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1.
Based on the authors' laboratory experience indicating that increased bacterial contamination in Mycobacterium avium ssp. paratuberculosis (MAP) cultures may be because of the addition of brain heart infusion broth (BHI) during the decontamination process, this study was designed to examine whether BHI is a required component for the isolation of MAP from ESP(R) broth cultures. Twenty-six National Veterinary Services Laboratory (NVSL) proficiency test samples supplied for the year 2005 were used for the comparison. Two paired sets of samples were processed in the experiment. In one set, the hexadecylpyridinium chloride monohydrate (HPC) and antibiotic brew were prepared in half strength BHI and for the other set, HPC and antibiotic brew were prepared in sterile distilled water. Culture of the 26 samples using the BHI/HPC decontamination method identified 13 (50%) positives, whereas culture using the water/HPC decontamination method identified 14 (54%) positives. The proportions of samples with a positive result did not differ significantly between the 2 decontamination methods. Although in most cases it took longer to identify a positive result by the BHI method, the difference between methods with respect to the number of days to a positive culture result was not statistically significant. Retrospective data collected from the Animal Disease Diagnostic Laboratory, Ohio also suggest that inclusion of BHI in the decontamination protocol may not have any effect on MAP recovery or contamination rate. Elimination of BHI from broth cultures may increase the sensitivity of MAP isolation, and reduce the cost of testing.  相似文献   

2.
The effect of cultural conditions on the production of leukotoxin by biotypes A and B of F. necrophorum was investigated. Biotypes A and B were grown in prereduced, anaerobically sterilized, brain-heart infusion (BHI) broth. The average leukotoxin titer of culture supernatant was 18 times higher from biotype A strains than from biotype B strains. Leukotoxin activity peaked during the late-log and early-stationary phases of growth, then declined precipitously in both biotypes. F. necrophorum biotype A was grown in different media (BHI, liver infusion, and Eugon broths), at various pH (6.6, 7.3, 7.7, and 8.2), incubation temperatures (30, 35, 39, and 43 degrees C), redox potentials (-352 to +375 mV), and iron concentrations (less than 0.2, 4.2, 42.1, and 361.4 microM). Anaerobic BHI broth with pH from 6.6 to 7.7 at 39 degrees C incubation temperature supported maximal F. necrophorum growth and leukotoxin production. The optimum redox potential for F. necrophorum growth was in the range of -230 to -280 mV. However, the presence of titanium III citrate or dithiothreitol (7.78 mM) in the medium decreased (P less than 0.05) the leukotoxicity of F. necrophorum. Low iron concentration (less than 0.2 microM) decreased (P less than 0.05) growth rate but not leukotoxin activity of F. necrophorum, whereas high iron concentration inhibited the leukotoxin activity.  相似文献   

3.
The in vitro development of buffalo oocytes up to the blastocyst stage was studied in serum-free, semidefined media containing bovine serum albumin, follicle-stimulating hormone (FSH), insulin, transferrin and selenium (ITS) and epidermal growth factor (EGF). In experiment 1, oocytes aspirated from abattoir-derived ovaries were cultured in eight serum-free, semidefined culture media containing different combinations of these four factors. In experiment 2, the maturation of buffalo oocytes and the development of the embryos were compared in a complex co-culture system and in the serum-free, semidefined media. Supplementation with FSH and EGF significantly (P < 0.05) increased the maturation rates of buffalo oocytes, and the yield of blastocysts was higher (P < 0.05) in media containing EGF and ITS. The yield of blastocysts was lower in the serum-free semidefined media (P < 0.05) than in the complex co-culture system.  相似文献   

4.
Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia. An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3-20kDa. However, conventional techniques to study bacterial protein expression, such as SDS-PAGE and 2-dimensional electrophoresis, typically focus on the 15-200kDa range. In this study we have evaluated Surface Enhanced Laser Desorption Ionisation-ProteinChip (SELDI) technology for the analysis of protein expression, in particular those of <20kDa, of APP grown under different environmental conditions. Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium. Optimum conditions for SELDI profiles included a sample size of 1 microg and the use of sinapinic acid as the energy absorbing matrix. In the <20kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33-66% pig serum were most similar to those grown in defined medium. The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences. The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20kDa mass range.  相似文献   

