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This page contains information on MASTiK for veterinary use.
The information provided typically includes the following:
  • MASTiK Indications
  • Warnings and cautions for MASTiK
  • Direction and dosage information for MASTiK


This treatment applies to the following species:
Manufacturer: ImmuCell

Mastitis Antibiotic Susceptibility Testing Kit


General Information

Despite great improvements in recent years in mastitis prevention, both clinical and subclinical mastitis remain the most common diseases of dairy cows. Most cases are due to the bacteria Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Klebsiella and E. coli. Early diagnosis and treatment of clinical mastitis with an effective antibiotic is usually a cost-effective approach. Besides the treatment expense, costs associated with clinical mastitis include unsalable milk during the treatment and withdrawal period, and most importantly, decreased milk production through the remainder of lactation.

Because traditional antibiotic susceptibility tests are usually slow (often taking several days) and often inaccurate (they are human not bovine tests), veterinarians and dairy farmers most commonly treat cows with clinical mastitis by guessing which antibiotic will work. These guesses are usually based on what has been working lately or simply intuition. Due to the emergence of many antibiotic resistant strains of mastitis-causing bacterial and ever-shrinking profit margin in dairying, there is a need for rapid, cost-effective, and accurate antibiotic susceptibility test for mastitis. MASTiK® is such a test, one that is performed in milk rather than laboratory media using bacteria that exists in the cow’s udder rather than a standardized concentration. The results should be used as an aid in choosing an approved antibiotic at an approved dosage for mastitis therapy.

Important Information: Please Read Carefully Before Proceeding

MASTiK® is a laboratory diagnostic test kit to be used only as an aid in selecting FDA approved antibiotics for mastitis therapy. Examples of approved over-the-counter antibiotics are penicillin, cephapirin and erythromycin. Examples of approved antibiotics which may be used with a veterinarian's prescription include hetacillin, pirlimycin, amoxicillin, and cloxacillin. The antibiotics listed above are available under a variety of trade name and must be used according to label directions. The dosage or frequency of treatment of these antibiotics cannot be increased without a veterinarian's prescription.

Oxytetracycline and sulfadimethoxine are examples of antibiotics which cannot be used to treat mastitis unless prescribed by a veterinarian in an extra-label fashion. These drugs are included in the MASTiK® panel solely for the veterinarian's information.

Use Of The Test

MASTiK® is a rapid in-vitro antibiotic susceptibility test for clinical mastitis in the bovine due to the following major mastitis-causing bacteria: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Klebsiella species, E. coli and Actinomyces pyogenes. Results are usually obtained in 12 hours or less, but range from 6 to 24 hours. Organisms which can cause mastitis much less frequently (and are usually unsuccessfully treated with antibiotics) such as Pseudomonas species, Prototheca, Mycoplasma species, and yeasts will not show evidence of growth in this test. A test result of no growth in the positive control well usually means antibiotic therapy is not indicated.

MASTiK® is not intended for use on specimens other than mastitic milk.

Samples to be tested must be aseptically drawn from quarters with mastitis. Affected cows may or may not be sick, but they must not have been recently treated with antibiotics by any route. Always take a sample from a cow before you treat her and refrigerate it in case you wish to run a test later. Chronic or subclinical cases can usually be successfully tested, although they will require a longer incubation time with the reagent before transferring to the test panel.

Principle Of The Test

A milk sample is drawn under sterile conditions from the affected quarter and mixed with a MASTiK® reagent (skim milk and the purple dye BCP, a pH color indicator) and incubated from 0 to 8 hours, usually 1-3 hours at 35°-37°C (95°-99°F) to allow bacterial multiplication. A small quantity of this mixture (0.05 ml) is then added to each of 16 wells of the MASTiK® panel containing 7 antibiotics at 2 concentrations each. The panel is covered with a clear adhesive seal and incubated at 35°-37°C until results are obtained. Since the major mastitis-causing bacteria ferment lactose, producing lactic acid, their growth will lower the pH of the milk media gradually changing the color from purple to yellow. If the antibiotic inhibits the growth of the bacteria, there will be little or no lactic acid produced and thus little or no change in color from the original purple.

