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1.
The spontaneous production of cell-wall-deficient filaments and protoplasts by a strain of Actinomyces hordeovulneris (UCD 81-332-9) in 10% sucrose L-form media is reported. Multiple mineral deposits were present within the variants at 48 hours. Electron microscopy revealed that these deposits were amorphous, dense, and at the inner face of the cytoplasmic membrane in wall-less protoplasts and also in filaments which had a thin wall of 10 nm. These cell-wall-deficient variants produced L-form colonies when cultured for an additional 48 hours on 10% sucrose-BYE L-form plates. The colonies were composed of only a few filaments and many vesicles which were negative with Dienes' stain. Silver substitution stains of UCD 81-332-9 cells that had been grown in L-form broth for 5 days revealed heavy calcification of all cells including protoplasts. Gram stains of L-form grown cells revealed the presence of long-beaded, infrequently branched gram-positive filaments similar to those observed in clinical specimens. The formation of cell wall-deficient variants with coincident mineralization is believed to be related to the phenomenon of sulfur granule formation in vivo.  相似文献   

2.
Nasal secretions, faecal samples and buffy coats were obtained from 102 cattle from a North Dakota dairy herd with a history of calf scours. Treated buffy coats, faecal samples and nasal secretions were inoculated into tetrathionate broth (TB), incubated at 37°C overnight, and plated onto brilliant green agar medium with novobiocin (BGAN). The TB was left at room temperature for 5 days and then used to inoculate fresh TB. The fresh TB was incubated at 37°C over night and plated onto BGAN medium. All the plates were incubated at 37°C over night and observed forSalmonella-like growth. Suspect colonies were further tested andSalmonella isolates were serotyped by the National Veterinary Services laboratory. Twenty-two of the 36 calves sampled harbouredS. typhimurium in their faeces, but no samples from cows were positive. NoSalmonella were isolated from the buffy coats, but 4 calves were shown to haveSalmonella in their nasal secretions. Extended enrichment of the faecal cultures in TB resulted in a significant increase inSalmonella isolations, although 2 samples were positive following the initial enrichment period and not after secondary enrichment. The typicalSalmonella isolate detected from this herd contained a transmissible R-plasmid encoding resistance to tetracycline, kanamycin, sulphisoxazole and ampicillin. This study confirmed that delayed secondary enrichment in TB is superior to primary enrichment for detection ofSalmonella from cattle.Abbreviations Ap ampicillin - BGAN brilliant green agar with novobiocin - Cm chloramphenicol - DSE delayed secondary enrichment - Gn gentamicin - Kn kanamycin - NA nalidixic acid - NADC National Animal Disease Center in Ames, Iowa - NVSL National Veterinary Services Laboratory in Ames, Iowa - PB polymyxin B - PE primary enrichment - Sm streptomycin - Su sulphisoxazole - TB tetrathionate broth - Tc tetracycline - TSI triple sugar iron agar  相似文献   

3.
An unstable L-form of Staphylococcus aureus was identified in milk samples from 3 quarters of 2 cows after treatment with cloxacillin. Milk samples incubated on standard 5% blood agar plates were culture-negative for 7 to 30 days after treatment, but S aureus was reisolated in 80% of 66 samples by additional culturing on enriched L-form media when incubated in 10% CO2 at 37 C. The organism was identified at various phases of reversion of L-form agar and was confirmed on transfer to blood agar plates.  相似文献   

4.
The ability of BACTEC radiometric 7H12 broth, Middlebrook 7H10 Tween broth, Middlebrook 7H10 agar, and Herrold's egg-yolk medium to provide early detection of Mycobacterium paratuberculosis was evaluated. The minimum detection times in days for the various media were: 7H12, 9; 7H10 agar, 23 (plate), 28 (slant); 7H10 Tween broth; 27; and Herrold's egg-yolk medium, 43 (plate), 49 (slant). The radiometric broths provided the earliest detection of M. paratuberculosis, and 3625 organisms ml-1 were required to produce a positive, radiometric growth-index reading. Of the non-radiometric plate and slant media evaluated, microscope examination of the translucent 7H10 agar plate resulted in the earliest detection and highest mean colony counts (387) as compared with Herrold's egg-yolk agar plate (208). Similar results were noted for 7H10 and Herrold's egg-yolk agar slants; however, accurate colony counts could not be determined because of confluent growth. All media were supplemented with 2 micrograms ml-1 of mycobactin J and excess amounts of this supplement inhibited the growth of M. paratuberculosis in radiometric 7H12 media.  相似文献   

