Initial and Persisting Staining power of Solutions of Iron-Hematoxylin Lake

A study was made of factors affecting the initial staining power and the stability of iron-hematoxylin lake solutions. The findings were applied to the preparation of a superior hematoxylin staining solution. This is made up as follows: in 50 ml. water dissolve, in order, 1.0 g. ferric ammonium sulfate [FeNE₄ (SO₄)₂⋅ 12H₂O], 0.8 ml. sulfuric acid, 50 ml. 95% ethyl alcohol, 0.5 g. hematoxylin. Filter the solution to remove the insoluble, white crust of the ferric ammonium sulfate. The solution stains well ten minutes after it has been made. Peak performance is attained within 5 hours, and is maintained for 4 to 8 weeks. Staining time is 3 to 30 minutes. Excess stain can be rinsed off the slide and section by immersion in water, after which destaining, if necessary, can be accomplished with a solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.18 ml. sulfuric acid. The slides may or may not be placed next in a neutralizing solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.5 g. sodium bicarbonate. They may then be passed through 50 ml. water, 50 ml. 95% ethyl alcohol on the way to alcoholic counterstaining solutions, or through water leading to aqueous counterstains.

The nuclear stain produced is black, intense and very sharp and has proved to be consistently excellent on a variety of animal and human tissues following a number of different fixatives.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

Differential Staining Of Tissue In The Block With Picric Acid And The Feulgen Reaction

The authors have found a modification of the Feulgen reaction to be a satisfactory stain for tissue in the block.

Pieces of fresh mammalian tissue not thicker than 5 mm. are fixed for approximately 48 hours at 25° C. in a mixture of equal parts of 5% aqueous sulfosalicylic acid and saturated aqueous picric acid. They are washed for 30 minutes in three ten-minute changes of distilled water and placed in Feulgen's staining solution diluted to one-half strength with distilled water. The staining solution is allowed to act for 24 hours (2 to 3 mm. thick blocks) up to 48 hours for 5 mm. thickness. After staining, the specimens are transferred to a mixture of sodium bisulfite, 0.5 g. and N hydrochloric acid, 5 ml. in' 100 ml. of distilled water. Two changes of IS to 30 min. each in the acid sulfite are given and these are followed by dehydration through 50%, 70% and 95% alcohol. One to two hours are allowed for each change except the last 95%, in which the stained tissue is allowed to remain overnight. The dehydration is completed in two changes of absolute alcohol with subsequent clearing in xylene and embedding in paraffin. Sections may be cut 10 μ or other thickness desired, mounted on slides, paraffin removed, and covered in the usual manner. Nuclei stain reddish violet against a lemon yellow background when the stain is typical. Orange G, 200 mg. per 100 ml. may be added to the fixing fluid if a more polychromatic effect is desired.     

Rapid spectrophotometric assay of dodecyl sulfate using acridine orange

A rapid assay that is useful for quantitating as little as 1 μg sodium dodecyl sulfate in a 100-μl sample is described. Except for trichloroacetic acid, a variety of other substances tested caused little or no interference with the assay. The method is based on the extraction of a dodecyl sulfate-acridine orange complex from aqueous solution into toluene. Both extraction and spectrophotometric measurement were performed in the same tube, resulting in high precision in replicate determinations and added safety in handling toluene solutions.     

Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective R_F values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

BEHAVIOR OF WATER IN CERTAIN HETEROGENEOUS SYSTEMS

In various models designed to imitate living cells the surface of the protoplasm is represented by guaiacol which acts in some respects like certain protoplasmic surfaces. The behavior of water in these models presents interesting features and if these occur in vivo, as appears possible, they may help to explain some of the puzzling aspects of water relations in the living organism. When sufficient trichloroacetic acid is added to a two-phase system of water and guaiacol the two phases fuse into one. The effect of the acid is due to its attraction for water and for guaiacol. This is shown by the following facts. During the addition of the acid the mole fraction of water in the guaiacol phase increases but the activity of water in the guaiacol phase falls off. The activity coefficient of water may fall to less than one twelfth the value it had before acid was added. The behavior of guaiacol presents a similar picture. During the addition of acid the mole fraction of guaiacol in the aqueous phase increases but the activity of the guaiacol in the aqueous phase presumably decreases. Its activity coefficient calculated on this basis may fall to about one ninth of the value it had before the acid was added. Somewhat similar results are obtained when acetone is substituted for trichloroacetic acid or when ethanol is substituted for trichloroacetic acid and ethylene chloride for guaiacol. As trichloroacetic acid increases the mutual solubility of guaiacol and water we find that guaiacol saturated with water and having a high vapor pressure of water can take up water from an aqueous solution of trichloroacetic acid with a low vapor pressure of water: acid passes from the aqueous to the guaiacol phase, thus raising the vapor pressure of water in the aqueous phase and lowering it in the guaiacol phase. Diffusion experiments present some interesting features. When an aqueous solution, A, of trichloroacetic acid is separated by a layer of guaiacol, B, from distilled water, C, under certain conditions water moves from A to C. This depends on the fact that acid moves in the same direction and appears to carry water with it. Similar but less striking results were obtained with acetone diffusing through guaiacol and with ethanol diffusing through ethylene chloride. These phenomena differ from "anomalous osmosis" through solid membranes if it depends, as many suppose, on the diffusion of electrolytes through pores. We therefore suggest the term "anaphoresis" for the phenomena described here. Measurements of the mutual solubilities of water, guaiacol, and trichloroacetic acid and of water, guaiacol, and acetone are given and are discussed in relation to the diffusion experiments. To give a complete picture of the process of diffusion we need to know the activities and concentrations in all parts of the system. The difficulties of achieving this are obvious. The solubility relations are such that a concentration gradient of trichloroacetic acid in guaiacol produces a concentration gradient of water in the same direction, but the activity gradient of water is in the opposite direction. Since in certain respects guaiacol acts like some protoplasmic surfaces it seems possible that similar phenomena may occur in living cells. If so these results have an obvious bearing on the movement of water in the organism and on methods of studying permeability. It becomes necessary to know to what extent a substance entering or leaving the cell appears to carry water with it in the manner here indicated. In certain of the diffusion experiments the water takes a circular path, passing out of the dilute solution at one point and back into it (as vapor) at another. This circular path recalls the situation in the kidney where the water continually passes out of the blood into the glomerulus and tubule and then back into the blood from the tubule (where the solution is more concentrated). In both cases the circular path of the water is an essential feature.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

