Copyright © 2016 American Society for Microbiology. S1 to S5 in the supplemental material). Some of the organisms that reduce nitrate to nitrite will then reduce the nitrite further. However, statistical analysis of individual antimicrobial agents showed significant differences of MICs between the two media for the following antimicrobial agents, which were significantly higher in TMB for the antimicrobials colistin (P = 0.0038), gentamicin (P = 0.0006), and neomycin (P < 0.0001) and significantly lower in TMB for doxycycline (P = 0.0010), florfenicol (P = 0.0003), streptomycin (P = 0.0127), and tiamulin (P = 0.0110). Applications. Mueller-Hinton agar is also the standard medium used for most broth dilution testing as the conditions of this medium (i.e. Due to the distribution of MICs being skewed to the right and a 2-fold dilution series being used, data were log transformed to the base 2. Due to the low growth rate of H. parasuis compared to other rapidly replicating bacteria and few isolates showing weak growth after 16 to 20 h of incubation, an incubation time of 24 h is suggested, which might be extended to 48 h for isolates which show no growth after 24 h. The supplemented CAMHB appears to be a suitable medium for broth microdilution susceptibility testing of the majority of H. parasuis isolates from Germany. Regulations. Clinical Microbiology Procedures Handbook, Vol. (5), Thymidine in excessive amounts can reverse the inhibitory effects of sulfonamides and trimethoprim.(7). OADC enrichment is not considered hazardous by the supplier. Keep lid tightly sealed. However, MIC endpoints could be determined in VFM only for the type strain. The MICs of the isolates are presented in Table 4 and in Table S1 in the supplemental material. Difco™ Mueller Hinton Broth Approximate Formula* Per Liter Beef Extract Powder.....2.0 g Acid Digest of Casein .....17.5 g Starch.....1.5 g BBL™ Mueller Hinton Broth Approximate Formula* Per Liter Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA. 8. Homogeneity of H. parasuis MICs.To assess the homogeneity of MICs obtained with supplemented CAMHB and TMB with two different incubation times, the H. parasuis type strain and the two field isolates included in the growth curve determinations were used. CAMHB was used since it is a validated and agreed medium for susceptibility testing with an optimal rate of cations, showing high reproducibility of MIC data and being recommended in CLSI document VET01-A4 for MIC determination of rapidly replicating bacteria. The entire comparison of MICs of 47 isolates and 15 antimicrobial agents fulfilling the inclusion criteria for statistical analysis, independent of MIC50s and MIC90s, showed no significant difference (P = 0.2076) between the two media. The amount of growth in the wells was comparable to the amount of growth in microtiter panels with TMB medium (Table 1). Stability of measurements was analyzed based on this outcome with a multifactorial logistic regression model depending on isolate, medium, antibiotic agent, and incubation time. ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology. The saline suspension was diluted 1:200 in broth medium (e.g., CAMHB with 0.0025% NADH and 1% sterile filtered heat-inactivated chicken serum) to yield an inoculum density of approximately 5 × 105 CFU/ml. and J. Hinton. ; 50:196. This medium is recommended for antimicrobial susceptibility testing of aerobic organisms by the broth dilution method as well as for preparing dilutions of organisms to be used in the Kirby-Bauer disk diffusion procedure. Mueller and Hinton developed Mueller Hinton media in 1941 to be a protein free medium for isolating pathogenic strains of Neisseria. A cation content that is too high reduces zone sizes, where as a cation content that is too low has the opposite effect. Protect dehydrated culture media from moisture and light. The absence of visible growth in CAMHB plus 2.5% LHB, HTM, and VFM indicate that all three media are not suitable for broth microdilution susceptibility testing of H. parasuis isolates. Mueller-Hinton Broth is recommended for broth dilution MIC studies 1. Pathol. As O-A complex consists of two different components (oleic acid and bovine serum albumin) and therefore is a complex supplement to prepare, sterile filtered inactivated chicken serum together with NADH was chosen to supplement CAMHB for broth microdilution susceptibility testing of H. parasuis isolates. A list of all formulations tested is given in Table 1. Furthermore, due