy handling of the disks without the need for antibiotic disk dispensers. Each antibiotic disk is impregnated with standard concentrations of antimicrobials and is labeled as described below. Antimicrobials include Amoxicillin/Clavulanic Acid (Augmentin), Cephalothin, Ciprofloxacin, Nitrofurantoin, and Trimethoprim/Sulfamethoxazole (SXT).

FORMULA

UriStar-AST™ rings are 45mm in diameter. Each ring contains five, high-quality 6mm disk tips, with precise amounts of antimicrobics isolated by a hydrophobic barrier. The disk tips are clearly marked on one side with letters designating the agent. The concentration of the drug is listed in the table below.

Antibiotic	CLSI Code	Concentration	UriStar-AST™ Disk Tip Label
Amoxicillin/Clavulanic Acid (Augmentin)	AmC-30	20ug/10ug	AmC
Cephalothin	CF-30	30ug	CF
Ciprofloxacin	CIP-5	5ug	CIP
Nitrofurantoin	Fm-300	300ug	F/M
Trimethoprim/Sulfamethoxazole	SXT	1.25ug/23.75ug	SXT

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2 to 8 degrees C. away from direct light. Rings should not be used if there is any sign of deterioration, discoloration, contamination, or if the expiration date has passed.

Return unused rings to the refrigerator as soon as possible after use. Store the rings in the original container containing the desiccant pack.

The expiration date applies to the product in its intact packaging when stored as directed.

This product has the following shelf life from the date of manufacture:

365 Days:

US50

UriStar-AST™

Refer to the keyword "Storage", in the Hardy Diagnostics software program HUGO™, for more information on storing culture media.

PRECAUTIONS

This product is for in vitro diagnostic use only and is to be used only by adequately trained and qualified laboratory personnel. Observe approved biohazard precautions and aseptic techniques. All laboratory specimens should be considered infectious and handled according to "standard precautions". The "Guideline for Isolation Precautions" is available from the Centers of Disease Control and Prevention at www.cdc.gov/ncidod/dhqp/gl_isolation.html.

For additional information regarding specific precautions for the prevention of the transmission of all infectious agents from laboratory instruments and materials, and for recommendations for the management of exposure to infectious disease, refer to CLSI document M29.

Sterilize all biohazard waste before disposal.

Refer to the keyword "Precautions", in the Hardy Diagnostics software program HUGO™, for more information regarding general precautions when using culture media.

Refer to the keyword "MSDS", in the Hardy Diagnostics software program HUGO™, for more information on handling potentially hazardous material.

PROCEDURE

Direct specimen testing is not recommended. It is recommended that isolated organisms, established isolation techniques and tests for purity be performed before inoculating the medium for disk diffusion testing. Direct inoculation will produce erroneous results.

Growth Method:⁽⁶⁾

1. Select three to five similar colonies of the same morphologic type from an agar plate culture. Touch the top of each colony with an inoculation needle or loop and transfer the growth into a tube containing 4-5ml of a suitable broth such as Tryptic Soy Broth (Cat. no. K89 or R30).

2. Incubate the broth cultures at 35 degrees C. for 2 to 6 hours to develop a turbidity that exceeds or is equivalent to the 0.5 McFarland standard (Cat. no. ML05).

3. Adjust the turbidity of the actively growing broth culture with sterile saline or broth to achieve a turbidity equivalent to a 0.5 McFarland standard. To perform this step accurately, use either a photometric device or, if performed visually, use adequate light to visually compare the inoculum tube and the 0.5 McFarland standard against a Wickerham Card (Cat. no. Z08) with a white background and contrasting black lines.

Note: Overnight broth cultures should not be used as inoculum.

Inoculation of Test Plates:⁽⁶⁾

1. Within 15 minutes after adjusting the turbidity of the inoculum suspension, dip a sterile swab into the properly adjusted suspension. Rotate the swab several times and press firmly against the upper inside wall above the fluid level. This will remove excess inoculum from the swab.

2. Inoculate the dried surface of a Mueller Hinton (MH) Agar (Cat. no. G45) by streaking the swab over the entire agar surface. Repeat this procedure by streaking two more times, rotating the plate approximately 60 degrees between streakings to obtain even distribution of inoculum.

3. The lid may be left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for any surface moisture to be absorbed before applying the ring with the drug-impregnated disks. Press each disk tip down to ensure complete contact with the agar surface.

4. Invert the plates and place the plates agar side up in a 35 +/- 2 degrees C. incubator. Do not incubate the plates in increased CO₂ atmosphere because the interpretive standards were developed using ambient air incubation and CO₂ will significantly alter the size of the inhibitory zones of some agents.

Note: Avoid extremes in inoculum density. Never use undiluted overnight broth cultures or other unstandardized inocula for streaking plates.

5. After 16 to 18 hours of incubation, examine the plate. If the plate was adequately streaked and the inoculum was correct, the resulting zones of inhibition will be uniformly circular and there will be a confluent lawn of growth. If individual colonies are apparent, the inoculum was too light and the test must be repeated. Measure the diameters of the zones of complete inhibition including the diameter of the disk. Measure the zones to the nearest whole millimeter, using sliding calipers or a ruler which is held on the back of the inverted petri plate.

6. The zone margin is the area showing no obvious, visible growth. Hold the petri plate a few inches above a black non-reflecting background illuminated with reflected light. Ignore faint growth of tiny colonies that can be detected only with a magnifying lens. When discrete colonies grow within a clear zone of inhibition, the test should be repeated with a pure culture or subculture of a single colony from the primary culture plate. If discrete colonies continue to grow within the zone of inhibition, measure the colony-free inner zone. Consult references for further information.

INTERPRETATION OF RESULTS

Compare recorded zone diameters with those listed in the Quality Control and Interpretive Zone Sizes section below. Results with a specific organism may be reported as Resistant, Intermediate, or Susceptible.

