A Gelatin Fixative for Paraffin Sections

Mayer's albumen fixative, of which the active principle is white of egg, is used almost universally for affixing paraffin ribbons to the slide. About eight years ago the writer's attention was called to a gelatin fixative which has proved to be so superior to albumen that he has used it almost exclusively ever since in the making of a great variety of botanical preparations, and has recommended it to a number of other workers whose experience with it subsequently has been just as satisfactory. The gelatin method was first described by Szombathy¹ and later discussed by Artschwager,² but it does not seem to have received the attention in the literature which its importance deserves. It certainly merits a wide spread use among both botanists and zoologists.     

Community structure and seasonal dynamics of planktonic ciliates along salinity gradients

The ciliate community structure and seasonal dynamics in a solar saltern of the Yellow Sea were studied based on 4 sampling dates and 8 stations with salinities from 27.7‰ to 311.0‰. The effects of the type and concentration of the fixative used (Lugol's and Bouin's) were tested at the first sampling date. Fixative type and fixative concentration had significant effects on ciliate abundance and biovolume, with 1% Lugol's giving the best results. A detailed investigation using live observations and protargol staining techniques revealed a total of 98 morphospecies from 8 sampling stations. There was obvious seasonal variation in species composition at most of the stations, but this tended to be less distinct with increasing salinity, as the dominant ciliate group shifted from oligotrichs to heterotrichs. Ciliate abundance varied from 4.40×10¹ to 2.11×10⁵ cells l^?1 and biomass ranged between 2.39 and 9.87×10³ μg C l^?1 (at a salinity of 147.6‰). Both abundance and biomass decreased abruptly when salinity exceeded 100–150‰. Statistical analyses suggested that the dynamics of ciliate abundance and biomass were regulated by both salinity and by season, but those of diversity and species richness were mainly controlled by salinity and both significantly decreased with increasing salinity.     

Optimizing specimen processing for ancient soft tissue specimens

Abstract

Despite many reports concerning processing of ancient soft tissues, scant attention has been paid to optimizing procedures for processing soft tissues that have been altered by taphonomic processes. To determine the best procedures, we investigated the rehydration solution, time of exposure to the solutions, fixative solution and exposure to heat. Processes were evaluated based on the minimum section thickness, degree of tissue fragmentation, definition of tissue architecture and penetration of stains. We found that in desiccated samples, tissue architecture was optimized by using Ruffer's solution for rehydration and Schaffer's solution as fixative, because these tissues require water restoration within the tissues due to their compacted character. Heating enhanced penetration of dyes in these specimens, which improved diagnosis. Saponified tissues that had suffered extensive decomposition were more labile and required slow water uptake. The best histological sections were obtained using Sandison's solution followed by fixation with formaldehyde and avoiding heat. To obtain the best results with paleohistological specimens, the procedure must be determined by the condition of the sample and by accounting for the nature of its damage.     

Preparation of tissues for localization of sex hormones by autoradiography

Summary Tissues of rats given ³H-oestradiol were prepared for autoradiography according to methods commonly used in light and electron microscopy.By formalin fixation large amounts of radioactive material were lost, both in the fixative and during dehydration. Altogether 78.6±7.5 per cent was extracted from uterine tissue, while 49.0±4.6 per cent was lost from liver tissue removed 15 minutes after the injection. Significantly more radioactivity was lost in the fixative from liver tissue than from uterine. In the former fixation accounted for about 60 per cent of the loss, whereas in the latter it was responsible for about 25 per cent.Osmium tetroxide fixation was found to retain the radioactivity of liver and uterine tissue almost completely. However, large amounts were invariably extracted during dehydration. Although only 3.9±1.2 per cent of the radioactivity of uterine tissue diffused into the fixative, 72.8±12.4 per cent was extracted during ethanol dehydration. A heavy loss was also registered when dehydration and infiltration were carried out in glycol methacrylate.Glutaraldehyde perfusion and postfixation with osmium tetroxide retained almost completely the radioactivity of uterine and pituitary tissue. Nevertheless, nearly all of it was extracted during ethanol/propylene oxide dehydration and Epon embedding.The methods studied are not adequate for accurate autoradiographic localization of oestradiol.This work was supported by grants from The Norwegian Cancer Society and by Nordisk Insulinfond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID : II. NEW DETERMINATIONS OF THE ISOELECTRIC POINT AND COMBINING CAPACITY OF A PURIFIED GELATIN.

