New Rapid Methods for Tissue Diagnosis

Two new methods applicable to the staining of fixed and fresh frozen tissue sections are presented herein. In addition certain improvements are suggested for the technic reported by Geschickter, Walker, Hjort and Moulton (1931). In brief the procedures are as follows:

1. The thionin eosinate method of Geschickter et al (1931). This procedure has been modified as follows:
   1. A mixture of diethylene glycol, 40 parts, ethylene glycol, 40 parts, and grain alcohol, 20 parts is superior to ethylene glycol, 80 parts, and ethyl alcohol, 20 parts, as a solvent for the compound stain in that the staining is intensified.
   2. Ethylene glycol monobutyl ether supplants diethylene glycol monobutyl ether because of its lower viscosity.
   3. Ethyl phthalate replaces butyl phthalate on account of a more satisfactory viscosity.
2. The methyl green eosinate procedure is the same as the modified thionin eosinate method except for the following variations:
   1. The staining time is increased to one minute.
   2. Decolorization and washing are reduced to about 15 seconds.
3. The hematoxylin-eosin method. After cutting, the tissue sections are carried thru the following steps:

Unfold in water; transfer to formalin (4 to 40%) for at least 30 seconds; stain in hematoxylin (Harris) for 30 to 60 seconds; wash in water, 5 seconds; decolorize in 0.1% HC1 or saturated aqueous picric acid, 5 seconds; wash in water, S seconds; float in 0.5% ammonia, 5 to 10 seconds; wash in water, 5 seconds; stain in 5% aqueous eosin, 15 seconds¹; wash in water, 5 to 10 seconds; dehydrate in a mixture of diethylene glycol, 30 parts, and ethyl alcohol, 70 parts, 5 to 10 seconds; dehydrate in ethylene glycol monobutyl ether, 5 to 10 seconds; clear in ethyl phthalate, 5 to 10 seconds; float on a glass slide, blot with photographic lintless blotter, place a drop of neutral gum damar on the section, and cover with glass cover slip.     

A Rapid Silver-on-the-Slide Method for Nervous Tissue

A progressive silver staining method is described, which permits microscopic examination of the sections during the staining process. After formaldehyde fixation, dehydration and embedding in paraffin or celloidin, fine fibers and synaptic endings may be demonstrated. After formaldehyde fixation and mordanting in 3% K₂Cr₂O₇, myelinated fibers and mitochondria are specifically stained.

The unique feature of this method is, that the silver solution (0.5% protargol) is mixed with the reducing solution: 1.6% Rochelle salts, containing traces of Ag NO₃, MgSO₄, and K₂S (U.S.P.). The sections are placed directly into this mixture, which is then warmed to 45-55° C. Sections are removed when progressive staining is completed, washed in water, dehydrated and mounted.

In the fiber stain, nerve fibers and synaptic endings are dark brown or black, and nuclear chromatin is deep brown, against a pale yellow background. When the myelin sheath procedure is followed, the fiber bundles are deep brown, and the intensity of the staining remains the same for specific tracts, aiding in their identification.     

An Apparatus for Rapid Attachment of Frozen Tissue to Cryostat Tissue Carriers

A brass cylindrical container 11.5 cm high and 7.5 cm in diameter was housed in an insulated wooden box. A 2.2 cm diameter hole was drilled in the centre of the removable brass lid on the underside of which a holder was attached for a cryostat tissue carrier. The container was filled with a mixture of acetone and solid CO₂ to within 1.5 cm of the lid. The frozen tissue was placed in a drop of water on a tissue carrier which was then lowered into the holder through the hole in the lid. The tissue carrier was rapidly cooled by the acetone-solid CO₂ mixture thus freezing the water and attaching the tissue to the carrier.     