5.
将新鲜原材料配制的普通肉汤、普通琼脂、马丁肉汤、马丁琼脂培养基以及含有指示剂的麦康凯琼脂培养基于室温（普通实验室环境,下同）和冷库（4℃,下同）两种条件存放54周,定期通过灵敏度试验和活菌计数试验检验其质量。通过试验观察到：普通肉汤、普通琼脂培养基于两种条件存放54周后,普通肉汤灵敏度仍能达到10-8以上,普通琼脂培养基中活菌数没有明显减少;马丁肉汤、马丁琼脂培养基于两种条件存放至26周,马丁肉汤灵敏度仍能达到10-8以上,马丁琼脂培养基中活菌数没有明显减少,但30周后CVCC428在马丁肉汤和马丁琼脂中生长繁殖能力明显下降,到46周以后无生长;麦康凯琼脂培养基于两种条件存放54周后,质控菌活菌数没有明显减少,但30周后培养基中指示剂颜色消退明显,菌落特异性显色反应不明显。通过以上各试验结果,可将普通肉汤、普通琼脂培养基保质期暂定为12个月,马丁肉汤、马丁琼脂、麦康凯琼脂培养基保质期暂定为6个月。  相似文献   

6.
To determine whether a strain of Salmonella typhimurium (UCD 1755) of equine origin had enterotoxin activity, 2 ml of a cell-free culture lysate of strain UCD 1755 and approximately 10(9) viable strain UCD 1755 organisms were inoculated into ligated small intestinal segments of rabbits. Intestinal segments inoculated with viable strain UCD 1755 organisms and those inoculated with a cell-free culture lysate of strain UCD 1755 had significant (P less than 0.05) accumulations of fluid 10 hours after inoculation when compared with ligated intestinal segments either inoculated with sterile brain-heart infusion broth or left empty. There was not a statistically significant difference between fluid accumulation of intestinal segments inoculated with viable strain UCD 1755 and that of segments inoculated with a cell-free culture lysate of strain UCD 1755. The responses of equine colonic mucosa to culture filtrates of 2 strains of salmonella typhimurium (UCD 1755 and SL 1027) and purified cholera toxin were studied in vitro. Isolated smaples of colonic mucosa were incubated for 4 hours at 37 C in Krebs-Henseleit bicarbonate buffer (KHB) alone, KHB plus culture lysate of strain UCD 1755, KHB plus culture lysate of strain SL 1027, and KHB plus 1 microgram of cholera toxin/ml. Cyclic adenosine monophosphate (cAMP) content of each sample was determined.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
Enrichment media (tetrathionate, selenite and Rapp ap ort broths) and selective media (desoxycholate citrate agar and brilliant green agar) were tested in different combinations to ascertain their capacity for isolation of salmonella bacteria. The material consisted of 299 samples of cattle faeces from two herds infected with salmonella (Table 1), and of 111 artificially contaminated samples of pig faeces (Table 3). The tetrathionate and selenite broths were equally useful for the material as a whole, whereas the results varied between different species of salmonella which is of great practical interest. The number of salmonella isolations was much lower when enrichment with Rappaport broth was used. The rate of salmonella isolations can often be increased by parallel enrichments with two different media. Of the selective agar media tested, brilliant green agar was superior to desoxycholate citrate agar.  相似文献   

