Thioglycollate Media
Thioglycollate Media
Fluid Thioglycollate Medium • NIH Thioglycollate
Broth • Sterility Test Broth • Thioglycollate Medium,
Brewer Modified • Fluid Thioglycollate Medium
with Beef Extract • Thioglycollate Medium without
Dextrose • Thioglycollate Medium (Fluid), without
Dextrose or (Eh) Indicator • Thioglycollate Medium
without Indicator (135C) • Fluid Thioglycollate
Medium, Enriched • Enriched Thioglycollate Medium
Thioglycollate Medium with Calcium Carbonate
Enriched Thioglycollate Medium with Calcium
Carbonate
Intended Use
Fluid Thioglycollate Medium (FTM) is used for the steril-
ity testing of biologics and for the cultivation of anaerobes,
aerobes and microaerophiles.
NIH Thioglycollate Broth and Sterility Test Broth (USP
Alternative Thioglycollate Medium) may be used for sterility
testing instead of FTM.
Thioglycollate Medium, Brewer Modified is used for the
cultivation of obligate anaerobes, microaerophiles and faculta-
tive organisms.
Fluid Thioglycollate Medium with Beef Extract is used in
cultivating microorganisms from normally sterile biological
products.
Thioglycollate Medium without Dextrose and Thioglycol-
late Medium (Fluid), without Dextrose or (Eh) Indicator are
used as bases for fermentation studies of anaerobes, as well
as for detecting microorganisms in normally sterile materials,
especially those containing mercurial preservatives.
Thioglycollate Medium without Indicator (135C) is an enriched
general-purpose medium for the recovery of a wide variety
of microorganisms, particularly obligate anaerobes, from
clinical specimens and other materials.
Fluid Thioglycollate Medium, Enriched and Enriched
Thioglycollate Medium are general-purpose media used in
qualitative procedures for the cultivation of fastidious, as well
as nonfastidious microorganisms, including aerobic and anaerobic
bacteria, from a variety of clinical and nonclinical specimens.
Enriched Thioglycollate Medium when supplemented with
sodium bicarbonate or a marble chip is used to prepare a
standardized inoculum by the growth method for antimicrobial
susceptibility testing of anaerobic bacteria.
Difco Manual Sect III Ta new.ind547 547
Thioglycollate Medium with Calcium Carbonate and Thiogly-
collate Medium, Enriched, with Calcium Carbonate are recom-
mended for the maintenance of stock cultures.
Fluid Thioglycollate Medium and NIH Thioglycollate Broth/
Sterility Test Broth meet United States Pharmacopeia (USP)
performance specifications. T
Summary and Explanation
Quastel and Stephenson1 found that the presence of a small
amount of a compound containing an –SH group (cysteine,
thioglycollic acid, glutathione) permitted “aerobic” growth of
Clostridium sporogenes in tryptic digest broth.
Falk, Bucca and Simmons2 pointed out the advantages of
using small quantities of agar (0.06-0.25%) in detecting
contaminants during sterility testing of biologicals. The value
of combining a small amount of agar and a reducing substance
was demonstrated by Brewer.3 Brewer’s experiments revealed
that in a liquid medium containing 0.05% agar, anaerobes
grew equally well in the presence or absence of sodium thio-
glycollate. Marshall, Gunnish and Luxen4 reported satisfactory
cultivation of anaerobes in Brewer’s Thioglycollate Medium
in the presence of a mercurial preservative. Nungester, Hood
and Warren5 and Portwood6 confirmed the neutralization of
the bacteriostatic effect of mercurial compounds by sodium
thioglycollate. Incorporation of casein peptone was introduced
by Vera.7 Malin and Finn8 reported the commonly used medium
containing thioglycollate is inhibitory to some organisms in the
presence of a carbohydrate. In 1941, the National Institutes
of Health specified the use of two thioglycollate media in
sterility testing, the Brewer Formula and the Linden Formula.9
The Linden Formula was later referred to as Modified Brewer
Thioglycollate Medium in which meat infusion was replaced by
plant (soy) peptones.10
547
3/16/09 4:57:02 PM
 Section III
T Thioglycollate Media, cont.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the
development and testing of media for industrial and clinical applications, per the referenced publications.

Identity Specifications
Difco™ Fluid Thioglycollate Medium Difco™ Thioglycollate Medium without Dextrose
Dehydrated Appearance: Light beige, free-flowing, homogeneous. Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 2.98% solution, soluble in purified water upon Solution: 2.4% solution, soluble in purified water upon
boiling. When hot, solution is light amber, boiling. When hot, solution is light amber, clear
clear. to very slightly opalescent.
Prepared Appearance: Light amber, slightly opalescent, 10% or less of Prepared Appearance: Light amber, slightly opalescent, 10% or less of
upper layer may be medium pink. After shaking upper layer is green.
solution becomes pink throughout. Reaction of 2.4%
Reaction of 2.98% Solution at 25°C: pH 7.2 ± 0.2
Solution at 25°C: pH 7.1 ± 0.2
Difco Thioglycollate Medium without Dextrose or
™

Difco™ NIH Thioglycollate Broth Indicator

Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution: 2.9% solution, soluble in purified water upon boil-
ing. When hot, solution is light amber, clear to very
slightly opalescent, may have a slight precipitate.
Prepared Appearance: Light amber, clear to very slightly opalescent,
may have a slight precipitate. Reaction of 2.4%
Reaction of 2.9%
Solution at 25°C: pH 7.1 ± 0.2
Difco™ Fluid Thioglycollate Medium with Beef
Extract
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution: 3.47% solution, soluble in purified water upon
boiling for 1-2 minutes. When hot, solution is
medium amber, clear.
Prepared Appearance: Medium amber with some opalescence, 10% or
less of upper layer may be pink. After shaking
solution becomes pink throughout.
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 2.4% solution, soluble in purified water upon
boiling. When hot, solution is light amber,
clear.
Prepared Appearance: Light amber, slightly opalescent.
Solution at 25°C: pH 7.2 ± 0.2
Difco™ Thioglycollate Medium without Indicator
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 2.9% solution, soluble in purified water upon
boiling. When hot, solution is light amber,
clear.
Prepared Appearance: Light amber, very slightly to slightly opalescent.
Reaction of 2.9%
Solution at 25°C: pH 7.2 ± 0.2
Continued

Reaction of 3.47%
Solution at 25°C: pH 7.2 ± 0.2

Fluid Thioglycollate Medium is recommended in the FDA

Bacteriological Analytical Manual (BAM)11 and the Official
Methods of Analysis of AOAC International12 for the exami-
nation of food, and for determining the phenol coefficient and
sporicidal effects of disinfectants. Fluid Thioglycollate Medium
is also specified for sterility checks on banked blood.13 It is
one of the media recommended in the USP for use in sterility
testing of articles purporting to be sterile; these formulations
meet the requirements of the USP growth promotion test.14
NIH Thioglycollate Broth and Sterility Test Broth, which are
the USP Alternative Thioglycollate Medium, are Fluid Thio-
glycollate Medium without the agar or indicator components.
They are used for the same sterility test procedures except that
anaerobic incubation is recommended rather than aerobic
incubation. They also meet the requirements of the USP growth
promotion test.14
Uninoculated Bacteroides vulgatus Fluid Thioglycollate Medium with Beef Extract is recommended
Tube ATCC™ 8482
by the Animal and Plant Health Inspection Service, USDA,15 in
the detection of viable bacteria in live vaccines. Thioglycollate
Medium without Dextrose and Thioglycollate Medium without
Dextrose or Indicator may be used with added carbohydrates
for fermentation studies.

