Brucella Media Brucella Agar • Brucella Agar with 5 ... - BVA Scientific

Section III
B
m Brilliant Green Broth, cont.
3. Cool to room temperature. Dispense 2 mL amounts onto
sterile absorbent pads.
4. Use rehydrated medium within 24 hours.
5. Test samples of the finished product for performance using
stable, typical control cultures.
Procedure
1. Inoculate a water sample using the membrane filtration
procedure.
2. Place the filter on a pad saturated with m Brilliant Green
Broth.
3. Incubate at 35 ± 2°C in a humid atmosphere for 18-24
hours.
4. After incubation, examine for growth and the color of the
colonies.
Expected Results
Salmonella species form pink to red colonies.
References
1. Murray, Baron, Pfaller, Tenover and Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
American Society for Microbiology, Washington, D.C.
2. Kauffmann. 1935. Z. Hyg. Infektionskr. 117:26.
3. Kabler and Clark. 1952. Am. J. Public Health 42:390.
4. Geldreich and Jeter. 1952. Abstr. Bacteriol. Proc. 52nd Gen. Meet. Soc. Am. Bacteriologists 1952.
Availability
Difco m Brilliant Green Broth
Cat. No. 249410 Dehydrated – 500 g
Brucella Media
Brucella Agar • Brucella Agar with 5% Horse Blood
Brucella Broth
104
Intended Use
Brucella Agar is a culture medium for the cultivation of Brucella
organisms. With the addition of 5% horse blood, the medium is
used in qualitative procedures for the isolation and cultivation
of nonfastidious and fastidious microorganisms from a variety
of clinical and nonclinical specimens.
Brucella Broth is used for the cultivation of Brucella species
and for the isolation and cultivation of a wide variety of
fastidious and nonfastidious microorganisms.
Summary and Explanation
Brucella Agar was developed for the cultivation of Brucella
species from diagnostic specimens, such as blood, and from
foods and other potentially contaminated material. Brucella
Agar with 5% Horse Blood plates are particularly useful for
the cultivation of the more fastidious aerobic and anaerobic
microorganisms,including streptococci, pneumococci, Listeria,
Neisseria meningitidis and Haemophilus influenzae.
Brucella Broth may be used for the isolation and cultivation
of a wide variety of microorganisms including nutritionally
fastidious specimens. 1 This medium is recommended for
the cultivation of Brucella species and was recommended as
one of several media suitable for use as the liquid medium
component of biphasic blood culture bottles. 1,2 It is also used
to cultivate Campylobacter spp. 3
Principles of the Procedure
Brucella Agar and Brucella Broth support the growth of
fastidious microorganisms due to their content of peptones,
dextrose and yeast extract. The peptones supply organic
nitrogen. The yeast extract is a potent source of the B-complex
vitamins. Dextrose is utilized as an energy source. Sodium
bisulfite is a reducing agent, and sodium chloride maintains
the osmotic equilibrium. Agar is the solidifying agent in
Brucella Agar.
In BBL Brucella Agar with 5% Horse Blood plates, the horse
blood supplies both the X and V factors which are growth
requirements for certain organisms; e.g., Haemophilus
influenzae. 3 Sheep and human blood are not suitable for this
purpose because they contain enzymes that inactivate the nicotinamide
adenine dinucleotide (NAD) which is the V factor. 4
Defibrinated horse blood may give hemolytic reactions different
than sheep blood. 5 Some streptococci (e.g., group D) give
hemolytic reactions on horse blood but not on sheep blood
and may be mistakenly reported as group A. If a hemolytic
reaction is obtained, the organism should be tested with a
Taxo A bacitracin (0.04 unit) disc and it also should be
grouped serologically or tested by the fluorescent antibody
method. 6 Beta-hemolytic streptococci and Haemophilus
haemolyticus may be differentiated by performing a Gram stain
on a smear prepared from the colony.
Formulae
BBL Brucella Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein ...................................... 10.0
Peptic Digest of Animal Tissue ................................. 10.0
Dextrose ..................................................................... 1.0
Yeast Extract .............................................................. 2.0
Sodium Chloride ........................................................ 5.0
Sodium Bisulfite ......................................................... 0.1
Agar ......................................................................... 15.0
BBL Brucella Broth
Consists of the same ingredients without the agar.
*Adjusted and/or supplemented as required to meet performance criteria.
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Brucella Media, cont.
User Quality Control
Identity Specifications
BBL Brucella Agar
Dehydrated Appearance:
Fine, homogeneous, free of extraneous
material.