Rapid Methods for High-Throughput Detection of Sulfoxides

Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.The growing demand for green catalytic processes has increased the utilization of enzymes as industrial biocatalysts for the synthesis of fine chemicals (6, 19, 20). As a consequence, there is a continuous search for novel or improved biocatalysts. In order to find an appropriate candidate for a process, various sources of enzymes must be screened for activity (23). Therefore, a sensitive, reproducible, accurate, and simple high-throughput screening method is a key prerequisite for the development of biocatalytic processes on an industrial scale (32, 39).Screening systems are divided into three different classes. The first class contains assays applicable to testing growing or resting microbial colonies for enzymatic activity directly on agar plates (23), for example, detection of epoxide hydrolase activity on butane oxide by use of safranin O. Oxidation of the 1,2-diol product by Escherichia coli modified the membrane potential and led to accumulation of the red dye in the colonies producing active enzyme (34). In another study, the spontaneous oxidation of substituted catechols to brown-red quinones was used to screen random libraries of whole cells expressing toluene monooxygenases (TMOs) for regioselective oxidation of substituted phenols (12, 30). The positive clones produced a red halo around the cells. These assays are high-throughput, simple procedures but often require a tailored substrate with a chromophore, such as bromonaphthol or azo-dye (23).The second class includes chromogenic and fluorogenic assays applicable in microtiter plates or microarray formats (23). Microtiter plates in 96- or 384-well format are particularly well suited for spectroscopic reading using either UV-visible or fluorescence plate readers. This class may be subdivided into the following four groups: (i) enzyme-coupled assays, such as the determination of dehydrogenase activity through formation of NADH from NAD and an absorbance change at a wavelength of 340 nm; (ii) assays using chromogenic and fluorogenic substrates, such as various synthetically labeled substrates that are commercially available for the determination of hydrolytic activity produced by lipases, phosphatases, glycosidases, amidases, etc.; (iii) assays using chromogenic and fluorogenic sensors, such as widely used pH indicators (16), that may be applied in any reaction that includes a change in pH; and (iv) microarray assays using a solid support, enabling screening of thousands of samples. The high-throughput potential of these methods was demonstrated by profiling of 40 different esterases and lipases across 35 different fluorogenic ester substrates, using only 50 μl of each enzyme solution and a submilligram quantity of each substrate for over 7,000 tests (2).The third class of enzymatic assays rely on product detection by instruments and include gas chromatography (GC), high-pressure liquid chromatography (HPLC), mass spectrometry, nuclear magnetic resonance (NMR) spectrometry, and infrared radiation assays that have been adapted for high throughput (22, 23, 33). Such assays require expensive and sophisticated equipment, but they allow working directly with the substrate of interest and are rapidly adapted once the instrument is available (23).Various chemical substances can be synthesized by bacteria and fungi, among which are the chiral sulfoxides (5, 10, 11, 24, 36). As natural products, chiral sulfoxides possess a wide range of biological activities, from flavor and aroma precursor activities to antimicrobial properties. In addition, they are efficient auxiliaries that lead to essential asymmetric transformations (3, 11). Furthermore, one of the most significant applications of chiral sulfoxides is in the pharmaceutical industry (3). The world''s best-selling antiulcer drug, (S)-omeprazole, is a chiral sulfoxide (11, 14). Although there have been numerous reports on chemical and biological methods for synthesizing chiral sulfoxides, little information exists about rapid high-throughput assays for sulfoxide determination. In this study, four colorimetric or fluorometric procedures were evaluated and adapted for screening of whole-cell libraries containing variants of TMOs. Three of the four methods were exploited successfully to a high-throughput format using 96-well microtiter plates, whereas one method was not suitable due to low sensitivity. The method based on acid activation of omeprazole proved very efficient, but no positive variants were found, whereas the one based on selective inhibition of horse liver alcohol dehydrogenase (HLADH), originally reported by Sprout and Seto (28), was useful for detecting mutants with high activity and enantioselectivity in the oxidation of methyl p-tolyl sulfide.     

柠檬马鞭草快速繁殖技术研究

选择柠檬马鞭草带腋芽的幼嫩茎段为外植体进行快速繁殖。结果表明,腋芽诱导的最适培养基是MS基本培养基附加6-BA0.5～1.0mg／L和IBA0．1～0．3mg／L．外植体腋芽能正常萌发生长,并迅速进入增殖状态,20d转接1次,增殖倍数存3倍以上,最适生根培养基是1／2MS基本培养基附加IBA0．5mg／L,诱导生根率达99％。将生根苗移入苗盆。30d的成活率在85％以上。     

Two New Rapid SNP-Typing Methods for Classifying Mycobacterium tuberculosis Complex into the Main Phylogenetic Lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.     