In MASTiK® the antibiotic susceptibility of the organisms is tested in approximately the same concentration of organisms as the environment (milk media) present in the udder to be treated. Thus the result is expected to produce a more realistic correlation between the in-vitro sensitivity test and the response to treatment. The uncommon microorganisms of mastitis which do not ferment lactose will not cause any change in color of the media.


MASTiK® is a rapid antibiotic susceptibility test for mastitis. The results may be obtained in 12 hours in most cases, with a range of 6 to 24 hours. The MASTiK® test principally consists of reagent vials containing milk media with pH indicator dye and test panels consisting of 16 polystyrene wells per panel coated in various antimicrobials in two concentrations, packaged separately. The box containing MASTiK® Reagent should be removed from the kit on arrival and stored at refrigeration temperature (2°-7°C or 35°-46°F) until used. The other kit components may be stored at room temperature.

Six different samples can be tested per plate against two concentrations of seven different antimicrobials as shown below.



Negative Control

Growth inhibitor

Positive Control



1.0, 4.0

*Oxacillin + 2% NaCl

10., 4.0


0.5, 4.0


0.25, 2.0


2.0, 16.0


0.5, 4.0


4.0, 16.0

*Oxacillin is the parent compound for Cloxacillin.

**Ampicillin is the parent compound for Amoxicillin and Hetacillin.

***Cephalothin is the parent compound for Cephapirin and Ceftiofur.


If the bacteria are sensitive to the parent compound it means that they will also be susceptible to the daughter compounds. For example, if test results indicate that oxacillin is effective, then it follows that coxacillin (its daughter compound) is also effective. The reverse may or may not be true.

These concentrations of antimicrobials are obtained when 50 µl of culture is added to the well. The lowest concentration well is at a therapeutic level achieved in milk when the cow is treated with commonly used doses. The highest concentration well is at a level that may be difficult to maintain throughout the therapeutic period when commonly used dosages are chosen.

The distribution of the controls and the concentrations of the antimicrobials in µg/ml after reconstitution with the treated sample are detailed in the MASTiK® record card.



6 Test Kit

18 Test Kit

1. Plate containing six panels of 16 wells per panel coated with antimicrobials



2. Adhesive seals



3. Pad of record cards



4. Sterile transfer pipettes



5. Flip-top collections tubes



6. Package insert



7. MASTiK® Reagent - vials contain 3ml milk media with indicator dye



Materials needed but not included with MASTiK®:

1. Alcohol swabs

2. Incubator (34°-39°C or 93°-102°F).

Storage And Shelf Life

The plates should be stored at room temperature (15-25°C or 59-77°F) away from direct sunlight or heat. Use the panels prior to the expiration date printed on the label. If the package has been damaged in any manner, the panels in the package should be discarded. A silica gel desiccant is included in each foil package.

MASTiK® Reagent must be stored at refrigerated temperature (2-7°C or 25-46°F). Use the MASTiK® Reagent prior to the expiration date printed on the label. If the reagent has not retained its blue-purple color (i.e. it appears yellow or green) it may be contaminated and should not be used.


1. Strict aseptic measures should be followed in collection of samples and running the test.

2. The test is for diagnostic use only.

3. Since microorganisms present in the samples tested with this product can be infectious, proper handling and disposal methods should be established. The used panels, reagent vials and the samples should be disposed of properly, according to state and local regulations.

4. Only personnel adequately trained in such methods should perform this type of diagnostic procedure.

The Mastik® Plate (six Test Panels Per Plate)

Test Procedure


The milk samples must be collected and handled under aseptic conditions at all time.

Sample Collection: (important Note: If The Sample Is Not Collected Under Sterile Conditions As Outlined In Steps 1-4, Contamination Will Make These Test Results Meaningless).