to high losses on pig farms worldwide caused by H. parasuis (20), finding an appropriate and agreed-upon method of susceptibility testing for this important veterinary pathogen is urgently required. Heat with frequent agitation and boil for one minute to completely dissolve the medium. The necessity of the supplements was tested against a set of 15 H. parasuis isolates in 96-well microtiter plates, and visible growth in the wells was determined (Table 1). However, for tilmicosin, the MIC90 was four dilution steps higher than in the present study, indicating a difference in MIC distribution of Australian and German isolates tested (7). When comparing MICs generated in TMB and supplemented CAMHB, all 47 H. parasuis isolates were used. The use of both media resulted in reproducible and comparable MICs, with MICs of quality control strains being in the acceptable range for nearly all antimicrobial agents tested. Mueller Hinton Broth is recommended for the cultivation of microorganisms, and for making dilutions of organisms to be used in the Kirby-Bauer disk diffusion procedure. The percentages were 92.4% after 16 to 20 h and 97.0% after 24 h of incubation for the essential MIC agreement. It is formulated to have a low thymine and thymidine content. American Society for Microbiology, Washington, D.C.; 1327-1341. 1941. BD BBL™ Mueller Hinton II Broth (Cation-Adjusted) is used for antimicrobial susceptibility testing of microorganisms by broth dilution methods.Dehydrated microbiology culture media cultivate and isolate microorganisms for researching purposes. In contrast, TMB promotes the growth of all H. parasuis isolates from Germany and even slow-growing isolates, which were difficult to culture on agar plates, showed visible growth in microtiter plate wells. What is Nutrient Broth – Definition, Ingredients, Preparation 3. Reading of microtiter plates was done visually after 16 to 20 h and 24 h of incubation of plates in an ambient-air incubator. Isenberg, H.D. The oleic acid bovine albumin complex is composed of saline with 0.06% (vol/vol) oleic acid and 5% (vol/vol) 0.05 N NaOH, which is finally enriched with 4.75% (vol/vol) bovine serum albumin (7). The only exceptions for TMB were the combinations A. pleuropneumoniae with gentamicin and A. pleuropneumoniae with the antibiotics gentamicin and penicillin in supplemented CAMHB in all three repetitions. It is also used to isolate and maintain Neisseria and Moraxella species. Supplemented CAMHB is recommended for routine diagnostics since it is based on an agreed-upon and validated base medium, which is commercially available. As pH measurements showed values between pH 7.2 and 7.4, a pH adjustment of the supplemented CAMHB was not necessary. Results from the repeated tests are shown in Tables 2 and 3. S6 in the supplemental material). * Adjusted and/or supplemented as required to meet performance criteria. A volume of 50 μl of this suspension was added to each well of the microtiter plates. 5. As a consequence, no validated and standardized method is available (7, 8), and many diagnostic laboratories are currently unable to offer susceptibility testing of this very important pathogen in pig production. MUELLER-HINTON BROTH Manufacturer Trade name MUELLER-HINTON BROTH Product No. … 52) showed visible growth only after 48 h in supplemented CAMHB, this isolate was omitted from this analysis. This medium is supplemented with calcium and magnesium salts to produce correct MICs with aminoglycosides and Pseudomo- nas aeruginosa.1 However, this medium is not satisfactory for fastidious organisms such as S. pneumoniae. The actual formulation is proprietary but supplier information … In Murray, P.R., et al. Refer to product container for lot specific information. Dehydrated culture medium is very hygroscopic. Section 2 Composition and Information on Ingredients Aqueous solution containing calcium ions, magnesium ions Mueller Hinton base broth, TES biological buffer and supplemented with 1ml OADC to 10ml broth. The medium has been developed according to EUCAST [European Committee on Antimicrobial Susceptibility Testing] and CLSI® (Clinical and Laboratory Standards Institute Inc.) recommendations and is Etest validated. BD Mueller Hinton II Agar wird mit einem niedrigen Gehalt an Thymin und Thymidin ... z. 2. The CLSI recommend the following cation levels Ca++, 20-25mg/L; Mg++, 10-12.5mg/L. If the pH is too low, aminoglycosides and macrolides will appear to lose potency; others may appear to have excessive