5.
The purpose of the present study was to identify Moraxella (M.)—like organisms recovered from calves suffering from respiratory disease down to species level by means of tDNA-intergenic spacer length polymorphism analysis (tDNA-PCR), and to perform antimicrobial susceptibility testing of these isolates using an agar dilution technique. A total of 16 isolates originating from 12 unrelated occasions were identified as Moraxella ovis, and tDNA fingerprinting showed clear delineation from other Moraxella species. The minimal inhibitory concentrations (in μg/mL) for 90% of the investigated isolates were ≤0.03 for ampicillin; 0.25 for ceftiofur; 0.5 for oxytetracycline; 8 for gentamicin; 64 for spectinomycin; 0.5/9.5 for the combination trimethoprim-sulfonamides; 4 for erythromycin; 8 for tilmicosin; 1 for florfenicol and 0.125 for enrofloxacin.  相似文献   

6.
The growth rates of 16 isolates of Tritrichomonas foetus from three distinct geographic regions were investigated in modified Diamond's medium, liver infusion broth medium and a commercially available culture kit. While some differences in growth characteristics were detected for different isolates and in the three different media, all isolates grew. Trichomonads reached peak concentrations from an initial concentration of 10(4) trichomonads/ml on Days 2, 3 and 4 in modified Diamond's medium, on Days 2-6 (excluding CAPTF102) in the commercial culture kit and on Days 2-7 in liver infusion broth medium. Viable parasites were detectable for longer periods in liver infusion broth medium and the commercial culture kit than in Diamond's medium. Peak concentrations for isolates tended to be higher in modified Diamond's medium than in liver infusion broth medium or the commercial culture kit. Results show that these three media are suitable for the growth of all 16 T. foetus isolates from three continents and suggest that these media could be used effectively throughout the world.  相似文献   

7.
Obligate anaerobes are susceptible in vitro to trimethoprim-sulfonamides. Clinical efficacy of this drug combination for treatment of infectious processes in which anaerobes are involved is uncertain. We hypothesize that this uncertainty is a result of thymidine in tissues and exudates which abrogates the inhibitory effect of trimethoprim-sulfonamides. We shown herein that species of anaerobic bacteria commonly encountered in infectious processes grew on thymidine-containing medium despite the presence of levels of trimethoprim-sulfamethoxazole (S x T) previously shown to be inhibitory. Biologic fluids and tissues, where anaerobic bacteria are commonly encountered in diseased states, were shown to contain thymidine levels that were sufficient to reverse the inhibitory effect of S x T upon these species of bacteria. These observations suggest that the use of trimethoprim-sulfonamides is not a rational choice for treatment of infectious processes in which an obligate anaerobe is a component.  相似文献   

8.
Out of 185 media formulated and studied for the growth of Sphaerophorus necrophorus, ten were selected for intensive investigation. Their ability to promote growth was compared photometrically. Medium 156 made up of Brewer's thioglycollate broth, yeast extract, L-cystine, and ascorbic acid, was found to yield a very heavy growth. The viability of S. necrophorus was maintained indefinitely by weekly subcultures. When not subcultured, all isolates remained viable for fifteen weeks and some isolates for 30 weeks at 37°C.

Twenty-nine isolates were tested. No variation in maximum growth was noticed.

Medium 156 is an optimum medium for S. necrophorus. It can be incubated aerobically without interfering with maximum growth. The medium does not deteriorate when stored for at least 12 weeks at room temperature.

