Presents Overview Part 1 MultiChrome

MultiChrome TM Veterinary Microbiology Culture System MultiChrome GP(+) Exp 3/10 Lot #123 MultiChrome GP(-) Procedures and Techniques For Performing In-house Cultures using Kacey MultiChrome™ Bi-pates. Gram PositiveGram Negative

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 degrees for 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 or loop containing the now diluted WST & Turbidity adjusted 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. 3) 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 4) Remove “Sensi-Ring” from the foil pouch with tweezers at the inner tab & place the “Sensi-Ring” facing down onto the MH plate tapping down in non-disk areas, label the specimen to be incubated. 5) Place the MH Inoculated plate into an incubator upside down, set timer & incubate for 24 hours. Remove & read inhibition zones with Kacey Clear Acetate Reader TRANSFER TO MULLER HINTON PLATE USING KACEY “WST” TUBES 1) Taking a Kacey Sterile Swab or Loop carefully dab into the three (3) different places containing the bacteria on the MultiChrome Bi-plate. 2) Immediately place the sterile Kacey Swab or Loop containing the bacteria into the Kacey Working Solution tube (WST which contains 1.0 ml of sterile 0.085%solution). Mix the Kacey Swab or Loop with a gentle twirling motion while in the WST tube for approximately 3-5 seconds. 3) Compare sample to the Kacey Turbidity Standard (white cap) KTS. Sample should match in turbidity, if not add more WST sol (green top) or more sample to match KTS tube. (see enclosed Turbidity Card). Quick reference only. Refer to detailed instruction manual for additional information. GN GP 24 Hrs WST Dilution Rotate Swab 3-5 sec 24 Hrs GN GP New Kacey Sterile Swab Compare to KTS tube (white top) & match turbidity by adding more WST(grn top) solution or more sample KTS White Tube Sample

BACTERIA AS A PATHOGEN In veterinary medicine we routinely see them affecting nine areas:  Outer Ear Infections  Urinary Tract Infections  Conjunctival infections  Genital infections  Wound/abscess  Skin Infections  Upper Respiratory Infections  Mastitis  Septicemia

MultiChrome™ Supplies For Each Test MultiChrome GP(+) Exp 3/10 Lot #123 MultiChrome GP(-) 1 MultiChrome™ bi-plate A sterile “Rayon”* swab included or sterile loop included with MultiChrome™ kit. Incubator: We recommend the Kacey Mini-Incubator with its individual timers for up to six cultures. *Always use Rayon swabs as chemicals in cotton can alter results.

Kacey Mini Incubator Up Close Small foot print (8”w x 13”hx12”d)  Precise Digital temperature control  Numbered trays fit both round & rectangle plates  Six individual timers  Audible/Visible alerts  Energy efficient, UL listed  Made in the USA  1year warranty Just Load… Press Timer...and Walk Away! MultiChrome GP(+) Exp 3/10 Lot #123 MultiChrome GP(-) Patent Pending

Pre-heat incubator. Pre heat incubator (98.6 F or 37 C default) which will take approximately 30 minutes. The Kacey Mini Incubator will automatically default to this temperature when plugged in F or 37C

Pre-heat MultiChrome™ plate. Remove a plate from the refrigerator and thaw MultiChrome™ plate for approx 9-10 min. Place plate upside down to avoid condensation landing on media. The Kacey Mini Incubator has a “Red” pre-heat timer button which allows you set a 10 minute warning. MultiChrome GN(-) MultiChrome GP(+) Exp 3/10 Lot #123

Obtain sample and inoculate MultiChrome™ plate. Using a sterile loop or rayon swab obtain a sample from the affected area. Streak both the Gram (+) and (-) chambers of the plate in a zigzag fashion. MultiChrome GN(-) MultiChrome GP(+) Exp 3/10 Lot #123

Incubate sample. Cover MultiChrome™ plate with lid and turn upside down. Place in incubator and incubate for 24 hours. On the Kacey Mini Incubator press one of six pre-set 24 hours timers and walk away. MultiChrome GN(-) MultiChrome GP(+) Exp 3/10 Lot # hours

Read sample. At 24 hours remove from incubator and read. Note whether the colony is a Gram (+) or (-) as you reference the following color chart. Also, keep in mind the types of bacteria most associated with different affected areas of the body (see the next slide). 24 hours

Commonly found bacteria Based on results, a further course of action can now be with taken to determine the most efficacious antibiotic(s) using the Sensi-Ring™ sensitivity system. 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

Color reference chart Gram Negative Gram Positive Strep (+) Mixed Staph/Strep (+) This is a partial list of the various bacteria seen on MultiChrome™. Some bacteria may look slightly different than shown, but the colors and Gram (+), (-) designation remain constant. This library will continue to grow for use a reference guide.

