Use of silica gel polymer for DNA extraction with organic solvents

Phenol and chloroform are the standard solvents used for DNA extraction. These solvents aid in the removal of protein and lipid from crude or partially purified cell extracts. Although the procedure is well established, the solvents are noxious, caustic, and unpleasant. We describe in this paper the use of a special blood collection tube to isolate the offensive organic solvents. With the use of silica gel polymer containing tubes, phenol, phenol:chloroform, or chloroform can be separated from the DNA containing aqueous phase in a rapid and safe manner. The method permits higher yields of DNA since the DNA is poured from the tube rather than aspirated with pipet.     

Affixing Polyester Wax Sections to Glass Slides by Celloidin and Cellulose Acetate Applied in Sequence

Sections cut from material embedded in polyester wax can be firmly attached as follows: One drop of a 2% solution of celloidin in amyl acetate is smeared on clean slides, and sections taken from the floatation water onto these slides are dried at room temperature. After drying the slides are immersed in a 2% solution of cellulose acetate in acetone for 1 min, transferred directly to absolute ethanol, through 50% ethanol, and into water. Sections affixed by this method and stained by either hematoxylin-eosin or toluidine blue schedules do not loosen and have negligible background staining.     

Gradient Plate Bioassay for Tyrothricin and its Application to Dentifrices

A bioassay for tyrothricin, based on a procedure used by Szybalski in bacterial resistance studies, was developed. Replacement of the tube dilution assay with this procedure represented economy in time and equipment, with a resultant increase in productivity. The procedure involved preparation of special agar plates which provided graded concentrations of tyrothricin along a horizontal axis. A culture dish was inclined, and a base layer of agar, without antibiotic, was poured to cover the bottom of the dish and allowed to harden. A second layer of agar, containing 1 ppm of tyrothricin, was poured and allowed to harden with the dish in a level position. Diffusion of the antibiotic from the top layer into the bottom layer yielded a concentration gradient. A third thin layer of agar seeded with Streptococcus faecalis was poured on the surface. After incubation, a bacterial growth front, representing the minimal effective concentration (MEC), developed. The MEC is expressed as distance (in millimeters) from the edge of the plate. Unknowns were directly related to a standard preparation for calculation of tyrothricin concentration.     

Rapid Removal of Tissue Culture Monolayers by Collodion for Subsequent Staining

Removing cultures from roller tubes before staining eliminates the destaining which often occurs when the cells are first stained and then removed by embedding in collodion. The cells are fixed in situ, dehydrated, and covered with collodion (Merk's flexible) for 10 min. The collodion is poured off, the fluid residue lining the tube allowed to dry for 10 min, and the tube is filled with tap water. The collodion cast containing the cells is loosened and removed, cut into strips, placed on slides and blotted into firm contact. The collodion is then dehydrated and dissolved with absolute alcohol followed by a 1:1 mixture of alcohol and ether. The slides can then be rehydrated and stained by conventional methods.     

体外~(14)C-尿素呼气试验检测胃内幽门螺杆菌感染研究

目的　建立诊断胃内幽门螺杆菌感染 (Hp)的体外 1 4 C-尿素呼气试验 (1 4 C- U BT)。方法　 47例 Hp阳性和 32例 Hp阴性患者接受测试 ,用口服微量胃液采集胶囊的办法收集胃液标本于一 10 m l无菌试管内 ,加入生理盐水 0 .5 m l和 18.5 k Bq1 4 C-尿素后立即加橡皮塞密封试管 ,室温放置反应 3h,注射器经橡皮塞注入 2 M H2 SO41.0 ml,使 1 4 CO2 释出。同一注射器回抽气体并立即注入装有 6 .5 ml的 1 4 CO2 搜集闪烁剂液闪瓶内搜集 1 4 CO2 ,最后在液体闪烁计数仪上作 1 4 C放射性测定。结果　 47例 Hp阳性病人 1 4 C放射性几何均数为 5 30 dpm,而 32例 Hp阴性者结果为 2 1dpm,二者相差 2 3倍 (Wilcoxon秩和检验 ,u=5 .5 976 ,P<0 .0 1)。以受试者工作特征曲线分析法得出判别阈值为 75 dpm ,对 Hp诊断的敏感性和特异性为 92 %(4 3/ 47)和 91% (2 9/ 32 )。结论　体外 1 4 C- UBT诊断 Hp感染具有高度的准确性 ,无放射性损伤之虞 ,可适用于临床诊断。     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