  相似文献   

9.
A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.  相似文献   

10.
The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.  相似文献   

11.
Studies using 86 media for maximum growth of Campylobacter fetus for antigen production showed that a diphasic medium (solid base with liquid overlay) was most suitable. The solid base was double strength cystine heart agar. The liquid overlay was thioglycollate medium of Brewer (135-C) without agar. This medium yielded maximum growth of C. fetus in six days with good motility, less clumping and less filament formation than all other media tried.  相似文献   

12.
The antimicrobial susceptibilities of 76 field isolates of Brachyspira hyodysenteriae from different states of Australia were tested in a newly developed broth dilution procedure. The antimicrobial agents used were tiamulin, valnemulin, tylosin, erythromycin, lincomycin and clindamycin. The results from the broth dilution susceptibility testing of 39 of the isolates were compared with results obtained for the same isolates using the agar dilution method. Amongst the isolates tested by broth dilution, 17 were from three farms and had been collected over a number of years. Their pulsed field gel electrophoresis pattern previously had been determined. The broth dilution technique was simple to use, less labor intensive than agar dilution, and gave clear end points. The results obtained using the two methods generally corresponded well, although in a few cases the MIC obtained by broth dilution were lower than those with agar dilution. For the 76 isolates tested by broth dilution, the MIC(90) (mg/l) was: tiamulin, 1; valnemulin, 0.5; tylosin>256; erythromycin>256; lincomycin, 64 and clindamycin, 16. Only minor differences in susceptibility patterns were found amongst isolates from different Australian states. Over all the isolates, and also amongst the isolates obtained from different years on the three farms, there was no trend for the susceptibility of the isolates to alter with time.  相似文献   

13.
The aim of this study was to compare and evaluate the time required to isolate Brachyspira hyodysenteriae and Brachyspira pilosicoli from porcine faeces. This was done using previously described selective media (spectinomycin) S400, (colistin, vancomycin and spectinomycin) CVS and (spectinomycin, vancomycin, colistin, spiramycin and rifampin with swine faecal extract) BJ, compared with the method based on blood agar modified medium, with spectinomycin and rifampin (BAM-SR), including a pre-treatment step. Fourteen spirochaetal strains were obtained in pure cultures after 5 days (48 h in BAM-SR primary plate and three passages every 24 h in brain heart infusion (BHI) without antibiotics) pre-treating simulated samples in brain heart infusion broth with spectinomycin (400 microg/ml) and rifampin (15 microg/ml), before streaking on the selective BAM-SR medium. Spirochaetes from samples in S400, CVS and BJ, with and without pre-treatment, were obtained in pure cultures only after repeatedly transferring on plates of the same selective medium requiring 15-18 days according to the strain. BAM-SR used after the pre-treatment step showed a detection limit ranging from 3.5 x 10(2) to 6.7 x 10(7) cells/g faeces and was the only method able to support the growth of spirochaetes after 48 h.  相似文献   

14.
The objectives of this study were to determine the effect of bacterial culture conditions on adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 in vivo to pig enterocytes and to compare the results with adherence in vitro to cultured HEp-2 and IPEC-J2 cells. Growth of O157:H7 in MacConkey broth (MB) resulted in almost no adherence to both HEp-2 and IPEC-J2 cells; prior exposure of the bacteria to pH 2.5 reduced adherence. There was greater adherence by bacteria from static cultures than by those from shaken cultures and by bacteria cultured in brain-heart infusion (BHI) plus NaHCO? (BHIN) than by bacteria cultured in BHI. In contrast, in pig ileal loops, bacteria cultured in MB adhered well to enterocytes, and prior exposure to pH 2.5 had no effect on adherence. Among several media tested for their effect on bacterial adherence in the pig intestine, MB and BHIN proved to be the best. Bacterial adherence was dose-dependent and was more extensive in the ileum than in the colon. This study demonstrated that there are remarkable differences between culture conditions that promote adherence of an EHEC O157:H7 strain in vitro and in vivo, that culture conditions profoundly affect adherence to epithelial cells in vitro and in vivo, and that pig ileal loops are better suited to adherence studies than are colon loops.  相似文献   

15.
The type strain WVU 1853 and field strains SG, N26 and A642 of Mycoplasma synoviae were examined for their requirement for nicotinamide-adenine dinucleotide (NAD) for in vitro growth. All the strains grew and could be repeatedly passaged in Frey broth medium supplemented with filter-sterilised NAD. In modified Frey broth medium from which NAD was omitted and broth medium for which the supplements including yeast extract and NAD were autoclaved, only strains N26 and A642 could be grown and passaged. The growth curves of strain N26 determined in broth media with and without NAD were similar. These results indicated that there are differences in NAD-requirement for in vitro growth among strains of M synoviae.  相似文献   

