Mueller Hinton II Broth Formulae
the isolation medium to Mueller Hinton Broth using standard
Difco™ Mueller Hinton Broth
For enrichment purposes, inoculate the specimen onto primary
media and then into the broth, according to recommended
BBL™ Mueller Hinton Broth
Incubate the tubes at 35°C under conditions appropriate for
Approximate Formula* Per LiterBeef Extract . 3.0
Expected Results *Adjusted and/or supplemented as required to meet performance criteria.
For broth dilution antimicrobial susceptibility testing, refer to
Directions for Preparation from Dehydrated Product
Growth in broth media is indicated by the presence of turbidity
1. Suspend the powder in 1 L of purified water:
compared with an uninoculated control. Difco™ Mueller Hinton Broth – 21 g; BBL™ Mueller Hinton Broth – 22 g. References
1. Mueller and Hinton. 1941. Proc. Soc. Exp. Biol. Med. 48:330. 2. National Committee for Clinical Laboratory Standards. 2000. Approved Standard: M7-A5.
2. Heat with frequent agitation and boil for 1 minute to com-
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. NCCLS, Wayne, Pa.
3. Jorgensen, Turnidge and Washington. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.),
Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
3. Autoclave at 116-121°C for 10-15 minutes (consult product
4. Forbes, Sahm and Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, Inc.,
5. Isenberg (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for
4. Check prepared medium to ensure the final pH is 7.3 ± 0.1
Availability
5. Test samples of the finished product for performance using
Difco™ Mueller Hinton Broth (Not cation-adjusted) Procedure
For a complete discussion on broth dilution antimicrobial
BBL™ Mueller Hinton Broth (Not cation-adjusted)
susceptibility testing, refer to the appropriate procedures
Prepared Tubes, 2 mL (K Tubes) – Pkg. of 10
Prepared Tubes, 2 mL (K Tubes) – Ctn. of 100
Organisms to be subcultured must first be isolated in pure
Prepared Tubes, 5 mL (C Tubes) – Pkg. of 10
culture on an appropriate solid medium. Transfer growth from
Prepared Tubes, 5 mL (C Tubes) – Ctn. of 100
Mueller Hinton II Broth (Cation-Adjusted) Mueller Hinton II Broth (Cation-Adjusted) with Lysed Horse Blood • Mueller Hinton II Broth (Cation-Adjusted) with 2% Sodium Chloride Intended Use
sulfamethoxazole and vancomycin according to the protocol
Mueller Hinton II Broth is intended for use in quantitative
procedures for susceptibility testing of rapidly-growing aerobic
Mueller Hinton II Broth with 2% Sodium Chloride (NaCl) is
and facultatively anaerobic bacteria isolated from clinical
for testing methicillin-resistant strains of Staphylococcus aureus
specimens. It is formulated to have a low thymine and thymi-
dine content and is adjusted to the calcium and magnesiumion concentrations recommended in NCCLS standard M7.1
Summary and Explanation
Mueller Hinton II Broth with Lysed Horse Blood is for use
The development of laboratory tests to determine the activity
in broth dilution antimicrobial susceptibility testing of Strep-
of antimicrobial agents has paralleled the development of these
tococcus pneumoniae with 11 antimicrobial agents; i.e.,
agents. In 1929, Fleming used a serial dilution technique to
cefaclor, cefotaxime, ceftriaxone, cefuroxime, chloramphenicol,
measure the lowest concentration of penicillin that prevented
erythromycin, imipenem, penicillin, tetracycline, trimethoprim/
growth of a test organism in broth.2 Ericsson and Sherrispublished an excellent review of the various methods for
Section III M Mueller Hinton II Broth, cont.
susceptibility testing and the relationship of dilution and
concentration at the site of infection that is two to four times
greater than the MIC value, while for urinary tract infections, apeak urine concentration of 10-20 times the MIC value should
Rammelkamp and Maxon were among the earliest to use
be achieved.9 However, effective antimicrobial therapy also
the tube dilution test to determine the in vitro antimicrobial
susceptibility of bacteria isolated from clinical specimens.4 Thedevelopment of this test resulted from the need to know why
Cation-adjusted Mueller Hinton Broth is the medium usually
some patients infected with S. aureus did not respond to peni-
used for dilution antimicrobial susceptibility tests. This me-
dium is supplemented with calcium and magnesium salts toproduce correct MICs with aminoglycosides and Pseudomo-
The tube dilution test (broth dilution) involves exposing bacteria
nas aeruginosa.1 However, this medium is not satisfactory for
to decreasing concentrations of antimicrobial agents in liquid
fastidious organisms such as S. pneumoniae. Cation-adjusted
media, usually by serial two-fold dilution. The mixture, consisting
Mueller Hinton Broth supplemented with 2-5% lysed horse
of microorganisms, nutrient medium and antimicrobial agent,
blood is the medium recommended for susceptibility testing of
is incubated at 35°C for 16-20 hours. The lowest concentra-
tion of antimicrobial agent at which no visible growth occursis defined as the minimal inhibitory concentration (MIC).