Efflux of Red Cell Water into Buffered Hypertonic Solutions

Buffered NaCl solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38°C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the supernatant fluid. The amount of water added to the hypertonic solutions by the red cells was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093°C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of -0.560 to -0.930°C. Beyond -0.930°C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of -2.001°C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity.     

Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma

Tryptophan metabolites with an indole ring are enriched by adsorption either as an ion pair with a trichloroacetic acid anion or as its undissociated form on porous polystyrene polymer (TSK 2000 S) from strongly acidic plasma deproteinized by trichloroacetic acid, and after washing with water, they are eluted with a 90% methanol solution. Following the removal of the solvent, the residue is dissolved in a small amount of water and then subjected to high-performance liquid chromatography (hplc) analysis. Using 0.2 ml of adsorbent, the recovery of the 500 pmol added for each of the tryptophan metabolites into 1.5 ml of deproteinized plasma is above 70%. This method is used for the analysis of normal rabbit and rat plasma. The hplc analysis, with native fluorescence detection, shows several peaks corresponding to tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and indole-3-propionic acid. Peak identification and cross reactivity were checked by the retention time with two hplc systems, fluorometric characterization, and electrochemical characterization. This method is easy and is simple enough for routine analysis.     

Modification of the Marchi Technic

The formula proposed by Swank and Davenport (1935) was modified and applied to human and macaque nervous material. Three groups of experiments were performed and the following observations were made. (1) Diluting the osmic acid component, without altering the relative concentration of the other constituents of the solution resulted in practically no staining of the degenerated fibers. (2) When all constituents of the staining solution were used in much lower concentration than previously suggested, enhancement of staining of the degenerating fibers occurred and the different structures of the normal tissue were more easily identified. (3) At low concentrations of osmic acid and potassium chlorate, the contrast was diminished and artifacts produced by increasing the concentration of acetic acid or formalin or both. The new formula, based on the present results, consists of osmic acid, 0.5%, 11 ml.; potassium chlorate, 1%, 16 ml.; formalin (cone), 3 ml.; acetic acid, 10%, 3 ml.; and distilled water to make 100 ml. (All solutions are aqueous). Good staining after a long period of fixation in formalin, following degeneration of 8-80 days, was obtained and the cost of staining solution greatly reduced.     

INTRAVENOUS NUTRITION—A Clinical Evaluation of a 50 Per Cent Dextrose in Water Solution,Containing 1 mg. of Hydrocortisone per 100 ml

A 50 per cent dextrose in water solution, containing 1 mg. of hydrocortisone per 100 ml., was used successfully in 70 patients for intravenous nutritional maintenance and repletion. There were no adverse systemic effects during or following 216 infusions. The only undesirable local reaction was the rare occurrence of pain in the arm when the concentrated solutions were given too rapidly. Glycosuria was minimal if the infusion rate did not exceed 0.85 gm. of glucose per kilogram of body weight per hour, particularly if 50 units of insulin were added to each 550 ml. bottle of 50 per cent dextrose. In patients without significantly elevated serum potassium content, 30 mEq. of potassium chloride, acetate or phosphate was added to each bottle to prevent hypokalemia.Preliminary observations suggest that this new solution may be given safely intravenously, without need for cutdowns or plastic catheters, if the needle is carefully inserted and well immobilized in the arm vein and the duration of the infusion is not too prolonged. Further studies on the effect of such high caloric supplementation plus protein hydrolysates in parenteral nutritional repletion and maintenance are indicated.     