1. Cooper''s gelatin purified according to Northrop and Kunitz exhibited a minimum of osmotic pressure and a maximum of opacity at pH 5.05 ±0.05. The pH of solutions of this gelatin in water was also close to this value. It is inferred that such gelatin is isoelectric at this pH and not at pH 4.70. 2. Hydrogen electrode measurements with KCl-agar junctions were made with concentrated solutions of this gelatin in HCl up to 0.1 M. The combination curve calculated from these data is quite exactly horizontal between pH 2 and 1, indicating that 1 gm. of this gelatin can combine with a maximum of 9.35 x 10^–4 equivalents of H⁺. 3. Conductivity titrations of this gelatin with HCl gave an endpoint at 9.41 (±0.05) x 10^–4 equivalents of HCl per gram gelatin. 4. E.M.F. measurements of the cell without liquid junction, Ag, AgCl, HCl + gelatin, H₂, lead to the conclusion that this gelatin in 0.1 M HCl combines with a maximum of 9.4 x 10^–4 equivalents of H⁺ and 1.7 x 10^–4 equivalents of Cl^- per gram gelatin.     

Simple Aids to Microscope Illumination

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

Pronase treatment increases the staining intensity of GABA-immunoreactive structures in the paravertebral sympathetic ganglia

Summary A novel tissue preparation technique for improving gamma-aminobutyric acid (GABA) immunocytochemistry has been developed. The influence of the glutaraldehyde concentration in the fixative and the effect of pronase treatment on the GABA immunostaining were tested. This method includes fixation with a high concentration of glutaraldehyde, gelatin embedding and treatment of the sections with pronase. In sympathetic (paravertebral) ganglia and their connectives, the most intense and specific immunoreaction was obtained with the following procedure: immersion fixation in 5% glutaraldehyde, infiltration and embedding in 15% gelatin, secondary fixation of the samples with 4% formaldehyde, floating frozen sections and digestion with 0.1% pronase for 15–20 min. With this technique, the GABA-containing structures (cells and nerve fibers with varicosities forming basket-like networks around some principal neurons) were selectively labeled. The data presented suggest that (1) a high concentration (5%) of glutaraldehyde in the primary fixative is necessary to preserve a large proportion of the GABA content; (2) this glutaraldehyde fixation partly masks the GABA immunoreactivity; and (3) this masking may be overcome by a proteolytic treatment preceding the immunostaining. This method has been extensively tested for the light microscopic visualization of GABA-containing tissue components in the sympathetic ganglion chain, but it may probably also be used for the immunocytochemical detection of other small molecules in other parts of the nervous system.     

MEASUREMENTS OF SOME CELLULAR CHANGES DURING THE FIXATION OF AMPHIBIAN ERYTHROCYTES WITH OSMIUM TETROXIDE SOLUTIONS

On average, 15 per cent of the total haemoglobin present in the blood of the newt Triturus cristatus was extracted during 45 minutes of fixation in Palade-Caulfield fixative. This extraction was reduced with fixatives buffered at pH 6.2 instead of pH 7.4. The addition of Ca⁺⁺ ions to a final concentration of 0.01 M in the fixative completely suppressed haemoglobin extraction. The effect of the pH, and the presence or absence of Ca⁺⁺ ions in the fixative, on the rate of haemoglobin extraction has been determined. During Palade-Caulfield fixation the average projected area of newt erythrocytes increased by 37 per cent, and after dehydration and embedding in Epon the average area was 25 per cent greater than that of the unfixed cell. Fixatives buffered at pH 6.2 and containing 0.01 M Ca⁺⁺ ions caused cellular shrinkage, with the average projected area decreasing by 10 per cent in the fixative. This shrinkage continued during dehydration, and the final average area of the erythrocytes in Epon was 26 per cent less than that of the unfixed cells. Similar measurements with erythrocytes of Amphiuma tridactylum showed that after Palade-Caulfield fixation the average cellular area was increased by 45 per cent, and after dehydration and embedding in Araldite it was 36 per cent greater than that of the unfixed cell. The average nuclear area increased by 35 per cent during fixation but after embedding it was 26 per cent greater than that of the unfixed nuclei. With a fixative at pH 6.2 containing 0.01 M Ca⁺⁺ ions, both the nucleus and the whole cell shrank during fixation. The nuclear area decreased by 20 per cent and the cellular area by 22 per cent. After dehydration and embedding in Araldite, the average nuclear area had decreased by 35 per cent and the cellular area by 40 per cent. It has been shown that OsO₄ fixation lowers the isoelectric points of haemoglobins and other proteins. This finding has been used in the interpretation of the observed cellular changes resulting from fixation.     