Thornsberry and McDougal reported that adding 2% sodiumchloride to cation-adjusted Mueller Hinton Broth improved the
The term “microdilution” appeared in the literature in 1970
reliability of MIC tests using oxacillin for detecting methicillin-
to describe the minimal inhibitory concentration tests
resistant S. aureus (MRSA).11 In addition, they recommend
performed with volumes of 0.1 mL or less of antimicrobial
the alternative direct inoculum standardization procedure (see
solution.5 Correlations between MIC values using microdilution
“Procedure,” step 2) and incubation of the inoculated MIC trays
and tube dilution methodologies have been reported to be
The qualitative disc diffusion antimicrobial susceptibility
Principles of the Procedure
procedure has been standardized since 1966.8 The rationale for
Acid hydrolysate of casein and beef extract provide nutrients
an MIC susceptibility test rather than the disc diffusion test is
for growth of test organisms. These ingredients are selected
that it gives quantitative information. It provides a relationship
for low thymine and thymidine content as determined by MIC
between the amount of antimicrobial agent required to inhibit
values with Enterococcus faecalis and sulfamethoxazole-
the growth of an organism in vitro and the achievable concentra-
trimethoprim (SXT). Calcium and magnesium ion concentra-
tions in the blood, urine, cerebrospinal fluid or bile, under
tions are adjusted to provide the amounts recommended by
various dosage conditions. It has been suggested that in the treat-
NCCLS to give the correct MIC values with aminoglycosides
ment of systemic infections, the drug dosage should yield a peak
and P. aeruginosa.1 The pH has been adjusted to the specifica-tion in NCCLS standard M7. User Quality Control
Mueller Hinton II Broth with Lysed Horse Blood contains thenutrients necessary to support the growth of S. pneumoniae. Identity Specifications BBL™ Mueller Hinton II Broth (Cation-Adjusted)
MRSA cultures often consist of two populations, one that is
susceptible and one that is resistant (so-called “occult resis-
tant” strains). The methicillin-resistant population grows more
slowly and prefers a high salt concentration as contained in
water upon boiling. Solution is paleto light yellow, clear to trace hazy.
Mueller Hinton II Broth with 2% NaCl. In addition, the lower
Pale to light yellow, clear to trace hazy.
pH of this medium (6.9) improves the stability of β-lactam
antibiotics during storage of prepared MIC test tubes or trays.12
Antimicrobial agents are prepared in serial two-fold dilutions
in Mueller Hinton II Broth (with or without lysed horse blood)
and are inoculated with the test culture to give a final concen-
Cultural Response
tration of 5 × 105 CFU/mL. Following incubation at 35°C,
BBL™ Mueller Hinton II Broth (Cation-Adjusted)
the presence of turbidity indicates growth of the organism.
Prepare the medium per label directions. Inoculate with approximately
The lowest concentration of antimicrobial agent showing no
105 of the test organisms, dispense into an antimicrobial susceptibility
growth is the MIC of that organism for that agent. The inter-
test system and incubate at 35 ± 2°C for 16-20 hours.
pretation as to whether the organism is susceptible, intermedi-
ORGANISM
ate, or resistant in its response to the agent is made by
comparing the MIC to those in the MIC interpretive standards
Mueller Hinton II Broth, cont.
Various factors have been identified as influencing broth dilution
II Broth with 2% NaCl. Suspensions of test organisms must
susceptibility tests. These include the medium, antimicrobial
be used within 15 minutes of standardization.
potency, inoculum concentration, pH, antimicrobial stability and
3. Inoculation of Antimicrobial Dilutions
mechanisms of resistance by the test organisms.3,14,15
The amount of inoculum depends on the procedure used.1The standardized inoculum prepared above will contain
approximately 1-2 × 108 CFU/mL. The final concentration
BBL™ Mueller Hinton II Broth (Cation-Adjusted)
in a well (or tube) should be 5 × 105 CFU/mL (not CFU/tube
If the volume of antimicrobial solution in the tube is
*Adjusted and/or supplemented as required with appropriate salts to provide 20-25 mg/L of calcium
1 mL, dilute the standardized inoculum 1:100 in Mueller
and 10-12.5 mg/L of magnesium and as additionally required to meet performance criteria.