MODIFIED ETHANOL INJECTION METHOD FOR LIPOSOMES CONTAINING β-SITOSTEROL β-D-GLUCOSIDE

A modified ethanol injection method for liposomes containing soybean phosphatidylcholine (SPC), cholesterol (Ch), β-sitosterol β-D-glucoside (Sit-G) and oleic acid (OA) was developed, that can produce homogeneous unilamellar liposomes without the use of sonication and dialysis. In this method, water is poured into a concentrated lipid-ethanol solution and then ethanol is removed in an evaporator. Dilution with water causes spontaneous formation of small and homogenous unilamellar vesicles from micellar aggregate. The size of liposomes can be controlled by the ratio of ethanol to water. OA and Sit-G were distributed at the surface of liposomes and were recognized by Concanavalin A, respectively. This easy and quick method for preparation of liposomes may be applicable in many areas.     

Distinctive diagnostic features of material aspirated from marrow in metabolic bone diseases

In a series of cases of Paget's disease of the bone, two types of cells not previously described were observed in material aspirated from bone marrow in areas of osteitis deformans. One type was mononuclear, the other was giant, multinucleated and syncytial. They have been identified as osteoblasts and osteoclasts, respectively. The identification was based mainly on correlation with the histologic picture of osteitis deformans and of normal-growing bones as seen in section studies. Both osteoblasts and osteoclasts were recovered in aspirated bone marrow material in other instances of metabolic bone diseases associated with increased bone repair and bone resorption-in hyperparathyroidism, osteoblastic malignant lesions, rickets, hemolytic anemia in children, and in normal infants in the growth zone of bone in the tibia. They were not seen in senile and postmenopausal osteoporosis. Recognition of osteoblasts and osteoclasts in smear preparations from aspirated bone marrow material may serve as a diagnostic aid in metabolic bone diseases. The differentiation of osteoblasts from neoplastic cells is important in cases in which metastatic cancer of the bone is suspected and x-ray findings are inconclusive.     

Direct and Repeatable Bone Marrow Chromosome Preparations from Living Mice

Using a 27 gauge hypodermic needle, bone marrow is aspirated from a lumbar vertebra into 0.1 ml of Hanks' salt solution. The aspirate is kept well mixed in 1% sodium citrate for 15 min, centrifuged, and the cell pellet fixed for 30 min in Clarke's 3:1 ethanol-acetic fixative. After removal of the fixative the cells are suspended in 0.05-0.1 ml of 60% acetic acid, centrifuged and resuspended in 0.03 ml of this fixative. Chromosome preparations are made by spreading the suspension on a slide heated to 60 C.     

Scrapie I. Transmission and Pathogenesis

Homogenized brain, extracts, and residues of brain from normal and scrapie sheep were inoculated into 116 sheep. Of 72 sheep inoculated with scrapie material 27 developed the disease, whereas four of 44 inoculated with ;normal' brain material showed symptoms similar to those of scrapie. The scrapie agent survived extraction for 18 hours with diethyl ether followed by water for 24 hours. Similarly the agent survived extraction in a Soxhlet apparatus with 95 per cent ethanol containing 2.0 per cent 4 M HC1 for 24 hours. There was no evidence of demyelination in sections of brain and spinal cord supporting the view that allergic encephalomyelitis is not responsible for the scrapie symptoms. Liver function, as measured by the bromsulfalein test, remained normal until just before death. These results preclude that liver dysfunction contributes to the disease but do not exclude the possibilities of other metabolic derangements.     