16.
A C Tanner  C C Wu 《Avian diseases》1992,36(3):714-717
A technique is described for determining the antimicrobial susceptibility of Mycoplasma gallisepticum, using the Sensititre broth microdilution system. Fourteen M. gallisepticum field isolates and one reference isolate (R-strain) were tested in duplicate against seven antimicrobials. Isolates were susceptible to oxytetracycline, furaltadone, and lincomycin/spectinomycin, but not to amoxycillin and apramycin. Susceptibility to erythromycin and tylosin varied. These data are in agreement with those reported by other workers using more traditional methods, but this adaptation of the broth microdilution technique eliminates any variation attributable to the time-consuming preparation of antimicrobial dilutions associated with these methods.  相似文献   

17.
为便于猪多杀性巴氏杆菌病活疫苗（E0630株）质量控制和降低生产成本,研制了ZJ-1和ZJ-2两种合成培养基,并进行了两种合成培养基与马丁肉汤的比较试验。结果表明,合成培养基ZJ-1培养活菌数高于ZJ-2和马丁肉汤。用合成培养基ZJ-1培养制备3批疫苗,并分别用兔进行安全检验和小鼠效力检验。3批疫苗对兔均2／2健活,对小鼠的保护力分别为9／10、9110、8／10,符合产品质量标准。本试验表明,运用合成培养基替代传统培养基是可行的。  相似文献   

18.
The microdilution antifungal method (CLSI BMD, M27-A3) was used for testing the antifungal susceptibility of Malassezia species. However, optimal broth media that allow sufficient growth of M. pachydermatitis and produce reliable and reproducible MICs using the CLSI BMD protocol are yet to be established. In this study, the susceptibility of M. pachydermatis isolates to ketoconazole (KTZ), itraconazole (ITZ) and fluconazole (FLZ) was evaluated in vitro by the CLSI BMD test using Christensen's urea broth (CUB) and mRPMI 1640 containing lipid supplementation, Sabouraud dextrose broth with 1% tween 80 (SDB), and Dixon broth (DXB). A FLZ-resistant M. pachydermatis was generated in vitro and tested under the same conditions. A good growth of M. pachydermatis incubated for 48 and 72h, respectively, was observed in CUB, SDB and DXB and not in mRPMI 1640 (p<0.001). No statistically significant differences were detected between the MIC values registered after 48h and 72h incubation. ITZ displayed lower MIC values than KTZ and FLZ regardless of the media employed. A large number of FLZ-resistant Malassezia strains (86.6%) was observed using DXB. A MIC>64mg/L was observed only when the FLZ-resistant M. pachydermatis isolate was tested in SDB. Based on the results obtained herein, culture in SDB, stock inoculum suspensions of 1-5×10(6)CFU/ml, and an incubation time of 48h are proposed as optimal conditions for the evaluation of the in vitro antifungal susceptibility of M. pachydermatis using a modified CLSI BMD protocol.  相似文献   

19.
Three strains of a new species of mycoplasma were recovered from pneumonic pig lungs, known free of Mycoplasma hyorhinis, by prolonged incubation in pig testicle cell cultures. The three strains produced a characteristic cytopathic effect in the cell cultures. A highly enriched meat-infusion-broth medium was evolved and permitted regular propagation of these organisms. Pneumonia could consistently be produced by intratracheal inoculation of pigs with the mycoplasma propagated in the enriched broth medium or in cell cultures. The mycoplasma were recovered from the lungs of experimentally infected pigs by inoculation into the broth medium. Comparative studies of the pneumonia producing mycoplasma and of M. hyorhinis were carried out in cell cultures, broth media, and in pigs infected experimentally by different routes. The morphological characteristics of the mycoplasma, grown in the different media, are described and illustrated.  相似文献   

20.
Abstract

The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.

Received August 1, 2011; accepted December 6, 2011  相似文献   

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