Evaluation of chromogenic medium (ORSAB) for routine identification of methicillin resistant Staphylococcus aureus (MRSA)

Performance of chromogenic medium (ORSAB) for routine detection of methicillin resistant S. aureus (MRSA) was evaluated on 510 specimens collected from patients suspected of MRSA infection or colonization. Addition of ORSAB plates to the routine protocol allowed MRSA identification in 24 hours from samples plating. In 18 samples MRSA colonies were identified only on ORSAB plates, those cases would have been missed by routine protocol alone.     

Disc Plate Method of Microbiological Antibiotic Assay: I. Factors Influencing Variability and Error 1

Several factors are investigated that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures. Of these factors the most serious is the unequal exposure of the individual plates at top or bottom of stacks to temperatures above and below room temperature. This unequal temperature exposure is avoided by novel handling and incubation procedures. A major variable, but one which can be controlled, is the varying time interval between pouring seeded agar and the time of applying the pads with antibiotic to the plates. This influence of time of setting and the effects of several other sequential operations are combined into a composite variable. This variable is then accounted for and normalized by interposing "external" reference plates set with a reference solution in the sequence of approximately 100 plates. No "internal" reference zones are employed. Such factors as volume of agar poured, wedge shape of agar in a dish, volumetric errors in dilutions, and timing considerations are studied and discussed. The results of this study form the basis for a test protocol which is presented in a following paper.     

Characterization of clones of HIV-1 infected HuT 78 cells defective in gag gene processing and of SIV clones producing large amounts of envelope glycoprotein

Single-cell clones of HIV-1 (FRE-3) or SIV/Mne infected HuT 78 cells were obtained by plating dilutions of virally infected HuT 78 cells on a monolayer of sheep choroid plexus cells in 96-well microtiter plates. Several of these clones produce HIV-1 virus mutants that accumulate the gag precursor polyprotein and lack a functional protease. These protease-deficient viruses are non-infectious and consist of aberrant "immature" virus particles as determined by electron microscopy. Several SIV mutants are also described that produce large amounts of either the envelope glycoprotein gp120 or the nucleic acid binding gag protein. These mutants are useful for the purification of these retroviral proteins, in developing assays of protease inhibitors, and in preparing SIV envelope protein vaccines.     

A Simple,Highly Efficient Plating Method for Trypanosomatids1

A simple technique for plating trypanosomatids includes use of plates with lower agar concentrations than those usually prescribed for plating bacteria and a simple system to prevent dehydration due to the long incubation time needed for formation of visible colonies. Consistently high plating efficiency was thus achieved. Colonies from Herpetomonas samuelpessoai and Crithidia deanei were clearly distinguishable from each other; their individual characteristics varied with plating conditions.     

Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution

Abstract Micrococcus luteus starved for 2–7 months in spent medium following growth to stationary phase in batch culture exhibited a culturability (as estimated by direct plating on nutrient agar plates) of < 0.001%. However, following a lag, some 70% of the cells could be lysed upon inoculation into and cultivation in fresh lactate minimal medium containing penicillin, showing the capability of a significant portion of the cells at least to enlarge (and thus potentially to resuscitate). When the viable cell count was estimated using the most probable number method, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, and little different from the viable cell count as assessed by plating on solid media. However, the apparent viability of these populations evidenced with the most probable number method was 1000–100 000-fold greater when samples were diluted into liquid media containing supernatants taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.     

Estimates of measurement uncertainty from proficiency testing schemes, internal laboratory quality monitoring and during routine enforcement examination of foods

