Variation in the concentration of divalent cations, primarily calcium an magnesium, affects results of aminoglycoside, tetracycline, and colistin tests with The percentages were 92.4% after 16 to 20 h and 97.0% after 24 h of incubation for the essential MIC agreement. Dehydrated culture medium is very hygroscopic. These ingredients are selected for low thymine and thymidine content as determined by MIC values with Enterococcus faecalisand sulfamethoxazole-trimethoprim (SXT). In brief, isolates were grown on chocolate agar for 24 to 48 h at 35°C ± 2°C in ambient air, colonies were suspended in 0.9% saline, and the suspension was adjusted to an initial concentration of approximately 103 to 104 CFU/ml. SUMMARY AND EXPLANATION This medium was originally formulated by Mueller and Hinton as a proteinfree medium for the purpose of isolating pathogenic - Neisseria. * Adjusted and/or supplemented as required to meet performance criteria. Results from MIC determinations of H. parasuis field isolates.Using both media, supplemented CAMHB and TMB, there was no difficulty in determining visible growth of all 47 H. parasuis isolates in the wells of microtiter plates and reading of MIC endpoints was feasible. It is also low in inhibitors for sulfonamides, trimethoprim and tetracycline. Protect dehydrated culture media from moisture and light. Hence, this study might contribute to establish a standardized broth microdilution susceptibility testing method for H. parasuis. Check prepared Mueller Hinton Broth to ensure the final pH is 7.3 +/- 0.1 at 25ºC. Ingredients : Gms/Litre: Beef extract: 2.0: Acid hydrolysate of casein: 17.5: Starch: 1.5: Agar: 17.0: Final pH 7.3 +/- 0.1 at 25ºC. CM0405 Date of issue - 09/10/2008. H. parasuis type strain DSM 21448 (Leibniz-Institute DSMZ, Braunschweig, Germany) and 46 H. parasuis field isolates were used for the present study. 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). A. Adapters; Alcohols; Aluminum Foil; Ampules; Apparel; Apparel - Cleanroom Autoclave at 121°C for 15 minutes. The amount of growth in the wells was comparable to the amount of growth in microtiter panels with TMB medium (Table 1). The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original light beige. 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 5. 413822 - 20 x 90 mm Ref. In this study, nearly all MICs of QC strains were in the acceptable range with both media used, although the QC ranges have been established in media other than TMB or supplemented CAMHB, and therefore, it was not expected to have all MICs in the published ranges. The opposite effects are possible if the pH is too high. It is formulated to have a low thymine and thymidine content. Supplemental material for this article may be found at https://doi.org/10.1128/JCM.01403-16 For supplementation of CAMHB, the medium was first tested with all TMB supplements, and then one supplement at a time was removed. Although in H. parasuis endemic herds, young piglets are usually protected by maternal immunity, several environmental stress factors may lead to disease as well as infections of naive populations. American Society for Microbiology, Washington, D.C.; 1327-1341. 1. When comparing MICs generated in TMB and supplemented CAMHB, all 47 H. parasuis isolates were used. ; 45:493-496. Heat with frequent agitation and boil for one minute to completely dissolve the medium. This study was financially supported by the BMEL through the DART project (grant number FKZ 2811HS010). Exp. In brief, colonies were selected from a 24-h agar plate, inoculated into 0.9% saline, and adjusted to a turbidity comparable to that of a 0.5 McFarland standard. Calcium and magnesium ion concentrations are adjusted to provide the amounts recommended by CLSI1to give the correct MIC values with aminoglycosides and Pseudomonas aeruginosa. Statistical analyses were carried out using SAS software, version 9.3 (SAS Institute, Cary, NC) (25). VFM is recommended in CLSI standard VET01-A4 for susceptibility testing of Actinobacillus pleuropneumoniae and Histophilus somni (17), while HTM and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 2.5% to 5% lysed horse blood (LHB) are CLSI-approved broth media for the testing of fastidious organisms, with HTM being recommended for Haemophilus influenzae and Haemophilus parainfluenzae (18). Keep lid tightly sealed. 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. 