Brucella Dye Tolerance Media

Cat. no. Z006 Basic Fuchsin 1:50,000, 20x125mm Tube, 20ml 20 tubes/box
Cat. no. Z036 Thionin 1:50,000, 20x125mm Tube, 20ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Brucella Dye Tolerance Media are recommended for use in the speciation of Brucella spp.

SUMMARY

Brucellosis is a zoonosis of worldwide importance, particularly in developing countries. The organism is highly endemic in the Mediterranean Basin, Middle East, Western Asia, Africa and Latin America. The incidence of brucellosis in industrialized countries has decreased significantly due to aggressive programs to eradicate infected bovines. Historically, brucellosis has been primarily associated with occupational exposure for farmers, butchers, veterinarians and laboratory workers. Recent infections have been associated with the ingestion of unpasteurized goat milk. Infection results from direct contact of abraded skin or mucosal surfaces, consumption of contaminated food products or inhalation. Aerosols are highly infectious requiring only 10-100 organisms. The incubation period ranges from five days to two months.

The genus Brucella consists of small, non-motile gram-negative coccobacilli or short rods that are pathogenic for humans and animals. Colonies on blood agar are usually 0.5 to 1mm in diameter. The organisms are arranged singly and less frequently in pairs, short chains or small groups. Bi-polar staining is usually not observed. The organisms are non motile and do not possess flagella. Brucella species are catalase positive and most strains are oxidase positive. Enhanced CO2 may be required for some strains especially on primary isolation. Historically there have been six recognized species: melitensis, abortus, suis, canis, ovis and neotomae. Three of the species contain multiple biovars: Brucella melitensis(3), Brucella abortus(7), and Brucella suis(5).

Four species can cause brucellosis in humans: Brucella melitensis, Brucella abortus, Brucella suis and Brucella canis (rare). The severity and duration of brucellosis depends partially on the species of Brucella involved. Brucella melitensis is the most serious and debilitating infection. Brucella suis is highly invasive and tends to localize causing suppuration and necrosis. Brucella abortus is less invasive and the disease and tissue damage caused is less severe. Brucella canis causes mild to localized infection and complications are rare. Brucella speciation and biovars are defined by host specificity, tolerance to the dyes fuchsin and thionin, CO2 requirement, rate of urease activity, agglutination in monospecific adsorbed rabbit antisera and susceptibility to Brucella Tbilisi phage.

In 1954, Brucella suis became the first agent weaponized by the United States in its offensive biological warfare program. Brucella is currently listed as a Category B agent that can be used for biowarfare, bioterrorism or biocriminal activities. As part of the Laboratory Response Network, Level B laboratories are responsible for the definitive identification of Brucella species.

Non-molecular speciation of Brucella includes tolerance to the dyes thionin and fuchsin. These dyes are incorporated into the medium at varying concentrations. The concentration of dyes typically used is 1:50,000 for each dye. Hardy Diagnostics Brucella Dye Tolerance Media can be used to aid in the speciation of Brucella spp.

FORMULA

Ingredients per liter of deionized water:*

Heart Infusion Agar 40.0gm
Basic Fuchsin or Thionin Acetate (Stock solution) 1.0gm

Final pH 7.5 +/- 0.3 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. contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

1. Place tubes of dye media and one tube of Brain Heart Infusion Agar (Cat. no. Q16) into a boiling waterbath to melt the media.

2. Temper tubes to 44 to 46ºC. Tighten the caps and invert to mix the dye. Pour the media containing the different dye concentrations into individually labeled plates. Also pour the Brain Heart Infusion Agar into an individually labeled plate. Allow to solidify.

3. Suspend a loopful of freshly grown (18-24 hour) bacterial culture into 1ml of sterile physiological saline (Cat. no K248).

4. Immerse a sterile cotton swab (Cat. no. HD258061WC) into the suspension, remove and inoculate the dye plates and one control plate (not containing dye) by making a single streak across each one of the plates.

5. Invert the plates and incubate at 35ºC. Examine daily for up to 4 days. Record presence or absence of growth.

INTERPRETATION OF RESULTS



Thionin Fuchsin Urease a
Dye concentration 1:50,000
(Z036)
1:50,000
(Z006)
< 5 min. > 5 min.
B. melitensis
(biovar 1-3)
+ + v +
B. abortus
(biovar 1)
- + - +
2 - - - + b
3 + + - +
4 - + - +
5 + + - +
6 + + - +
7 + + - +
B. suis
(biovar 1)
+ - + c

2 + - + c

3 + + + c

4 + + + c

5 + - + c

B. canis + - d + c

a. Warm urea slant inoculated heavily.                              +   =   positive
b. Rare strains are urease negative.                                   -   =   negative
c. Usualy immediate or instant reaction.                            v   =   variable
d. Some strains grew at this concentration.                   

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, waterbaths, Brain Heart Infusion Agar (Cat. no. Q16), Saline, 0.85% (Cat. no. K248), other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Hardy Diagnostics tests Brucella Dye Tolerance dilutions for sterility, pH and fill volume.

USER QUALITY CONTROL

Physical Appearance

Basic Fuchsin dilution (Z006) should appear opaque, and light to medium amber with a fuchsia to reddish-purple or bright pink layer at the top of the tube. The media may appear layered with the dye more concentrated at the top of the tube.

Thionin dilution (Z036) should appear opaque, and light to medium amber with a teal to turquoise layer at the top of the tube. The media may appear layered with the dye more concentrated at the top of the tube.

REFERENCES

1. Diagnostic Procedures for Bacteriology, Mycology and Parasitology, 6th ed. 1981. American Public Health Association.

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

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Benenson, Abram S. 1995. Control of Communicable Diseases Manual, 16th ed., American Public Health Association, Washington D.C.

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