DISTINCTIVE DIAGNOSTIC FEATURES OF MATERIAL ASPIRATED FROM MARROW IN METABOLIC BONE DISEASES

In a series of cases of Paget''s disease of the bone, two types of cells not previously described were observed in material aspirated from bone marrow in areas of osteitis deformans. One type was mononuclear, the other was giant, multinucleated and syncytial. They have been identified as osteoblasts and osteoclasts, respectively. The identification was based mainly on correlation with the histologic picture of osteitis deformans and of normal-growing bones as seen in section studies.Both osteoblasts and osteoclasts were recovered in aspirated bone marrow material in other instances of metabolic bone diseases associated with increased bone repair and bone resorption—in hyperparathyroidism, osteoblastic malignant lesions, rickets, hemolytic anemia in children, and in normal infants in the growth zone of bone in the tibia. They were not seen in senile and postmenopausal osteoporosis.Recognition of osteoblasts and osteoclasts in smear preparations from aspirated bone marrow material may serve as a diagnostic aid in metabolic bone diseases.The differentiation of osteoblasts from neoplastic cells is important in cases in which metastatic cancer of the bone is suspected and x-ray findings are inconclusive.     

Ethanol Causes Protein Precipitation—New Safety Issues for Catheter Locking Techniques

Objective

The ethanol lock technique has shown great potential to eradicate organisms in biofilms and to treat or prevent central venous catheter related infections. Following instillation of ethanol lock solution, however, the inherent density gradient between blood and ethanol causes gravity induced seepage of ethanol out of the catheter and blood influx into the catheter. Plasma proteins so are exposed to highly concentrated ethanol, which is a classic agent for protein precipitation. We aimed to investigate the precipitating effect of ethanol locks on plasma proteins as a possible cause for reported catheter occlusions.

Methods

Plasma samples were exposed in-vitro to ethanol (concentrations ranging from 7 to 70 v/v%) and heparin lock solutions. In catheter studies designed to mimic different in-vivo situations, the catheter tip was placed in a plasma reservoir and the material contained within the catheter was analyzed after ethanol lock instillation. The samples underwent standardized investigation for protein precipitation.

Results

Protein precipitation was observed in plasma samples containing ethanol solutions above a concentration of 28%, as well as in material retrieved from vertically positioned femoral catheters and jugular (subclavian) catheters simulating recumbent or head down tilt body positions. Precipitates could not be re-dissolved by dilution with plasma, urokinase or alteplase. Plasma samples containing heparin lock solutions showed no signs of precipitation.

Conclusions

Our in-vitro results demonstrate that ethanol locks may be associated with plasma protein precipitation in central venous catheters. This phenomenon could be related to occlusion of vascular access devices locked with ethanol, as has been reported. Concerns should be raised regarding possible complications upon injection or spontaneous gravity induced leakage of such irreversibly precipitated protein particles into the systemic circulation. We suggest limiting the maximum advisable concentration of ethanol to 28 v/v% in catheter lock solutions.     

The Glyoxal BIS(2-Hydroxyanil) Method Modified for Localizing Insoluble Calcium Salts