1. After washing hands thoroughly wash the teat with udder wash, water and towel dry.

2. Discard first stripping of milk.

3. Vigorously swab teat end with alcohol swab and let air dry.

4. Flip up cap from collection tube. While holding both tube and teat at a 45° angle, squirt at least 2ml (one stripping) of milk into the tube and cap the tube. Refrigerate the sample if a delay of more than 6 hours is anticipated before testing the sample. Freeze if the anticipated delay is more that 4 days.

Testing Of Samples (see Below For Quick Steps)

5. Open The Cap Of A Mastik® Reagent Vial And Pour Approximately 1ml Of The Mastitis Sample Into The Reagent Vial Up To The 4ml Line On The Label. Do Not Use The Pipettes Provided In The Kit To Add The Milk To The Reagent Vial. Recap The Reagent Vial, Invert To Mix And Label It Appropriately. If The Sample Is From A Highly Acute Case And The Cow Is Very Sick, Skip Step 6 And Proceed To Step 7.

6. Incubate The Reagent Vial At 35-37°c (95-99°f) For 1-3 Hours. The Sample-reagent Mixture Should Retain Its Blue-purple Color. If The Sample-reagent Mixture Begins To Change Color (from Purple To Blue-green Or Yellow), Pour Approximately 1ml Of The Sample-reagent Mixture Into A Fresh Reagent Vial And Proceed To The Next Step. Note: If The Sample Is From A Case Of Subclinical Or Chronic Mastitis Instead Of Clinical Or Acute Mastitis, Then Prolong The Initial Incubation Time To 6-8 Hours.

7. Remove The Plate From The Foil Package. Six Samples Can Be Run Concurrently From This Plate. For Running One Sample, Remove One Panel From The Plate. (note: For Easier Handling Place The Test Panel In An Empty Panel Frame). Return The Unused Panels To The Foil Pouch And Store Away From Light. Orient The Panel With The Square Plastic End Tab Towards You.

8. Mix By Inverting The Incubated Sample-reagent Vial Containing The Milk Sample Several Times. Using A Separate Sterile Disposable Transfer Pipette For Each Sample, Add 2 Drops Of Sample-reagent Mixture To Each Of The 16 Wells Of The Test Panel. Do Not Touch Wells With The Pipette. Solids, Flakes, Or Clots Should Be Avoided. Place Leftover Pre-incubated Vial In Refrigerator.

9. Remove The Clear Plastic Adhesive Sheet From Its Paper Backing And Cover All Wells.

10. Incubate The Plate At 35-37°c Or 95-99°f.

11. When Handling The Panel, Be Sure Not To Turn It Upside Down. Examine The Panel For Color Change After 4 To 8 Hours Of Incubation And Thereafter Every 4 To 8 Hours Until The Color Of The Positive Control Well Has Changed From Purple To Yellow. Do Not Incubate The Panel More Than 24 Hours In Total. The Bottom Of The Panel Should Be Checked For Color Changes By Holding The Panel Level Over Your Head. Most Panels Will Be Ready To Read In 8 To 12 Hours. In General, The More Acute The Case, The Faster The Results.

reading The Test Results

12. Do not turn the plate over. Hold it level over your head and examine the bottoms of the wells for color changes. Note the position of the square plastic tab located at the bottom of the panel. Be sure to match the positions on the panel with the record sheet when recording the results. The test results are ready to read if growth has occurred in the positive control well causing it to change color from purple to yellow. Well A1 of each panel is the positive control well and contains no antibiotics. Yellow in this well is recorded on the MASTiK® Record Card as positive. No color change in the positive control well indicates the probable presence of non-lactose fermenting organisms or the absence of living bacteria.

13. Well A2 of each panel is a negative control well and should always inhibit growth. Therefore, the negative control well should always be purple or the original color of the sample. Purple in this well should be recorded on the MASTiK® Record Card as negative. Purple color in all wells, including both controls, indicates the probable presence of non-lactose fermenting organisms or the absence of living bacteria.

