1. Presents

2. Sensi-Ring™ Antibiotic Sensitivity Detection System Procedures and Techniques For Performing In-house Antibiotic Sensitivities On Cultured Kacey MultiChrome™ Bi-pates. “Choosing the right drug for the right bug”

3. Why use Kacey Sensi-Ring™?  In house rapid identification (24 hours) of bacterial antibiotic sensitivity using 8 of the most common veterinary antibiotic choices per ring.  Cost Effective: Less than 1/3 the cost of a commercial laboratory like Antech and Idexx.  Specific Sensi-Ring for both Gram Positive and Gram Negative  System specific Sensi-Ring: - Urinary Tract Infection - Ear Infection - Skin and Wound Infection

4. Sensi-Ring™ Supplies For Each Test For added convenience we also offer a Culture & Sensitivity Starter Kit enough for four complete tests with all the supplies you need. Product #20241

5. Step 1: Obtaining live cultured bacteria from a Kacey MultiChrome™ bi-plate. Using the yellow loop, take 3 or 4 passes through the bacterial growth on the MultiChrome™. For bacterial colonies in both chambers see next slide.

6. Step 1 (con’t) : Procedure For MultiChrome bi-plates with bacteria on both the Gram Positive and Negative side. In this case, you will prepare two Kacey Maxi Mueller Hinton plates as indicated. One for the (+) and one for the (-). To each MH plate you will add the appropriate Sensi-Ring disk Example: place a Gram (+) in the MH plate with Gram (+) and Gram (-) in the MH plate with Gram (-). Then incubate each.

7. Step 2: Mixing with Saline (WST) to dilute Insert the yellow loop into the saline and twirl to make a cloudy solution. This is an important step to avoid overgrowth of a bacterial colony.

8. Step 3: Removing the diluted sample with enclosed Rayon* Swab Take a sample using the applicator tip of the swab. * Do not use cotton swabs even if sterile. Cotton will release a chemical that will alter the results.

9. Step 4: Inoculate diluted bacteria onto Kacey Maxi* Mueller Hinton plate Using the diluted bacteria solution on the soaked swab, evenly distribute the sample onto a warmed Kacey Maxi Muller Hinton plate (37 degrees 15-20 min). Streak the plate as shown below. 11-5, 1-7, 9-3 o’clock *Kacey Maxi Mueller Hinton plates are “true” 100mm plates. Most commercially sold Muller Hinton plates are 88mm and will not work with this system.

10. Step 5: Apply the Sensi-Ring™ Pick the appropriate Sensi-Ring™ ie. Gram Positive, Gram Negative, UTI or Skin/wound depending on samples origin. Grasp the tab on the inside of the ring and lower onto the agar upside down with lettering facing agar, centering the ring with tweezers.

11. Step 6: Tamp down Sensi-Ring™ Make sure that all of the disks touch the agar plate to allow antibiotic diffusion.

12. Step 7: Incubate Upside Down Place Kacey Maxi Mueller Hinton plate now inoculated upside down to prevent any contamination. Place into a preheated Kacey incubator at 37 degrees for 24 hours. 24 hours

13. Step 8: Remove and read after 24 hours Carefully remove plate from incubator keeping it upside down so that Sensi-Ring letters can be seen. Use gloves. Remember this is a live bacterial colony.

14. Step 9: Use the Kacey Zone Reader Card Place Kacey Maxi Mueller Hinton Plate upside down with antibiotic letters facing up. Place Zone Reader over the plate and align the notch of the Reader to the notch of the Sensi-Ring. Concentric circles should now encircle all of the disks. Read the Inhibition Zone in mm for each disk. Notch

15. Step 10: Log Test Results Using the Patient Lab Report form supplied log inhibition zones for each antibiotic disk.

16. Step 11: Treat with appropriate antibiotic Based on results, a course of action can now be taken with the most efficacious antibiotic(s). The following is a list of commonly found Aerobic Bacteria in small animal practice: Gram Positive: Staphylococci, Streptococci, Enterococci Gram Negative: E coli, Proteus, Klebsiella, Pseudomonas Ear: Staphylococci, Pseudomonas, Proteus, E. coli, Enterococci, Streptococci UTI: Staphylococci, Proteus, E. coli, Klebsiella, Enterococci, occasionally Pseudomonas Skin & Wound: Staphylococci, Streptococci, Pseudomonas, Enterococci, Proteus