548

Difco Manual Sect III Ta new.ind548 548 3/16/09 4:57:06 PM


Cultural Response
Difco™ Fluid Thioglycollate Medium
Prepare the medium per label directions. Inoculate and incubate at 30-35 C
for 18-48 hours (up to 72 hours, if necessary). To test for growth promotion
according to the USP/EP, inoculate using organisms marked with (*) and
incubate aerobically at 30-35°C for up to 5 days.
INOCULUM USP/EP
ORGANISM ATCC™ CFU RECOVERY GROWTH
Clostridium novyi 7659 10-102 Good N/A
Clostridium perfringens 13124 10-10 2
Good N/A
Staphylococcus aureus 25923 10-102 Good N/A
Bacillus subtilis* 6633 10-102 N/A Growth
Bacteroides vulgatus* 8482 10-102 N/A Growth
Clostridium sporogenes* 11437 10-102 N/A Growth
Clostridium sporogenes* 19404 10-102 N/A Growth
Kocuria rhizophila* 9341 10-102 N/A Growth
Pseudomonas aeruginosa* 9027 10-102 N/A Growth
Staphylococcus aureus* 6538 10-102 N/A Growth
Mercurial Neutralization Test – To perform, add 1% Merthiolate™* to
medium, inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P
and Streptococcus pyogenes ATCC 19615, and incubate at 30-35°C for
18-48 hours. Recovery of organisms indicates that Merthiolate has been
neutralized.
Difco™ NIH Thioglycollate Broth
Prepare the medium per label directions. Inoculate duplicate tubes and
incubate at 30-35°C for 18-48 hours (up to 72 hours, if necessary) under
anaerobic conditions (tight caps).
ORGANISM ATCC™ INOCULUM CFU RECOVERY Staphylococcus aureus 25923 10-102 Good
Bacteroides vulgatus 8482 10-102 Poor to good
Clostridium sporogenes 11437 10-102 Poor to good
Clostridium sporogenes 19404 10-10 2
Poor to good
Mercurial Neutralization Test – To perform, add 1% Merthiolate™ to
medium, inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P
and Streptococcus pyogenes ATCC 19615, and incubate at 30-35°C for
18-48 hours. Recovery of organisms indicates that Merthiolate has been
neutralized.
*Merthiolate is a trademark of Eli Lilly and Company.
Thioglycollate Medium without Indicator (135C) is the medium
of choice for diagnostic work because the lack of indicator avoids
possible toxicity to organisms.11 This medium supports a minimal
inoculum with early visibility of growth.
When used as an enrichment broth to support plated media,
thioglycollate media are often supplemented with hemin and
vitamin K1.16 Fluid Thioglycollate Medium, Enriched is BBL™
Fluid Thioglycollate Medium supplemented with vitamin
K1 and hemin. Enriched Thioglycollate Medium is BBL
Thioglycollate Medium without Indicator-135C supple-
mented with vitamin K1 and hemin. Enriched broth media
are recommended for use in the isolation and cultivation of
fastidious or slow growing, obligately anaerobic microorganisms
present in clinical materials.17,18 They are also recommended for
the isolation and cultivation of a wide variety of aerobic and
facultatively anaerobic microorganisms. Enriched Thioglycollate
Medium is prepared with an anaerobic head space and is
provided in screw-capped tubes in accordance with CDC
recommendations.17 Vitamin K1 and hemin have been shown
Difco Manual Sect III Ta new.ind549 549
Thioglycollate Media, cont.
Difco™ Fluid Thioglycollate Medium with Beef Extract
°
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Bacillus subtilis 6633 10-102 Good
Bacteroides vulgatus 8482 10-102 Good
Candida albicans 10231 10-102 Good
Clostridium chauvoei 10092 10-10 2
Good
Clostridium perfringens 13124 10-102 Good
Clostridium sporogenes 19404 10-102 Good
Kocuria rhizophila 9341 10-102 Good
Mercurial Neutralization Test – To perform, add 1% Merthiolate™ to medium,
inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P and Strepto-
coccus pyogenes ATCC 19615, and incubate at 30-35°C for 18-48 hours.
Recovery of organisms indicates that Merthiolate has been neutralized.
Difco™ Thioglycollate Medium without Dextrose,
Thioglycollate Medium without Indicator* or Thio-
glycollate Medium without Dextrose or Indicator*
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Bacteroides fragilis 25285 10-102 Poor to fair†
Bacteroides vulgatus 8482 10-102 Poor to fair†
Clostridium novyi 7659 10-10 2
Good
Clostridium sporogenes 11437 10-102 Good
*Mercurial Neutralization Test – To perform, add 1% Merthiolate™ to
medium, inoculate (103 CFU) with Staphylococcus aureus ATCC 6538P
and Streptococcus pyogenes ATCC 19615, and incubate at 30-35°C for
T
18-48 hours. Recovery of organisms indicates that Merthiolate has been
neutralized.
† Poor to good for Thioglycollate Medium without Indicator.
Continued
to be required by certain anaerobes for growth.19,20 The addi-
tion of calcium carbonate enhances the maintenance of stock
cultures by neutralizing acids produced during growth.16 The
Enriched Thioglycollate Medium (Broth) recommended by the
CLSI for inoculum preparation for susceptibility tests of anaer-
obes consists of Enriched Thioglycollate Medium (Thioglycollate
Medium without Indicator [135] with 1 µg/mL of Vitamin K1
and 5 µg/mL of hemin) supplemented with 1 mg/mL of sodium
bicarbonate or a marble chip to neutralize acids produced during
growth of the test organisms.21
Principles of the Procedure
Dextrose, peptone, L-cystine and yeast extract provide the
growth factors necessary for bacterial replication. Sodium
chloride provides essential ions. Sodium thioglycollate is a
reducing agent that prevents the accumulation of peroxides
which are lethal to some microorganisms. The L-cystine is also
a reducing agent, since it contains sulfhydryl groups which
inactivate heavy metal compounds and maintain a low redox
potential, thereby supporting anaerobiosis. Methylene blue is
549
3/16/09 4:57:07 PM
 Section III
T Thioglycollate Media, cont.