Solution:
4.3% solution, soluble in purified
water upon boiling. Solution is light
to medium, tan to yellow, clear to
slightly hazy, may contain small
amount of sediment.
Prepared Appearance: Light to medium, tan to yellow, clear
to slightly hazy.
Reaction of 4.3%
Solution at 25°C: pH 7.0 ± 0.2
BBL Brucella Broth
Dehydrated Appearance:
Fine, homogeneous, free of extraneous
material.
Solution:
2.8% solution, soluble in purified
water upon heating. Solution is pale
to medium, tan to yellow, clear to
slightly hazy.
Prepared Appearance: Pale to medium, tan to yellow, clear
to slightly hazy.
Reaction of 2.8%
Solution at 25°C: pH 7.0 ± 0.2
Cultural Response
BBL Brucella Agar
Prepare the medium per label directions without (plain) and with 5%
defibrinated horse blood (HB). Inoculate and incubate at 35 ± 2°C for
3 days with 3-5% CO 2
(incubate S. aureus without CO 2
).
INOCULUM RECOVERY RECOVERY
ORGANISM ATCC CFU PLAIN WITH HB
Brucella abortus 11192* 10 3 -10 4 Good Good
Brucella melitensis 4309* 10 3 -10 4 Good N/A
Brucella suis 4314* 10 3 -10 4 Good N/A
Staphylococcus aureus 25923 10 3 -10 4 Good N/A
Streptococcus pneumoniae 6305 10 3 -10 4 N/A Good
Streptococcus pyogenes 19615 10 3 -10 4 N/A Good
*Minimally one strain of Brucella should be used for performance testing. If these strains are
not available, verify performance with a known isolate.
BBL Brucella Broth
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 7 days with 3-5% CO 2
(incubate S. pyogenes for 66-72
hours without CO 2
).
ORGANISM ATCC INOCULUM CFU RECOVERY
Brucella abortus 11192* < 10 3 Growth
Brucella melitensis 4309* < 10 3 Growth
Brucella suis 4314* < 10 3 Growth
Streptococcus pyogenes 19615 < 10 3 Growth
*Minimally one strain of Brucella should be used for performance testing. If these strains are
not available, verify performance with a known isolate.
B
Precautions 7
1. Biosafety Level 2 practices, containment equipment and
facilities are recommended for activities with clinical specimens
of human or animal origin containing or potentially
containing pathogenic Brucella spp.
2. Biosafety Level 3 practices, containment equipment and
facilities are recommended for all manipulations of cultures
of the pathogenic Brucella spp. and for experimental
animal studies.
Directions for Preparation from
Dehydrated Product
1. Suspend the powder in 1 L of purified water:
BBL Brucella Agar – 43 g;
BBL Brucella Broth – 28 g.
Mix thoroughly.
2. For the agar, heat with frequent agitation and boil for
1 minute to completely dissolve the powder. For the broth,
heat slightly, if necessary, to obtain solution.
3. Autoclave at 121°C for 15 minutes.
4. For preparation of blood plates, add 5 to 10% sterile
defibrinated blood to sterile agar which has been cooled to
45-50°C.
5. Test samples of the finished product for performance using
stable, typical control cultures.
Procedure
Agar (without or with added blood)
Use standard procedures to obtain isolated colonies from
specimens.
Since many pathogens require carbon dioxide on primary
isolation, incubate plates at 35 ± 2°C for 24-72 hours in an
aerobic atmosphere supplemented with carbon dioxide.
Broth
For liquid specimens, use a sterile inoculating loop to transfer
a loopful to the broth medium. Swab specimens may be
inserted into the broth after the inoculation of plated media.
Incubate tubes for up to 7 days at 35 ± 2°C in an aerobic atmosphere
with or without supplementation with carbon dioxide.
For the preparation of biphasic blood culture bottles, aseptically
add sterile Brucella Broth to a blood culture bottle containing
solidified sterile Brucella Agar, with increased agar at a final
concentration of 2.5%. The bottles should contain 5-10% CO 2
and be vented. Blood cultures should be incubated at 35°C for
up to 30 days with subcultures prepared every 4 to 5 days. 1,2
Expected Results
Agar (without or with added blood)
After incubation, most plates will show an area of confluent
growth. Because the streaking procedure is, in effect, a
“dilution” technique, diminishing numbers of microorganisms
are deposited on the streaked areas. Consequently, one or more
105

Section III
B
Brucella Media, cont.
of these areas should exhibit isolated colonies of the organisms
contained in the specimen. Further, growth of each
organism may be semi-quantitatively scored on the basis of
growth in each of the streaked areas.
