Micro-Manipulation of Cells in Mammals

An arrangement of apparatus for applying micro-manipulative procedures to cells in living mammals is described. It was found satisfactory for manipulation of pericapillary cells and capillary endothelium in the greater omentum of the cat.

An animal board, a Bausch and Lomb triple purpose micro-projector and a double Fitz micro-manipulator were mounted on a spring platform. The micro-needles were drawn from Pyrex rods by hand. The stage of the microscope was modified to protect the condenser from fluids. Warm saline solution was carried to the exposed omentum thru flexible rubber tubing.

The use of a micro-dissection chamber which immobilized the part of the omentum under manipulation is explained. The construction of this chamber is shown by diagrams. A camera support was bolted to the base of the micro-manipulator.

The arrangement of apparatus is shown by a photograph.     

Marchi's Staining Method: II. Fixation

Following experimental lesions, spinal cords of cats and rabbits were fixed with acid, neutral, and alkaline solutions. Staining was limited to a chromate-osmic (Marchi's) solution and a chlorate-osmic solution. The following conclusions were drawn:

The presence of an acid in the fixative caused normal myelin sheaths to stain. This effect was reduced by washing tissues before staining, by adding acetic acid to the stain, or by employing a non-formalin fixative. It was, however, at no time entirely obviated.

A study was made of the granular deposits which occur in nearly all Marchi preparations and which are especially confusing if very light backgrounds are obtained.

The staining reactions of the granular deposits were very similar to those of degenerating myelin but some suppression of the granules was obtained by adding KCIO₃ to the formalin fixative.     

Book Reviews

Nicotinic Acetylcholine Receptor: Structure and Function (NATO Advanced Science Institutes Series H: Cell Biology, Vol. 3) Edited by A. MAELICKE Springer-Verlag, Berlin

The Chemical Basis of Radiation Biology C. Von Sonntag Taylor & Francis

Advances in Free Radicals in Disease Corongiu, F.P., Tomasi, A. and Vannini

Modern Biological Theories of Aging Aging Series Vol. 31. WARNER, H.R., BUTLER, R.N., SPROTT, R.L. and SCHNEIDER     

An Improvement in Staining Technic for Protozoa

A modification of Donaldson's iodine-eosin stain for staining intestinal protozoa is presented. This modification consists of using high dilutions of colloidal iodine (Chandler)² instead of Lugol's solution as well as high dilutions of eosin. A better resolution of the external and internal structures is brought about by the new method.

The procedure is as follows: A portion of the fecal material to be examined is suspended in a 0.6% salt solution; the suspension should be of a consistency so that one drop will make a satisfactory microscope mount under a cover glass. To ten parts of this suspension, in a test tube, is added one part of the stain which is prepared as follows:—

10 parts of distilled water

6 parts of a suspension of colloidal iodine (Chandler) containing 4% iodine—20% iodine suspensoid, Merck

1 part of a 10% water solution of anilin red, Merck (eosin yellowish)

Technicians will find, because iodine in the form of colloidal iodine is readily released to the organisms, that the use of this material is far superior to Lugol's solution hi carrying out the technic for staining intestinal protozoa in the study of fresh mount preparations. Not only are organisms more deeply stained with iodine but by eosin as well, even when employed in high dilutions.     

Book Review

LEGGETT, W. F. Ancient and Medieval Dyes. 5 × 8 in. 96 pp. Cloth. Chemical Publishing Co., Brooklyn, N. Y. 1944. $2.25.

Microtechnic In General. McCARTNEY, J. E. A new immersion oil “polyric”. J. Path. & Bact., 56, 265-6. 1944.

NICKERSON, MARK. A dry ice freezing unit for rotary microtomes. Science, 100, 177-8. 1944.

Dyes And Thedx Biological Uses. BERGEIM, FRANK H., and BRAKER, WILLIAM. Homosulfanilamides. J. Amer. Chem. Soc., 66, 1459. 1944.

CALDWELL, W. T., TYSON, F. T., and LAUER, LOTHAR. Substituted 2-sulfonamido-5-aminopyridines. II. J. Amer. Chem. Soc., 66, 1479. 1944.

JOHNS, C. K. Dye concentration in resazurin tablets. Amer. J. Pub. Health, 34, 955-8. 1944.

SMITH, WINSLOW WHITNEY. Relative sensitivity of different phases of growth curve of Bact. salmonicida to alkaline acriflavine. Proc. Soc. Exp. Biol. & Med., 56, 240-2. 1844.