Identity Specifications Cultural Response

BBL™ Fluid Thioglycollate Medium BBL™ Fluid Thioglycollate Medium
Dehydrated Appearance: Medium fine to fine, homogeneous, free of Prepare the medium per label directions. Inoculate and incubate
extraneous material. aerobically at 30-35°C for up to 5 days. To test for growth promotion
Solution: 2.95% solution, soluble in purified water according to the USP/EP, test with all of the organisms listed.
upon boiling. When hot, solution is pale to ORGANISM ATCC™ INOCULUM CFU RESULT
light, yellow to tan, clear.
Bacillus subtilis 6633 <102 Growth
Prepared Appearance: Pale to light, yellow to tan with light pink to
rose indicator on top, moderately hazy to Bacteroides vulgatus 8482 <102 Growth
hazy. Clostridium sporogenes 11437 <10
2
Growth
Reaction of 2.95% Clostridium sporogenes 19404 <102 Growth
Solution at 25°C: pH 7.1 ± 0.2
Kocuria rhizophila 9341 <102 Growth
BBL™ Sterility Test Broth Pseudomonas aeruginosa 9027 <102 Growth
Dehydrated Appearance: Fine, homogeneous, free of extraneous
Staphylococcus aureus 6538 <102 Growth
material.
Solution: 2.85% solution, soluble in purified water BBL™ Sterility Test Broth
upon boiling. When hot, solution is light Prepare the medium per label directions. Inoculate and incubate at 30-35°C
to medium, yellow to tan, clear to slightly for 7 days. Incubate organisms marked with (*) anaerobically (with tight
hazy. caps).
Prepared Appearance: Light to medium, yellow to tan, clear to slightly
ORGANISM ATCC™ INOCULUM CFU RESULT
hazy.
Bacillus subtilis 6633 ≤103 Growth
Reaction of 2.85%
Solution at 25°C: pH 7.1 ± 0.2 Bacteroides vulgatus* 8482 ≤102 Growth
BBL™ Thioglycollate Medium, Brewer Modified Candida albicans 10231 ≤103 Growth
Dehydrated Appearance: Fine, homogeneous, free of extraneous Clostridium sporogenes* 11437 ≤102 Growth
material. Kocuria rhizophila 9341 ≤103 Growth
Solution: 3.85% solution, soluble in purified water Streptococcus pneumoniae 6305 ≤103 Growth
upon boiling. When hot, solution is light to
medium, yellow to tan, clear. BBL™ Thioglycollate Medium, Brewer Modified
Prepared Appearance: Light to medium, yellow to tan with medium Prepare the medium per label directions. Inoculate and incubate at
green indicator color at the top, moderately 35 ± 2°C for 7 days under appropriate atmospheric conditions.
hazy to hazy.
ORGANISM ATCC™ INOCULUM CFU RESULT
Reaction of 3.85%
Solution at 25°C: pH 7.2 ± 0.2 Bacteroides fragilis 25285 ≤103 Growth
Staphylococcus aureus 25923 ≤103 Growth
BBL™ Thioglycollate Medium, Fluid, without Dextrose
or Eh Indicator Streptococcus pyogenes 19615 ≤103 Growth
Dehydrated Appearance: Fine, homogeneous, free of extraneous
material and may contain tan specks. BBL™ Thioglycollate Medium, Fluid, without
Dextrose or Eh Indicator
Solution: 2.4% solution, soluble in purified water upon
Prepare the medium per label directions. Inoculate and incubate at
boiling. When hot, solution is pale to light,
35 ± 2°C for 7 days under appropriate atmospheric conditions.
yellow to tan, clear.
Prepared Appearance: Pale to light, yellow to tan, slightly to mod- ORGANISM ATCC™ INOCULUM CFU RESULT
erately hazy. Clostridium sporogenes 11437 ≤10
3
Growth
Reaction of 2.4% Staphylococcus aureus 25923 ≤103 Growth
Solution at 25°C: pH 7.1 ± 0.2
BBL™ Thioglycollate Medium without Indicator – 135C BBL Thioglycollate Medium without Indicator – 135C
™

Prepare the medium per label directions. Inoculate and incubate as indicated
Dehydrated Appearance: Fine, homogeneous, free of extraneous
below.
material.
Solution: 3.0% solution, soluble in purified water upon INOCULUM INCUBATION
ORGANISM ATCC™ CFU TIME/TEMP RESULT
boiling. When hot, solution is pale to light,
tan to yellow, clear. Bacteroides fragilis 25285 ≤103 7 days/35 ± 2°C Satisfactory
Prepared Appearance: Pale to light, tan to yellow, moderately hazy Campylobacter jejuni
to hazy. subsp. jejuni 33291 ≤103 7 days/40-44°C Satisfactory
Reaction of 3.0% Saccaharomyces
Solution at 25°C: pH 7.0 ± 0.2 cerevisiae 9763 ≤103 7 days/25 ± 2°C Satisfactory
Staphylococcus
aureus 25923 ≤103 7 days/35 ± 2°C Satisfactory
Streptococcus
pyogenes 19615 ≤103 7 days/35 ± 2°C Satisfactory

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Difco Manual Sect III Ta new.ind550 550 3/16/09 4:57:09 PM