RESISTANT implies that isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules, and/or that demonstrate MICs or zone diameters that fall in the range where specific microbial resistance mechanisms (e.g. beta-lactamases) are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.

INTERMEDIATE includes isolates with antimicrobial agent MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates. Intermediate implies clinical efficacy in body sites where the drugs are physiologically concentrated (e.g. quinolones and beta-lactams in urine) or when a higher than normal dosage of a drug can be used (e.g. beta lactams). This category also included a buffer zone, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.

SUSCEPTIBLE implies that isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the recommended dosage is used for the site infection. Note: Enterococcus spp., cephalosporins, aminoglycosides (except for high-level resistance screening), clindamycin, and trimethoprim/sulfamethoxazole may appear active in vitro, but are not effective clinically, and isolates should not be reported as susceptible.

LIMITATIONS

Disk performance and results depend not only on disk potency, but on use of proper inoculum and control cultures, functional plated media, proper storage conditions and other factors.

The test applies primarily to rapidly growing aerobic pathogens isolated from urine cultures.

Refer to the keyword "Troubleshooting Guide", in the Hardy Diagnostics software program HUGO™, if problems are encountered with obtaining the correct zone sizes.

Refer to the keyword "Limitations", in the Hardy Diagnostics software program HUGO™, for more information regarding general limitations on culture media.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, slides, staining supplies, culture and susceptibility test media, 0.5 McFarland Standard (Cat. no. ML05), Wickerham Card (Cat. no. Z08), calipers, microscopes, incinerators, and incubators, etc., are not provided.

QUALITY CONTROL AND INTERPRETIVE ZONE SIZES

Control tests using prescribed cultures should be included each day susceptibility testing is performed or weekly if satisfactory performance can be documented according to the CLSI standard.⁽⁶⁾ Typical zone sizes of E. coli ATCC^® 25922, Staphylococcus aureus ATCC^® 25923, P. aeruginosa ATCC^® 27853 and E. coli ATCC^® 35218 (beta-lactamase-producing strain) are given in the chart below. Quality control acceptance is specific to the procedure, control organisms and antimicrobic agent combination. Additionally, E. faecalis ATCC^® 33186 is recommended for evaluating new lots of Mueller Hinton Agar for low thymine and thymidine content (see CLSI document M2).

Test Organisms	Incubation			Zone Diameter Interpretive Stds (mm)			QC Zone Diameter Limits (mm)
	Time	Temp	Atmos	RES R	INTER I	SUS S	E. coli ATCC® 25922	S. aureus ATCC® 25923	P. aeruginosa ATCC® 27853	E. coli ATCC® 35218
Amoxicillin/Clavulanic Acid, 20/10ug: (AmC-30)							18-24	28-36	-	17-22
for Staphylococcus spp. for Enterobacteriaceae	24hr	35°C	Aerobic	<19 <13	- 14-17	>20 >18
Cephalothin, 30ug: (CF-30)							15-21	29-37	-	-
for Staphylococcus spp. for Enterobacteriaceae	24hr	35°C	Aerobic	<14 <14	15-17 15-17	>18 >18
Ciprofloxacin, 5ug: (CIP-5)							30-40	22-30	25-33	-
for Staphylococcus spp. for Enterococcus spp. for Enterobacteriaceae for P. aeruginosa for Acinetobacter spp.	24hr	35°C	Aerobic	<15 <15 <15 <15 <15	16-20 16-20 16-20 16-20 16-20	>21 >21 >21 >21 >21
Nitrofurantoin, 300ug: (Fm-300)							20-25	18-22	-	-
for Staphylococcus spp. for Enterococcus spp. for Enterobacteriaceae	24hr	35°C	Aerobic	<14 <14 <14	15-16 15-16 15-16	>17 >17 >17
Trimethoprim/Sulfamethoxazole, 1.25/23.75ug: (SXT)							23-29	24-32	-	-
for Staphylococcus spp. for Enterobacteriaceae for Burkholderia cepacia for S. maltophilia for Acinetobacter spp.	24hr	35°C	Aerobic	<10 <10 <10 <10 <10	11-15 11-15 11-15 11-15 11-15	>16 >16 >16 >16 >16

REFERENCES

1. Bauer, A.W., et al. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am J. Clin. Pathol.; 45:493-496.

2. Ryan, K.J., et al. 1970. Disc sensitivity testing. Hospital Practice; 5:91-100.

3. Federal Register. Rules and regulations. Antibiotic susceptibility discs. Fed Regist.; 37:20525-20529, 1972. Erratum; 38:2756, 1973.

4. Ericsson, H.M., et al. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta. Pathol. Microbiol. Scand., Section B, Suppl.; 217:1-90.

5. World Health Organization Expert Committee on Biological Standardization. 1977. Technical report series 610. W.H.O., Geneva.

6. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS). Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard M2-A9. CLSI, Wayne, PA.

7. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS). Performance Standards for Antimicrobial Susceptibility Testing, Informational Supplement. Approved Standard M100. CLSI, Wayne, PA.

8. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS). Methods for dilution Antimicrobial Susceptibility Tests for bacteria that grow aerobically. Approved Standard M7. CLSI, Wayne, PA.

9. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. Approved Standard M11. CLSI, Wayne, PA.

10. Bushby, S.R.M. 1973. Trimethoprim-sulfamethoxazole. In vitro microbiological aspects. J. Infect. Dis.; p. 10-30.

11. Hitchings. 1974. Trimethoprim-sulfamethoxazole: Microbiological, pharmacological and clinical considerations. University of Chicago Press, Chicago.

12. York, M.K., et al. 1996. Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci. J. Clin. Microbiol.; 34:249-253.

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

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