VAN ARENDONK, A. M., and SHOULE, H. A. Dialkylaminoalkyl derivatives of substituted quinolines and quinaldines. J. Amer. Chem. Soc., 66, 1284. 1944.

WHEELER, KEITH, and DEGERING, E. F. Preparation and properties of certain derivatives of sulfamide. J. Amer. Chem. Soc., 66, 1242. 1944.

Animal Microtechnic. BOARDMAN, EDWARD T. Methods for collecting ticks for study and delineation. J. Parasitology, 30, 57-9. 1944.

DICKIE, MARGARET M. A new differential stain for mouse pituitary. Science, 100, 297-8. 1944.

GOVAN, A. D. TELFORD. Fat staining by Sudan dyes suspended in watery media. J. Path. & Bact., 56, 262-4. 1944.

LILLIE, R. D., and ASHBURN, L. L. Supersaturated solutions of fat stains in dilute isopropanol for demonstration of acute fatty degenerations not shown by Herxheimer technic. Arch. Path., 36, 432. 1943.

MULLEN, J. P. A convenient and rapid method for staining glycogen in paraffin sections with Best's carmine stain. Amer. J. Clin. Path., Tech. Sect., 8, 9-10. 1944.

NYKA, W. A method for staining the rickettsiae of typhns in histological sections. J. Path. & Bact., 56, 264. 1944.

POPPER, HANS, GYORGY, PAUL, and GOLDBLATT, H. Fluorescent material (ceroid) in experimental nutritional cirrhosis. Arch. Path., 37, 161. 1944.

SMALL, C. S., and SCHULTZ, M. A. Sustaining faded tissue sections. Amer. J. Clin. Path., Tech. Sect., 7, 66-7. 1943.

YOFFEY, J. M., and PARNELL, J. The lymphocyte content of rabbit bone marrow. J. Anat., 78, 109-12. 1944.

ZIEGLER, E. E. Hematoxylin-eosin tissue stain. An improved, rapid, and uniform technic. Arch. Path., 37, 68. 1044.

Plant Microtechnic. HAASIS, FERDINAND W. Staining rubber in ground or milled plant tissues. Ind. and Eng. Chem., Anal. Ed., 16, 480. 1944.

PARRIS, G. K. A simple nuclear stain and staining technique for Helminthosporia. Phytopathology, 34, 700. 1944.

Microorganisms. DARZINS, E. Rickettsienstudien. Zentbl. Bakl., Abt. I, Orig., 151, 18-20. 1943.

GOHAR, M. A. A staining method for Corynebacterium diphtheriae. J. Bact., 47, 575. 1944.

GRAY, P. H. H. Two-stain method for direct bacteria count. J. Milk Techn., 6, 76. 1943.     

Normal Butyl Alcohol Technic for Animal Tissues with Special Reference to Insects

N-butyl alcohol is substituted in dehydration for the higher ethyl alcohols. No special clearing is necessary as n-butyl is miscible with paraffin.

The greatest advantage of this method is the elimination of both hardening agents (the higher percentage ethyl alcohols and xylol or benzol). Another advantage is the great time toleration of the processes of dehydration and infiltration. For example, tissues have been kept without deleterious effects in n-butyl alcohol for a year before infiltration. Also, aphids which have been kept in a hot (58°C.) paraffin bath for as long as four weeks, have sectioned well. For small insects and vertebrate tissues about five days proved necessary to insure satisfactory infiltration.

N-butyl alcohol was found to give better results than many other technics in serial sectioning of lightly chitinized insects, and in the preparation of embryological and other vertebrate tissues. This technic has been used as a routine method by beginning students in animal microtechnic with better success than attended the usual methods.     

Book Reviews

The Biology of Nitric Oxide, vol. 3. Physiological and Clinical Aspects; vol. 4. Enzymology, Biochemistry and Immunology, Portland Press, 1994

Mitochondrial Diseases Editors: L. Ernster, R. Luft and S. Orrenius Publishers: Elsevier

Biothiols in Health and Disease Editors: Lester Packer & Enrique Cadenas Publishers: Marcel Dekker, Inc.     

ESCODD: European Standards Committee on Oxidative DNA Damage

“There can be no more important goal in a clinical laboratory than that of ensuring that the results produced have the precision and accuracy necessary to aid diagnosis and treatment.”

