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Mueller_hinton_ii_broth_cation_adjusted.pdf

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

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