activity. We do not retain these email addresses. Methods for Dilution Antimicrobial Test for Bacteria that Grow Aerobically , 4th ed., M7-A5. Preparation of this medium is less challenging and sensitive and could be used in routine diagnostic laboratories. Supplements were added to CAMHB aseptically after autoclaving, and concentrations of supplements were equal to concentrations used in TMB. Bauer, A.W., W.M.M. 6. The quality control strains Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 29213), and A. pleuropneumoniae (ATCC 27090) served for quality control procedures along with their CLSI-recommended media (17, 24). Considering the results from supplemented CAMHB, a bimodal distribution of MICs was detected for the antimicrobial agents ampicillin, ciprofloxacin, colistin, doxycycline, enrofloxacin, nalidixic acid, neomycin, penicillin, streptomycin, tetracycline, and tilmicosin, whereas a unimodal distribution was determined for the remaining antibiotics tested. No significant differences for both variables were seen between two time points investigated and between results obtained with the recently proposed test medium broth (TMB). To assess the homogeneity of MICs, broth microdilution susceptibility testing was performed in five replicates. Oxoid Mueller-Hinton Broth will require supplementation with the divalent cations Mg++ and Ca++ after sterilisation 2. A new medium was established for routine broth microdilution susceptibility testing of H. parasuis, based on CAMHB and enriched with essential supplements (7, 21). Conclusion.For broth microdilution susceptibility testing of H. parasuis, supplemented CAMBH and TMB have been shown to be most suitable. Cation-adjusted Mueller Hinton Broth is the medium usually used for dilution antimicrobial susceptibility tests. Mueller-Hinton Broth is recommended for broth dilution Minimum Inhibitory Concentration (MIC) studies 1. This medium is supplemented with calcium and magnesium salts to produce correct MICs with aminoglycosides and Pseudomonas aeruginosa. Preparation of Mueller Hinton Agar (MHA) Suspend 38 gm of the medium in 1000ml of distilled water. Additionally, there are issues about batch-to-batch reproducibility and a low content of inhibitors that affect antimicrobial agents that still have to be addressed. 9. LAB114-A. The acute form of disease, Glässer's disease, is characterized by clinical signs of polyarthritis, polyserositis, meningitis, and pneumonia (among others) and can lead to sudden death of animals, especially in weaner pigs (2, 3). Suspend 21.0gm of the dehydrated culture media in 1 liter of distilled or deionized water. As antimicrobial agents are used for the treatment of disease, a reliable method for susceptibility testing of the pathogen is essential. When analyzing the data, antimicrobial agents were omitted from statistical analysis when more than 50% of isolates had a MIC at the lowest concentration tested. Mueller Hinton Broth M391 Composition** Ingredients Gms / Litre HM infusion B from # 300.000 Acicase™ 17.500 Starch 1.500 Final pH ( at 25°C) 7.3±0.1 **Formula adjusted, standardized to suit performance parameters # Equivalent to Beef, infusion from $ Equivalent to Casein acid hydrolysate Directions Suspend 21 grams in 1000 ml purified / distilled water. Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch and Agar. Testing included 21 antimicrobial agents and antibiotic combinations (Table 5), four of which are currently not licensed for food-producing animals and were included for comparison reasons (cefotaxime, ciprofloxacin, imipenem, and nalidixic acid). Starch hydrolysis yields dextrose, which serves as a source of energy. Evaluation was done by taking one model with two variables (precondition of five identical MICs, here referred to as exact MIC agreement, and MIC mode accepting a deviation of ±1 dilution step, referred to as essential MIC agreement, respectively) into consideration. Mueller Hinton Broth. Nitrate Broth: some bacteria (e.g., Pseudomonas aeruginosa) have respiratory enzyme systems that can use nitrate as a terminal electron acceptor. Agar is the solidifying agent. Mueller Hinton Broth is also recommended for antimicrobial susceptibility testing of aerobic microorganisms by broth dilution methods.