T.P. Whitehead 1976     

An Inexpensive Microscope Lamp for Critical Illumination

A microscope lamp with light of adjustable intensity, which will afford critical illumination of a quality satisfactory for the most exacting cytological work, can be constructed for about $7.50. The essential parts needed are: auto headlight bulb, radio power transformer, radio “potentiometer”, and simple plano-convex lens. A ground glass is not used with 4 and 2 mm. objectives, a solid source of light being obtained by placing the bulb so that one filament coil is directly behind the other. This light source is brought to a focus by the condensing lens at a field diaphragm and this image is projected by the microscope condenser into the plane of focus of the microscope.     

Acid Versus Neutral Formalin Solution as a Neurological Fixative

The fixing action of 10% formalin solution alone and with formic, acetic, propionic, butyric, lactic, monochloracetic, dichloracetic, or trichloracetic acid was studied by means of stains with silver, osmic acid and cresyl violet. The following conclusions were reached:

1. In general, better fixation and staining was obtained with acid than without.

2. Less difference was seen in comparing one acid with another than was expected before the experiments were made.

3. Propionic, butyric, and dichloracetic showed no promise of having practical value.

4. Formic and monochloracetic acids as modifiers gave superior stains with osmic acid, while silver and cresyl violet stains of the same material were about equal to those made from formalin-acetic fixed material.

5. Lactic acid caused somewhat more distortion of tissue elements than the others but was compatible with good staining.

6. Acetic acid was most effective in concentrations of 3 to 5% while the stronger acids such as formic, monochloracetic, lactic and trichloracetic were effective in concentrations of 0.5 to 1%.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Book Review

Salmon, M. V. Practical phase-contrast microscopy. The Microscope, 6, 177-88. 1947.

Evans, Titus C. Radioautographs in which the tissue is mounted directly on the photographic plate. Proc. Soc. Exp. Biol, and Med, 64, 813-15. 1947.

Tolbert, B. M., and Branch, G. E. K. The spectra of the doubly charged positive ions of some p,p'-diaminotriphenylmethane dyes. J. Amer. Chem. Soc, 69, 1083-81. 1947.

Van Wyk, J. J., and Clark, W. M. The luminosity and chromaticity of indicators as a function of pH. J. Amer. Chern. Soc, 69, 1296-1301. 1947.

Hamre, Christopher J. Hematopoiesis in the bone marrow of rats recovering from nutritional anemia. J. Lab. & Clin. Med, 32, 756-76. 1947.

Limarzi, Louis R. Evaluation of bone marrow concentration techniques. A modified method for the simultaneous preparation and staining of blood and bone marrow films. J. lab. & Clin. Med, 32, 782-40. 1947.

Heath, O. V. S. Role of starch in light-induced stomatal movement, and a new reagent for staining stomatal starch. Nature, 159, 647-8. 1947.

Cohen, H. A. A new quick method for staining Treponema pallidum. Acta Med. Orientalia, 6, 99-100. 1947.

Henry, H., and Stacey, M. Histochemistry of the Gram-staining reaction for micro-organisms. Proc. Roy. Soc, Ser. B. Biol. Sci, 133, 891-406. 1946.

Para, M. Silver impregnation of spirochetes in tissue sections. Arch. Path, 42, 649. 1946.

Ruiz, Merino, J. A method of staining capsules. Rev. Sanidad e Hig. Publ (Madrid), 20, 1112. 1946.     

Book Reviews

Oxygen, Gene expression and Cellular Function, Vol 105 Lung Biology in Health and Disease L. B. Clerch and D. J. Massaro Marcel Dekker, 1997

Food and Free Radicals Edited by Midori Hiramatsu, Toshikazu Yoshikawa and Masayosu Inoue Plenum Press, 1997, ISBN 0-306-45493-9 vii + 169 pages

Preventing Coronary Heart Disease: The Role of Antioxidants, Vegetables and Fruit Ed Lesley Rogers and Imogen Sharp National Heart Forum 1997 The Stationery Office London

Inducible Gene Expression Volume 1 Environmental Stresses and Nutrients Volume 2 Hormonal Signals Ed by PA Baeuerle, Birkhauser Verlag AG, Basel, 1997

Antioxidants in Science, Technology, Medicine and Nutrition Gerald Scott Albion Chemical Science Series, Ellis Horwoood Publishing Ltd, Chichester, UK, 1997

Flavonoids in Health and Diseases Eds C A Rce-Evans and L Packer Marcel Dekker Inc, New York, 1997     

Marchi's Staining Method: Studies of Some of the Underlying Mechanisms Involved

Spinal cord of cat and rabbit was stained, after experimental lesions, by variations of Marchi's method. The following conclusions were drawn:

1. The presence of an oxidizing agent (K₂Cr₂O₇, NaIO₃, or KCIO₃) in the osmic acid solution is of primary importance and a preliminary oxidation in Mueller's fluid is unnecessary or even detrimental.