an indicator of the level of oxidation/reduction in the medium;
increased oxidation raises the Eh, causing the methylene blue
indicator to become green. Resazurin is an oxidation-reduction
indicator, being pink when oxidized and colorless when
reduced. The small amount of agar assists in the maintenance
of a low redox potential by stabilizing the medium against
convection currents, thereby maintaining anaerobiosis in the
lower depths of the medium. The USP lists 5.5g/L of dextrose
in the formulations for Fluid Thioglycollate Medium and
Alternative Thioglycollate Medium; some of the following
formulations include the anhydrous form of dextrose (5.0g/L).
Vitamin K1 is a growth requirement for some strains of
Prevotella melaninogenica18 and is reported to enhance the
growth of some strains of Bacteroides species and gram-
positive nonsporeformers.22 Hemin is the source of the X
factor, which stimulates the growth of many microorganisms.
Calcium carbonate neutralizes acids produced during growth,
which helps to maintain the viability of fastidious organisms;
e.g., pneumococci, gram-negative cocci, Clostridium perfringens
and other acid-sensitive bacteria.
Dipotassium phosphate is a buffering agent.
Formulae
Difco™ Fluid Thioglycollate Medium
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 15.0 g
Yeast Extract................................................................ 5.0 g
Dextrose...................................................................... 5.5 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.5 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
Resazurin..................................................................... 1.0 mg
BBL™ Fluid Thioglycollate Medium
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 15.0 g
Yeast Extract................................................................ 5.0 g
Dextrose (anhydrous)................................................... 5.0 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.5 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
Resazurin..................................................................... 1.0 mg
Difco™ NIH Thioglycollate Broth
Approximate Formula* Per Liter
Casitone.................................................................... 15.0 g
Yeast Extract................................................................ 5.0 g
Dextrose...................................................................... 5.5 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.5 g
Sodium Thioglycollate.................................................. 0.5 g
BBL Sterility Test Broth
™ Approximate Formula* Per Liter
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 15.0 g
Yeast Extract................................................................ 5.0 g
Dextrose (anhydrous).................................................. 5.0 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.5 g
Sodium Thioglycollate.................................................. 0.5 g
Difco Manual Sect III Ta new.ind551 551
Thioglycollate Media, cont.
BBL™ Thioglycollate Medium, Brewer Modified
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 17.5 g
Papaic Digest of Soybean Meal..................................... 2.5 g
Dextrose.................................................................... 10.0 g
Sodium Chloride.......................................................... 5.0 g
Sodium Thioglycollate.................................................. 1.0 g
Dipotassium Phosphate................................................ 2.0 g
Methylene Blue............................................................ 2.0 mg
Agar............................................................................ 0.5 g
Difco™ Fluid Thioglycollate Medium with Beef Extract
Approximate Formula* Per Liter
Beef Extract................................................................. 5.0 g
Yeast Extract................................................................ 5.0 g
Pancreatic Digest of Casein........................................ 15.0 g
Dextrose ..................................................................... 5.5 g
Sodium Chloride......................................................... 2.5 g
L-Cystine...................................................................... 0.5 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
Resazurin..................................................................... 1.0 mg
Difco™ Thioglycollate Medium without Dextrose
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 15.0 g
Yeast Extract................................................................ 5.0 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.25 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
Methylene Blue............................................................ 2.0 mg
Difco™ Thioglycollate Medium without Dextrose
or Indicator
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 15.0
Yeast Extract................................................................ 5.0
Sodium Chloride.......................................................... 2.5
g
g
g
T
L-Cystine...................................................................... 0.25 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
BBL™ Thioglycollate Medium, Fluid, without Dextrose
or Eh Indicator
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 20.0 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.5 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
Difco™ Thioglycollate Medium without Indicator
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 15.0 g
Yeast Extract................................................................ 5.0 g
Dextrose...................................................................... 5.0 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.25 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.75 g
BBL™ Thioglycollate Medium without Indicator – 135C
Pancreatic Digest of Casein........................................ 17.0 g
Papaic Digest of Soybean Meal..................................... 3.0 g
Dextrose...................................................................... 6.0 g
Sodium Chloride.......................................................... 2.5 g
L-Cystine...................................................................... 0.25 g
Sodium Thioglycollate.................................................. 0.5 g
Agar............................................................................ 0.7 g
Sodium Sulfite.............................................................. 0.1 g
*Adjusted and/or supplemented as required to meet performance criteria.
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 Section III
T Thioglycollate Media, cont.
Directions for Preparation from
Dehydrated Product
1. Suspend the powder in 1 L of purified water:
Difco™ Fluid Thioglycollate Medium – 29.8 g;
BBL™ Fluid Thioglycollate Medium – 29.5 g;
Difco™ NIH Thioglycollate Broth – 29 g;
BBL™ Sterility Test Broth – 28.5 g;
BBL™ Thioglycollate Medium, Brewer Modified – 38.5 g;
Difco™ Fluid Thioglycollate Medium w/Beef Extract – 34.7 g;
Difco™ Thioglycollate Medium w/o Dextrose – 24 g;
Difco™ Thioglycollate Medium w/o Dextrose or Indicator
– 24 g;
BBL™ Thioglycollate Medium, Fluid, w/o Dextrose or Eh
Indicator – 24 g;
Difco™ Thioglycollate Medium w/o Indicator – 29 g;
BBL™ Thioglycollate Medium w/o Indicator – 135C – 30 g.
Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder.
3. Follow label directions specific for each medium.
4. Autoclave at 121°C for 15 minutes, or as directed on the
label.
5. Store fluid thioglycollate media at 15-30°C. If more than 30%
of the medium is pink prior to use, reheat once (100°C) to
drive off absorbed oxygen.
6. Test samples of the finished product for performance using
stable, typical control cultures.
Precautions
Do not reheat the media more than once; continued reheating
gives rise to toxicity.
Procedure
Follow the procedures outlined in the references and, where
applicable, in product package inserts.
Expected Results
After incubation, growth is evidenced by the presence of
turbidity compared to an uninoculated control. Strict aerobes
tend to grow in a thin layer at the surface of the broth;
obligate anaerobes will grow only in that portion of the broth
below the upper oxidized layer.
Limitation of the Procedure16
Anaerobes can be overgrown by more rapidly growing fac-
ultative organisms. Examine and Gram stain broth if plating
medium reveals no growth. Never rely on broth cultures
exclusively for isolation of anaerobes. Some anaerobes may be
inhibited by metabolic products or acids produced from more
rapidly growing facultative anaerobes.
References
1. Quastel and Stephenson. 1926. J. Biochem. 20:1125.
2. Falk, Bucca and Simmons. 1939. J. Bacteriol. 37:121.
3. Brewer. 1940. JAMA 115:598.
4. Marshall, Ginnish and Luxen. 1940. Proc. Soc. Exp. Biol. Med. 43:672.
5. Nungester, Hood and Warren. 1943. Proc. Soc. Exp. Biol. Med. 52:287.
552
Difco Manual Sect III Ta new.ind552 552
6.
7.
Portwood. 1944. J. Bacteriol. 48:255.
Vera. 1944. J. Bacteriol. 47:59.
8.
9.
Malin and Finn. 1957. J. Bacteriol. 62:349.
Linden. 1941. Fluid thioglycollate medium for the sterility test. National Institutes of Health, Bethesda,
Md.
10. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,
vol. 1. Williams & Wilkins, Baltimore, Md.
11. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Inter-
national, Gaithersburg, Md.
12. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
13. Federal Register. 1992. Fed. Regist. 21:640.2.17.
14. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The
national formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc.,
Rockville, Md.
15. Federal Register. 1992. Fed. Regist. 21:113.26.
16. Reischelderfer and Mangels. 1992. In Isenberg (ed.), Clinical microbiology procedures handbook,
vol. 1. American Society for Microbiology, Washington, D.C.
17. Dowell, Lombard, Thompson and Armfield. 1977. Media for isolation, characterization, and
identification of obligately anaerobic bacteria. CDC laboratory manual. Center for Disease
Control, Atlanta, Ga.
18. Chapin. 2007. In Murray, Baron, Jorgensen, Landry and Pfaller (ed.), Manual of clinical microbiology,
9th ed. American Society for Microbiology, Washington, D.C.
19. Gibbons and MacDonald. 1960. J. Bacteriol. 80:164.
20. Wilkins, Chalgren, Jimeniz-Ulate, Drake and Johnson. 1976. J. Clin. Microbiol. 3:359.
21. Clinical and Laboratory Standards Institute. 2003. Approved standard: M11-A5. Methods for
antimicrobial susceptibility testing of anaerobic bacteria, 5th ed. CLSI, Wayne, Pa.
22. Finegold, Sutter, Attebery and Rosenblatt. 1974. In Lennette, Spaulding and Truant (ed.), Manual of
clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
Availability
Difco™ Fluid Thioglycollate Medium
AOAC BAM BS12 COMPF EP USDA USP
Cat. No. 225640 Dehydrated – 100 g
225650 Dehydrated – 500 g
225620 Dehydrated – 2 kg
225630 Dehydrated – 10 kg
BBL™ Fluid Thioglycollate Medium
AOAC BAM BS12 COMPF EP USDA USP
Cat. No. 211260 Dehydrated – 500 g
211263 Dehydrated – 5 lb (2.3 kg)
211264 Dehydrated – 25 lb (11.3 kg)
221195 Prepared Tubes, 8 mL (K Tubes ) – Pkg. of 10*
221196 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100*
220888 Prepared Tubes, 20 mL (A Tubes) – Pkg. of 10
220889 Prepared Tubes, 20 mL (A Tubes) – Ctn. of 100
299108 Prepared Bottles, 100 mL (serum) – Pkg. of 10
299417 Prepared Bottles, 100 mL (septum screw cap) –
Pkg. of 10
257217 Sterile Pack Bottles (double bagged), 100 mL –
Pkg. of 10
299112 Prepared Bottles, 500 mL (septum screw cap) –
Pkg. of 10
257407 Prepared Bottles, 200 mL (flip off cap and stopper) –
Pkg. of 10
Europe
Cat. No. 257408 Prepared Bottles, 300 mL – Pkg. of 10
257409 Prepared Bottles, 500 mL – Pkg. of 4
257406 Prepared Bottles, 600 mL – Pkg. of 4
257370 Prepared Bottles (ETO), 100 mL – Ctn. of 44
257426 Prepared Tubes (ETO), 18 mL – Ctn. of 60
257246 Prepared Bottles, 100 mL
(flip off cap and stopper) – Pkg. of 25
257249 Prepared Bottles, 100 mL (125 mL capacity,
screw cap) – Pkg. of 25
257275 Prepared Bottles, 100 mL (150 mL capacity,
screw cap) – Pkg. of 25
257422 Prepared Bottles (wide mouth), 150 mL –
Pkg. of 25
257264 Sterile Pack Bottles (double bagged), 100 mL –
Pkg. of 10
257290 Sterile Pack Bottles (double bagged), 800 mL –
Pkg. of 4
257097 Sterile Pack Bottles (ETO), 100 mL – Ctn. of 44
257317 Sterile Pack Bottles (wide mouth), 150 mL –
Pkg. of 25
3/16/09 4:57:12 PM