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. 1966. 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 … 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. It is formulated to have a low thymine and thymidine content. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA. 8. Prepared Food & Ingredients Produce Seafood & Aquaculture ... Mueller Hinton Broth - LAB114. 3. and J. Hinton. Mueller Hinton Broth is recommended for preparing suspensions of microorganisms for disk diffusion sensitivity testing. Ich optimiere Ihren persönlichen Einkauf. This could be the reason for the deviation of a few MICs from the acceptable range of the QC strain A. pleuropneumoniae. Test ranges of antimicrobial agents included in this study. Batch-Nr. Thermo Scientific™ Remel Mueller Hinton Broth is for the cultivation of a wide variety of microorganisms. 1752 N St. NW Barry and Fay. - Hier erfahren Sie mehr - Mehr aus: Bio-, Organische Chemikalien *Packung * Pflichtfelder. Trypticase soy agar or tryptone soya agar (TSA) and Trypticase soy broth or tryptone soya broth (TSB) with agar are growth media for the culturing of bacteria. 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. Due to the explorative nature of the experiments, no multiple adjustments were performed and comparison-wise P values were reported. The MIC determinations were repeated in five independent experiments. In a recent study, VFM was reported to be unsuitable for broth microdilution susceptibility testing of Australian H. parasuis isolates, and TMB was proposed (7). | Ref. B. BD TrypticaseTM Soy Broth (Casein­Soja-Pepton-Bouillon) oder Mueller-Hinton-II-Bouillon (Kationen-adjustiert) für die Zubereitung eines Standardinokulums und sterile Bouillon oder Salzlösung für die Verdünnung des Inokulums.7 Gemäß EUCAST: 0,9%ige Kochsalzlösung (Mengen von 5 mL) zur … Comparing MIC50s and MIC90s of supplemented CAMHB and TMB, slight differences were detected. To assess the homogeneity of MICs, broth microdilution susceptibility testing was performed in five replicates. Clinical Microbiology Procedures Handbook, Vol. Mueller Hinton Broth II Cation Adjusted 2.2 Safety Data Sheet acc. Refer to product container for lot specific information. 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. 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. Mueller Hinton Broth, not cation-adjusted, is a dehydrated culture medium that is not supplemented with calcium or magnesium ions. Bauer, A.W., W.M.M. 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). As antimicrobial agents are used for the treatment of disease, a reliable method for susceptibility testing of the pathogen is essential. Mueller Hinton Broth contains beef infusion, casamino acids and starch. The isolates originated from diseased pigs and were collected at pig farms from different geographic regions in Germany between 2011 and 2013. All field isolates were provided by diagnostic laboratories in Germany and included 25 serotyped isolates of serovars 1 (n = 2), 2 (n = 2), 4 (n = 2), 5 (n = 2), 6 (n = 2), 8 (n = 2), 9 (n = 1), 10 (n = 2), 11 (n = 2), 12 (n = 2), 13 (n = 2), 14 (n = 2), and 15 (n = 2). 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. It is recommended that procedures are accurately followed to ensure reliable results. Growth of H. parasuis in broth media.To determine the adequacy of the broth media for susceptibility testing, type strain DSM 21448 and three H. parasuis field isolates of serovars 5 (two isolates) and 14 (one isolate) were chosen. Therefore, the aim of this study was to develop a method that could be easily performed by diagnostic laboratories and that appears suitable for a harmonized susceptibility testing. 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. pH, cation concentration, and thymidine contents) are well maintained.. Mueller Hinton agar (MHA) can be purchased from commercial suppliers or can also be prepared from the dehydrated medium. Starch hydrolysis yields dextrose, which serves as a source of energy. ; 50:196. Applications. 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. A pH outside the range of 7.3 +/- 0.1 may adversely affect susceptibility test results. 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. 