14. Record the result of the test wells as S, R, or I on the MASTiK® Record Card.

PURPLE = NO GROWTH = S (Susceptible)

YELLOW = GROWTH = R (Resistant)

Intermediate = Slight Growth = I (partial growth)

Any test well that has not changed color and is similar in color to the negative control well is recorded on the MASTiK® Record Card as S (Susceptible). Any well that has changed color toward yellow and is similar in color to the positive control well is indicative of growth and recorded as R (Resistant). A well intermediate in color between the positive and negative control wells is recorded as I (Intermediate).


If color changes are not dramatic, continue incubation another 4-8 hours but not more than 24 hours total and re-read results.

When running multiple panels simultaneously, read each panel independent of the other panels based on the appropriate changes in the control wells as described above.

Interpretation Of Test Results

MASTiK® is an in-vitro susceptibility test and the test results should be used as an aid only, in selecting the drug of choice for treatment. The concentration of the antimicrobials in the lowest concentration well is at a therapeutic level that is usually achieved in milk when the cow is treated with the commonly used dosages; the highest concentration well is at a level that is sometimes higher than the concentration achieved by usual therapeutic dosages. Thus, in general, when choosing an antimicrobial for therapy, the best choice is one in which both of its wells displayed no growth (retained the original purple color). The second best choice would be the drug in which only the lowest concentration well showed growth (changed in color from purple to yellow).

When all wells, including the positive and negative controls, remain purple, infection with non-lactose fermenting organisms such as Mycoplasma bovis, Pseudomonas aeruginosa, yeast or Prototheca should be suspected.

Use In Dry Treatment Selection

If you are depending on dry treatment to cure subclinical mastitis, it makes sense, especially in high producing cows, to use an antibiotic to which the bacteria are susceptible. If you are using the same dry treatment on all quarters of all cows, chances are it will work no better than using the same antibiotic all the time to treat all cases of clinical mastitis.

If a cow is near drying off and you know she has subclinical mastitis from culture results or somatic cell counting, identify the problem quarter(s) by culture or CMT* test. You may then run MASTiK® with a simple modification; increase the incubation time of the sample-reagent mixture outlined in step 6 to 6-8 hours.


MASTiK® susceptibility test is effective only with lactose fermenting bacteria. It does not reveal the sensitivity of non-lactose fermenting organisms. Good correlations between the test results and treatment response is expected but can not be assumed in all cases.

* Contact ImmuCell to obtain CMT.


Problem: No Color Changes After Recommended Incubation Time

1. No bacteria in sample (case is not bacterial mastitis).


Do standard culture and identification.

2. Bacteria in sample are totally engulfed by leukocytes present in the milk.


Re-sample cow and repeat test.

Note that most commonly used antibiotics cannot enter these cells are not indicated in these cases.

3. Organism in sample is not Staphylococcus, Streptococcus, Actinomyces, or coliform.


Culture and identify organism.

4. Not enough bacteria in sample. Occurs most often when testing subclinical or chronic cases.


Continue incubating and check test every 4 hours. Problem often avoided if preincubation (step 6) period is extended to 6-8 hours.

Laboratory Note: MASTiK® may be run from colonies isolated on agar by standardizing the inoculum.

5. Slow growing bacteria.


Continue incubation of panel and check for color changes every 4 hours.

6. Cow was treated with an antibiotic before sample was taken.


Sample only cases not treated within past 48 hours. Always aseptically sample the cow before initiating treatment.

Problem: Overgrowth - All Wells Change Color.

1. Occurs in cases with unusually high numbers of bacteria, especially if panel is not checked every 4 hours.


Re-run the test with the remainder of the original sample that has been refrigerated. Skip step 6 (2 hour pre-incubation) and immediately fill panel. If the quantity left is insufficient to fill the panel (less than 1 ml) add a small amount of MASTiK® Reagent, incubate for 30 minutes, and then fill panel.

2. Can occur with contaminated sample.


Must use proper sampling technique. Must handle sample and fill plate using aseptic technique.

Problem: Color Changes Not Dramatic.


Continue incubation.

Problem: Skips - Growth Occurs In High Concentration Well, But Not In Low Concentration Well Of The Same Drug.