8.
To assess the effect of pooling fecal samples on the sensitivity of detection of E. coli O157:H7, 12 calves, inoculated orally with 10(8)cfu per calf of nalidixic acid resistant E. coli O157:H7, were used to provide positive fecal samples. After inoculation, calves were sampled twice weekly. Negative fecal samples were from calves at a local dairy. Samples from inoculated calves were incubated without pooling or were mixed with known negative fecal samples in a 1:4 ratio or a 2:3 ratio (positive:negative) for detection of E. coli O157:H7. Samples were enriched 6h in Gram negative broth with vancomycin, cefixime, and cefsoludin, underwent immunomagnetic separation with Dynabeads, and were plated onto sorbitol MacConkey agar with cefixime, and tellurite (SMACct). Morphologically typical colonies were plated onto blood agar, incubated overnight at 37 degrees C and an indole test was performed on each colony. Indole positives colonies were plated on SMAC agar with 20 microg/ml nalidixic acid (SMACnal). Colonies that grew on SMACnal were confirmed by O157 agglutination. Sensitivity of detection in non-pooled samples was 77%. Samples pooled 1:4 and 2:3 with negative samples were 55 and 52% sensitive, respectively. Pooling decreased sensitivity of detection for E. coli O157:H7 in bovine fecal samples (P<0.01). A deterministic binomial probability model was developed to assess the probability of detecting pens of cattle shedding E. coli O157 using a pooling protocol or individual samples. Pooling decreased sensitivity of detection at low pen prevalence compared to individual samples but was similar at high prevalence.  相似文献   

9.
In a comparison of two commonly used membrane filters for enumerating fecal coliform bacteria it was demonstrated that Seitz type M filters recovered statistically more colonies of bacteria than did Millipore HAWG 047S1 filters from pure cultures of Escherichia coli incubated at 44 °C. The membranes were grown on 0.4 % Teepol agar. On incubation at 37°C no significant discrepancy was found. As a reference method was used pour plating in plate count agar (Difco). It was demonstrated that incubation at 44°C did not per se inhibit propagation of fecal coliforms. Both types of filters examined were sterilized by the manufacturers with ethylene oxide. The discrepancy found can therefore not be due to sterilization procedures.  相似文献   

10.
The isolation and identification of Yersinia enterocolitica from minced meat on CIN agar medium is still one of the major problems in food microbiology because of the low selectivity of cefsulodin-irgasan-novobiocin (CIN) agar. A total of 198 minced meat samples were collected from commercial establishments (butcher shops and supermarkets) in seven German cities in order to investigate the sensitivity and specificity of three identification techniques suitable for the differentiation of Y. enterocolitica within the rich background flora on CIN agar plates. As expected isolation of Y. enterocolitica from minced meat on CIN agar medium after 72 h enrichment in peptone, sorbitol and bile salts (PSB) broth was difficult because all plates were abundantly covered with numerous 'typical'Yersinia-like colonies of bull's eye appearance as well as with atypical colonies. Based on the phenotype of the colonies it was possible to detect colonies showing Yersinia-like growth on CIN agar in 52 samples (26%). For identification of Y. enterocolitica the API 20E system (bioMerieux, Nürtingen, Germany), the Yersinia identification kit (Merlin, Bornheim-Hersel, Germany) and a 16S rRNA based PCR assay were compared. Only in one sample (0.5%) a Y. enterocolitica strain was detected by all methods. Of the three identification systems tested for routine laboratory diagnostics the API 20E system was found to be the most suitable tool to identify Y. enterocolitica colonies within the rich background flora from minced meat samples on CIN agar plates.  相似文献   

11.
Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa > or = 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.  相似文献   

12.
A stable L-form of Haemophilus pleuropneumoniae (Shope) was isolated from primary pig kidney cell tissue cultures which had been inoculated 28 days previously with glycine induced spheroplasts of this organism.

H. pleuropneumoniae was definitely cytopathic in primary pig kidney cell cultures, producing cell rounding, cytoplasmic vacuolation and nuclear enlargement with peripheral condensation of nuclear DNA. By contrast, the effect of spheroplasts was much less distinct, producing only loss of cytoplasmic granularity coincident with apparent loss of some cytoplasmic RNA, and slight nuclear enlargement.

Both the organism and its L-form were shown to be related by cultural methods, antibiotic sensitivity tests, immunofluorescence and immunodiffusion.

The L-form remained stable after 90 serial passages on agar and 45 in broth, each medium being capable of supporting the growth of both forms of the organism.