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. VFM is recommended in CLSI standard VET01-A4 for susceptibility testing of Actinobacillus pleuropneumoniae and Histophilus somni , while HTM and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 2.5% to 5% lysed horse blood (LHB) are CLSI-approved broth media for the testing of fastidious organisms, with HTM being recommended for Haemophilus influenzae and … Bacterial isolates. Therefore, a new medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was developed. 1941. R38. Contact us for support › Back to top. As the third H. parasuis field isolate (no. to 29 CFR 1910.1200 App D Muller Hinton II Cation-Adjusted Broth Version number: GHS 2.0 Replaces version of: 2016-02-18 (M5860 1) Revision: 2018-07-03 United States: en Page: 1 / 9. Diol. Results of these experiments showed that supplementation of sterile filtered inactivated chicken serum or O-A complex is necessary to obtain visible growth of H. parasuis isolates in CAMHB (Table 1). In this study, colony count determinations during culturing in four different media detected growth of H. parasuis field isolates in VFM and TMB, whereas only growth of type strain DSM 21448 could be detected in HTM and CAMHB plus 2.5% LHB. For this purpose, which of the supplements of TMB were essential to provide sufficient growth of preferably all H. parasuis isolates and serovars was evaluated. Five out of 21 MIC50s were up to two dilution steps higher when using TMB, while it was seen that one MIC50 was one dilution step lower. Isolates were cultured on chocolate agar with 10% defibrinated horse blood in ambient air at an incubation temperature of 35°C ± 2°C for 24 to 48 h. To confirm that the field isolates were H. parasuis, a PCR assay amplifying an 821-bp internal fragment of the 16S small subunit rRNA gene was used as described earlier (20). Copyright © 2016 American Society for Microbiology. 6. Antimicrobial susceptibility testing Clinical. Pathol. 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. S1 to S5 in the supplemental material). Subsequently, statistical analysis was performed to examine the homogeneity of MIC data and to investigate the difference between supplemented CAMHB and TMB. 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. Inoculum size, rate of growth, medium formulation and pH, length of incubation and incubation environment, drug diffusion rate, and measurement of endpoints can affect results. If a nonapproved medium is used for broth microdilution susceptibility testing, validation of the medium has to undergo an extensive procedure. Manual of Clinical Microbiology, 7th ed. 413824 - 100 x 90 mm With VFM, visible growth could be detected only for seven isolates, although the H. parasuis type strain and all three field isolates showed a sufficient increase in CFU/ml during growth curve determination. J. Clin. At least 15 different H. parasuis serovars have been identified so far, with the most common serovars being 4 and 5 (4, 5). Am. Phone: (202) 737-3600, Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback, Print ISSN: 0095-1137; Online ISSN: 1098-660X, Towards a Standardized Method for Broth Microdilution Susceptibility Testing of, Sign In to Email Alerts with your Email Address. Supplementation of CAMHB and determination of visible growth of 15 H. parasuis isolates. P. aeruginosa 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. For the MIC mode accepting a deviation of ±1 dilution step, 90.9% of MICs showed homogeneous MICs for supplemented CAMHB, while 98.5% of the MICs were homogeneous for TMB (P = 0.0567). The proposed method enables broth microdilution susceptibility testing of H. parasuis isolates in routine diagnostics and might contribute to establishment of a standardized method for this important veterinary pathogen. Importantly, NADH-containing HTM is recommended in CLSI document M07-A9 (18) for susceptibility testing of fastidious organism such as Haemophilus influenzae and is therefore a CLSI-verified supplement. However, the results from QC strains indicate that there is no effect of medium components on the activity of antimicrobial agents. That protects against toxic material in the field, delivering up-to-date and authoritative coverage of both basic and Microbiology... Available, a pH outside the range of 7.3 +/- 0.1 may affect! Has the opposite effect between pH 7.2 and 7.4, a pH adjustment of the experiments, multiple. For the antibiotic combination trimethoprim-sulfamethoxazole and supplemented CAMHB was not necessary of supplemented CAMHB was not necessary of &. 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