Mastitis Triplate (TKT Agar/ MacConkey Agar/ Blood Agar)

Cat. no. J312 Mastitis Triplate (TKT Agar / MacConkey Agar / Blood Agar),
15x100mm Triplate, 7ml/section
10 plates/bag

INTENDED USE

Hardy Diagnostics Mastitis Triplate (TKT Agar / MacConkey Agar / Blood Agar) is recommended as a general purpose growth and selective isolation media for the isolation of organisms responsible for mastitis in dairy populations.

SUMMARY

Bovine mastitis, the inflammation of the mammary gland in dairy cattle, is mainly caused by infections from bacteria. Over 130 different microorganisms have been isolated from bovine mastitic milk with Staphylococcus , Streptococcus , Mycoplasma , and coliform species being the most common etiologic agents. (6) Hardy Diagnostics Mastitis Triplate, a three sectioned plate containing TKT Agar, MacConkey Agar, and Blood Agar, is designed to aid in the growth and presumptive identification of mastitis related organisms. The Blood Agar is used to grow all organisms as well as demonstrate hemolysis patterns. MacConkey Agar is used for the selective isolation and differentiation of coliforms, while TKT Agar is used in the selective isolation and differentiation of Streptococcus species. (3-7)

Tryptic Soy Agar (TSA) is the basal medium for the Blood Agar portion of the Mastitis Triplate. Sheep blood has been added to facilitate the growth of most organisms and for the observation of hemolytic reactions. The absence of reducing sugars and carbohydrates allows hemolysis to occur without hindrance. (1-2) Staphylococcus aureus will appear round and shiny with golden-yellow colonies demonstrating a zone of partial to complete hemolysis, while Streptococcus species will demonstrate alpha-, beta-, or non-hemolytic patterns and are often white to gray in color. (3-7)

MacConkey Agar is added to the Mastitis Triplate to selectively isolate and differentiate coliforms. The MacConkey Agar formulation in use is a modification of the original Neutral Red Bile Salt Agar developed by MacConkey. (9) In addition to containing sodium chloride, the modified formula has a lowered agar content and an adjusted concentration of bile salts and neutral red. Bile salts and crystal violet are added to inhibit the growth of most gram-positive organisms, while the differentiation of enteric microorganisms is achieved by the combination of the neutral red indicator and lactose. (1-2) Lactose-fermenting organisms, like E.coli and Klebsiella species, form pink colonies surrounded by a zone of bile salt precipitation. The color change is due to the production of acid, which changes the neutral red pH indicator from colorless to red. Acid production is also responsible for the formation of bile salt precipitation. Non-lactose-fermenters, like Pseudomonas species, develop into transparent, colorless colonies with no precipitated zone. (1-4)

TKT Agar contains selective agents to suppress the normal bovine flora, including gram-negative bacilli and staphylococci, thereby allowing the isolation and presumptive identification of mastitis related streptococci. TKT Agar also has esculin to differentiate group D streptococci from Streptococcus agalactiae. (3-7) When esculin is hydrolyzed by organisms it forms dextrose and esculetin, which react with a compound in the media to produce a darkening or blackness around the colonies. (1,2) Therefore on TKT Agar; S. agalactiae , which is incapable of esculin hydrolysis, should appear beta-hemolytic with no darkening of the media, while S. uberis should appear non-hemolytic with obvious darkening of the media. (3-7)

In order to further differentiate and presumptively identify Streptococcus agalactiae from bovine samples, beta toxin is added to the TKT media. Hardy Diagnostics Beta Toxin (Cat. no. Z306) is a simplified and modified version of the traditional CAMP procedure. Beta Toxin contains extracted S. aureus beta-hemolysin and is added to the TKT media to evaluate the CAMP reaction on primary isolation. The modified CAMP procedure has demonstrated a high degree of reliability in the identification of S. agalactiae . (4-7) In this method the inoculated plate is observed for CAMP reaction after overnight incubation. A positive CAMP reaction of TKT media is noted by an enhanced zone of beta-hemolysis and presumptively indicates of S. agalactiae. (3-7) The combination of esculin and beta toxin in the TKT formulation can be used to further differentiate non-esculin degrading CAMP-positive S. agalactiae from occasional CAMP-positive isolates of S. uberis, which are capable of esculin hydrolysis. As well as having selective and differential properties, TKT Agar is also highly nutritious. Organic nitrogen, particularly amino acids and long-chained peptides are supplied by the combination of meat peptones and beef extract, while osmotic equilibrium is maintained by sodium chloride. Sheep blood has been added to facilitate growth and to detect hemolytic activity of streptococcal isolates.

FORMULA

Ingredients per liter of deionized water:

TKT Agar:
Meat Peptone 10.0gm
Beef Extract 5.0gm
Sodium Chloride 5.0gm
Esculin 1.0gm
Thallium Acetate 0.333gm
Crystal Violet 1.3mg
Sheep Blood 60.0ml
Beta Toxin 25.0ml
Agar 13.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

MacConkey Agar:
Peptone 17.0gm
Lactose 10.0gm
Sodium Chloride 5.0gm
Proteose Peptone 3.0gm
Bile Salts 1.5gm
Neutral Red 30.0mg
Crystal Violet 1.0mg
Agar 13.5gm

Final pH 7.1 +/- 0.2 at 25ºC.