AIMS: To determine the levels of measurement uncertainty (MU) obtained in proficiency testing and routine microbiological analyses of foods and to compare these with estimates of MU obtained for results of analyses obtained in collaborative interlaboratory studies of microbiological methods. METHODS AND RESULTS: Raw data submitted by participants in the Food Examination Proficiency Assessment Scheme were obtained from the Central Science Laboratory (York). Internal quality monitoring data were obtained from Health Protection Agency (HPA) laboratories, together with data from routine food examinations undertaken in HPA laboratories. The data sets were analysed to determine the relative standard deviations of reproducibility (RSD(R)), based on log(10) colony count values, and thence the relative measures of expanded uncertainty. Analysis of proficiency test data showed extreme values of RSD(R) up to +/-30% depending upon the organism, the laboratory and the method of examination. RSD(R) values on routine samples averaged around +/-12% but ranged up to +/-41% in a few instances. Internal quality assessments for different organisms ranged up to +/-27%, depending upon the particular organism and examination procedure. The results show little difference in uncertainty for counts obtained using different plating systems (e.g. pour plates, spread plates or spiral plating) on the same dilutions of the same food samples. The data are compared with estimates of microbiological uncertainty derived in interlaboratory studies. CONCLUSIONS: The estimates of uncertainty ranged widely, both within and between laboratories, and appeared to bear little relationship to the foodstuff under examination. The extent of MU associated with many routine microbiological examinations is generally no worse than those produced in inter-laboratory trials, although notable exceptions were seen. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the levels of MU may have wide impact on the establishment of international standard methods for microbiological examination of foods and the ability to set realistic microbiological criteria.     

Homology of posterior interray plates in crinoids: a review and new perspectives from phylogenetics,the fossil record and development

Despite their importance for understanding phylogeny, character evolution and classification, well-constrained homology relationships for posterior plating in crinoids have only recently been attempted. Here, we re-evaluate posterior plate homologies in all major crinoid lineages using development, fossil ontogenies and phylogenetic evidence. Based on these lines of evidence, we change terminology for some posterior plates to correct misnomers and make recommendations for updated terminology of others to better reflect homology. Among pentacrinoids (disparids, hybocrinids, eucladids, flexibles and articulates) the relative position of posterior interray plates, not their topology, reflects homology. From proximal to distal, pentacrinoid posterior plates are the radianal, anal X and right sac plate, regardless of the total number of plates in the adult calyx. Camerate posterior plating contrasts with pentacrinoids, but insufficient data are available to resolve homology relationships between these two clades. More examples of early post-larval ontogeny are needed in camerates and other Palaeozoic crinoids.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

FuGENE 6 Transfection Reagent: the gentle power

FuGENE 6 Transfection Reagent has been commercially available since 1997. Since that time, its popularity has increased due to its ease of use, minimal to no cytotoxicity, and the high level of transfection in many different cell lines. FuGENE 6 Transfection Reagent is gentle on the cells. Adherent cells can be trypsinized and transfected by the DNA:FuGENE 6 reagent complex prior to plating, making it a strong candidate for high throughput applications. Additionally, low cell numbers can be transfected in 96-well plates. As with most reagents, the complex formation step is critical and special handling is required because the reagent is supplied in 80% ethanol. For example, contact with plastic must be avoided as inhibitors of transfection can leach from some plastics. We investigated parameters that have been reported to affect the transfection efficiency including the use of common antibiotics, passage level of the cells, and length of time for complex formation. These parameters are often cell line dependent and can be optimized to increase transfection efficiency for a specific cell line.     

Efficient method for the detection of microbially-produced antibacterial substances from food systems

A novel method for the isolation of microbially-derived inhibitory substances from food sources was developed. The method involves an enrichment step coupled to a killing assay which is initially carried out in multiwell plates. The technique has advantages in that large numbers of samples can be tested in parallel. The assay can be completed in less than 60 h and is more sensitive than direct plating due to the enrichment step. This novel screening approach was compared with the standard direct plating approach in an effort to identify the antimicrobial potential of a number of Kefir grains. Kefir grains were incubated in 10% reconstituted skim milk for 20 h at 32 degrees C to enable production of any potential biopreservatives. Following overnight incubation, fermentates were aliquoted into multi-well plates and a known number of indicator cells was added to each well. The fermentates were incubated for a further 20 h and counts were carried out to determine whether a reduction in indicator cell numbers had occurred. A reduction in cell-forming units indicated the presence of an inhibitory substance and these inhibitory fermentates were selected for further investigation. Using the protocol outlined, Kefir fermentates capable of inhibiting Listeria innocua DPC1770 and Escherichia coli O157:H45 were identified.     