混合甲醛固定液固定大肠癌淋巴结标本的最佳免疫组化效果研究

目的:探讨混合甲醛固定液固定大肠癌淋巴结标本的最佳免疫组化效果。方法:采用不同pH值(6.0、7.0、8.0)的混合甲醛固定液对39枚大肠癌淋巴结标本进行不同时间(6 h、6 h-12 h、1 d-7 d)的固定处理。以细胞角蛋白20(CK20)为目标抗原,运用OIympusdp 70图像采集分析仪抽选出混合甲醛固定液最佳免疫组化染色的pH值及固定时间。结果:经pH值为7.0混合甲醛固定液处理后,阳性率为92.31%,高于经pH值为6.0、8.0的混合甲醛固定液处理后的76.92%、74.36%,且经pH值为7.0、8.0处理后的阳性率比较有统计差异(P0.05)。混合甲醛固定液的固定时间在6 h-12 h时的阳性率为94.87%,高于固定时间为6 h、1 d-7 d处理的30.77%、76.92%(P0.05)。结论:对于大肠癌淋巴结标本,以CK20为目标抗原,选择pH值为7.0的混合甲醛固定液固定6 h-12 h能够得到质量较佳的免疫组化染色效果。     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : I. MEMBRANE POTENTIALS.

1. It is shown that a neutral salt depresses the potential difference which exists at the point of equilibrium between a gelatin chloride solution contained in a collodion bag and an outside aqueous solution (without gelatin). The depressing effect of a neutral salt on the P.D. is similar to the depression of the osmotic pressure of the gelatin chloride solution by the same salt. 2. It is shown that this depression of the P.D. by the salt can be calculated with a fair degree of accuracy on the basis of Nernst''s logarithmic formula on the assumption that the P.D. which exists at the point of equilibrium is due to the difference of the hydrogen ion concentration on the opposite sides of the membrane. 3. Since this difference of hydrogen ion concentration on both sides of the membrane is due to Donnan''s membrane equilibrium this latter equilibrium must be the cause of the P.D. 4. A definite P.D. exists also between a solid block of gelatin chloride and the surrounding aqueous solution at the point of equilibrium and this P.D. is depressed in a similar way as the swelling of the gelatin chloride by the addition of neutral salts. It is shown that the P.D. can be calculated from the difference in the hydrogen ion concentration inside and outside the block of gelatin at equilibrium. 5. The influence of the hydrogen ion concentration on the P.D. of a gelatin chloride solution is similar to that of the hydrogen ion concentration on the osmotic pressure, swelling, and viscosity of gelatin solutions, and the same is true for the influence of the valency of the anion with which the gelatin is in combination. It is shown that in all these cases the P.D. which exists at equilibrium can be calculated with a fair degree of accuracy from the difference of the pH inside and outside the gelatin solution on the basis of Nernst''s logarithmic formula by assuming that the difference in the concentration of hydrogen ions on both sides of the membrane determines the P.D. 6. The P.D. which exists at the boundary of a gelatin chloride solution and water at the point of equilibrium can also be calculated with a fair degree of accuracy by Nernst''s logarithmic formula from the value pCl outside minus pCl inside. This proves that the equation x² = y ( y + z) is the correct expression for the Donnan membrane equilibrium when solutions of protein-acid salts with monovalent anion are separated by a collodion membrane from water. In this equation x is the concentration of the H ion (and the monovalent anion) in the water, y the concentration of the H ion and the monovalent anion of the free acid in the gelatin solution, and z the concentration of the anion in combination with the protein. 7. The similarity between the variation of P.D. and the variation of the osmotic pressure, swelling, and viscosity of gelatin, and the fact that the Donnan equilibrium determines the variation in P.D. raise the question whether or not the variations of the osmotic pressure, swelling, and viscosity are also determined by the Donnan equilibrium.     