Broth
Growth in the tubes is indicated by the presence of turbidity
compared with an uninoculated control.
If growth appears, cultures should be examined by Gram
stain and subcultured onto appropriate media; e.g., Trypticase
Soy Agar with 5% Sheep Blood and/or Brucella Agar and
Chocolate II Agar, Eosin Methylene Blue Agar, Levine or
MacConkey II Agar.
References
1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,
vol. 1. Williams & Wilkins, Baltimore, Md.
2. Moyer, Holcomb and Hausler. 1991. In Balows, Hausler, Herrmann, Isenberg, and Shadomy (ed.),
Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C.
3. Chapin and Murray. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.). Manual of
clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
4. Krumweide and Kuttner. 1938. J. Exp. Med. 67:429.
5. Vera and Power. 1980. In Lennette, Balows, Hausler and Truant (ed.), Manual of clinical microbiology,
3rd ed. American Society for Microbiology, Washington, D.C.
6. Vera. 1971. Health Lab. Sci. 8:176.
7. U.S. Public Health Service, Centers for Disease Control and Prevention, and National Institutes of
Health. 1999. Biosafety in microbiological and biomedical laboratories, 4th ed. HHS Publication
No. (CDC) 93-8395. U.S. Government Printing Office, Washington, D.C.
Availability
BBL Brucella Agar
ISO USDA
Cat. No. 211086 Dehydrated – 500 g
221547 Prepared Plates with 5% Horse Blood – Pkg. of 20*
221548 Prepared Plates with 5% Horse Blood –
Ctn. of 100*
BBL Brucella Agar with 5% Horse Blood//
MacConkey II agar with MUG
Cat. No. 298303 Prepared I Plate Dishes – Ctn. of 100*
BBL Brucella Broth
USDA
Cat. No. 211088 Dehydrated – 500 g
296185 Dehydrated – 5 lb (2.3 kg)
*Store at 2-8°C.
Brucella Media for Anaerobes
Brucella Agar with 5% Sheep Blood, Hemin and
Vitamin K 1 • Brucella Laked Sheep Blood Agar with
Kanamycin and Vancomycin (KV)
Intended Use
Brucella Agar with 5% Sheep Blood, Hemin and Vitamin K 1 is
used for the isolation and cultivation of fastidious, obligately
anaerobic microorganisms.
Brucella Laked Sheep Blood Agar with Kanamycin and Vancomycin
(KV) is used for the selective isolation of fastidious
and slow growing, obligately anaerobic bacteria from the same
specimen.
Summary and Explanation
The isolation of obligately anaerobic bacteria from clinical and
nonclinical materials requires the use of selective, nonselective
and enrichment media. 1 Brucella Agar with 5% Sheep Blood,
Hemin and Vitamin K 1 is an enriched, nonselective medium
for the isolation and cultivation of a wide variety of obligately
anaerobic microorganisms. Nonselective media are used to
isolate organisms present in low numbers and to provide an
indication of the numbers and types of organisms present in
the specimen or sample.
Kanamycin and vancomycin are included in Brucella Laked
Blood KV Agar for use in selective isolation of gram-negative
anaerobes, especially Bacteroides. The combination of kanamycin
and vancomycin for this purpose was first described by
Finegold et al. 2 Vancomycin, however, may inhibit
Porphyromonas asaccharolytica. 3
Principles of the Procedure
Brucella Agar supports the growth of fastidious microorganisms
due to its content of peptones, dextrose and yeast extract. The
sheep blood, hemin and vitamin K 1 , provide essential nutrients
for certain obligate anaerobes. 4
The addition of the antimicrobial agents, kanamycin and
vancomycin, renders Brucella Laked Blood KV Agar selective
for gram-negative microorganisms. The kanamycin inhibits
protein synthesis in susceptible organisms, whereas the
vancomycin inhibits gram-positive bacteria by interfering with
cell wall synthesis. 5 The laked blood improves pigmentation
of the Prevotella melaniogenica - P. asaccharolytica group.
Procedure
These media should be reduced immediately prior to inoculation
by placing under anaerobic conditions for 18-24 hours. 6
An efficient and easy way to obtain suitable anaerobic
conditions is through the use of BBL GasPak EZ anaerobic
systems or an alternative system. 7
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