细菌革兰氏染色探究式实验教学的设计和 实施效果分析

细菌革兰氏染色效果受多种因素的影响,基于探究式教学法的理论原则,本文将固定方式和脱色时间两个关键因素设计成梯度对比实验,通过结果的直观差异性使教学效果显著提高.更为重要的是,学生在分组和组内实验探索过程中,通过内在好奇心和外在具体任务的双重驱动,主体的创新精神得到激发,情感价值体验得到满足,创新意识、动手能力、思维品质和协作精神都得到了明显的提升.实践证明,针对特定问题巧妙设计探索引导实验,不仅可以增强学生的实践动手能力,提升教学效果,而且可以充分调动学生的主体积极性,激发创新意识和培养严谨的科研思维品质.     

Use of urinary gram stain for detection of urinary tract infection in childhood

In this study, urinary culture, urinary Gram stain, and four tests within the urinalysis, leukocyte esterase, nitrite, microscopyfor bacteria, and microscopyforpyuria, were examined in 100 children with symptoms suggesting urinary tract infection. Our purpose was to determine the validity of the urinary Gram stain compared with a combination of pyuria plus Gram stain and overall urinalysis (positiveness of nitrite, leukocyte esterase, microscopy for bacteria, or microscopy for white blood cell). Of 100 children, aged two days to 15 years, 70 (70 percent) had a positive urinary culture: 40 girls (57 percent) and 30 boys (43 percent). Escherichia coli was the most common isolated agent. The sensitivity and specificity of the urinary Gram stain were 80 percent and 83 percent, and that of the combination of pyuria plus Gram stain 42 percent and 90 percent, and that of the overall urinalysis 74 percent and 3.5 percent respectively. Our findings revealed that neither method of urine screen should substitute for a urine culture in the symptomatic patients in childhood.     

Accuracy of tracheal aspirate gram stain in predicting Staphylococcus aureus infection in ventilator-associated pneumonia

Background

The Gram stain can be used to direct initial empiric antimicrobial therapy when complete culture is not available. This rapid test could prevent the initiation of inappropriate therapy and adverse outcomes. However, several studies have attempted to determine the value of the Gram stain in the diagnosis and therapy of bacterial infection in different populations of patients with ventilator-associated pneumonia (VAP) with conflicting results. The objective of this study is to evaluate the accuracy of the Gram stain in predicting the existence of Staphylococcus aureus infections from cultures of patients suspected of having VAP.

Methods

This prospective single-center open cohort study enrolled 399 patients from December 2005 to December 2010. Patients suspected of having VAP by ATS IDSA criteria were included. Respiratory secretion samples were collected by tracheal aspirate (TA) for standard bacterioscopic analysis by Gram stain and culture.

Results

Respiratory secretion samples collected by tracheal aspirates of 392 patients were analyzed by Gram stain and culture. When Gram-positive cocci were arranged in clusters, the sensitivity was 68.4%, specificity 97.8%, positive predictive value 88.1% and negative predictive value 92.8% for predicting the presence of Staphylococcus aureus in culture (p < 0.001).

Conclusions

A tracheal aspirate Gram stain can be used to rule out the presence of Staphylococcus aureus in patients with a clinical diagnosis of VAP with a 92.8% Negative Predictive Value. Therefore, 7.2% of patients with Staphylococcus aureus would not be protected by an empiric treatment that limits antimicrobial coverage to Staphylococcus aureus only when Gram positive cocci in clusters are identified.     

Mechanism of gram variability in select bacteria.