Difco™ NIH Thioglycollate Broth (USP Alternative
Thioglycollate Medium)
USP
Cat. No. 225710 Dehydrated – 500 g
BBL™ Sterility Test Broth (USP Alternative Thioglycollate
Medium)
USP
Cat. No. 211651 Dehydrated – 500 g
BBL™ Thioglycollate Medium, Brewer Modified
Cat. No. 211716 Dehydrated – 500 g
Difco Fluid Thioglycollate Medium with Beef Extract
™
Cat. No. 269720 Dehydrated – 500 g
269710 Dehydrated – 10 kg
Difco™ Thioglycollate Medium without Dextrose
Cat. No. 236310 Dehydrated – 500 g
Difco™ Thioglycollate Medium without Dextrose or
Indicator
Cat. No. 243210 Dehydrated – 500 g
BBL™ Thioglycollate Medium, Fluid, without Dextrose
or Eh Indicator
Cat. No. 211727 Dehydrated – 500 g
221398 Prepared Tubes (K Tubes) – Ctn. of 100*
Difco™ Thioglycollate Medium without Indicator
Cat. No. 243010 Dehydrated – 500 g
Thiol Broth
Intended Use
Thiol Broth is used for cultivating organisms from body fluids
and other materials containing penicillin, streptomycin or
sulfonamides.
Summary and Explanation
Szawatkowski1 and Shanson and Barnicoat2 reported Thiol
Broth to be superior in supporting the growth of Bacteroides
species in blood cultures. Thiol Broth was used to study the
optimum incubation period of blood culture broths.3 Media
containing thiol and thioglycollate are recommended for
recovery of nutritionally variant streptococci (NVS).4
Thiol Broth is cited in the first edition of Clinical Microbiology
Procedures Handbook5 as a medium specific for anaerobic
bacteria in blood cultures.
Principles of the Procedure
Peptones and yeast extract provide nitrogen, vitamins and
amino acids in Thiol Broth. Dextrose is a carbon source.
Sodium chloride maintains osmotic balance. Para-aminobenzoic
acid is a preservative. Sodium thioglycollate and L-cystine are
rich in sulfhydryl (-SH) groups, which neutralize the bacterio-
static and bactericidal effects of penicillin, streptomycin and
sulfonamides.
Difco Manual Sect III Ta new.ind553 553
Thiol Broth
BBL™ Thioglycollate Medium without Indicator – 135C
BS12 CMPH2
Cat. No. 211720 Dehydrated – 500 g
221199 Prepared Tubes, 8 mL (K Tubes) – Pkg. of 10*
221200 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100*
221797 Prepared Tubes, 10 mL (D Tubes) – Pkg. of 10*
221798 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100*
221047 Prepared Tubes, 20 mL (A Tubes) – Ctn. of 100*
BBL™ Fluid Thioglycollate Medium, Enriched
Cat. No. 297642 Prepared Tubes (K Tubes) – Ctn. of 100*
BBL™ Enriched Thioglycollate Medium
BS12 CLSI CMPH2 MCM9
Cat. No. 221741 Prepared Tubes, 5 mL (K Tubes) – Pkg. of 10*
221742 Prepared Tubes, 5 mL (K Tubes) – Ctn. of 100*
221787 Prepared Tubes, 8 mL (K Tubes) – Pkg. of 10*
221788 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100*
297289 Prepared Tubes, 10 mL (D Tubes) – Pkg. of 10*
297292 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100*
BBL™ Thioglycollate Medium with Calcium
Carbonate Chip
Cat. No. 298518 Prepared Tubes (K Tubes) – Ctn. of 100
BBL™ Enriched Thioglycollate Medium with
Calcium Carbonate
Cat. No. 297264 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100*
*Store at 2-8°C.
T
Formula
Difco™ Thiol Broth
Approximate Formula* Per Liter
Proteose Peptone No.3............................................... 10.0 g
Pancreatic Digest of Casein.......................................... 4.35 g
Gelatin......................................................................... 1.0 g
Yeast Extract................................................................ 5.0 g
Dextrose...................................................................... 0.2 g
Sodium Chloride.......................................................... 5.0 g
L-Cystine, Disodium .................................................... 2.4 g
Sodium Thioglycollate.................................................. 1.0 g
p-Aminobenzoic Acid................................................... 0.05 g
*Adjusted and/or supplemented as required to meet performance criteria.
Directions for Preparation from
Dehydrated Product
1. Suspend 29 g of the powder in 1 L of purified water. Mix
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
pletely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using
stable, typical control cultures.
Procedure
For a complete discussion on processing and interpretation of
blood cultures and other specimens, refer to appropriate refer-
ences.5,6
553
3/16/09 4:57:13 PM

amino acids and longer-chained peptides. Sodium chloride main-
tains osmotic equilibrium. Defibrinated sheep blood supplies
nutrients necessary to support the growth of fastidious organ-
isms and to detect hemolytic reactions while also inhibiting the
growth of Haemophilus haemolyticus, a bacterium commonly
found in nose and throat specimens that is indistinguishable from
beta-hemolytic streptococci.1 Gentamicin is an aminoglycoside
antibiotic that inhibits the growth of gram-negative bacteria.
Agar is the solidifying agent.
Procedure
Use standard procedures to obtain isolated colonies from speci-
mens. Incubate the plates in an inverted position (agar side up)
at 35°C in a CO2-enriched atmosphere for 18-48 hours.
Expected Results
Staphylococci and gram-negative bacteria are inhibited. Circular,
flat, translucent colonies surrounded by zones of alpha hemolysis
may be presumptively identified as Streptococcus pneumoniae.
However, when the colonies are young, they may be dome-
Bacto™ Tryptic Soy Broth/Trypticase™
Soy Broth (Soybean-Casein Digest Medium)
Trypticase™ Soy Broth with 6.5% Sodium Chloride
Trypticase™ Soy Broth with 5% Fildes Enrichment
Bacto™ Tryptic Soy Broth without Dextrose
Intended Use
Tryptic (Trypticase) Soy Broth (Soybean-Casein Digest Medium)
is a general purpose medium used in qualitative procedures for
the cultivation of fastidious and nonfastidious microorganisms
from a variety of clinical and nonclinical specimens.
Trypticase Soy Broth with 6.5% Sodium Chloride is used to
differentiate Enterococcus spp. from the Streptococcus bovis
group of streptococci.
Trypticase Soy Broth with 5% Fildes Enrichment is used for
the cultivation of fastidious organisms; e.g., Haemophilus
influenzae.
Tryptic Soy Broth without Dextrose, a low carbohydrate for-
mulation of Tryptic Soy Broth, is used for cultivating fastidious
and nonfastidious microorganisms.
Tryptic (Trypticase) Soy Broth meets United States Pharma-
copeia (USP), European Pharmacopoeia (EP) and Japanese
Pharmacopoeia (JP)1-3 performance specifications, where
applicable.
Difco Manual Sect III Tb new.ind579 579
Tryptic Soy Broth
shaped and may be confused with viridans streptococci, which
will also grow on this medium. Gram staining, biochemi-
cal tests and serological procedures should be performed to
confirm findings.
References
1. Spellerberg and Brandt. 2007. In Murray, Baron, Jorgensen, Landry and Pfaller (ed.), Manual of
clinical microbiology, 9th ed. American Society for Microbiology, Washington, D.C.
2. Dilworth, Stewart, Gwaltney, Hendley and Sande. 1975. J. Clin. Microbiol. 2:453.
3. Sondag, Morgens, Hoppe and Marr. 1977. J. Clin. Microbiol. 5:397.
Availability
BBL™ Trypticase™ Soy Agar with 5% Sheep Blood
(TSA II) with Gentamicin
Cat. No. 297457 Prepared Plates – Pkg of 20*
*Store at 2-8°C.
T
Summary and Explanation
Tryptic (Trypticase) Soy Broth (TSB) is a nutritious medium that
will support the growth of a wide variety of microorganisms,
including common aerobic, facultative and anaerobic bacteria
and fungi.4-7 This formulation is included in the USP as a medium
for use in performing microbial enumeration tests and tests for
specified microorganisms when testing nonsterile pharmaceutical
products.1
TSB was chosen by the USDA Animal and Plant Health Inspection
Service for detecting viable bacteria in live vaccines.8 TSB is rec-
ommended for testing bacterial contaminants in cosmetics9,10 and
complies with established standards in the food industry.10-16
Because of its capacity for growth promotion, TSB is also recom-
mended for use as the inoculum broth for disc diffusion and agar
dilution antimicrobial susceptibility testing as standardized by
the Clinical and Laboratory Standards Institute (CLSI).17,18
Trypticase Soy Broth with 6.5% Sodium Chloride is used to
differentiate the enterococcal species from the S. bovis group of
streptococci by the 6.5% NaCl tolerance test.19
Trypticase Soy Broth supplemented with 5% Fildes Enrichment
provides growth factors necessary for the cultivation of fastidi-
ous organisms.20
579
3/16/09 4:59:31 PM
 Section III
T Tryptic Soy Broth, cont.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™/Bacto™ and BBL™ brands may reflect differences
in the development and testing of media for industrial and clinical applications, per the referenced publications.