Studies of the cultivable flora of normal human feces

Highly diluted feces, obtained from healthy adult individuals, was plated on blood-agar plates which were incubated both aerobically and anaerobically. From the anaerobic plates containing 30 to 60 colonies, every colony was subcultured. Nearly all isolates were obtained in pure culture and partially characterized. It was found thatBacteroides species were the most predominant organisms, being present in numbers approximating 10¹⁰ per gram wet weight. Selected bacteria present in lower numbers were determined by plating appropriate dilutions of feces on selective media. It was found that coliforms, streptococci and lactobacilli were regularly present in concentrations of 10⁶ ? 10⁸ organisms per gram wet weight material, whileVeillonella, Streptococcus salivarius, Bacteroides melaninogenicus and staphylococci were present in lower numbers. Fusobacteria were only found in one sample, whileNeisseria were not detected in any of the samples. Wet mounts of fecal material, inspected by darkfield microscopy, did not reveal the presence of spirochetes. Anaerobes outnumbered facultative bacteria by a factor of 40, indicating that the human adult fecal flora is predominantly anaerobic. Total microscopic counts indicate that bacteria comprise approximately 30% of the mass of human feces.     

Shiga-toxigenic Escherichia coli O157 and non-Shiga-toxigenic E. coli O157 respond differently to culture and isolation from naturally contaminated bovine faeces

AIM: To quantify the effect of enrichment, immunomagnetic separation (IMS), and selective plating procedures on isolation of Shiga-toxigenic Escherichia coli O157 (STEC O157) and non-Shiga-toxigenic Escherichia coli O157 (non-STEC O157) from naturally contaminated bovine faeces. METHODS AND RESULTS: Two broth enrichment times, two IMS strategies, and two selective plating media were evaluated. STEC O157 and non-STEC O157 strains were often isolated from the same faecal specimen and responded differently to the isolation protocols. A large-volume IMS system was more sensitive than a conventional small-volume IMS method, but was also more expensive. STEC O157 was more frequently isolated from 6 h enriched broth and ChromAgar plates containing 0.63 mg l(-1) potassium tellurite (TCA). Non-STEC O157 was more frequently isolated from un-enriched broth and ChromAgar plates without tellurite (CA). CONCLUSIONS: The combination of 6-h enrichment in Gram-negative broth containing vancomycin, cefixime and cefsuludin, large volume IMS and selective plating on TCA maximized STEC O157 recovery from naturally contaminated cattle faecal specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: The pairing of proper enrichment with a specific plating procedure is key for STEC O157 recovery from naturally contaminated bovine faeces. Incorporating tellurite into an E. coli O157 detection strategy may select for the subset of E. coli O157 that contains the Shiga-toxin genes.     

SARS-CoV细胞空斑试验

目的建立适用于生物安全三级实验室操作的SARS．CoV空斑试验方法。方法SARS－CoVl0倍系列稀释接种Vero－E6细胞,用0．6％琼脂糖覆盖中性红着色（琼脂糖．中性红法）或覆盖1％甲基纤维素,4％甲醛固定,结晶紫染色（甲基纤维素－结晶紫法）,空斑计数。结果琼脂糖－中性红法病毒滴度为6．14Lg pfu／mL,甲基纤维素－结晶紫法病毒滴度为6．57Lg pfu／mL。结论二种空斑试验方法相比,病毒滴度无明显差异。甲基纤维素－结晶紫空斑试验法形成的空斑比琼脂糖－中性红法清晰、操作简单安全、结果可长期保存。     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood.

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

Selective plating medium for enumeration and recovery of a hexachlorocyclohexane-degrading Pseudomonas Ptm+ strain from soil

A double selective plating medium has been developed to enumerate a Pseudomonas Ptm+ strain capable of assimilating hexachlorocyclohexane (HCH). The ability of the isolate to utilize HCH and/or streptomycin as sole sources of carbon was chosen to impart selectivity. In trials when serial 10-fold soil dilutions were spread on HCH plus streptomycin mineral agar and incubated, the normal microflora did not grow. When soil containing an HCH degrading Pseudomonas was cultured, only the target colonies developed. This double selective plating method is suitable for environmental monitoring of HCH scavengers as the medium is sensitive, specific and practical.     