Thermal resistance and inactivation of Enterobacter sakazakii isolates during rehydration of powdered infant formula

Enterobacter sakazakii may be related to outbreaks of meningitis, septicemia, and necrotizing enterocolitis, mainly in neonates. To reduce the risk of E. sakazakii in baby foods, thermal characteristics for Korean E. sakazakii isolates were determined at 52, 56, and 60 degrees C in saline solution, rehydrated powdered infant formula, and dried baby food. In saline solution, their D-values were 12-16, 3-5, and 0.9-1 min for each temperature. D-values increased to 16-20, 4-5, and 2-4 min in rehydrated infant formula and 14-17, 5-6, and 2-3 min in dried baby food. The overall calculated z-value was 6-8 for saline, 8-10 for powdered infant formula, and 9-11 for dried baby food. Thermal inactivation of E. sakazakii during rehydration of powdered infant formula was investigated by viable counts. Inactivation of cultured E. sakazakii in infant formula milk did not occur for 20 min at room temperature after rehydration with the water at 50 degrees C and their counts were reduced by about 1-2 log CFU/g at 60 degrees C and 4-6 log CFU/ml with the water at 65 and 70 degrees C. However, the thermostability of adapted E. sakazakii to the powdered infant formula increased more than two times. Considering that the levels of E. sakazakii observed in powdered infant formula have generally been 1 CFU/100 g of dry formula or less, contamination with E. sakazakii can be reduced or eliminated by rehydrating water with at least 10 degrees C higher temperature than the manufacturer-recommended 50 degrees C.     

Ensiled alkali-treated straw. I. Effect of level and type of alkali on the composition and digestibility in vitro of ensiled barley straw

In three experiments barley straw chopped to 5 cm nominal particle length was ensiled in laboratory silos for 90 days after treatment with alkali. In the first two experiments, NaOH was added at 0, 1.05, 2.10, 3.15 or 4.20 g per 100 g straw dry matter (DM) (Experiment 1) or at 0, 2.5, 5.0, 7.5 and 10.0 g per 100 g straw DM (Experiment 2) in solutions at either 60 ml or 120 ml solution per 100 g straw DM. Digestibility in vitro of organic matter (OM) and digestible OM in DM (DOMD) increased with increasing level of NaOH. The effect of volume of solution on digestibility was small. The pH of the straws decreased during storage. The content of neutral detergent fibre decreased as the level of NaOH increased, but there was relatively little change in the contents of acid detergent fibre or acid detergent lignin. Lactate and acetate were detected in all silages, and butyrate was present in silages made from straws treated with less than 5 g NaOH per 100 g straw DM. On opening the silos little moulding was seen and the temperature of the straws remained close to ambient in both experiments throughout 16 days of subsequent exposure to air.In the third experiment, the comparative effects of Ca(OH)₂ and KOH were studied alone and in combination ($\text{50}\text{50}$ by weight) with NaOH. KOH mixed with NaOH gave levels of DOMD in vitro similar to those obtained with NaOH alone. Ca(OH)₂, whilst improving DOMD, was slightly less effective than the other alkalis.The optimum level of alkali for the treatment of barley straw prior to ensiling appeared to be 7.5 g/100 g straw DM. At this level of addition, DOMD in vitro would be expected to be about 65%. Ca(OH)₂ is worth further attention as an alternative to NaOH.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

温特曲霉转富马酸为L-苹果酸的初步研究*

从大量霉菌中选育到一株具有较高富马酸酶活性的温特曲霉(Aspergillus wentii) A5-61。在摇瓶培养条件下,32℃ 96小时,产L-苹果酸达10.49g/100ml,对富马酸的转化率达90.80%。利用菌体细胞,进行酶转化试验,结果表明：1.6g湿菌体接入25ml含富马酸10.0%（用NaOH中和至pH7.0）的转化液中,35℃16～24小时,连续转化三次,分别产生L—苹果酸9.61g/100ml、9.73g/100ml、6.93g/100ml。对菌体整体细胞酶学性质的研究表明,其最适反应温度35℃,最适反应pH7.0,Cu2＋对该酶有明显的抑制作用,该酶的Km＝0.154mol/L,Vmax＝0.0571mol/L·h。     

A New Method Using a Modified Mayer's Hemalum at pH 6 for Demonstrating Mucous Neck Cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

A new method using a modified Mayer's hemalum at pH 6 for demonstrating mucous neck cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

Extraction of adenine nucleotides from cultured endothelial cells

The goal of this work was to find a method suitable for the extraction of adenine nucleotides from cultured vascular endothelial cells. Extraction of cell monolayers with 80% methanol in water yielded extracts with a higher content of ATP than did extraction of cells with perchloric acid, trichloroacetic acid, or boiling water. The optimal extraction solution was 80% methanol with 0.5 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or EDTA, heated to 70 degrees C immediately before use. Extraction of nucleotides by this solution was rapid and the recovery of exogenous ATP added during the extraction process was generally greater than 90%. An aqueous methanol or ethanol solution may be applicable for the extraction of nucleotides and other metabolites from cultured animal cells, dispersed cells, and frozen, powdered tissues.