TO enable staining of insoluble calcium salts with glyoxal bis(2-hydroxyanil) (GBHA), the original solution containing 2 ml of 0.4% GBHA in absolute ethanol, and 0.3 ml of aqueous 5% NaOH, and limited to staining only soluble calcium salts, was modified as follows: 1, 2 ml of 0.4% GBHA in absolute ethanol in 0.6 ml of 10% aqueous NaOH; 11, 0.1 gm GBHA in 2 ml of 3.4% NaOH in 75% ethanol. To prevent diffusion and loss of calcium, the tissues were processed by the freeze-substitution or freeze-dry method and sections stained without removing the paraffin. Modification I is effective only when 1 or 2 drops placed on the section are evaporated gradually to dryness, concentrating the GBHA and NaOH on the insoluble calcium salts. Modification II is effective when dried or poured on the the section and allowed to stain for 5 min. The stained slides are immersed for 15 min in 90% ethanol saturated with KCN and Na₂CO₃ for specificity to calcium; rinsed and counterstained in 95% ethanol containing 0.1% each of fast green FCF and methylene blue; rinsed and dehydrated in ethanol; deparaffinized and cleared in xylene; and mounted in neutral synthetic resin. Although the modified methods tested on models failed to stain reagent grade CaCO₃ and Ca₃(PO₄)₂ crystals completely, apatite in developing vertebrae and calcified plaques in soft tissues were stained intensely red. The distribution of gross deposits of insoluble calcium salt in tissue sections corresponded with that shown in adjacent sections by the alizarin red S, ferrocyanide, and von Kossa methods. The modified GBHA method revealed smaller quantities of insoluble as well as soluble calcium salts discretely within cells where the other methods failed; also, calcium in cytoplasm of hypertrophied cartilage cells of developing vertebrae, and in cytoplasm of renal tubular cells of magnesium-deficient rats, not described previously, was demonstrated.     

A Department Devoted to Abstracts of Books and Papers from Other Journals Dealing With Stains and Microscopic Technic in General

TO enable staining of insoluble calcium salts with glyoxal bis(2-hydroxyanil) (GBHA), the original solution containing 2 ml of 0.4% GBHA in absolute ethanol, and 0.3 ml of aqueous 5% NaOH, and limited to staining only soluble calcium salts, was modified as follows: 1, 2 ml of 0.4% GBHA in absolute ethanol in 0.6 ml of 10% aqueous NaOH; 11, 0.1 gm GBHA in 2 ml of 3.4% NaOH in 75% ethanol. To prevent diffusion and loss of calcium, the tissues were processed by the freeze-substitution or freeze-dry method and sections stained without removing the paraffin. Modification I is effective only when 1 or 2 drops placed on the section are evaporated gradually to dryness, concentrating the GBHA and NaOH on the insoluble calcium salts. Modification II is effective when dried or poured on the the section and allowed to stain for 5 min. The stained slides are immersed for 15 min in 90% ethanol saturated with KCN and Na₂CO₃ for specificity to calcium; rinsed and counterstained in 95% ethanol containing 0.1% each of fast green FCF and methylene blue; rinsed and dehydrated in ethanol; deparaffinized and cleared in xylene; and mounted in neutral synthetic resin. Although the modified methods tested on models failed to stain reagent grade CaCO₃ and Ca₃(PO₄)₂ crystals completely, apatite in developing vertebrae and calcified plaques in soft tissues were stained intensely red. The distribution of gross deposits of insoluble calcium salt in tissue sections corresponded with that shown in adjacent sections by the alizarin red S, ferrocyanide, and von Kossa methods. The modified GBHA method revealed smaller quantities of insoluble as well as soluble calcium salts discretely within cells where the other methods failed; also, calcium in cytoplasm of hypertrophied cartilage cells of developing vertebrae, and in cytoplasm of renal tubular cells of magnesium-deficient rats, not described previously, was demonstrated.     

The latex serologic test for syphilis-a new screening procedure

A new macroscopic screening test for syphilis, the Latex-sts test, is extraordinarily simple. After inactivation of the patient's serum for 30 minutes at 56 degrees C the test is performed by mixing the patient's serum with latex particles coated with cardiolipin and a protein fraction obtained from the non-pathogenic Reiter strain of Treponema pallidum. Two to three minutes after mixing, the result of the test is observed on a ringed serologic plate. The sensitivity, specificity and reproducibility of the new test are equivalent to those of the qualitative Venereal Disease Research Laboratory tube test. The advantages of the Latex-sts are that it can be done in a short time, it is simple and it requires a minimum of laboratory equipment. The coated latex particles are stable for 12 months.     

