Staining Flagellate Protozoa by Various Silver-Protein Compounds

Seven samples of silver protein (Protargol type) were tested on flagellate protozoa from the alimentary tract of frogs, golden hamsters, and termites. The samples consisted of one of Protargol (Winthrop's prewar German), one of Protargol S (Winthrop-Stearns, Inc., present manufacture, Commission Certified), and five of the experimental batches (4Z, 20, 20B, 22, and 36) by H. A. Davenport and collaborators (1952). The pre-war Protargol is rated best and batch 22 second best for staining of all flagellates. Protargol S gave uniform, but only fair results, with all organisms, while batch 20B, better than Protargol S in several instances, was poorer in a few. Thus Protargol S and batch 20B are rated third, as about equal. Batch 4Z is rated fourth; and batch 20, which stained some species, fifth. Batch 36 stained no protozoa. The tests show that while the present Protargol S is usable for protozoa, it is still inferior to the old German variety. Since some of the experimental batches, none of which was made by exactly the same process, gave promising results, further study of the relation between manufacturing process and subsequent staining reaction should be fruitful.     

Preparation and Test In G Of Silver-Protein Compounds

Silver derivatives of the protargol type were made from 13 different commercial peptones. The peptone samples were purified by precipitating with an ethanol concentration of 75%, reprecipitating once, and discarding any water-insoluble material. A 20-22% solution of the purified material in water, allcalinized with 2 ml. of strong ammonia per 100 ml., was precipitated by adding an equal volume of 25% aqueous AgNO₃, and stood in a refrigerator overnight. Thorough washing of the precipitate with distilled water, draining, and then dissolving it in a purified peptone solution (30-35% aqueous), whose volume was 0.6 that of the solution used for silver precipitation, gave the soluble silver derivative. Ammonia was added to facilitate solution and the final pH adjusted to 8.0-8.4. The concentrated solution was dehydrated either with acetone or in a dessicator under reduced pressure and ground to a powder. Staining tests on neurological tissue showed that the Pharmaceutical peptone (Cudahy Packing Co., Omaha, Nebr.) and the Bacteriologic peptone (Wilson Laboratories, Chicago, 111.) gave the best preparations for staining nerve fibers, and that these were essentially equal to the formerly available German made Protargol.     

Cytological staining of protozoa: a case study on the impregnation of hypotrichs (Ciliophora: spirotrichea) using laboratory-synthesized protargol

Protargol (silver proteinate) impregnation is a common method used to identify and characterize ciliated protozoa. Unfortunately, chemical companies have stopped producing the ‘strong’ protargol powder used in this method. Based on an in-house protocol for its synthesis published in 2013, more than 10 batches of protargol powder were produced and subsequently applied in taxonomic studies. During these studies, the protocol for protargol powder synthesis was slightly modified and employed a peptone not originally listed in the 2013 protocol. This modification improved the results of the impregnation protocol. Protargol preparations of hypotrichs were optimized by adjusting the pH during staining rather than during the synthesis. The pH was adjusted to 7.5–7.6, and an acetone developer was used. While the conditions used in this study are not completely comparable to those using the commercially produced protargol, access to this information could help researchers investigate the diversity of ciliates, particularly hypotrichs.     

The Use of Protein Silver for Staining Protozoa

When commercially prepared silver products suitable for staining protozoa by the Bodian silver technic apparently became unavailable, a substitute for Protargol was prepared as follows: 0.9 g. of gelatin is dissolved by heat in 100 ml. of distilled water; to this 0.1 g. of silver nitrate is added at 60°C; this solution is poured into Columbia staining dishes (10 ml.) in which one or two drops of M/10 sodium hydroxide have been added. Copper is not used in the impregnating bath. Smears fixed in Hollande's or Schaudinn's fixatives are bleached and impregnated for 36 hours or more at 35°C. Impregnated smears are reduced with a mixture of hydroquinone and sodium sulfite, and toned with gold chloride as recommended by Kirby (1945).     

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. I. Staining Properties of Commercial Samples and Their Component Dyes

SYNOPSIS. Studies on the composition of commercial Giemsa stain and its effect upon staining quality are reported. These studies were supplemented by observations on the preparation of the components of Giemsa stain and their staining properties in aqueous solution, in Nocht's solution, and in laboratory prepared Giemsa stains containing one azure component. Five groups of commercial batches were differentiated on the basis of their staining reactions on thick and thin films of bovine blood containing Babesia bigemina and B. argentina. Spectrophotometric and chromatographic analysis showed that four groups differed in the proportions of the thiazine components present, while the fifth-group did not appear to be Giemsa stain. Comparison of their staining effects with those obtained with each component in laboratory prepared stains indicated that the major effects of commercial batches on both blood cells and parasites were due to the thiazine component or components in highest proportions, with satisfactory staining of protozoa associated with those batches containing high proportions of methylene blue and azure B and low proportions of the remaining thiazine components.
The function of each component of Giemsa stain is defined and the need for the proper balancing of thiazine eosinates with free azure is shown. Close correlation was obtained between analysis by spectrophotometry and chromatography and direct staining tests when samples initially with low MX values were re-examined spectrophotometrically after removal of their methylene violet content. The existence of a leuco form of eosin is reported and its possible significance to the Romanowsky effect is discussed.     

