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Material Safety Data Sheet

Required Products

Organisms

Organisms in the Clinical sector this product works with:

Dehydrated Culture Media

Sector: Clinical

BRUCELLA MEDIUM BASE

Code: CM0169

For the preparation of serum-dextrose-antibiotic medium for the cultivation and isolation of Brucella using Brucella Selective Supplement SR0083 or SR0209. Without antibiotics, it may be used in conjunction with the Cruickshank dyestrip method for differentiation between strains.

Typical Formula*

gm/litre

Peptone

10.0

`Lab-Lemco’ powder

5.0

Glucose

10.0

Sodium chloride

5.0

Agar

15.0

pH 7.5 ± 0.2 @ 25°C

 

* Adjusted as required to meet performance standards

Directions
Suspend 45 g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and add 5% of inactivated Horse Serum (i.e. Horse Serum held at 56°C for 30 minutes). Mix well before pouring.

BRUCELLA SELECTIVE SUPPLEMENTS

Vial contents (each vial is sufficient for 500ml of medium)
Code: SR0083
Code: SR0209

Polymyxin B

2,500 IU
2,500 IU

Bacitracin

12,500 IU
12,500 IU

Cycloheximide

50 mg

Nalidixic acid

2.5 mg
2.5 mg

Nystatin

50000 IU
50000 IU
Vancomycin
10 mg
10 mg

Natamycin

25 mg

Directions
Reconstitute supplement as directed. Incubate for 10-15 minutes at 35°C. Mix thoroughly and immediately add the vial contents to 500 ml of sterile Oxoid Brucella Medium Base, cooled to 50°C together with 5-10% v/v sterile inactivated horse serum SR0035 and 1-5% w/v of a filter-sterilised 10% solution of dextrose. Mix well and pour into sterile petri dishes.

Description
Brucella Medium Base may be used to prepare the serum-dextrose-antibiotic medium described by Jones and Brinley Morgan1 for the cultivation and isolation of Brucella, including fastidious types. Brucella medium with antibiotics has advantages over the media described by Kuzdas and Morse2 and Renoux3 in that it will support growth of fastidious types and it is more effective as a selective medium. During investigations, Jones and Brinley Morgan showed that serum-glucose agar with antibiotics gave excellent growth of all Brucella strains and permitted better growth of Brucella abortus biotype 2 - a strain which had been difficult or impossible to cultivate.

Technique
The addition of dyes (i.e. malachite green and gentian violet) as selective agents, is not recommended, as it may result in poor growth of many Brucella strains. Where a non-selective medium is required, the medium may be employed with the addition of serum only (i.e. without antibiotics): for subsequent differentiation between strains of Brucella. This medium is recommended for use in conjunction with the Cruickshank dyestrip method4:
1. Impregnate filter paper strips with 1:200 Basic Fuchsin or 1:600 Thionin. Dry.
2. Place the strips parallel on the surface of the serum-dextrose agar and then cover with a thin layer of the same medium. Then allow the medium to solidify.
3. Make stroke inoculations of the Brucella strains to be tested, at right angles to the strips.
4. Incubate in 10% carbon dioxide for 2-3 days at 35°C.
5. Examine. Resistant strains grow right across the strip, but sensitive strains show inhibition of growth up to 10mm from the strip. Typical growth patterns are then as follows:

 

Basic Fuchsin 1:200

Thionin 1:600

Brucella abortus

growth

no growth

Brucella melitensis

growth

growth

Brucella suis

no growth

growth

However, there are exceptions to the above and it is therefore advisable to base identification on many characteristics5.

The slow growth of Brucella species, combined with their requirement for highly nutritious media means that selective agents must be incorporated to prevent overgrowth of contaminant organisms from milk or veterinary tissues.

Media containing bacteriostatic dyes are inhibitory to strains of Brucella abortus biotype 2 and other fastidious strains. Antibiotics used in place of dyes enabled growth of all biotypes Brucella species to appear on selective media1. However, Leech et al.6 showed that the serum-glucose-antibiotic formulation1 was not sufficiently selective and was less efficient than guinea-pig inoculation.

Barrow and Peel7 modified a selective medium devised by Mair8. This contained both antibiotics and gentian violet. Failure of some strains of Brucella abortus to grow confirmed their sensitivity to very low concentrations of the dye recognised by Mair.

Farrell9 developed a highly selective antibiotic-containing nutrient medium which incorporated bacitracin 25iu/ml, vancomycin 20mg/ml, cycloheximide 100mg/ml and nystatin 100iu/ml, in a serum-glucose agar base.

In comparative trials10 the medium was shown to give a rate of isolation equivalent to that achieved by guinea-pig inoculation. It also supported the growth of Brucella abortus biotype 2 strains.

Brucella Selective Supplement SR0083 is based on the superior formulation of Farrell. Its greater efficiency at suppressing bacterial contamination than either serum glucose agar or Barrow and Peel’s Medium was shown in a further trial11.
1. For direct culture of Brucella species from milk transfer the samples to sterile tubes and hold overnight at 40°C.
2. Withdraw an aliquot of gravity cream with a spiral wire and spread over a plate of supplemented agar with a bent sterile glass rod.
3. Incubate the plates at 35°C in an atmosphere containing 10-20% (v/v) carbon dioxide and examine every two days for ten days.
4. Brucella colonies appear as 1-2mm diameter convex colonies with round entire edges, and may be identified by slide agglutination.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared agar plates at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

Positive control: (w/o antibiotics)

Expected results

Bordetella bronchiseptica ATCC® 4617 *

Good growth; small clear colonies

Negative control: (with antibiotics)

 

Staphylococcus aureus ATCC® 25923 *

No growth

* This organism is available as a Culti-Loop®

References
1. Jones Lois M. and Brinley Morgan W.J. (1958) Bull. Wld Hlth Org. 19. 200-203.
2. Kuzdas C.D. and Morse E.V. (1953) J. Bact. 66(4). 502-504.
3. Renoux G. (1954) Ann. Inst. Pasteur 87(3). 325-333.
4. Cruickshank J.C. (1948) J. Path. Bact. 60. 328-329.
5. Stableforth A.W. and Jones Lois M. (1963) Internat. Bull. Bact. Nomen. Taxon. 13. 145-158.
6. Leech F.B., Vessey M.P., Macrae W.D., Lawson J.R., MacKinnon D.J. and Brinley Morgan W.J. (1984) Animal Disease: Survey No 4 HMSO.
London
. p17.
7. Barrow G.I. and Peel M. (1967) Mon. Bull. Minist. Hlth 26. 192-196.
8. Mair N.S. (1955) Mon. Bull. Minist. Hlth 14. 184-191.
9. Farrell I.D. (1969) PhD Thesis, University of Liverpool.
10. Farrell I.D. and Robinson L. (1972) J. Appl. Bact. 35. 625-630.
11. Hunter D. and Kearns M. (1977) Br. Vet. J. 133. 486-489.

 
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