  相似文献   

13.
Tissues of mycoplasma infected chicks and turkey poults were cultured and subcultured on mycoplasma agar. Usually, colonies which grew on the agar initially inoculated could be subcultured, but sometimes they could not. At other times, colonies were not seen on the agar initially inoculated but appeared on the subcultured plate.  相似文献   

14.
[目的]为了解导致原料乳变质的微生物种类及其产蛋白酶活力。[方法]对广东省恒远市某规模化牧场提供的蛋白水解、脂肪上浮的变质原料乳稀释,涂布在MPC琼脂平板上。将平板于6.5 ℃恒温培养箱培养10 d,挑取形态不同的单菌落在LB液体培养基复壮增菌,提取其DNA,利用16S rDNA方法对分离的菌株进行鉴定,并对该菌株的蛋白酶活力及高温钝化后酶残留率进行测定。[结果]MPC琼脂板菌落形态单一,原料乳中分离鉴定出的两株菌均为霍氏假单胞菌,该菌株在28 ℃下对乳平板的水解圈为（15.35±0.56）mm,经135 ℃处理5 s,其蛋白酶残留率为43.63%。[结论]变质原料乳中分离到两株霍氏假单胞菌,并且该菌可产生热不敏感的蛋白酶,该酶的存在会造成原料乳中蛋白质水解,导致UHT乳在货架期内出现蛋白水解、胀包等质量问题。  相似文献   

15.
The isolation and identification of Yersinia enterocolitica from minced meat on CIN agar medium is still one of the major problems in food microbiology because of the low selectivity of cefsulodin–irgasan–novobiocin (CIN) agar. A total of 198 minced meat samples were collected from commercial establishments (butcher shops and supermarkets) in seven German cities in order to investigate the sensitivity and specificity of three identification techniques suitable for the differentiation of Y. enterocolitica within the rich background flora on CIN agar plates. As expected isolation of Y. enterocolitica from minced meat on CIN agar medium after 72 h enrichment in peptone, sorbitol and bile salts (PSB) broth was difficult because all plates were abundantly covered with numerous ‘typical’Yersinia‐like colonies of bull's eye appearance as well as with atypical colonies. Based on the phenotype of the colonies it was possible to detect colonies showing Yersinia‐like growth on CIN agar in 52 samples (26%). For identification of Y. enterocolitica the API 20E system (bioMerieux, Nürtingen, Germany), the Yersinia identification kit (Merlin, Bornheim‐Hersel, Germany) and a 16S rRNA based PCR assay were compared. Only in one sample (0.5%) a Y. enterocolitica strain was detected by all methods. Of the three identification systems tested for routine laboratory diagnostics the API 20E system was found to be the most suitable tool to identify Y. enterocolitica colonies within the rich background flora from minced meat samples on CIN agar plates.  相似文献   

16.
Species of anaerobic bacteria isolated from clinical veterinary specimens were tested for susceptibility to trimethoprim-sulfonamides by the broth-disk elution technique. Three different media were used for each organism: prereduced anaerobically sterilized (PRAS) brain-heart infusion broth (BHI), thioglycollate broth, and a semidefined PRAS medium. Susceptibility results from these media were compared with those determined by interpreting the minimal inhibitory concentration obtained using an agar dilution technique. Results from broth-disk testing in semidefined medium agreed in 68.7% of the cases, in 53.7% for thioglycollate broth, and in 36.9% for BHI. The greatest deviation between techniques occurred with isolates belonging to the genus Bacteroides, followed by those of the genus Clostridium and those of the genus Fusobacterium. This deviation was directly proportional to increasing concentrations of thymidine in the BHI and thioglycollate broths but not with the semidefined medium. We conclude that the broth-disk elution method for measuring susceptibility of obligate anaerobes to trimethoprim-sulfonamides is unsuitable.  相似文献   

17.
Treponema hyodysenteriae growth under various culture conditions   总被引：5，自引：0，他引：5  
The influence of various culture conditions on the growth of Treponema hyodysenteriae was determined. Six different anaerobically prepared culture broths were tested for the ability to support growth of strains B78, B204 and B169. Each medium contained glucose (0.2%) and 10% (v/v, final concn.) heat-inactivated fetal calf serum. Brain-heart infusion (BHIS), heart infusion (HS) and veal infusion (VS) broths gave the highest cell yields of the spirochete with the shortest incubation times. Vigorous mixing of the cultures and the introduction of O2 (1%, final concn.) into the culture atmosphere were necessary for optimum growth. Although BHIS broth was found to be the best for routine cultivation of the 3 strains, HS broth was more suitable for investigating the physiology of growing cells, inasmuch as cell growth in this medium was limited unless a growth substrate was added. Glucose, fructose, sucrose, galactose, trehalose, N-acetyl-glucosamine, glucosamine, mannose, maltose and pyruvate were growth substrates for all 3 strains. During the growth of B204 cells in HS broth under N2:O2 (99:1), glucose and O2 were consumed and CO2, H2, acetate and butyrate were produced. In HS agar-containing medium, cells of strains B78 and B204 formed spreading colonies typical in appearance to those of other spirochetes.  相似文献   