An improved synthesis of (20R,22R)-cholest-5-ene-3beta,20,22-triol, an intermediate in steroid hormone formation and an activator of nuclear orphan receptor LXR alpha.

Asymmetric dihydroxylation of (20(22)E)-cholesta-5,20(22)-dien-3beta-ol acetate (2a), prepared from pregnenolone, gave a 1:1 mixture (67% yield) of (20R,22R)-cholest-5-ene-3beta,20,22-triol 3-acetate (3a) and its 20S,22S isomer 3b. Highly purified 3a and 3b were obtained by semipreparative silver ion high performance liquid chromatography. Saponification of 3a and 3b gave (20R,22R)-cholest-5-ene-3beta,20,22-triol (4a) and its 20S,22S isomer 4b. This simple approach provided the natural isomer 4a more efficiently than previously described chemical or enzymatic syntheses. Full 1H and 13C nuclear magnetic resonance data were presented for triols 4a and 4b and their synthetic precursors. Side-chain conformations of 2a, its 20(22)Z isomer, 4a, and 4b were studied by molecular mechanics and nuclear Overhauser effect difference spectroscopy.     

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikata's modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikata's modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.     

Standardization of the Feulgen-Schiff technique

Summary Four fuchsin analogues (Pararosaniline, Rosaniline, Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches.Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 m.Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

Phosphotungstic Acid-Eosin Combined with Hematoxylin as A Stain for Negri Bodies in Paraffin Sections

Experimentation with the Papanicolaou stain in this laboratory led to the discovery that the eosin, combined with phosphotungstic acid, was responsible for differential staining of Negri bodies. Eosin prepared as in EA 36 was used, but without the light green and Bismarck brown. Paraffin sections of hippocampus from brains of animal affected with rabies were fixed in 10% formol or in a mixture of 2 volumes of saturated aqueous HgCl₂ and 1 volume of absolute alcohol. They were stained first with hematoxylin and then with eosin. This procedure gave better results than staining with other types of eosin or by the original EA 36 mixture. The Negri bodies were well stained and their structure easily visible. The best results were obtained from material fixed with the HgCl₂ solution.     

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikata's modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikata's modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.     

Production of Bacteriophage T7

Bacteriophage T7 was grown with Escherichia coli B as the host organism in 3- and 20-liter vessels. Under the best growth conditions devised, the yields of T7 in the culture lysates averaged 1.33 x 10(12) and 0.95 x 10(12) plaque-forming units per ml, respectively, compared with the best previously reported yields of 10(11) to 3 x 10(11) plaque-forming units per ml in 1-liter batches grown in the presence of air, or double this in similar batches grown in the presence of oxygen. The bacteriophage was purified by a simple method which gave average yields of 143 mg/liter and 131 mg/liter from the 3- and 20-liter batches, respectively. The efficiency of plating of the final material ranged from 18 to 42%. The purified bacteriophage is a convenient source of monodisperse deoxyribonucleic acid, the molecular weight of which is about 25 x 10(6).     

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage

Beef burgers were stored at 4°C in a vacuum in nisin-activated antimicrobial packaging. Microbial ecology analyses were performed on samples collected between days 0 and 21 of storage to discover the population diversity. Two batches were analyzed using RNA-based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The active packaging retarded the growth of the total viable bacteria and lactic acid bacteria. Culture-independent analysis by pyrosequencing of RNA extracted directly from meat showed that Photobacterium phosphoreum, Lactococcus piscium, Lactobacillus sakei, and Leuconostoc carnosum were the major operational taxonomic units (OTUs) shared between control and treated samples. Beta diversity analysis of the 16S rRNA sequence data and RNA-DGGE showed a clear separation between two batches based on the microbiota. Control samples from batch B showed a significant high abundance of some taxa sensitive to nisin, such as Kocuria rhizophila, Staphylococcus xylosus, Leuconostoc carnosum, and Carnobacterium divergens, compared to control samples from batch A. However, only from batch B was it possible to find a significant difference between controls and treated samples during storage due to the active packaging. Predicted metagenomes confirmed differences between the two batches and indicated that the use of nisin-based antimicrobial packaging can determine a reduction in the abundance of specific metabolic pathways related to spoilage. The present study aimed to assess the viable bacterial communities in beef burgers stored in nisin-based antimicrobial packaging, and it highlights the efficacy of this strategy to prolong beef burger shelf life.     