Facile synthesis of mono-6-amino-6-deoxy-alpha-, beta-, gamma-cyclodextrin hydrochlorides for molecular recognition, chiral separation and drug delivery

We describe a protocol for the synthesis of mono-6-amino-6-deoxy-cyclodextrin hydrochloride (CD-NH3Cl), applicable to alpha-, beta- and gamma-cyclodextrin. These structurally simplest, highly water-soluble cationic cyclodextrins can be widely used in molecular recognition, chiral separation and drug delivery studies. Starting from commercially available chemicals, CD-NH3Cl is synthesized in four steps: (i) selective tosylation of cyclodextrin by the use of p-toluenesulfonyl chloride to afford mono-6-(p-toluenesulfonyl)-6-deoxy-cyclodextrin (Ts-CD); (ii) azide substitution of Ts-CD with sodium azide to afford mono-6-azido-6-deoxy-cyclodextrin (CD-N3); (iii) reduction of CD-N3 with triphenylphospine followed by hydrolysis to prepare mono-6-amino-6-deoxy-cyclodextrin (CD-NH2); and (iv) treatment of CD-NH2 with hydrochloric acid to afford the titled CD-NH3Cl with good yield. The overall protocol requires approximately 2 weeks.     

Olfactory Thresholds of the U.S. Population of Home-Dwelling Older Adults: Development and Validation of a Short,Reliable Measure

Current methods of olfactory sensitivity testing are logistically challenging and therefore infeasible for use in in-home surveys and other field settings. We developed a fast, easy and reliable method of assessing olfactory thresholds, and used it in the first study of olfactory sensitivity in a nationally representative sample of U.S. home-dwelling older adults. We validated our method via computer simulation together with a model estimated from 590 normosmics. Simulated subjects were assigned n-butanol thresholds drawn from the estimated normosmic distribution and based on these and the model, we simulated administration of both the staircase and constant stimuli methods. Our results replicate both the correlation between the two methods and their reliability as previously reported by studies using human subjects. Further simulations evaluated the reliability of different constant stimuli protocols, varying both the range of dilutions and number of stimuli (6–16). Six appropriately chosen dilutions were sufficient for good reliability (0.67) in normosmic subjects. Finally, we applied our method to design a 5-minute, in-home assessment of older adults (National Social Life, Health and Aging Project, or NSHAP), which had comparable reliability (0.56), despite many subjects having estimated thresholds above the strongest dilution. Thus, testing with a fast, 6-item constant stimuli protocol is informative, and permits olfactory testing in previously inaccessible research settings.     

Procedure for Isolation and Enumeration of Vibrio parahaemolyticus,

An evaluation of criteria used in the identification of Vibrio parahaemolyticus showed that cultural responses varied with respect to growth in broth with 10% NaCl, type of hemolysis, reactions in triple sugar-iron-agar, and serological reactions. With few or no exceptions, cultures were positive for cytochrome oxidase, utilized glucose fermentatively, were sensitive to pteridine (0/129) and novobiocin, and failed to grow in Trypticase soy broth (TSB) without NaCl. A procedure employing a direct plating technique, with or without prior enrichment, was designed for the isolation and enumeration of V. parahaemolyticus. The plating medium consisted of 2.0% peptone, 0.2% yeast extract, 1.0% corn starch, 7% NaCl, and 1.5% agar, with the pH adjusted to 8.0. The enrichment broth was TSB with 7% NaCl. Dilutions of food homogenates were either spread directly on the plates or inoculated into enrichment broth. TSB enrichments were incubated at 42 C for 18 hr. A loopful of the TSB tubes then was streaked onto the direct plating medium. Incubation of plates was at 42 C for 24 to 48 hr. Smooth, white to creamy, circular, amylase-positive colonies were then picked as suspect V. parahaemolyticus. Confirmation of gram-negative, fermentative, oxidase-positive, pleomorphic rods sensitive to pteridine 0/129 was made by a fluorescent-antibody technique. With this procedure, a satisfactory quantitative recovery of known V. parahaemolyticus from inoculated seafoods was made possible. V. parahaemolyticus was nto isolated from other salted foods.