THE COMBINATION OF SALTS AND PROTEINS. I

1. It has been found that the ratios of the total concentrations of Ca, Mg, K, Zn, inside and outside of gelatin particles do not agree with the ratios calculated according to Donnan''s theory from the hydrogen ion activity ratios. 2. E.M.F. measurements of Zn and Cl electrode potentials in such a system show, however, that the ion activity ratios are correct, so that the discrepancy must be due to a decrease in the ion concentration by the formation of complex ions with the protein. 3. This has been confirmed in the case of Zn by Zn potential measurements in ZnCl₂ solutions containing gelatin. It has been found that in 10 per cent gelatin containing 0.01 M ZnCl₂ about 60 per cent of the Zn⁺⁺ is combined with the gelatin. 4. If the activity ratios are correctly expressed by Donnan''s equation, then the amount of any ion combined with a protein can be determined without E.M.F. measurements by determining its distribution in a proper system. If the activity ratio of the hydrogen ion and the activity of the other ion in the aqueous solution are known, then the activity and hence the concentration of the ion in the protein solution can be calculated. The difference between this and the total molar concentration of the ion in the protein represents the amount combined with the protein. 5. It has been shown that in the case of Zn the values obtained in this way agree quite closely with those determined by direct E.M.F. measurements. 6. The combination with Zn is rapidly and completely reversible and hence is probably not a surface effect. 7. Since the protein combines more with Zn than with Cl, the addition of ZnCl₂ to isoelectric gelatin should give rise to an unequal ion distribution and hence to an increase in swelling, osmotic pressure, and viscosity. This has been found to be the case.     

THE COLLOIDAL BEHAVIOR OF PROTEINS

1. It is well known that neutral salts depress the osmotic pressure, swelling, and viscosity of protein-acid salts. Measurements of the P.D. between gelatin chloride solutions contained in a collodion bag and an outside aqueous solution show that the salt depresses the P.D. in the same proportion as it depresses the osmotic pressure of the gelatin chloride solution. 2. Measurements of the hydrogen ion concentration inside the gelatin chloride solution and in the outside aqueous solution show that the difference in pH of the two solutions allows us to calculate the P.D. quantitatively on the basis of the Nernst formula See PDF for Equation if we assume that the P.D. is due to a difference in the hydrogen ion concentration on the two sides of the membrane. 3. This difference in pH inside minus pH outside solution seems to be the consequence of the Donnan membrane equilibrium, which only supposes that one of the ions in solution cannot diffuse through the membrane. It is immaterial for this equilibrium whether the non-diffusible ion is a crystalloid or a colloid. 4. When acid is added to isoelectric gelatin the osmotic pressure rises at first with increasing hydrogen ion concentration, reaches a maximum at pH 3.5, and then falls again with further fall of the pH. It is shown that the P.D. of the gelatin chloride solution shows the same variation with the pH (except that it reaches its maximum at pH of about 3.9) and that the P.D. can be calculated from the difference of pH inside minus pH outside on the basis of Nernst''s formula. 5. It was found in preceding papers that the osmotic pressure of gelatin sulfate solutions is only about one-half of that of gelatin chloride or gelatin phosphate solutions of the same pH and the same concentration of originally isoelectric gelatin; and that the osmotic pressure of gelatin oxalate solutions is almost but not quite the same as that of the gelatin chloride solutions of the same pH and concentration of originally isoelectric gelatin. It was found that the curves for the values for P.D. of these four gelatin salts are parallel to the curves of their osmotic pressure and that the values for pH inside minus pH outside multiplied by 58 give approximately the millivolts of these P.D. In this preliminary note only the influence of the concentration of the hydrogen ions on the P.D. has been taken into consideration. In the fuller paper, which is to follow, the possible influence of the concentration of the anions on this quantity will have to be discussed.     