Identity Specifications
Bacto™ Tryptic Soy Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution: 3.0% solution, soluble in purified water upon
warming. Solution is light amber, clear.
Prepared Appearance: Light amber, clear.
Reaction of 3.0%
Solution at 25°C: pH 7.3 ± 0.2
Difco™ Tryptic Soy Broth (prepared bottles)
Appearance: Light to medium tan yellow, clear to trace hazy.
Reaction at 25°C: pH 7.3 ± 0.2
Bacto Tryptic Soy Broth without Dextrose
™

Dehydrated Appearance: Light beige, free-flowing, homogeneous.

Solution: 2.75% solution, soluble in purified water upon
warming. Solution is light amber, clear to very
slightly opalescent.
Prepared Appearance: Light amber, clear to very slightly opalescent.
Reaction of 2.75% Uninoculated Staphylococcus
Tube epidermidis
Solution at 25°C: pH 7.3 ± 0.2 ATCC™ 12228

Cultural Response
Bacto™ Tryptic Soy Broth
Prepare the medium per label directions. Inoculate and incubate at
30-35°C for 18-72 hours (up to 5 days for A. brasiliensis and C. albicans).
Prepare duplicate cultures of A. brasiliensis, B. subtilis and C. albicans
and incubate at 20-25°C for up to 3 days (up to 5 days for A. brasiliensis
and C. albicans).
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Neisseria meningitidis 13090 10-100 Fair to good
Staphylococcus epidermidis 12228 10-100 Good
Streptococcus pneumoniae 6305 10-100 Good
Streptococcus pyogenes 19615 10-100 Good
Aspergillus brasiliensis (niger) 16404 <100 Growth (30-35°C)
Aspergillus brasiliensis (niger) 16404 <100 Growth (20-25°C)
Bacillus subtillis 6633 <100 Growth (30-35°C)
Bacillus subtillis 6633 <100 Growth (20-25°C)
Candida albicans 10231 <100 Growth (30-35°C)
Candida albicans 10231 <100 Growth (20-25°C)
Escherichia coli 8739 <100 Growth
Pseudomonas aeruginosa 9027 <100 Growth
Salmonella enterica
subsp. enterica serotype
Typhimurium 14028 <100 Growth
Difco™ Tryptic Soy Broth (prepared bottles)
Inoculate and incubate at 30-35°C for 18-24 hours (up to 3 days for
B. subtilis). For (*) cultures incubate at 20-25°C for up to 3 days (up to
5 days for A. brasiliensis).
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Aspergillus brasiliensis (niger)* 16404 10-100 Growth (20-25°C)
Bacillus subtillis 6633 10-100 Growth (30-35°C)
Bacillus subtillis* 6633 10-100 Growth (20-25°C)
Candida albicans* 10231 10-100 Growth (20-25°C)
Escherichia coli 8739 10-100 Growth
Pseudomonas aeruginosa 9027 10-100 Growth
Salmonella enterica
subsp. enterica serotype
Typhimurium 14028 10-100 Growth
Staphylococcus aureus 6538 10-100 Growth
Bacto™ Tryptic Soy Broth without Dextrose
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C
for 18-48 hours.
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Neisseria meningitidis 13090 30-300 Fair to good
Staphylococcus epidermidis 12228 30-300 Good

Staphylococcus aureus 6538 <100 Growth Streptococcus pneumoniae 6305 30-300 Good
Streptococcus pyogenes 19615 30-300 Good
Continued

Tryptic Soy Broth without Dextrose, a modification of TSB, is
a basal medium to which carbohydrates may be added for use
in fermentation studies. Phenol red and other indicators may
also be added.
Principles of the Procedure
Enzymatic digests of casein and soybean provide amino acids
and other complex nitrogenous substances. Dextrose is an energy
source. Sodium chloride maintains the osmotic equilibrium.
Dibasic potassium phosphate acts as a buffer to control pH.

580

Difco Manual Sect III Tb new.ind580 580 3/16/09 4:59:36 PM

 Tryptic Soy Broth, cont.

Identity Specifications
BBL™ Trypticase™ Soy Broth
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution: 3.0% solution, soluble in purified water upon warming. Solution is light, tan to yellow, clear to slightly hazy.
Prepared Appearance: Light, tan to yellow, clear to slightly hazy.
Reaction of 3.0%
Solution at 25°C: pH 7.3 ± 0.2
BBL Trypticase Soy Broth (prepared bottles)
™ ™

Appearance: Light to medium tan yellow, clear to trace hazy.