18.
A cloth-ELISA (C-ELISA) antigen capture assay for the detection of Brucella melitensis and B. abortus was developed. Segments (6-mm squares) of hydrophobic polyester cloth coated with diluted serum from a B. abortus-infected cow were incubated with saline suspensions of heat-killed Brucella cells, or with cultures of bovine or sheep blood or bovine tissue homogenates that had been inoculated with B. abortus or B. melitensis added to trypticase soy broth (TSB) and incubated for 2-3 days. The captured antigen was detected by a bovine anti-Brucella antibody-horseradish peroxidase conjugate system. The enrichment culture technique detected as few as three Brucella colony-forming units (c.f.u.) in 0.5 ml of bovine blood and was positive in cultures in which the Brucella concentration had reached 3 X 10(6) c.f.u. ml-1 (after 2 or 3 days incubation). The combined enrichment-cloth-ELISA method gave complete correlation with cultural isolation and results were available 3 days before colonies appeared in conventional culture. Hydrophobic cloths have potential use in diagnostic procedures since they provide simple, rapid and economical assays.  相似文献   

19.
The whole-cell proteins of ten strains of Rhodococcus equi isolated from horses, pigs, or humans, including the type strain ATCC 6939, were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein profiles of seven different capsular serotypes and the type strain were very similar when bacteria were cultured under the same conditions. Protein profiles were largely unaffected by incubation at two temperatures (30 degrees C, 37 degrees C) or times (12 h, 48 h). There were generally minor differences in protein profiles between strains grown in different media (brain heart infusion, nutrient, minca broths, tryptic soy-blood agar) with the marked exception of a prominent diffuse 17.5 kd protein which was expressed in nutrient broth. This protein was not produced by the type strain and was lost on repeated passage in vitro (50th, 100th passage) in two of three other strains examined.  相似文献   

20.
Solid and liquid media designed to support growth of cell wall deficient variants were evaluated for the presence of endogenous variants. The media compared in this study consisted of brain heart infusion base containing 10% sucrose, 3% NaCl + 5% sucrose, 3% NaCl, or 5% NaCl + 20% sucrose, and supplemented with filtrates of 10–20% horse serum - Sources A, B, C, suppliers of slaughterhouse (Type I) serum; and S, a supplier of standing herd (Type II) serum - and yeast extract. Three types of yeast supplements used in this study were aqueous extracts of: (1) fresh, or (2) active dry yeast; and (3) 0.5% commercial dehydrated yeast extract. Liquid control media prepared from one supplier gave rise to a pleomorphic Gram positive rod on extended incubation ( > 10 days) in spite of rigorous controls for asepsis during preparation and use. The second and more serious problem concerned repeated encounters with ‘pseudocolony’-like structures in work with clinical material. Correlation with severity of disease, different rates of appearance, and promotion of development by high osmolarity, led to a comparison of this phenomenon in control Type I- vs. Type II-BYE medium. Type I gave rise to numerous ‘pseudocolony‘- like bodies within 3 weeks, while Type II media containing standing herd serum developed < 20 per plate. Media variations known to promote growth of L-phase variants and mycoplasmas were noted to affect time of appearance and numbers of atypical colonies in control, uninoculated Type II-BYE. These included serum level, quality of yeast extract, pH, osmolar supplements, and the O2 tension. The development of these structures in only certain Type II-BYE cultures of clinical fluids does not support the view that disturbance of an agar surface, or the presence of cells (or nuclei) promotes ‘growth’. No atypical colony-like structures or amber bodies developed in plates incubated for 7 weeks in the presence of vapors of phenol + formaldehyde, nor during an additional 10-day interval in a normal gaseous environment. It is suggested that all horse sera tested in this study contained unclassified atypical L-forms with unusual resistance to heat and chemicals.  相似文献   

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