Gram stains were performed on strains of Actinomyces bovis, Actinomyces viscosus, Arthrobacter globiformis, Bacillus brevis, Butyrivibrio fibrisolvens, Clostridium tetani, Clostridium thermosaccharolyticum, Corynebacterium parvum, Mycobacterium phlei, and Propionibacterium acnes, using a modified Gram regimen that allowed the staining process to be observed by electron microscopy (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Furthermore, since a platinum salt replaced the iodine mordant of the Gram stain, energy-dispersive X-ray spectroscopy could evaluate the stain intensity and location by monitoring the platinum signal. These gram-variable bacteria could be split into two groups on the basis of their staining responses. In the Actinomyces-Arthrobacter-Corynebacterium-Mycobacterium-Propionibacterium group, few cells became gram negative until the exponential growth phase; by mid-exponential phase, 10 to 30% of the cells were gram negative. The cells that became gram negative were a select population of the culture, had initiated septum formation, and were more fragile to the stress of the Gram stain at the division site. As cultures aged to stationary phase, there was a relatively slight increase toward gram negativity (now 15 to 40%) due to the increased lysis of nondividing cells by means of lesions in the side walls; these cells maintained their rod shape but stained gram negative. Those in the Bacillus-Butyrivibrio-Clostridium group also became gram negative as cultures aged but by a separate set of events. These bacteria possessed more complex walls, since they were covered by an S layer. They stained gram positive during lag and the initial exponential growth phases, but as doubling times increased, the wall fabric underlying the S layer became noticeably thinner and diffuse, and the cells became more fragile to the Gram stain. By stationary phase, these cultures were virtually gram negative.     

Mechanism and use of the commercially available viability stain, BacLight.

BACKGROUND: BacLight (Molecular Probes, Eugene, OR, USA) is a popular fluorescence-based two-component stain for determining bacterial cell viability. The main purpose of this work was to fully elucidate the mechanism and to determine why it is sometimes reported that cells stain simultaneously live and dead. METHODS: Solutions of DNA were stained with the two components, propidium iodide (PI) and SYTO9, in different combinations, and fluorescence spectra were collected. RESULTS: K(PI) and K(SYTO9) were approximately 3.7 x 10(5)/M and 1.8 x 10(5)/M. SYTO9 emissions were stronger and overlapped those of PI. Fluorescence resonance energy transfer from SYTO9 to PI was observed. It was, even under normal conditions, possible for DNA bound SYTO9 to have a component in the red region equal to that of DNA bound PI. Potentially confusing emissions were also found to occur when PI was not in sufficient excess to saturate nucleic acid (>0.4 M PI to 1 M DNA base pairs). CONCLUSIONS: The mechanism is a combination of displacement of SYTO9 by PI and quenching of SYTO9 emissions by fluorescence resonance energy transfer. Confusing results can occur if the relative intensities of the stains or the concentration of PI relative to nucleic acid are not properly accounted for.     

A case study: oligonucleotide purification from gram to hundred gram scale

This article describes the purification and scale-up of ISIS 2302, a 20-mer phosphorothioate oligonucleotide by anion-exchange (AX) chromatography. The key operating parameters were optimized at gram scale and further scaled up to hundred gram. SOURCE 30Q, a high efficiency polymeric chromatographic media was used for both the small and large-scale work. High length-based purity and yield were maintained at scale-up. This purification is one of the largest demonstrations of AX purification of phosphorothioate oligonucleotide.     

Characterization of guanylate kinase from gram positive and gram negative microorganisms; preliminary results

Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions. Guanylate kinase (GMPK) is an essential enzyme for the biosynthesis of GTP and dGTP by catalyzing the phosphoryl transfer from ATP to (d)GMP resulting in ADP and (d)GDP. Despite the similar fold of the monomer there is an important difference between GMPKs from prokaryotes and eukaryotes: eukaryotes GMPK are monomers while prokaryotes GMPK are dimmers, tetramers or hexamers. For this reason bacterial GMPKs are possible targets for new antibacterial drugs. Finding new targets for antibacterial therapies is a prior subject in today's medical research. The purpose of this work was to characterize guanylate kinases from both gram positive and gram negative pathogenic bacteria. We started with GMPK from Enterococcus faecalis as gram positive microorganism and Pseudomonas aeruginosa as gram negative representative.     

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.