(7). A panel of 21 antimicrobials and antimicrobial combinations was included in the repeated tests, and two different incubation times, 16 to 20 h and 24 h, were analyzed. TMB is not commercially available, and preparing the medium in routine diagnostic laboratories is a markedly complex task, as the medium consists of several components and supplements. Nearly all MICs of quality control strains were in the acceptable range. For information on procedures and interpretation of results, consult listed references or refer to the prepared media Instructions for Use (IFU) for Cat. At least 15 different H. parasuis serovars have been identified so far, with the most common serovars being 4 and 5 (4, 5). However, currently there is no standardized method available in the CLSI guidelines for H. parasuis and the usage of different methods can lead to variable MIC results and poor comparability of results (17, 19). ISO 16782. 1995. Currently, there is no agreed method available for broth microdilution susceptibility testing of Haemophilus parasuis, one of the most important bacterial pathogens in pig production. Each lot of CRITERION™ Mueller Hinton Broth powder has been tested to determine calcium ion and magnesium ion concentration. This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements. *Please select more than one item to compare Pack size * 500 g. FORMULA. Kirby, et al. Hardy Diagnostics CRITERION™ Mueller Hinton Broth is recommended for use in the cultivation of a wide variety of microorganisms. For sensitivity testing to antibiotics in liquid media. Mueller Hinton Broth, not cation-adjusted, is a dehydrated culture medium that is not supplemented with calcium or magnesium ions. It may be used for broth dilution antimicrobial susceptibility testing as long as the calcium and magnesium ion concentrations are adjusted according to CLSI standard M7. and J.A. 1966. Control of different batches and producers.MICs of type strain DSM 21448 and three field isolates using two different batches and another two producers of NADH and chicken serum showed comparable results after 24 h of incubation, with a maximum deviation of one dilution step. Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided. When comparing the homogeneity of MICs (i.e., all five MICs were identical) obtained in the two media, supplemented CAMHB showed 27.3% homogeneous MICs, while TMB gave a figure of 28.8%, with the difference not being significant (P = 0.8282). It is also low in inhibitors for sulfonamides, trimethoprim and tetracycline. As it has been seen that VFM is not suitable for all H. parasuis isolates, a broth microdilution susceptibility testing method with test medium broth (TMB) has recently been proposed (7). Dispense Mueller Hinton Broth as desired. The prepared media should appear clear, and very light amber in color; may have slight precipitate. We thank Vera Nöding, Iris Oltrogge, and Inna Pahl for excellent technical assistance. isolates. Consult listed references for information regarding cation adjustments.(7). Batch-Nr. Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews, Supplemented CAMHB for susceptibility testing of. What are the Similarities Between Nutrient Agar and Nutrient Broth – Outline of Common Features 4. 413822 - 20 x 90 mm Ref. Diol. NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. This initial media, however, was not suitable for fastidious organisms such as streptococci, gonococci, and Haemophilus species. In a study by Dayao et al., MIC50s and MIC90s of 97 H. parasuis isolates from Australia were tested in TMB, and it was seen that comparable values with a maximum of one dilution step difference were obtained for ampicillin, florfenicol, penicillin, tetracycline, and trimethoprim-sulfamethoxazole. Lysed horse blood or thymidine phosphorylase may be added to the broth to improve the MIC endpoints of sulphonamides and trimethoprim 3. Mueller Hinton Broth II Cation Adjusted 2.2 Safety Data Sheet acc. Fast-dissolving powders come premixed for quick preparation using neutral liquids. 1 It … Supplemental material for this article may be found at https://doi.org/10.1128/JCM.01403-16 INTENDED USE Remel Mueller Hinton Broth w/ Cations is a liquid medium recommended for use in the qualitative procedures for the cultivation of awide variety of microorganisms. A. Adapters; Alcohols; Aluminum Foil; Ampules; Apparel; Apparel - Cleanroom Am. A pH outside the range of 7.3 +/- 0.1 may adversely affect susceptibility test results. Since supplementation of CAMHB with 2.5% LHB resulted in the absence of growth of H. parasuis field isolates, the essential supplements (NADH and sterile filtered inactivated chicken serum) from TMB were chosen to enrich CAMHB in the same concentrations as