Hinton II Broth (0.1 mL to a 10-mL tube of broth). Directions for Preparation from
Add 1.0 mL of the adjusted inoculum to each tube con-
Dehydrated Product
taining an antimicrobial agent and 2.0 mL to a sterile
1. Suspend 22 g of the powder in 1 L of purified water. Mix
2. Heat with frequent agitation and boil for 1 minute to com-
In this method, the antimicrobial dilutions are made in
sterile plastic trays with round or conical-shaped wells.
3. Autoclave at 116-121°C for 10 minutes. DO NOT OVER-
The volume is either 0.05 or 0.1 mL in each well. If the
volume in the well is 0.1 mL, dilute the inoculum 1:10
4. Test samples of the finished product for performance using
and add 0.005 mL of the inoculum per well, using a
replicator. One well in each tray should contain 0.1 mLof broth without any antimicrobial agent (growth
Procedure
Mueller Hinton II Broth (Cation-Adjusted) may be used for
If a dropper (0.05 mL) is used for the inoculum and the
inoculum preparation for MIC tests and for preparation of
volume of antimicrobial solution is 0.05 mL, this
antimicrobial dilutions for the microdilution or macrodilution
results in a 1:2 dilution. Therefore, dilute the inoculum
procedure. Details for the preparation of antimicrobial agents
1:100 and add 0.05 mL to each well to obtain the final
concentration of 5 × 105 CFU/mL (5 × 104 CFU/well). Add 0.05 mL of broth without any antimicrobial agent
1. Inoculum Standardization (for rapidly growing bacteria)
(growth control well). After the trays are inoculated,
a. Using aseptic technique, pick 3-5 isolated colonies of
cover with tape or a tight-fitting lid to prevent evapo-
the same organism from an 18- to 24-hour Trypticase™
Soy Agar with 5% Sheep Blood (TSA II) plate and
inoculate into 5 mL of Mueller Hinton II Broth.
Incubate the tubes or trays (stacked no more than four high)
b. Incubate 2-6 hours at 35°C. Periodically check turbidity
at 35°C for 16-20 hours for Mueller Hinton II Broth,
against the 0.5 McFarland turbidity standard.
20-24 hours for Mueller Hinton II Broth with Lysed Horse
• If comparable, go to step 3, Inoculation of Antimi-
Blood (S. pneumoniae) and a full 24 hours for Mueller
Hinton II Broth with 2% Sodium Chloride (MRSA). Do
• If too turbid, dilute aseptically with additional
not use a CO incubator. To prevent drying out, the trays
Mueller Hinton II Broth and repeat turbidity check.
should be covered with plastic tape, a tight fitting lid, or
If turbidity is comparable to the standard, go to step
3, Inoculation of Antimicrobial Dilutions.
Control cultures should be included each time a suscepti-
• If not turbid enough, continue incubation. When
bility test is performed or weekly if satisfactory performance
turbidity is comparable to the standard, go to step
can be documented according to the NCCLS standard.1 The
3, Inoculation of Antimicrobial Dilutions.
correct quality control MIC ranges will be found in M100
Suspensions of test organisms must be used within 15 minutes
2. Alternative Direct Inoculum Standardization (for rapidly
Expected Results
growing bacteria, S. pneumoniae and MRSA)
The minimal inhibitory concentration (MIC) of an antimicrobial
A stationary phase culture may also be used. In this method,
agent for a specific organism is the lowest concentration which
skip step number 1b and simply suspend enough colonies
will inhibit the growth of the organism. Growth is indicated by
in the broth to equal the turbidity of the 0.5 McFarland
turbidity or sediment. Some microorganisms when tested against
standard. For S. pneumoniae, use Mueller Hinton II Broth
trimethoprim/sulfamethoxazole or sulfonamides alone do not
with Lysed Horse Blood. For MRSA, use Mueller Hinton
always give clear-cut end points. In the case of doubling dilutions
Section III M Mueller Hinton II Broth, cont.
of trimethoprim/sulfamethoxazole, there may be a “trailing” of
References
growth. Such a pattern typically shows an obvious reduction in
1. National Committee for Clinical Laboratory Standards. 2000. Approved standard: M7-A5.
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed.
the amount of growth and, then, either small pellets (usually less
2. Fleming. 1929. Br. J. Exp. Pathol. 10:225.
than 1 mm in diameter) in the rest of the wells, or an obvious
3. Ericsson and Sherris. 1971. Acta Pathol. Microbiol. Scand. Sect B Suppl. 217:1.
reduction in the amount of growth and then a slight but detect-
4. Rammelkamp and Maxon. 1942. Proc. Soc. Exp. Biol. and Med. 51:386. 5. Gavan and Town. 1970. Am. J. Clin. Pathol. 53:880.
