革兰氏染色三步法与质量控制

革兰氏染色(Gram stain),是细菌学中一个经常使用和十分重要的方法,自从1884年微生物学家Gram氏发明著名的革兰氏染色法以后,100多年来虽然经过后来学者的几次改进,但都仍然沿用着Gram氏原来的四步法,基本原理也没有改变。最近Allen氏对Ziehl-Neelsen抗酸菌染色法的改进,是一个良好的启示,使我们开始了革兰氏染色三步法的研究并取得了成功。现将我们建立的革兰氏染色三步法与质量控制报告如下。 1 材料和方法 1.1 结晶紫染色液 甲液:结晶紫2g;95％乙醇20ml。 乙液:草酸铵0.8g;蒸馏水80ml。 甲乙二液先分别溶解,然后混合在一起,过滤除去残渣后装入滴瓶中备用。     

An Automated Double Staining Procedure for Bone and Cartilage

Differential skeletal staining is an important part of developmental toxicologic studies. Traditionally these studies have required time-consuming differentiation of one or both stains used and careful attention to the maceration step to prevent specimen destruction. We present a fully automated protocol which does not require differentiation of either dye and incorporates a controlled maceration step which is highly reproducible. This has resulted in high quality staining that is reproducible, stable, and can be done in volume with minimal personnel time. The process involves the staining of skinned, eviscerated specimens fixed in 95% ethanol. Using an automated tissue processor, the specimen is stained in alcian blue for 24 hr, macerated in 3% potassium hydroxide for 24 hr and stained with murexide for 24 hr. The specimens are cleared and preserved in glycerol. Within three days specimens have red stained bone and blue stained cartilage. The procedure was developed using 20-day-old Sprague-Dawley rat fetuses to evaluate the feasibility of using the procedure for teratology studies involving the fetal skeleton. Evenly stained specimens can be examined within three days and stored for years without loss of staining.     

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5‐chloromethylfluorescein diacetate

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5‐chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

Electron microscopy and image analysis of the mitochondrial outer membrane channel,VDAC

The channel protein in the outer membrane ofNeurospora crassa mitochondria, VDAC, forms extended planar crystals on the membrane. The arrays, which are induced by phospholipase A₂, are polymorphic, varying from parallelogram (P) to near-rectangular (R) geometry with increased phospholipase treatment. Computer-based analysis of projection images of negatively stained VDAC arrays indicates that the protein forms a transmembrane channel in the P array. Comparison of average images of arrays embedded in different negative stains suggests that the bore of the channel is 2–2.5 nm. The locations of functionally important lysine clusters on VDAC are inferred from the effects of succinylation on projection images of arrays negatively stained with phosphotungstate. Projection images of unstained frozen-hydrated arrays indicate the general shape of the channel and suggest each channel is formed by one 31-kDa VDAC polypeptide.     

Electrophoresis in the presence of Coomassie brilliant blue R-250 stains polyacrylamide gels during protein fractionation

A method of staining polyacrylamide gels in which the dye is electrophoresed together with the sample is proposed. The method cuts short and simplifies the conventional electrophoresis procedure by eliminating the separate poststaining step. In the gels run in the presence of sodium dodecyl sulfate, the method produces protein staining patterns which are quantitatively identical to the ones obtained by conventional staining procedure. Additional advantages of the method are easy control over the degree of staining and homogenous staining independent of the gel thickness and concentration of the dye.     

Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains.

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P less than 0.005) for epididymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan-Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membranes and documented use of the Bryan-Akruk acrosomal stain for evaluation of sperm cell populations for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.     

The Sodium Channel Has Four Domains Surrounding a Central Pore

The voltage-gated sodium channel generates the action potential. This 300-kDa protein has four homologous regions, which are also homologous to the voltage-sensitive tetrameric potassium channel. We isolated sodium channels fromElectrophorus electricuselectroplax by detergent solubilization and immunoaffinity chromatography and studied their structure by electron microscopy of negatively stained specimens. Different projections were aligned, classified, and averaged. In side view, the channel protein exhibits the shape of a truncated cone, 14 nm in height. One end has a diameter of 12 nm and is asymmetric, while the other is more symmetric and has a diameter of 7–10 nm. In top views, the sodium channel appears to consist of four domains of different size and to have a stain-filled pore in the center.     

Coomassie brilliant blue staining of lipids on thin-layer plates

Coomassie brilliant blue staining of lipids on silica gel thin-layer chromatography plates is described. This stain proved to be useful for the wide-range detection of simple and complex lipids on thin-layer plates. It can stain several classes of lipids, including cholesterol, cholesterol esters, glycerides, phospholipids, ceramides, and neutral and acidic glycosphingolipids. It stains the spots evenly without a corrosive reagent, and is simple to use and suitable for storage. The visual detection limits of this stain for lipids were 0.05 to 0.5 microgram.     

Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate

A silver stain technique using treatment with potassium permanganate for visualisation of proteins and DNA separated by gel electrophoresis was developed and applied both to the very thin (thickness 0.1-0.2 mm) and to the usual 1-2 mm thick gels. The technique is reproducible, only stable chemicals are used and it is shortened to about 1 h even for the 1 mm gel (30 min for the 0.2 mm gel). It is applicable to gels thicker than 2 mm with somewhat longer washes. Amounts as low as 2 X 10(-10) g of protein per band have been detected. Protein bands of different colours are obtained. The technique has been used with success in continuous, discontinuous and 2D-gel systems. Bands have been detected which were not observed when the other silver staining techniques were used.