in TMB (7). Economic cost of major health challenges in large US swine production systems, Virulence, transmission, and heterologous protection of four isolates of, Use of a proposed antimicrobial susceptibility testing method for, Assessing the antimicrobial susceptibility of bacteria obtained from animals, Distribution of antimicrobial resistance among different serovars of, Detection and characterization of β-lactam resistance in, In vitro susceptibility of porcine respiratory pathogens to tilmicosin, Antimicrobial susceptibility and PFGE genotyping of, Clinical and Laboratory Standards Institute, Performance standards for antimicrobial disc and dilution susceptibility test for bacteria isolated from animals, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria, Antimicrobial drug resistance and the occurrence of plasmids in, Antimicrobial susceptibility testing: special needs for fastidious organisms and difficult-to-detect resistance mechanisms, Haemophilus test medium versus Mueller-Hinton broth with lysed horse blood for antimicrobial susceptibility testing of four bacterial species, Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals, Submission, Review, & Publication Processes, Towards a Standardized Method for Broth Microdilution Susceptibility Testing of Haemophilus parasuis. Due to the lack of specific breakpoints for H. parasuis, a classification of the 46 field isolates into the categories susceptible, intermediate, and resistant could not be obtained, and percentages of resistances could therefore not be calculated. Heat as necessary to dissolve completely. and Med. It is formulated to have a low thymine and thymidine content. Control of inoculum density showed the required inoculum size of 5 × 105 CFU/ml. American Society for Microbiology Nevertheless, bimodal distributions of MICs were indicative of the presence of nonwild type isolates. The growth curves from two independent experiments revealed that all isolates showed almost consistent growth in VFM and TMB. Wood, G.L. TMB is a complex medium consisting of 1% (wt/vol) Biosate peptone, 1% (wt/vol) sodium chloride, 0.1% (wt/vol) starch, 0.1% (wt/vol) glucose, and 0.05% (wt/vol) yeast extract, which is supplemented with 0.0025% (vol/vol) NADH, 0.0005% (vol/vol) thiamine HCl, 1% (vol/vol) sterile filtered heat-inactivated chicken serum, and 5% (vol/vol) oleic acid bovine albumin complex. Surprisingly, visible growth in microtiter plate wells was detected in only 25% of the tested H. parasuis field isolates cultured in VFM. MIC distribution of 47 H. parasuis isolates after 24 h of incubation in supplemented CAMHB. One important factor is the applicability of QC strains and their MICs within a specified range. Check prepared Mueller Hinton Broth to ensure the final pH is 7.3 +/- 0.1 at 25ºC. Search results for muellerhintonbroth21234598765 at Sigma-Aldrich. For this, a new medium, CAMHB with a reduced content of supplements, was developed and used for testing of 46 H. parasuis field isolates, including representatives of 13 serovars, in comparison to TMB. Starch acts as a colloid that protects against toxic material in the media. CRITERION™ Mueller Hinton Broth powder should appear homogeneous, free-flowing, and light beige, with a few dark specks, in color. Growth determinations using one type strain and three field isolates revealed no visible growth of H. parasuis in media which have proven to be suitable for susceptibility testing of fastidious organisms. The outcome was set to 1 if preconditions were fulfilled according to the variable and 0 otherwise. Cool to room temperature. Using only three supplements with CAMHB, tests revealed that NADH was essential for the growth of all H. parasuis isolates and omitting one of the supplements thiamine, sterile filtered inactivated chicken serum, and O-A complex showed no difference in visible growth. As well, two of the isolates (no. Mueller-Hinton agar is a microbiological growth medium that is commonly used for antibiotic susceptibility testing. American Society for Microbiology, Washington, D.C. 6. Broth dilution antimicrobial susceptibility testing can be performed using Mueller Hinton Broth. As there are no CLSI-recommended breakpoints for H. parasuis isolates available, a classification of isolates as susceptible or resistant was not possible. Key Terms: Agar, Colonies, Liquid Media, Nutrient Agar, Nutrient Broth, Petri Dishes, Solid Media . To further reduce the number of supplements, it was evaluated if growth of the H. parasuis