Blood Agar:
Pancreatic Digest of Casein 15.0gm
Peptic Digest of Soybean Meal 5.0gm
Sodium Chloride 5.0gm
Sheep Blood 50.0ml
Agar 5.4gm

Final pH 7.3 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Sample Collection: It is important that a milk sample be taken from the mammary gland and not from dust or fecal particles on the udder surface. To ensure the pathogen is from the milk, the teat surface and orifice should be wiped with seventy percent (70%) ethyl alcohol. It is also essential to obtain a sample before the cow has been treated with antimicrobial agents. Consult listed references for additional information on specimen collection. (4)

Method of use:

1. Allow the Mastitis Triplate (TKT Agar / MacConkey Agar / Blood Agar) to warm to room temperature before inoculating the surface of the medium with the milk samples.

2. After overnight incubation at 35ºC, observe plates for growth, hemolysis, and esculin hydrolysis reactions as noted in appropriate reference literature. (1-8)

INTERPRETATION OF RESULTS

TKT Agar:
TKT Agar should be examined for growth, hemolytic reactions, CAMP reaction, and esculin-hydrolysis. Growth on TKT indicates the presence of Streptococcus species. Colonies should be evaluated for alpha-, beta-, and non-hemolytic patterns. Additionally, a positive CAMP reaction, associated with an enhanced zone of beta-hemolysis, should be observed. Esculin hydrolysis, as indicated by a darkening around the colonies, is also important for colony differentiation. S. uberis and other group D streptococci capable of esculin hydrolysis will blacken the media, while non-hydrolytic esculin organisms, like S. agalactiae will remain unchanged. Therefore, the occasional CAMP-positive isolates of S. uberis can be differentiated from CAMP-positive strains of S. agalactiae based on the presence of blackening in the media. Consequently, organisms demonstrating enhanced beta-hemolysis that are not surrounded by a darkening in the media can be presumptively identified as S. agalactiae . Consult listed references for the identification of colony morphology and further biochemical tests required for identification. (1,2,4,6)

MacConkey Agar:
MacConkey Agar is examined for growth, colony morphology, and lactose-fermentation. Growth indicates the presence of gram-negative organisms. Well isolated colonies of lactose-fermenting bacteria will appear pink to red in color and are surrounded by a zone of bile salt precipitation. Non-lactose-fermenting colonies, appear transparent and colorless, with no zone of bile salt precipitation. Consult listed references for further procedures for identification of isolates. (1,2,4,6)

Blood Agar:
Blood Agar should be examined for growth, colony morphology, as well as for colonies with alpha-, beta-, and non-hemolytic patterns. Blood Agar is a nutritious general all purpose media intended to growth most mastitis pathogens. Consult listed references for further procedures for the identification of isolates. (1-6)

LIMITATIONS

TKT Agar is used in conjunction with other tests to identify cultures of S. agalactiae. It is necessary to confirm, with other biochemical or molecular tests, the identification of all organisms suspected of being S. agalactiae.

The concentration of bile salts in MacConkey Agar is relatively low in comparison to other enteric media. If gram-negative organisms are suspected, the parallel use of more selective media for gram-negative enterics, such as HE Agar (Cat. no.G63) or XLD Agar (Cat. no. G65) is recommended in order to increase the chances of pathogen isolation.

Some strains of Proteus may swarm on MacConkey Agar. Serial subculturing may be required to assure adequate isolation of mixed flora samples.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, 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
TKT Agar:
Streptococcus agalactiae
ATCC ® 12386
A 24hr 35°C Aerobic Growth; beta-hemolytic, CAMP-positive demonstrating enhanced beta-hemolysis, esculin-negative
Streptococcus equi subsp. zooepidemicus
ATCC ® 700400
A 24hr 35°C Aerobic Growth; non-hemolytic, CAMP-negative, esculin-positive
Streptococcus dysgalactiae
ATCC ® 43078
A 24hr 35°C Aerobic Growth; non-hemolytic, CAMP-negative, esculin-negative
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic No Growth
Staphylococcus aureus
ATCC ® 25923
B 24hr 35°C Aerobic No Growth
MacConkey Agar:
Escherichia coli
ATCC ® 25922
A 24hr 35°C Aerobic Growth; pink to red colonies, with bile salt precipitate surrounding the colonies
Proteus mirabilis
ATCC ® 12453
A 24hr 35°C Aerobic Growth; colorless colonies, with no swarming
Streptococcus agalactiae
ATCC ® 12386
B 24hr 35°C Aerobic No Growth
Blood Agar:
Streptococcus agalactiae
ATCC ® 12386
A 24hr 35° Aerobic Growth; beta-hemolytic
Streptococcus equi subsp. zooepidemicus
ATCC ® 700400
A 24hr 35°C Aerobic Growth; non-hemolytic
Streptococcus dysgalactiae
ATCC ® 43078
A 24hr 35°C Aerobic Growth; non-hemolytic

User Quality Control

PHYSICAL APPEARANCE

Mastitis Triplate:

REFERENCES

1. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

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

3. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

4. Quinn, P.J., et al. 1994. Clinical Veterinary Microbiology . Wolfe Publishing, London, England.

5. National Mastitis Council. 1999. Laboratory Handbook on Bovine Mastitis . NMC Inc., Madison, WI.

6. Carter, G.R., et al. 1995. Essentials of Veterinary Microbiology , 5th ed. Williams & Wilkins, Philadelphia, PA.

7. A Practical Look at Contagious Mastitis, www.nmconline.org /contmast, 04/18/02.

8. National Mastitis Council. 1999. Laboratory Handbook on Bovine Mastitis , NMC Inc., Madison, WI.

9. MacConkey, A.T. 1905. Lactose-fermenting bacteria in faeces. J. Hyg. ; 5:333-379.


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