2. Acetic acid added to Marchi's fluid, accentuates the action of the oxidizing agent in restraining the staining of normal myelin.

3. Too high concentration of oxidizing agent or of acid may inhibit staining of degenerating myelin.

4. Marchi's and Busch's methods have been modified as follows: Fix one day in 10% formalin and transfer without washing to the staining mixture, either A or B. Staining mixture A: Marchi's fluid plus 1 to 3% glacial acetic acid. B: An aqueous solution containing KCIO₃ 0.25%, osmic acid 0.33%, and acetic acid 1%. Stain about one week. These methods worked on spinal cord and medulla, but cannot be recommended for brain.

5. The detrimental effects of long post mortem autolysis or of prolonged fixation in formalin may be counteracted to some degree by increasing the concentration of the acid in Marchi's fluid up to 5% or of the KCIO₃ up to 0.4% in the modified Busch's fluid.     

Biological Stain Commission Membership, May 1949

Officers

President, H. J. Conn, Box 269, Geneva, N. Y. (representing Society of American Bacteriologists.)

Vice-President, W. F. Windle, University of Pennsylvania, Philadelphia, Pa. Secretary, S. I. Kornhauser, University of Louisville Medical School, Louisville

Ky. Treasurer, E. H. Stotz, University of Rochester, School of Medicine, Rochester

N. Y. (representing American Chemical Society.)     

A New Hematological Stain: I. Constituents and Methods of Use

The stain proposed by the author is quickly and simply prepared by mixing equal parts of the following permanent stock solutions:

The mixed stain is usable, for at least eight months, and is applicable to practically all hematological purposes: blood smears, fixed sections, frozen sections, and touch preparations. In the technics utilized to produce its action on preparations treated in different ways, the only variants are the methods for treating the cells, while the stain itself remains totally unchanged for all purposes. For blood smears fixed with methyl alcohol, the technic consists merely of pouring the stain on the slide, leaving it for 5 to 7 minutes, and then washing it off. On sections, a further process of differentiation with acid acetone is rapidly carried out. The various types of granules, including megakaryocyte and platelet granules, are clearly demonstrated. For frozen sections, the technic is extremely rapid, yet yields excellent differentiation.     

Haemoglobin and Myoglobin as Inhibitors of Hydroxyl Radical Generation in a Model System of “Iron Redox” Cycle

Methionine was oxidized to ethylene by an “Iron Redox” system containing H₂O₂, Fe-EDTA and ascorbate. generating hydroxyl radicals or another species of similar reactivity. Oxy or met forms of haemoglobin and myoglobin were found to inhibit methionine oxidation. Methionine oxidation was elevated in the “Iron Redox” system by increasing ascorbic acid concentration. However, in the presence of metmyoglobin or methaemoglobin, the increases in ascorbic acid did not lower the haemproteins' inhibitory effects but rather increased them.

The pro-oxidative or anti-oxidative activities of haemproteins in biological oxidative reactions seem to be dependent on compartmentalization and on the presence and concentrations of reducing compounds and H₂O₂.     

Commentary: The Role of Iron-Sulfur Clusters in in vivo Hydroxyl Radical Production

The in vivo production of HO- requires iron ions, H₂O₂ and O₂- or other oxidants but probably does not occur through the Haber-Weiss reaction. Instead oxidants, such as O₂-, increase free iron by releasing Fe(II) from the iron-sulfur clusters of dehydratases and by interfering with the iron-sulfur clusters reassembly. Fe(II) then reduces H₂O₂, and in turn Fe(III) and the oxidized cluster are re-reduced by cellular reductants such as NADPH and glutathione. In this way, SOD cooperates with cellular reductants in keeping the iron-sulfur clusters intact and the rate of HO- production to a minimum.

O₂- and other oxidants can release iron from Fe(II)-containing enzymes as well as copper from thionein. The released Fe(III) and Cu(II) are then reduced to Fe(II) and Cu(I) and can then participate in the Fenton reaction.

In mammalian cells oxidants are able to convert cytosolic aconitase into active IRE-BP, which increases the “free” iron concentration intracellularly both by decreasing the biosynthesis of ferritin and increasing biosynthesis of transferrin receptors.

The biological role of the soxRS regulon of Escherichia coli, which is involved in the adaptation toward oxidative stress, is presumably to counteract the oxidative inactivation of the iron clusters and the subsequent release of iron with consequent increased rate of production of HO.