1. Uneven distribution of bacterial numbers when filling plate.


One skip on a panel occasionally is acceptable and can be ignored.

2. Flakes (coated with high numbers of bacteria) were pipetted into wells.


Avoid pipetting clumps and flakes from sample.

Problem: Sample Began To Change Color In Sample Vial After Pre-incubation (step 6) And Before Filling Panel.


Add approximately one ml of the mixture to a fresh vial of MASTiK® Reagent.

Immediately fill the plate and incubate it.

Check the plate every 2 hours for color changes.

Treatment Failure

Although MASTiK® is highly accurate, some cases may not respond to the recommended antibiotic. Some causes for this phenomenon include:

1. Sample was contaminated.

2. Pathogenic bacteria were intracellular, not in milk.

3. Case is due to a pathogen often unresponsive to antibiotics such as Pseudomonas, Actinomyces, yeasts, Prototheca, or Mycoplasma.

4. The quarter is infected with two or more pathogens growing at different rates.

5. Course of treatment was too short or infrequent.

6. Case was due to chronic infection with Staphylococcus aureus.

7. Test was misread.

8. Antibiotic administered by the intramammary route did not disperse and distribute evenly throughout gland due to low volume of dose, improper vehicle, antibiotic kinetics, or inflammatory by-products.

9. Drug was administered improperly.

10. Administered drug (intramammary) was contaminated.

11. Sample did not contain a major mastitis pathogen.

12. Cow was too clinically ill.

Some of the above pitfalls can be avoided if the sample is simultaneously cultured and identified while running the MASTiK® test.

Examples Of Mastitis Preparation Trade Names


Trade Name


A. Erythromycin (ERY)



Gallimycin Dry Cow™




B. Oxacillin (OXA)

(daughter compound: cloxacillin)






Fort Dodge

C. Ampicillin (AMP)

(daughter compounds: amoxicillin, hetacillin)




Fort Dodge

D. Penicillin (PEN)

or penicillin combination products


G.C. Hanford







E. Cephalothin (CEP)

(daughter compounds: cephapirin, ceftiofur)


Fort Dodge


Fort Dodge


Fort Dodge


Fort Dodge

F. Pirlimycin (PIR)



Quick-step Chart

1. Pour Approximately 1ml Of Mastitis Sample Into Mastik Reagent Tube (fill To 4ml Line On Label). Recap, Invert To Mix And Label Tube. If Case Is Acute And Highly Severe, Skip Step 2 And Proceed To Step 3.

2. Incubate Sample-reagent Mixture For 1-3 Hours (6-8 Hours If Mastitis Is Chronic Or Subclinical) At 95-99°f (35-37°c).

3. Remove Plate From Foil Pouch. Remove The Panels Needed. Using Supplied Pipette, Add 2 Drops Of Mixture To Each Of The 16 Test Wells (2 Columns Of 8 Wells Per Sample).

4. Remove Clear Plastic Adhesive Sheet From Paper Backing And Cover All 16 Wells.

5. Incubate Panel At 95-99°f (35-37°c).

6. Examine Panel For Color Changes Every 4-8 Hours Until Positive Control Well Has Changed From Purple To Yellow.

7. Hold Panel Overhead And Record Colors On Mastik® Record Sheet, Making Sure You Match The Positions Of The Wells With The Sheet. The Negative Control Well Should Have Retained Its Original Purple Color. For Other Wells, Record Yellow As Resistant And Purple As Susceptible.

MASTiK® is a registered trademark of ImmuCell Corporation.

ImmuCell, 56 Evergreen Drive, Portland, ME 04103


Rev: 08/03

Nac No.


Telephone:   207-878-2770
Order Desk:   800-466-8235
Fax:   207-878-2117
Every effort has been made to ensure the accuracy of the MASTiK information published above. However, it remains the responsibility of the readers to familiarize themselves with the product information contained on the US product label or package insert.

Copyright © 2018 North American Compendiums. Updated: 2018-01-04