Bacterial pathogen probability charts for Canine and Feline: These reference charts suggest the highest and lowest percentage for infection of various parts of the body by common pathogenic bacteria. Once identified proceed with accepted veterinary antibiotic protocols for treatment of pathogens. Species: Canine PercentageProbability BacteriaUTIEarBoneSkinGenitalWoundBloodURI Staph Intermedius10-15%25-30%40-50%60-70%15-25%25-35% 30-35% E. Coli>50%10-20% 20-30%30-35%20-30%35-45%15-20% Enterococcus faecalis<10%NGS10-20%NGS Proteus mirabilis15%20-25%10-20%<10%NGS10-20%NGS Obligate anerobesNGS 25-35%10-20%NGS PseudomonasNGS15-25%NGS<10% 10-20% <10% Klebsiella10-15%NGS 20-35%<10% PasturellaNGS 10-25%NGS 15-20% NGS= Not Generally Seen Species: Feline PercentProbability Bacteria Conjunctival GenitalUTIWoundURISkin Pasturella10-20%15-25%10-15%30-40%>50% E. ColiNGS30-35%40-45%10-20%15-20%20-30% Proteus mirabilisNGS 10-15%<10% PseudomonasNGS<10% NGS<10%NGS Obligate anerobesNGS10-25%NGS25-35%NGS KlebsiellaNGS Chlamydia psittoci50-75%NGS B-hemolytic strep15-25%NGS NGS= Not Generally Seen Probability reference charts taken from “Target: The Antimicrobial Reference Guide to Effective Treatment”, second edition, revised. Author: Dr. David Aucoin, DVM, Diplomate of the American College of Veterinary Clinical Pharmacology. Data on file, Kacey® Asheville, N.C

Treatment. Many organisms can be easily identified and treated after the use of the MultiChrome™ system without further testing. However, for organisms that have proven resistant to typical antibiotic regimens it may be necessary to conduct a Sensitivity test. Kacey Diagnostics has made “Sensitivity” testing easy, accurate and cost effective as the MultiChrome™ by introducing the Sensi-Ring system in the next section.

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 degrees for 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 or loop containing the now diluted WST & Turbidity adjusted 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. 3) 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 4) Remove “Sensi-Ring” from the foil pouch with tweezers at the inner tab & place the “Sensi-Ring” facing down onto the MH plate tapping down in non-disk areas, label the specimen to be incubated. 5) Place the MH Inoculated plate into an incubator upside down, set timer & incubate for 24 hours. Remove & read inhibition zones with Kacey Clear Acetate Reader TRANSFER TO MULLER HINTON PLATE USING KACEY “WST” TUBES 1) Taking a Kacey Sterile Swab or Loop carefully dab into the three (3) different places containing the bacteria on the MultiChrome Bi-plate. 2) Immediately place the sterile Kacey Swab or Loop containing the bacteria into the Kacey Working Solution tube (WST which contains 1.0 ml of sterile 0.085%solution). Mix the Kacey Swab or Loop with a gentle twirling motion while in the WST tube for approximately 3-5 seconds. 3) Compare sample to the Kacey Turbidity Standard (white cap) KTS. Sample should match in turbidity, if not add more WST sol (green top) or more sample to match KTS tube. (see enclosed Turbidity Card). Quick reference only. Refer to detailed instruction manual for additional information. GN GP 24 Hrs WST Dilution Rotate Swab 3-5 sec 24 Hrs GN GP New Kacey Sterile Swab Compare to KTS tube (white top) & match turbidity by adding more WST(grn top) solution or more sample KTS White Tube Sample

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.

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 Kacey Mueller Hinton Plate Loop Rayon Swab Red Top Turbidity Tube Appropriate Sensi-Ring™ Kacey Micro Incubator

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.

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.

Comparing Sample to Standard Compare your red top tube against the Standard (white top tube) which comes which every kit. They should match closely in turbidity. You can place the barcode card behind them to compare. If sample is too opaque, add more WST solution from dropper enclosed. Sample Standard White Tube Kacey C&S Turbidity Card Directions On Back Place Behind Tubes

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 degrees for 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 or loop containing the now diluted WST & Turbidity adjusted 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. 3) 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 4) Remove “Sensi-Ring” from the foil pouch with tweezers at the inner tab & place the “Sensi-Ring” facing down onto the MH plate tapping down in non-disk areas, label the specimen to be incubated. 5) Place the MH Inoculated plate into an incubator upside down, set timer & incubate for 24 hours. Remove & read inhibition zones with Kacey Clear Acetate Reader TRANSFER TO MULLER HINTON PLATE USING KACEY “WST” TUBES 1) Taking a Kacey Sterile Swab or Loop carefully dab into the three (3) different places containing the bacteria on the MultiChrome Bi-plate. 2) Immediately place the sterile Kacey Swab or Loop containing the bacteria into the Kacey Working Solution tube (WST which contains 1.0 ml of sterile 0.085%solution). Mix the Kacey Swab or Loop with a gentle twirling motion while in the WST tube for approximately 3-5 seconds. 3) Compare sample to the Kacey Turbidity Standard (white cap) KTS. Sample should match in turbidity, if not add more WST sol (green top) or more sample to match KTS tube. (see enclosed Turbidity Card). Quick reference only. Refer to detailed instruction manual for additional information. GN GP 24 Hrs WST Dilution Rotate Swab 3-5 sec 24 Hrs GN GP New Kacey Sterile Swab Compare to KTS tube (white top) & match turbidity by adding more WST(grn top) solution or more sample KTS White Tube Sample

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

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.

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 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.

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.

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

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

Remove and read after 24 hours Carefully remove plate from incubator keeping it upside down so that Sensi-Ring letters can be seen.

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

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

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

MRSA & MRSP Can be detected with Sensi-Ring™ Skin and Wound

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

UTI and Ear samples

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

Disposal of plate & clean up Once read, plates 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 (coming soon) 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.

Identify your top performing clinic/hospital for use as reference Purchase of one of the following components per month Sign official agreement Agreement can be cancelled by either party with a 30 day notice in writing of the cancellation Incubator Promotion

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