Effects of Fixation and Tissue Processing on Immunohistochemical Demonstration of Specific Antigens

Identification of biomarkers in archival tissues using immunochemistry is becoming increasingly important for determining the diagnosis and prognosis of tumors, for characterizing preinvasive neoplastic changes in glandular tissues such as prostate, for evaluating the response of tumors and preinvasive neoplastic changes to certain therapies (i.e., as a surrogate intermediate end point), for selecting patients who are candidates for specific therapies (e.g., immunotherapy) and for retrospective studies. For detecting specific biomarkers it is important to understand the limitations imposed by the fixation methods and processing of the tissues. This study was designed to determine the effects of fixation on the detection in archival paraffin blocks of selected antigens postulated to be important in tumor biology. We evaluated the antigens TGFα, p185^erbB-2, broad spectrum keratins, p53, and TAG-72 (B72.3). Fixatives evaluated included standard preparations of neutral buffered formalin, acid formalin, zinc formalin, alcoholic formalin, ethanol, methanol, and Bouin's fixative. We found that in general neutral buffered formalin is the poorest fixative for maintaining antigen recognition by immunohistochemistry and that no single fixative was best for all antigens. The dehydrating (coagulant) fixatives (e.g., ethanol and methanol) preserved immunorecognition of p53 and broad spectrum keratins best while the slow cross-linking fixatives (e.g., unbuffered zinc formalin) were best for demonstrating TGFα and p185^erbB-2. Fixatives other than neutral buffered formalin produced equivalent recognition of the epitope of TAG-72 by B72.3. In formalin fixed archival tissues, only a portion of the antigen signal can be detected by routine immunohistologic methods.     

THE IONIC ACTIVITY OF GELATIN

2.5 and 1.25 per cent gelatin have been titrated potentiometrically in the absence of salts and in the presence of two concentrations (0.0750 and 0.0375µ) of NaCl, MgCl₂, K₂SO₄, and MgSO₄. The data have been used to calculate values of ± S = v^z – (v – 1)^z, where v^z = v ² – (v ² – v) r_x/18. The maximum and minimum values of S with NaCl were used to calculate the mean distance (r_x) between like charges in gelatin. This is found to be 18 Å.u. or over (between acid or basic groups) which agrees with the probable value and the titration index dispersion. Thus the data with NaCl are shown to be normal and to obey the equation found to hold for simple weak electrolytes; namely, pK'' – pK = Sa See PDF for Equation where S is related to the valence and distance by the above equations. Using the NaCl data as a standard the deviations (ΔS) produced by the other salts are calculated and are found to agree quantitatively with the deviations calculated from equations derived for the simple weak electrolytes. This shows that in gelatin, as in the simple electrolytes, the deviations are related to the "apparent valences" (values which are a function of the true valence and the distance between the groups). The maximum "apparent valences" of gelatin are 2.4 for acid groups (in alkaline solution) and 1.8 for basic groups (in acid solution). These values correspond to the hypothetical condition of zero distance between the groups. They have no physical significance but have a practical utility first as mentioned above, and second in that they may be used in the unmodified Debye-Hückel equation to give the maximum effect of gelatin on the ionic strength. The true effect is probably even lower than these values would indicate. The data indicate that gelatin is a weak polyvalent ampholyte having distant groups and that the molecule has an arborescent structure with interstices permeated by molecules of the solvent and other solutes. The size and shape probably vary with the pH.     

Effects of water on the mechanical properties of gelatin films

The mechanical properties of gelatin films were studied in relation to the effect of water, and compared with those of collagen films. The S-shaped sorption isotherm was separated into an adsorption curve C₁ and dissolution curve C₂. From the C₂ curve, the interaction parameter χ₁ of Flory–Huggins' equation was calculated. The χ₁ of gelatin were larger than those of collagen at low relative humidities (RH), while they coincided with each other at high RH. It was found that a composite curve was made by shifting stress relaxation curves obtained at different humidities along the log time axis. The shift factor for the formation of the composite curve was analyzed by Fujita–Kishimoto's equation, which was based on the free volume theory. The parameter β, which expressed the extent of the contribution of sorbed water to the increase in the free volume of the system, was 0.05 in the range of C₂ from 0 to 0.08 (0–65% RH). This value was much smaller than 0.16 for collagen. The value was 0.16 in the range of C₂ higher than 0.08, which was equal to that of the collagen. Dynamic shear modulus G′, loss modulus G″, and tan δ were determined as functions of RH. The gelatin film extended more than 100% at 73% RH under the very small stress of about 10⁷ dyn/cm². This corresponds to the region where β changes from 0.05 to 0.16, although such a phenomenon was not observed in the collagen film. The wide-angle X-ray pattern of extended gelatin was similar to that of renatured collagen fiber.     