Reaction at 25°C: pH 7.3 ± 0.2

Cultural Response
BBL™ Trypticase™ Soy Broth BBL™ Trypticase™ Soy Broth (prepared bottles)
Prepare the medium per label directions. Inoculate tubes and incubate at Inoculate and incubate at 35-37°C for 48 hours. Incubate (*) cultures at
30-35°C for up to 3 days (up to 5 days for A. brasiliensis and C. albicans). 30-35°C for up to 3 days. Incubate (**) cultures at 20-25°C for up to 3
Prepare duplicate cultures of A. brasiliensis, B. subtilis and C. albicans days (up to 5 days for A. brasiliensis and C. albicans).
and incubate at 20-25°C for up to 3 days (up to 5 days for A. brasiliensis
ORGANISM ATCC™ INOCULUM CFU RECOVERY
and C. albicans).
Escherichia coli 25922 <100 Growth
ORGANISM ATCC™ INOCULUM CFU RECOVERY
Staphylococcus aureus 25923 <100 Growth
Aspergillus brasiliensis (niger) 16404 <100 Growth (30-35°C)
Aspergillus brasiliensis (niger)** 16404 <100 Growth (20-25°C)
Aspergillus brasiliensis (niger) 16404 <100 Growth (20-25°C)
Bacillus subtillis* 6633 <100 Growth (30-35°C)
Bacillus subtilis 6633 <100 Growth (30-35°C)
Bacillus subtillis** 6633 <100 Growth (20-25°C)
Bacillus subtilis 6633 <100 Growth (20-25°C)
Candida albicans** 10231 <100 Growth (20-25°C)
Candida albicans 10231 <100 Growth (30-35°C)
Pseudomonas aeruginosa* 9027 <100 Growth (30-35°C)
Candida albicans 10231 <100 Growth (20-25°C)
Staphylococcus aureus* 6538 <100 Growth (30-35°C)
Escherichia coli 8739 <100 Growth
Pseudomonas aeruginosa 9027 <100 Growth
Salmonella enterica
subsp. enterica serotype
Typhimurium 14028 <100 Growth T
Staphylococcus aureus 6538 <100 Growth

The addition of 6.5% sodium chloride to Trypticase Soy Broth Formulae

permits the differentiation of salt-tolerant enterococci, which
are resistant to the high salt content, from the salt-intolerant
S. bovis group and other streptococcal species. At this concen-
tration, the sodium chloride is a selective agent that interferes
with membrane permeability and osmotic and electrokinetic
equilibria.4
Fildes Enrichment is a peptic digest of sheep blood that supplies
the X (hemin) and V (nicotinamide adenine dinucleotide, NAD)
factors necessary for the growth of H. influenzae.
Dextrose is omitted from the formula for Tryptic Soy Broth
without Dextrose to permit use of the medium in fermentation
studies. The carbohydrate concentration used most frequently
in fermentation reactions is 0.5% or 1%.
Tryptic Soy Broth and Trypticase Soy Broth are provided as
prepared media in a variety of bottle styles. In addition, Tryptic
Soy Broth is provided as a Sterile Pack Bottle; i.e., the bottle has
been terminally sterilized inside of autoclavable double-bags. All
varieties of bottled TSB conform with requirements for Ready-
Bacto™ Tryptic Soy Broth (Soybean-Casein Digest
Medium)
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 17.0 g
Papaic Digest of Soybean............................................. 3.0 g
Dextrose...................................................................... 2.5 g
Sodium Chloride.......................................................... 5.0 g
Dipotassium Phosphate................................................ 2.5 g
BBL™ Trypticase™ Soy Broth (Soybean-Casein Digest
Broth)
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 17.0 g
Papaic Digest of Soybean............................................. 3.0 g
Sodium Chloride.......................................................... 5.0 g
Dipotassium Phosphate................................................ 2.5 g
Dextrose...................................................................... 2.5 g
Bacto™ Tryptic Soy Broth without Dextrose
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 17.0 g
Enzymatic Digest of Soybean Meal............................... 3.0 g
Sodium Chloride.......................................................... 5.0 g
Dipotassium Phosphate................................................ 2.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

To-Use Media as described in the USP.

581

Difco Manual Sect III Tb new.ind581 581 3/16/09 4:59:37 PM

 Section III
T Tryptic Soy Broth, cont.
Directions for Preparation from
Dehydrated Product
1. Suspend the powder in 1 L of purified water:
Bacto™ Tryptic Soy Broth – 30 g;
BBL™ Trypticase™ Soy Broth – 30 g;
Bacto™ Tryptic Soy Broth without Dextrose – 27.5 g.
Mix thoroughly.
2. Warm gently until solution is complete.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using
stable, typical control cultures.
Sample Collection and Handling
For clinical specimens, refer to laboratory procedures for details
on specimen collection and handling.5,7,17-19
For food, dairy or cosmetic samples, follow appropriate stan-
dard methods for details on sample collection and preparation
according to sample type and geographic location.9-16
For pharmaceutical samples, refer to the USP for details on
sample collection and preparation for testing of nonsterile
products.1
Procedure
For clinical specimens, refer to appropriate standard references
for details on testing protocol to obtain isolated colonies from
specimens using Tryptic/Trypticase Soy Broth.17-19
For food, dairy or cosmetic samples, refer to appropriate
standard references for details on test methods using Tryptic/
Trypticase Soy Broth.9-16
For pharmaceutical samples, refer to USP General Chapters <61>
and <62> for details on the examination of nonsterile products
and performing microbial enumeration tests and tests for specific
organisms using Tryptic/Trypticase Soy Broth.1
Swab specimens may be inserted into the medium after inocula-
tion of appropriate plated media. For liquid specimens, use a
sterile inoculating loop to transfer a loopful of the specimen to
the broth medium. Specimens known or suspected to contain
obligate anaerobes should be inoculated near the bottom of
the tube.
Incubate the tubes and bottles with loosened caps at 35 ± 2°C
aerobically with or without supplementation with carbon
dioxide. Tubed and bottled media intended for the cultivation of
anaerobes should be incubated under anaerobic conditions. An
efficient and easy way to obtain suitable anaerobic conditions
is through the use of BD GasPak™ EZ anaerobic systems or
equivalent alternative system. Examine for growth after 18-24
hours and 42-48 hours of incubation.
Expected Results
Growth in broth media is indicated by the presence of turbidity
compared to an uninoculated control. Broth cultures should be
held for at least a week before discarding as negative.
582
Difco Manual Sect III Tb new.ind582 582
References
1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville,
Md.
2. European Directorate for the Quality of Medicines and Healthcare. 2008. The European pharma-
copoeia, 6th ed., Supp. 1, 4-1-2008, online. European Directorate for the Quality of Medicines
and Healthcare, Council of Europe, 226 Avenue de Colmar BP907-, F-67029 Strasbourg Cedex 1,
France.
3. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed.,
online. Japanese Ministry of Health, Labour and Welfare.
4. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol.
1. Williams & Wilkins, Baltimore, Md.
5. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby Inc.,
St. Louis, Mo.
6. Fredette and Forget. 1961. The sensitivity of several media to small inocula. Extract from a paper
presented at the Canadian Society of Microbiology Annual Meeting, June 12-15. Kingston, Ontario,
Canada.
7. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
American Society for Microbiology, Washington, D.C.
8. Federal Register. 1992. Fed. Regist. 21:113.26.
9. Curry, Joyce and McEwen. 1993. CTFA microbiology guidelines. The Cosmetic, Toiletry and Fragrance
Association, Inc., Washington, D.C.
10. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
11. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
American Public Health Association, Washington, D.C.
12. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
13. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
14. Health Canada. The compendium of analytical methods, online. Food Directorate, Health Products
and food Branch, Health Canada, Ottawa, Ontario Canada.
15. U.S. Department of Agriculture. Microbiology laboratory guidebook, online. Food Safety and Inspec-
tion Service, USDA, Washington, D.C.
16. International Organization for Standardization. 1996. Microbiology of food and animal feeding stuffs
– Horizontal method for the detection and enumeration of Listeria monocytogenes – Part 1: Detection
method. ISO 11290-1, 1st ed., 1996-12-15. ISO, Geneva, Switzerland.
17. Clinical and Laboratory Standards Institute. 2006. Approved Standard M2-A9: Performance standards
for antimicrobial disk susceptibility tests, 9th ed., CLSI, Wayne, Pa.
18. Clinical and Laboratory Standards Institute. 2006. Approved Standard M7-A7: Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed., CLSI, Wayne, Pa.
19. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
20. Fildes. 1920. Br. J. Exp. Pathol. 1:129.
Availability
Bacto™ Tryptic Soy Broth (Soybean-Casein Digest Medium)
AOAC BAM BS12 CCAM CLSI CMPH2 COMPF EP EPA ISO
JP MCM9 SMD USDA USP
Cat. No. 211824 Dehydrated – 100 g†
211825 Dehydrated – 500 g†
211822 Dehydrated – 2 kg†
211823 Dehydrated – 10 kg†
290612 Prepared Bottles (wide mouth), 90 mL –
Pkg. of 10†
290613 Prepared Bottles (wide mouth), 100 mL –
Pkg. of 10†
257213 Sterile Pack Bottles (double bagged), 100 mL –
Pkg. of 10
Europe
Cat. No. 257423 Prepared Tubes, 13 mL – Pkg. of 25†
254960 Prepared Bottles (double-strength), 50 mL –
Pkg. of 25
257248 Prepared Bottles, 100 mL – Pkg. of 10†
257265 Prepared Bottles (double bagged), 100 mL –
Pkg. of 10†
257276 Prepared Bottles, 100 mL (screw cap) –
Pkg. of 25†
257247 Prepared Bottles, 100 mL (tear off seal
with stopper) – Pkg. of 25†
257307 Prepared Bottles (ETO), 100 mL – Pkg. of 44†
257316 Prepared Bottles (wide mouth), 150 mL –
Pkg. of 25†
257412 Prepared Bottles, 300 mL – Pkg. of 10†
257413 Prepared Bottles, 500 mL – Pkg. of 4†
257414 Prepared Bottles, 600 mL – Pkg. of 4†
257291 Prepared Bottles (double bagged), 800 mL –
Pkg. of 4†
3/16/09 4:59:39 PM