17. Step 12: Disposal of plate & clean up Once read, plate should be sprayed with a bactericidal agent, covered with its lid taped shut, placed in a plastic bag and disposed of in your bio-hazard bin. If you have a Kacey Sanitizer unit follow its instructions. Place lid back on the plate, tape it shut, bag it and dispose of it in your bio-hazard bin. Dispose of all used supplies in your bio-hazard bin. Spray your counters down and wash your hands in an aseptic manor. Remember, these are live cultures.

18. Alternate Bacteriology Protocol Inoculating Mueller Hinton Plate First Without Using the MultiChrome Culture Plates With Kacey Culture and Sensitivity system is it possible to first directly inoculate a Maxi Mueller Hinton plate, incubate for 24 hours and determine most efficacious antibiotic before actually identifying the pathogenic bacteria. A sample can then be drawn from the center area of the Sensi-Ring™ on the Maxi Mueller Hinton plate and transferred to a MultiChrome™ plate for incubation and identification of the bacteria(s).

19. Urine Samples: Spin down urine and do cytology on the sample of the sediment. - If bacteria is present, directly inoculate sediment onto a Kacey Maxi Mueller Hinton plate with a sterile Rayon swab enclosed smeared evenly over the surface as described in step 4. Ear Samples: Do a cytology on each ear swab. - If bacteria is present, directly inoculate the Kacey Maxi Mueller Hinton plate with sample smeared evenly over the surface as described in step 4. Alternate Bacteriology Protocol Inoculating Mueller Hinton Plate First Without Using the MultiChrome Culture Plates

20. Sensi-Rings™ case samples Case study: 2 year old canine which cultured on the MultiChrome™ both a large colony of Gram Positive Staph in pink and a smaller Strep colony in blue. The colonies when tested for sensitivity using the Sensi-Ring™ indicated sensitivity to a variety of antibiotics not previously used. Staph Strep

21. UTI and Ear samples

22. Sensi-Rings™ Ability to identify different strains of bacteria. Sensi-Rings™ used to identify antibiotic sensitivity on different strains of cultured Staphylococci.

23. Sensi-Rings™ Quick Reference Card Kacey Diagnostics Quick Reference For Culture & Sensitivity Study INNOCULATION OF CULTURE BI-PLATE 1) Remove the plate from refrigerator & pre-heat in the incubator @37 degrees for 15-20 minutes prior to inoculating the Kacey MultiChrome bi- plate. 2) Using a sterile inoculating loop ( 20uL) for liquids and a Kacey Sterile Swab for solids,inoculate the sample onto both sides of the Kacey Multichrome Bi-plate, by utilizing a zigzag streaking motion. 3) Replace clear lid and place Kacey MultiChrome Bi-plate in the incubator upside down (inverted position) and incubate at 37°C +/- 2 degree C for no less then 24 hours. The plate should be examined after 24 hours, but no later than 48 hours after incubation. INOCULATION OF MULLER HINTON (MH) PLATE 1) Using the Kacey Swab containing the now diluted WST dilution streak the 1st time the entire periphery of the Mueller Hinton plate making a 360° circle 2) Streak a 2nd time again the Muller Hinton Plate with a fresh sample of the WST dilution usingbroad strokes starting at theto 3 o’clockposition. 2) Streak a 3rd time again the Muller Hinton plate with a fresh sample of the WST dilution using wide broad strokes starting at the 11 to 5 o’clock position 3) Remove “Sensi-Ring” from the foil pouch with tweezers at the inner tab & place the “Sensi-Ring” on to MH plate tapping down in non-disk areas, label the specimen to be incubated. 4) Place the MH Inoculated plate into an incubator upside down, set timer & incubate for 24 hours. Remove & read inhibition zones with Kacey Digital Reader. 1 2 3 TRANSFER TO MULLER HINTON PLATE USING KACEY “WST” TUBES 1) Taking a Kacey Sterile Swab carefully dab on to three (3) different places containing the bacteria on the MultiChrome Bi-plate. 2) Immediately place sterile Kacey Swab containing the bacteria into a Kacey Working Solution tube (WST containing 1.0 ml of 0.085%solution). 3) Mix the Kacey Swab with a gentle twirling motion while in the WST tube for approximately 3-5 seconds. Use the same Kacey Swab for inoculation of the Muller Hinton plate in step #3. Quick reference only. Refer to detailed instruction manual for additional information. GN GP 1 2 3 WST Dilution Rotate Swab 3-5 sec GN GP 24 Hrs New Kacey Sterile Swab