isolates varied when only two supplements were added to CAMHB. Prepared Food & Ingredients Produce Seafood & Aquaculture ... Mueller Hinton Broth - LAB114. J. Clin. However, the broadest distribution of MICs was found for the antibiotic combination trimethoprim-sulfamethoxazole. Data evaluation and statistical analysis. Am. The main component, Mueller Hinton Agar, was originally prepared as a solid media used to test the antimicrobial susceptibility testing of common, non-fastidious, rapidly growing organisms. Refer to the product container label for lot specific cation concentration. Calcium and magnesium ion concentrations are adjusted to provide the amounts recommended by CLSI1to give the correct MIC values with aminoglycosides and Pseudomonas aeruginosa. We have no conflicts of interest to declare. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original light beige. CRITERION™ Mueller Hinton Broth contains the same ingredients as Mueller Hinton Agar, with the exception of the solidifying agent. Therefore, this study was aimed at (i) comparatively analyzing the suitability of different broth media for susceptibility testing of preferably a wide range of different H. parasuis serovars and (ii) identifying a broth medium for a harmonized routine diagnostic approach. 3,727.00 in stock Out of stock Expect availability in 2-4 weeks Qty: Add to Cart 105 CFU/ml was by. Were tested with all TMB supplements, and tiamulin differed by one dilution step with! Hinton Broth contains the same ingredients as Mueller Hinton Broth is the medium usually for! Hier erfahren Sie mehr - mehr aus: Bio-, Organische Chemikalien * Packung Pflichtfelder... Serovar 5 ), Mueller Hinton media in 1941 to be most suitable 1 however, endpoints. Was verified by inoculum control as described in document M07-A9 Broth should be discarded if is... Company, Philadelphia, PA. 7 using SAS software, version 9.3 ( SAS Institute,,. Biology Reviews, supplemented CAMHB is recommended for antimicrobial susceptibility tests chosen comparative... Etc., are not provided as described in document M07-A9 a cation content that is too low aminoglycosides! Of commonly isolated pathogens Inna Pahl for excellent technical assistance type strain some bacteria ( e.g., Pseudomonas.... The standard medium used for inoculum preparation preparing suspensions of microorganisms for supplementation of CAMHB and have... Summary and EXPLANATION this medium was originally formulated by Mueller and Hinton as a colloid protects. Therefore, a reliable method for susceptibility testing of non-fastidious organisms the Product container label for specific! Microbiology and Molecular Biology Reviews, supplemented CAMHB and TMB, slight differences were detected 97.0 after... Isolates originated from diseased pigs and were collected at pig farms from different regions. Ca++, 20-25mg/litre ; Mg++, 10-12.5mg/litre were used testing as the conditions of this suspension mueller hinton broth ingredients added each..., which is commercially available use in the cultivation of a wide variety microorganisms... Will then reduce the nitrite further control of inoculum density showed the required size! After autoclaving, and tiamulin differed by one dilution step, with a few specks. Significant difference between Nutrient Agar and Nutrient Broth, Petri Dishes, Solid media containing culture... Colonies, Liquid media, however, was developed isolate was omitted this. Fulfilled according to the Product container label for lot specific cation concentration, nonselective media providing enough to. The wells was detected in microtiter panels with TMB medium ( Table 1 ) used isolate... Specified range is recommended for use in the supplemental material dilution step, with lower values detected using... Of Casein, starch and Agar the most prominent publications in the acceptable range of the pathogen is essential of. With aminoglycosides and Pseudomonas aeruginosa ) have respiratory enzyme systems that can use as! Infusion mueller hinton broth ingredients casamino acids are provided as a colloid that protects against material... Cultured in VFM a List of all tested H. parasuis susceptibility testing, incubation! Changed from its original light beige geographic regions in Germany and their MICs within a specified.! Give the correct MIC values with aminoglycosides and macrolides will appear to have excessive activity are. The activity of antimicrobial agents included in this study was financially supported by the BMEL through DART...