BBL™ Trypticase™ Soy Broth (Soybean-Casein Digest Broth)
AOAC BAM BS12 CCAM CLSI CMPH2 COMPF EP EPA ISO
JP MCM9 SMD USDA USP
Cat. No. 211768 Dehydrated – 500 g†
296264 Sterile, Dehydrated – 500 g
211771 Dehydrated – 5 lb (2.3 kg)†
211772 Dehydrated – 25 lb (11.3 kg)†
295634 Prepared Tubes, 1 mL (K Tubes) – Ctn. of 100
221815 Prepared Tubes, 2 mL (K Tubes) – Ctn. of 100
221715 Prepared Tubes, 5 mL (K Tubes) – Pkg. of 10
221716 Prepared Tubes, 5 mL (K Tubes) – Ctn. of 100
221092 Prepared Tubes, 8 mL (K Tubes) – Pkg. of 10
221093 Prepared Tubes, 8 mL (K Tubes) – Ctn. of 100
299936 Prepared Tubes, 10 mL (D Tubes) – Ctn. of 100†
221823 Prepared Tubes, 15 mL (A Tubes) – Ctn. of 100
299749 Prepared Tubes, 20 mL (A Tubes) – Ctn. of 100†
297811 Prepared Tubes, 21 mL (A Tubes) – Pkg. of 10
297380 Prepared Bottles, 30 mL – Each
299107 Prepared Bottles, 100 mL (serum bottle) –
Pkg. of 10†
299416 Prepared Bottles, 100 mL (septum screw cap) –
Pkg. of 10†
257411 Prepared Bottles, 200 mL (flip cap with stopper) –
Pkg. of 10
299113 Prepared Bottles, 500 mL – Pkg. of 10†
Trypticase™ Soy Broth with 0.15% Agar
Intended Use
Trypticase Soy Broth (TSB) with 0.15% Agar is a general-
purpose medium for cultivation of fastidious and nonfastidious
microorganisms, especially anaerobic bacteria.
Summary and Explanation
TSB is a nutritious medium that supports the growth of a wide
variety of microorganisms, including common aerobic, anaero-
bic and facultative bacteria and fungi.1-3 The addition of agar
enhances the cultivation of some microorganisms, particularly
anaerobes from root canal and other clinical specimens.1
Principles of the Procedure
TSB contains enzymatic digests of casein and soybean meal to
provide nitrogenous substances. Dextrose is a source of energy,
and sodium chloride provides osmotic equilibrium.
Supplementing TSB with agar is useful for the cultivation of
anaerobes. The agar in the medium retards the absorption of
oxygen by reducing convection currents in the medium.
Procedure
Liquid media for anaerobic incubation should be reduced prior
to inoculation by placing the tubes, with caps loosened, under
anaerobic conditions for 18-24 hours. An efficient and easy way
to obtain suitable anaerobic conditions is through the use of
BD GasPak™ EZ anaerobic systems or an alternative anaerobic
system.4
The organisms to be cultivated must first be isolated in pure
culture on an appropriate plated or slanted medium.
Difco Manual Sect III Tb new.ind583 583
TSB with 0.15% Agar
Mexico
Cat. No. 252605 Prepared Tubes, 10 mL
252736 Prepared Tubes, 5 mL
BBL™ Trypticase™ Soy Broth with 6.5% Sodium Chloride
Cat. No. 211351 Prepared Tubes (K Tubes) – Ctn. of 100
BBL™ Trypticase™ Soy Broth with Fildes Enrichment
Cat. No. 221403 Prepared Tubes (K Tubes) – Pkg. of 10*
221404 Prepared Tubes (K Tubes) – Ctn. of 100*
Bacto™ Tryptic Soy Broth without Dextrose
BAM
Cat. No. 286220 Dehydrated – 500 g
286210 Dehydrated – 10 kg
*Store at 2-8°C.
†QC testing performed according to USP/EP/JP performance specifications.
Using a sterile inoculating loop or needle, transfer fresh growth
from the plate or slant to the tubed medium to achieve the
desired concentration of viable organisms. Specimens known or
suspected to contain obligate anaerobes should be inoculated
near the bottom of the tube.
T
Tubed media intended for isolation and cultivation of anaerobes
should be incubated under anaerobic conditions for up to
7 days.
Expected Results
Growth in tubes is indicated by the presence of turbidity
compared to an uninoculated control.
If growth appears, cultures should be examined by Gram
staining and subculturing onto appropriate media; e.g., a
Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) plate
and/or Chocolate II Agar plate, EMB Agar or MacConkey II
Agar plate, etc. If anaerobes are suspected, subcultures should
be incubated anaerobically, as in a GasPak EZ anaerobic
system.
References
1. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,
vol. 1. Williams & Wilkins, Baltimore, Md.
2. Fredette, Auger and Forget. 1961. Can . Med. Assoc. J. 84:164.
3. Forbes, Sahm, and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed.
Mosby, Inc., St. Louis, Mo.
4. Seip and Evans. 1980. J. Clin. Microbiol. 11:226.
Availability
BBL™ Trypticase™ Soy Broth with 0.15% Agar
Cat. No. 298263 Prepared Tubes (K Tubes), 9 mL – Ctn. of 100
583
3/16/09 4:59:40 PM