24. Kacey Diagnostics MICROBIAL ZONE INHIBTION INTERPRETIVE CHART* w/ Zone Diameter Interpretive Standards for common veterinary Antibiotics Amikacin ……………………………………………. Amoxicillin ………………………………………….. Amoxi Clavulante ………………………………….. Augmentin-(Clavamox)……………………………. Azithromycin-(Zithromax)…………………………. Cephalexin-(Keflex)……………………………….. Cefapodoxime …………………………………….. Ceftazdime …………………………………………. Ciprofloxacin –(Cipro)…………………………….. Clindamycin –(Clincaps)………………………….. Cotrimoxazole (trimethoprim/sulfa)……………… Choloramphenicol-(Chloromycetin)……………… Cefalothin-(Keflin)…………………………………. Doxycycline-(Vibramycin)………………………… Enrofloxacin-(Baytril)………………………………. Gentamycin-(Gentocin)……………………………. Neomycin …………………………………………... Oxacillin …………………………………………….. Penicillin…………………………………………….. Polymycin ………………………………………….. Ticarcillin-(Ticar)……………………………………. Tobramycin-(Tobrex)………………………………. Trimethoprim/Sulfa-(Bactrim Septra Primor)……. 30ug 13ug 30ug 10ug 30ug 5ug 30ug 25ug 30ug 5ug 10ug 30ug 1ug 10ug 300ug 75ug 10ug 25ug =14 =13 =14 =17 =14 =15 =13 =10 =17 =14 =12 =17 =6 =12 =10 =28 =8 =14 =12 =10 15-16 14-17 15-17 18-20 15-17 16-20 14-17 11-15 18-20 15-17 13-15 18-20 7-9 13-16 11-12 —— 9-11 15-19 13-14 11-15 =17 =18 =21 =18 = 19 = 16 = 21 = 18 = 16 = 21 = 10 =17 =13 = 29 =12 = 20 = 15 = 16 AK A AMC AUG ATH CFX CPD CAZ CIP CD TS C KF DXT ENF GM NE OX PG PB TC TN T/S Antimicrobial Agent Name Sensi Code Disc Potency Resistant Intermediate Susceptible *These are the most widely accepted standard procedures used by the US Food and Drug administration (FDA) and the World Health Organization (WHO). These standard procedure values were adopted as a consensus standard by the Clinical and Laboratory Standard Institute (CLSI) formerly known as NCCL and are periodically updated. The latest CLSI documentation should always be consulted for current recommendations. The list of abbreviations for the antibiotics has been selected as standardization by KACEY® for worldwide sales, distribution, nomenclature and identification of the Kacey Sensi-Rings. It is highly recommended that any bacterial agent whose etiology and treatment is beyond the scope of this system be sent to a qualified veterinary reference lab for more detailed testing. It is also recommended that any sample sent the lab be obtained from the middle of the Sensi Ring on the Kacey Maxi Muller Hinton Plate". Sensi-Rings™ & MultiChrome™ are trademarks Kacey® © 2013 Rev 10.3.2013 All Rights Reserved Sensi-Rings™ Quick Reference Card (con’t)

25. 1854 A Hendersonville Rd Asheville, NC 28803 Ph: 1(828)685-3569 Fax: 1(828)685-3571 www.kaceyinc.org “Kacey Products Providing Better Animal Care” Kacey Diagnostics © 2014 All Rights Reserved Rev 12.30.12 “MultiChrome”, “Sensi-Rings” are trademarks of Kacey® Inc.