AMPHOTERIC COLLOIDS : I. CHEMICAL INFLUENCE OF THE HYDROGEN ION CONCENTRATION.

1. It has been shown in this paper that while non-ionized gelatin may exist in gelatin solutions on both sides of the isoelectric point (which lies for gelatin at a hydrogen ion concentration of C_H = 2.10^–5 or pH = 4.7), gelatin, when it ionizes, can only exist as an anion on the less acid side of its isoelectric point (pH > 4.7), as a cation only on the more acid side of its isoelectric point (pH < 4.7). At the isoelectric point gelatin can dissociate practically neither as anion nor as cation. 2. When gelatin has been transformed into sodium gelatinate by treating it for some time with M/32 NaOH, and when it is subsequently treated with HCl, the gelatin shows on the more acid side of the isoelectric point effects of the acid treatment only; while the effects of the alkali treatment disappear completely, showing that the negative gelatin ions formed by the previous treatment with alkali can no longer exist in a solution with a pH < 4.7. When gelatin is first treated with acid and afterwards with alkali on the alkaline side of the isoelectric point only the effects of the alkali treatment are noticeable. 3. On the acid side of the isoelectric point amphoteric electrolytes can only combine with the anions of neutral salts, on the less acid side of their isoelectric point only with cations; and at the isoelectric point neither with the anion nor cation of a neutral salt. This harmonizes with the statement made in the first paragraph, and the experimental results on the effect of neutral salts on gelatin published in the writer''s previous papers. 4. The reason for this influence of the hydrogen ion concentration on the stability of the two forms of ionization possible for an amphoteric electrolyte is at present unknown. We might think of the possibility of changes in the configuration or constitution of the gelatin molecule whereby ionized gelatin can exist only as an anion on the alkaline side and as a cation on the acid side of its isoelectric point. 5. The literature of colloid chemistry contains numerous statements which if true would mean that the anions of neutral salts act on gelatin on the alkaline side of the isoelectric point, e.g. the alleged effect of the Hofmeister series of anions on the swelling and osmotic pressure of common gelatin in neutral solutions, and the statement that both ions of a neutral salt influence a protein simultaneously. The writer has shown in previous publications that these statements are contrary to fact and based on erroneous methods of work. Our present paper shows that these claims of colloid chemists are also theoretically impossible. 6. In addition to other physical properties the conductivity of gelatin previously treated with acids has been investigated and plotted, and it was found that this conductivity is a minimum in the region of the isoelectric point, thus confirming the conclusion that gelatin can apparently not exist in ionized condition at that point. The conductivity rises on either side of the isoelectric point, but not symmetrically for reasons given in the paper. It is shown that the curves for osmotic pressure, viscosity, swelling, and alcohol number run parallel to the curve of the conductivity of gelatin when the gelatin has been treated with acid, supporting the view that these physical properties are in this case mainly or exclusively a function of the degree of ionization of the gelatin or gelatin salt formed. It is pointed out, however, that certain constitutional factors, e.g. the valency of the ion in combination with the gelatin, may alter the physical properties of the gelatin (osmotic pressure, etc.) without apparently altering its conductivity. This point is still under investigation and will be further discussed in a following publication. 7. It is shown that the isoelectric point of an amphoteric electrolyte is not only a point where the physical properties of an ampholyte experience a sharp drop and become a minimum, but that it is also a turning point for the mode of chemical reactions of the ampholyte. It may turn out that this chemical influence of the isoelectric point upon life phenomena overshadows its physical influence. 8. These experiments suggest that the theory of amphoteric colloids is in its general features identical with the theory of inorganic hydroxides (e.g. aluminum hydroxide), whose behavior is adequately understood on the basis of the laws of general chemistry.     