Alcian Blue Pyridine Variant–a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

Alcian Blue Pyridine Variant-a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

Staining Neural End Feet and Mitochondria After Postchroming and Carbowax Embedding

Spinal cord of cat and rabbit was fixed by perfusion with 10% formalin in physiological salt solution followed by a 2-day immersion in 10% aqueous formalin. Further treatment (postchroming) consisted of a 5-day immersion in: K₂Cr₂O₇, 5 gm; CrFl₃-4H₂O, 2 gm; distilled water, 100 ml; followed by 5% aqueous K₂Cr₂O₇ at 38–40°C for 2–4 wks. After thorough washing, blocks were embedded by infiltration first with polyethylene glycol 1000 M. E. and then with Nonex 63B (Gemec Chemicals Co., London, E. C. 2), and casting in the Nonex. Sections were stained, either mounted or unmounted, by modifications of the Bielschowsky-Gros method, and mounted sections by Weigert-Pal's hematoxylin or by Silver's Protargol method. All 3 methods gave apparently complete staining of pericellular end feet and showed also an abundance of mitochondria. Cytologic preservation was much better than that seen after the usual procedures for this type of staining. Retention of lipoid material in the sections is considered to be the cause of efficient staining of end feet and mitochondria.     

Erfahrungen mit der Feulgenfärbung für quantitative cytochemische DNS-Untersuchungen

Zusammenfassung Es wird über quantitative photometrische DNS-Messungen an 1500 menschlichen Lymphozyten und Granulozyten mit dem Zeiss Cytophotometer UMSP I nach Feulgenfärbung berichtet. Die Färbungen wurden mit frischem und altem Schiffschem Reagenz durchgeführt. Außerdem wurde das Schiffsche Reagenz mit verschiedenen Pararosanilinpräparaten angesetzt.Wir erhielten eine identische Anfärbung der DNS, wenn das Schiffsche Reagenz noch nicht alt war und wenn das Schiffsche Reagenz mit demselben Pararosanilinpräparat angesetzt wurde.Die Präparate blichen durch Lagerung aus. Die geringste Bleichung erhielten wir bei Verwendung von Schiffschem Reagenz mit einem Pararosanilin acridinfrei von Chroma.

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Summary The DNA- content of 1500 human lymphocytes and granulocytes was measured with the Zeiss cytophotometer UMSP I after Feulgen staining. The Feulgen staining was carried out with freshly prepared as well as aged Schiffs reagent. In addition we used several batches of Schiffs reagent prepared with various Pararosanilin preparations (Merck, Bayer, Chroma).We got an identical staining of DNA in all samples stained with fresh Schiffs reagent or with Schiffs reagent, prepared from the same batch of Pararosanilin. A fading of the Feulgen stained nuclei was observed after storing the samples for several months. Of all samples the ones stained with Pararosanilin Chroma were most resistant to fading.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.

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Für ihre gewissenhafte Mitarbeit danke ich meiner technischen Assistentin Fräulein R. Schmelzer.     

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.     

Inter-comparison of 14C-labelled bicarbonate solutions prepared by different institutes for measurement of primary productivity in natural waters and monoalgal cultures

An intercomparative study was carried out to investigate possibleeffects on primary productivity measurements when using NaH¹⁴C0₃solutions prepared by different methods. Five different ampoulebatches coded A, B, C, D and E were tested. Three of the batches(A, B and D) had been produced by direct dilution of industriallyproduced NaH¹⁴CO₃, of high specific activity. A and D were dilutedwith distilled water added carrier, whereas no information onhow batch B was diluted could be obtained. Batch E was preparedby trapping ¹⁴CO₂, gas — released by strong HCl from Ba¹⁴CO₃— in sodium hydroxide. In the case of batch C, the processof manufacture was not known. The tests were carried out ondifferent phytoplankton material with low algal density. Twobatches (B and C) showed significant inhibitory effects on P_calc(–5–44%), on the slope of the ascending part ofthe light adaptation curve (), and on P_max. Batch A showed minor,but still significant effects. The four batches A, B, C andD carried rather high amounts of non-volatile rest activity(between 13 and 194 d.p.m./µCi), which made measurementsof the release of extracellular dissolved organic carbon (EOC)almost impossible. This phenomenon,per se, would if uncor-rectedproduce considerably higher per cent EOC release in low-productivewaters, as has been reported by many authors. As to the standardizationof the working solutions, two batches (C and D) showed a pooraccuracy (16 and 18% deviation, respectively) when tested atthe C14C, and two batches (A and B) showed unacceptably highvariability between ampoules of the same batch. The study indicatesthat it is not recom-mendable to use working solutions preparedby direct dilution of industrially produced NaH¹⁴CO₃ of highspecific activity without prior testing of possible effectson algal photosynthesis. It is recommended that the specificactivity of the working solution be measured if it is not exactlyspecified by the manufacturer by a ‘Certificate of Quality’.