Rapid Diagnosis of Bacteremia

Early appropriate treatment of bacteremia is important in minimizing morbidity and mortality. Standard blood culture methods are not optimal since several days are often required for recovery and identification of organisms which may be present in the blood. The use of a membrane filter technique allows one to grow any organisms present in blood much more rapidly than by broth or pour plate culture. Furthermore, growth is in the form of typical colonies on the surface of solid media, and a series of rapid diagnostic tests may be used to provide speedy identification. Use of membrane filters also facilitates removal by washing of normal antibacterial factors and antimicrobial drugs which may be present in blood. Although the filter technique yielded the most rapid growth, broth culture and whole blood pour plates yielded more positive cultures and use of all three systems was necessary for maximal recovery of organisms in blood cultures. Data on quantitative aspects of bacteremia in the antimicrobial era are also presented. The number of low level bacteremias (10 colonies/ml or less) is surprisingly high. This is particularly true for gram-negative bacilli; antimicrobial therapy at the time of culture undoubtedly influenced these results greatly. Finally, suggestions are given for a much simpler and more efficient membrance filter blood culture technique.     

Rapid Diagnosis of Bacteremia

Early appropriate treatment of bacteremia is important in minimizing morbidity and mortality. Standard blood culture methods are not optimal since several days are often required for recovery and identification of organisms which may be present in the blood. The use of a membrane filter technique allows one to grow any organisms present in blood much more rapidly than by broth or pour plate culture. Furthermore, growth is in the form of typical colonies on the surface of solid media, and a series of rapid diagnostic tests may be used to provide speedy identification. Use of membrane filters also facilitates removal by washing of normal antibacterial factors and antimicrobial drugs which may be present in blood. Although the filter technique yielded the most rapid growth, broth culture and whole blood pour plates yielded more positive cultures and use of all three systems was necessary for maximal recovery of organisms in blood cultures. Data on quantitative aspects of bacteremia in the antimicrobial era are also presented. The number of low level bacteremias (10 colonies/ml or less) is surprisingly high. This is particularly true for gram-negative bacilli; antimicrobial therapy at the time of culture undoubtedly influenced these results greatly. Finally, suggestions are given for a much simpler and more efficient membrance filter blood culture technique.     

Rapid Methods for Extracting Autolysins from Bacillus subtilis

Two procedures are described for the extraction of autolysins from whole cells. One method uses 5 M LiCl at 4 C. The amount of enzyme obtained by this method is six times more than that obtained by autolysis of cell walls and fourteen times more than that obtained by extracting cell walls with LiCl. With the other method, cells are extracted with 2% Triton X-100. This is less efficient than the LiCl method but yields about one-half the amount of enzyme obtained by cell wall autolysis and about the same amount as obtained by extracting cell walls with salt. Both procedures yield autolysin with multiple pH optima. Autolysins can be extracted from several bacterial species by either the LiCl or the detergent method. The data suggest that these techniques have sufficient sensitivity to detect small differences in autolytic activity among mutants and various organisms and are also suitable for large-scale isolation of autolysin for purification and characterization studies.     

岩生报春的组织培养与快速繁殖

1植物名称岩生报春（Primula saxatilis Kom.）。2材料类别种子。3培养条件（1）种子萌发培养基：1/2MS;（2）无菌苗增殖培养基：MS;（3）丛生芽诱导和增殖培养基：MS＋6-BA 2.5 mg·L-（-1）（单位下同）＋NAA 1.0;（4）生根培养基：MS＋NAA 0.1。上述各培养基均添加7 g·L-（-1）琼脂和30 g·L-（-1）蔗糖,pH 5.8～6.0。培养温度为20～23℃,光照强度为20～25μmol·m-（-2）·s-（-1）,光照时间为14 h·d-（-1）。     

Rapid Methods for Biochemical Testing of Anaerobic Bacteria

Rapid biochemical tests for nitrate, indole, gelatin, starch, esculin, and o-nitrophenyl-beta-D-galactopyranoside were performed on 112 strains of anaerobic bacteria. All tests were incubated under aerobic conditions, and results were recorded within 4 h. The tests for nitrate, indole, and starch showed a 95% or greater correlation when compared to the standard biochemical tests. Tests for esculin and gelatin showed an agreement of 86 and 77%, respectively. PathoTec test strips for nitrate, indole, esculin, o-nitrophenyl-beta-D-galactopyranoside, Voges-Proskauer, and urease were also tested and showed encouraging results.