Microtubules, Microfilaments, and Membranes in Phagocytosis: Structure and Function of the Oral Apparatus of the Ciliate Climacostomum virens

Climacostomum virens uses oral membranelles to drive suspended food particles into its buccal cavity. The cavity leads to a buccal tube which extends into the cell by as much as half a cell length. The inner end of this tube is delimited by a haplokinety (two rows of basal bodies). Internal to this zone is the cytostome and cytopharynx where food vacuoles form. The buccal tube is encircled by a ring of fibrous material, the cytostomal cord, in the region of the cytostome immediately below the haplokinety. Ribbons of postciliary microtubules extend from the kinetosomes of the haplokinety, attach to the cytopharyngeal membrane, and pass under the cytostomal cord. They become broader and expand into the cytoplasm. Cytopharyngeal vesicles pass between the microtubular ribbons and fuse with the cytopharyngeal membrane to generate membrane for forming food vacuoles. The cytopharyngeal vesicles contain a material which is secreted into the forming food vacuoles. Ciliates continue to feed after incubation in a medium containing cycloheximide, indicating that they draw on a pre-existing pool of membrane when forming the food vacuole.     

Polyoxyethylene Sorbitan Monopalmitate (Tween 40) as a Vehicle for oil Red O Fat Stain

An oil red O fat stain is prepared by dissolving 250 mg of the dye in 100 ml of a 1% Tween 40 solution in 30% alcohol, and incubating the mixture at 60°C for 24 hr. The solution is then filtered at room temperature under vacuum through medium porosity frittedglass. Frozen sections cut from material fixed in CaCl₂-CdCl₂-formalin (1%:1%:10%) are placed in the stain for not less than 4 hr. After washing in the alcoholic-Tween solvent, they are mounted on glass slides from distilled water with Farrants' medium. The resulting preparations appear to be permanent, for in a 2-yr test they have remained free from stain crystalization and the fat particles are still discrete and dark red.     

Frozen Section Micro-Incineration

A method for micro-incineration of frozen sections is described. Material containing diffusible or soluble salts is cut on the freezing microtome and the sections are placed into xylol and mounted out of xylol onto Corex D slides previously filmed with glycerin-gelatin medium. Material containing non-diffusible or insoluble salts can be fixed in 10% formalin before sectioning. Sections of the fixed material are dehydrated thru 50, 70, and 95% ethyl alcohol and mounted out of absolute alcohol onto Corex D slides previously fumed with glycerin-gelatin medium. After mounting by either procedure the sections are incinerated in an electric furnace and the temperature of incineration is dependent on the type of tissues to be incinerated and the character of the salts present. The method is time saving and when no fixation is required the whole procedure can be carried out in one hour.     

Molluscum contagiosum. A case report with fine needle aspiration cytologic diagnosis and ultrastructural features

BACKGROUND: Cytomorphologic and ultrastructural features of molluscum contagiosum, a rare skin lesion of viral etiology, are presented. CASE: A 4-month-old female was referred for fine needle aspiration cytology of papules over the back and chest wall. A Giemsa-stained preparation of whitish material aspirated from the chest wall nodule showed numerous large, intracytoplasmic, basophilic bodies that pushed the host cell nucleus to the periphery, giving a signet-ring appearance to a few cells. A cytologic diagnosis of molluscum contagiosum was suggested. On electron microscopy numerous intracytoplasmic viral particles were demonstrated, thus confirming the cytologic diagnosis. CONCLUSION: In clinically unsuspected cases, the cytologic diagnosis of molluscum contagiosum can be suggested by demonstrating pathognomonic molluscum bodies in aspirated material.     

A bright field/fluorescent stain for aluminum: its specificity,validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.