able graduation in the size of the pellets. In these cases, the MIC
6. Harwick, Weiss and Fekety. 1968. J. Lab Clin. Med. 72:511. 7. Marymount and Wentz. 1966. Am. J. Clin. Pathol. 45:548.
end point should be identified as the lowest concentration of
8. Bauer, Kirby, Sherris and Turck. 1966. Am. J. Clin. Pathol. 45:493. 9. Petersdorf and Plorde. 1963. Ann. Rev. of Med. 14:41.
antimicrobial agent beyond which there is no further reduction
10. Thornsberry. 1991. In Balows, Hausler, Herrmann, Isenberg and Shadomy (ed.), Manual of clinical
in the size of the pellet or amount of turbidity.
microbiology, 5th ed. American Society for Microbiology, Washington, D.C.
11. Thornsberry and McDougal. 1983. J. Clin. Microbiol. 18:1084. 12. Nickolai, Lammel, Byford, Morris, Kaplan, Hadley and Brooks. 1985. J. Clin. Microbiol. 21:366.
An organism may be susceptible, intermediate or resistant
13. National Committee for Clinical Laboratory Standards. 2002. Performance standards for antimicro-
bial susceptibility testing; 12th informational supplement, M100-S12(M7). NCCLS, Wayne, Pa.
for a given antimicrobial agent depending on the MIC value.
14. Thornsberry, Gavan and Gerlach. 1977. Cumitech 6, New developments in antimicrobial agent
susceptibility testing. Coord. ed., Sherris. American Society for Microbiology, Washington, D.C.
Interpretive standards for MIC values with various drugs may
15. Jorgensen, Turnidge and Washington. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.),
be found in NCCLS document M100 (M7)1 or may be
Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
16. Koneman, Allen, Janda, Schreckenberger and Winn. 1997. Color atlas and textbook of diagnostic
obtained from the drug manufacturer.
microbiology, 5th ed. Lippincott-Raven Publishers, Philadelphia, Pa.
17. Forbes, Sahm and Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, Inc.,
NOTE: Informational supplements to NCCLS Document M7,containing revised tables of antimicrobial agents and interpretive
Availability
standards are published periodically. The latest tables should be
BBL™ Mueller Hinton II Broth (Cation-Adjusted)
consulted for current recommendations. The complete standard
BS10 CMPH MCM7 NCCLS
and informational supplements can be ordered from the National
Committee for Clinical Laboratory Standards, 940 West
Prepared Tubes, 5 mL (K Tubes) – Pkg. of 10
Prepared Tubes, 5 mL (K Tubes) – Ctn. of 100
Valley Road, Suite 1400, Wayne, PA 19087-1898. Telephone:
Refer to other texts for additional information on antimicrobial
BBL™ Mueller Hinton II Broth (Cation-Adjusted) with Lysed Horse Blood
Prepared Tubes, 10 mL (K Tubes) – Pkg. of 10
BBL™ Mueller Hinton II Broth (Cation-Adjusted) with 2% Sodium Chloride Muller Kauffmann Tetrathionate Broth Base Intended Use Principles of the Procedure
Muller Kauffmann Tetrathionate Broth Base is used for enriching
Muller Kauffmann Tetrathionate Broth Base contains peptone
Salmonella from water, foodstuffs and fecal samples prior to
and beef extract as sources of carbon, nitrogen, vitamins and
minerals. Oxgall and added brilliant green are selective agentswhich inhibit gram-positive and other gram-negative organ-
Summary and Explanation
isms. Calcium carbonate is the buffer. Sodium thiosulfate is a
Muller1 recommended Tetrathionate Broth as a selective
medium for the isolation of Salmonella. Kauffmann2 modifiedthe formula to include oxbile and brilliant green as selective
agents to suppress bacteria such as Proteus spp. Difco™ Muller Kauffmann Tetrathionate Broth Base
The British Standard Specification specifies Brilliant Green
Tetrathionate Broth for isolating Salmonella from meat and meat
products and from poultry and poultry products.3 It is also a
recommended selective broth for isolating Salmonella from
animal feces and sewage-polluted water.4 Using more than one
selective broth increases the isolation of Salmonella from
*Adjusted and/or supplemented as required to meet performance criteria.
samples with multiple serotypes.5Muller Kauffmann Tetrathionate Broth Base conforms withISO/DIS 3565.3
Vi è indubbiamente un legame molto stretto tra i canti popolari e la città di Chioggia, un rapporto connesso alle caratteristiche strutturali, urbanistiche, sociali ed antropologiche della città, che va alle sue radici. Uno degli aspetti caratterizzanti del centro lagunare è costituito infatti dalla sua popolosità, un dato da intendere sia in senso quantitativo, relativo al considerevole i