REACTIONS OF VALONIA AND OF HALICYSTIS TO COLLOIDS

From the results of these tests it is clear that both Halicystis and Valonia have a high degree of tolerance for animal peptone, and a very high degree of tolerance for animal proteose and for egg albumen. The products of bacterial growths fostered by these proteins have a deleterious effect upon both species of algae; but, if it were possible to prevent bacterial growth entirely and at the same time supply proper food, it is probable that Halicystis and Valonia would show normal growth indefinitely in the presence of these three colloids. This is not true where exposure is made to yeast nucleic acid dissolved in sea water containing 0.00093 gm. per cc. of NaOH. Valonia is markedly less tolerant of this medium (perhaps of NaOH rather than the colloid used) than Halicystis. Such differential effects, however, reach a high point in the case of the solutions of diphtheria toxin and of edestin. Halicystis has a very high tolerance for diphtheria toxin, and Valonia a very low tolerance. In the case of edestin, the relationship is reversed. Here Halicystis has a very low tolerance, and Valonia a very high tolerance. In fact, it may be said that diphtheria toxin has no appreciable effect upon Halicystis, and edestin a very slight effect upon Valonia; while diphtheria toxin is extremely toxic to Valonia, and edestin is extremely toxic to Halicystis. We can offer no suggestions, at present, as to the way in which these effects are produced. It is probable that the very thin protoplasmic layer of these species, which is certainly no thicker than 8µ, is sufficient to obstruct the passage of proteins having large molecules, like egg albumen, with a degree of efficiency that is extraordinary. In the tests we have reported, areas of from 20 sq. cm. to 40 sq. cm. have been submitted to the action of a relatively high concentration of egg albumen for several days without permitting the passage of sufficient amounts to give definable tests either with Spiegler''s or with Tanret''s method,— presumably less than 1 part in 250,000. In the tests of the proteins having much smaller molecules (though the size may not be the explanation), there is some probability that the membranes exhibit a little permeability. The peptone and the proteose of animal origin, or biuret-positive substances derived from them, apparently pass the protoplasmic membranes occasionally in quantities sufficient to give biuret tests. The most probable case of protein passage, however, was that of the proteose of the scarlet runner bean, where specific detection of less than 1 part per 80,000 was possible. In this instance the proteose appeared to pass membranes that were healthy and were functioning normally. But since the cells of the algae had to be destroyed in making the tests, one cannot maintain this point. All one can say is that protein passage was indicated in carefully examined cells of both species, where no breaks in the protoplasmic membrane were discernible, and where samples of the treated cells behaved normally after treatment.     

Biochemical Composition of the Eggs of the Freshwater Snail Lymnaea stagnalis and Oviposition-induced Restoration of Albumen Gland Secretion

Summary

Galactogen and protein form the main constituents of the eggs of Lymnaea stagnalis. The amount of galactogen per egg is fairly constant, irrespective of the size of the egg mass or the age of the snail.

The restoration of the albumen gland, which produces the perivitelline fluid for the eggs, was studied in long-day (16 hr light-8 hr dark) snails after spontaneous oviposition. The wet wt of the gland and its galactogen and protein contents are markedly increased within 8 hr and reach a maximum at 32 hr after oviposition. These maxima correspond to the levels determined in snails that did not lay eggs for at least 1 to 2 days. The amounts of galactogen and of protein in the albumen gland are linearly related to the wet wt of this gland.

The restoration period of the albumen gland almost covers the mean egglaying interval. This implies synchronized cycles of albumen storage and egg formation.

The estimated amount of galactogen, released by the albumen gland during egg mass formation, is in accordance with that deposited in the eggs. In contrast, the wet wt of the eggs is 4.6 times higher than that of the released secretory material. Since after oviposition water uptake by the eggs in the egg mass is negligible, the perivitelline fluid, which is released by the albumen gland and surrounds the egg cell, must be diluted in the reproductive tract of the snail prior to oviposition.     

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer''s solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco''s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10⁵ cells/cm². The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.