Filter Paper Squares Research Articles

Colorimetric Paper-Based Analytical Devices (PADs) Backed by Chemometrics for Pd(II) Detection.

This paper presents the development of cheap and selective Paper-based Analytical Devices (PADs) for selective Pd(II) determination from very acidic aqueous solutions. The PADs were obtained by impregnating two cm-side squares of filter paper with an azoic ligand, (2-(tetrazolylazo)-1,8 dihydroxy naphthalene-3,6,-disulphonic acid), termed TazoC. The so-obtained orange TazoC-PADs interact quickly with Pd(II) in aqueous solutions by forming a complex purple-blue-colored already at pH lower than 2. The dye complexes no other metal ions at such an acidic media, making TazoC-PADs highly selective to Pd(II) detection. Besides, at higher pH values, other cations, for example, Cu(II) and Ni(II), can interact with TazoC through the formation of stable and pink-magenta-colored complexes; however, it is possible to quantify Pd(II) in the presence of other cations using a multivariate approach. To this end, UV-vis spectra of the TazoC-PADs after equilibration with the metal ions solutions were registered in the 300-800 nm wavelength range. By applying Partial Least Square regression (PLS), the whole UV-vis spectra of the TazoC-PADs were related to the Pd(II) concentrations both when present alone in solution and also in the presence of Cu(II) and Ni(II). Tailored PLS models obtained with matrix-matched standard solutions correctly predicted Pd(II) concentrations in unknown samples and tap water spiked with the metal cation, making the method promising for quick and economical sensing of Pd(II).

Droplet Distribution in a University Dental Clinic Setting: The Importance of High-Volume Evacuation.

The purpose of this study is to compare droplet distribution during a piezoelectric ultrasonic debridement procedure using either high-volume or slow-volume evacuation. Droplet distribution during a crown preparation with slow-volume evacuation is also examined. Fluorescein dye is added to the water reservoir and the procedures are performed by a single operator for 15 min on a dental manikin with artificial upper and lower teeth. Placement of filter paper squares (10 cm × 10 cm) in radiating lines away from the oral cavity of the dental manikin allows for visualization of droplet dispersion. Results show minimal difference in the spread of the droplets between the two evacuators during the debridement procedure; however, the slow-volume evacuator produces a higher concentration of droplets than the high-volume evacuator. An even higher concentration of droplets in the vicinity of the dental chair is observed during the crown preparation procedure. This study recommends the use of a high-volume evacuator where possible during professional debridement and crown preparation to reduce contamination around the dental chair from potentially pathogenic microorganisms.

Effect of the type and concentration of cellulose and temperature on metabolite formation by a fermentative thermophilic consortium

In this study, we hypothesized that anaerobic biodegradation of cellulose is influenced by cellulose type and concentration, temperature, and their interactions. Cellulose biodegradation by an anaerobic consortium was tested in thermophilic batch experiments that combined cellulase action, hydrolysis, and fermentation. Initially, the main constituents in the inocula were Thermoanaerobacter, Clostridium, and Acetivibrio spp. Four types of cellulose and a range of concentrations were used as feedstock with pathways involving hydrolysis and glycolysis to produce H2, CO2, acetate, and ethanol. Long fibrous cellulose, two types of microcrystalline cellulose, and filter paper squares were tested at several concentrations between 2 and 20 g/l as substrates. The yields ranged between 0.1 and 2.9 mmol H2 and 0.7–2.6 mmol ethanol per g cellulose. The rates ranged between 0.01 and 0.2 mmol H2, 0.03–0.2 mmol CO2, and 0.01–0.05 mmol ethanol per g cellulose·h. Statistical analyses indicated that the rates and yields of metabolite production were influenced by two-way interactions between the temperature, type, and concentration of cellulose. The results suggest that two-way interactions between experimental variables may impact the outcomes in cellulose bioconversion studies.

Mortality of Solenopsis invicta Workers (Hymenoptera: Formicidae) After Indirect Exposure to Spores of Three Entomopathogenic Fungi

Mortality caused by indirect exposure to Metarhizium brunneum and Beauveria bassiana (GHA and NI8) to the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), workers was evaluated. Groups of 50 workers were placed in one side of dual-box arenas. The opposite side of the arenas was lined with filter paper squares previously sprayed with unformulated purified spores (106 spores/ml) suspended in 0.2% Ethal TDA 3, HLB 8 of the three fungal strains, or untreated filter paper squares as the control. Daily observations were done for 1 wk to determine mortality. Dead ants from each treatment and control were collected, surface cleaned, and placed in PDA media and incubated at 27°C, 60% RH for 7 d to detect fungal growth. The presence of fungal growth in the dead ants confirmed that fungal spores infected workers while walking on the treated paper. In the M. brunneum and B. bassiana GHA treatments, 51.35 and 56.68% of the workers died, respectively, during days 1 and 2. However, only 9.47 and 35.96% of the mortality could be explained by fungal infection by M. brunneum and B. bassiana GHA, respectively. Most of the mortality observed in the B. bassiana NI8 treatment (84.48%) occurred later (between days 4–6) and most of this mortality occurring during day 4 (89.06%) could be explained by B. bassiana infection. Overall mortality was significantly higher in the B. bassiana NI8 treatment than the other two fungi tested and control. Potential application of these fungal strains for fire ant control are discussed.

Effects of ethanol treatment time on bacterial recovery

95% Ethanol is commonly found to be one of the main active ingredients in most alcohol wipes, which are commonly used to disinfect the skin before a vaccination. The objective of this study was to determine the effect of contact time on the recovery of bacteria from a surface. Overnight cultures of E. coli were spotted glass slides and allowed to dry. Filter paper squares saturated with 95% ethanol were applied to the bacterial spots for varying amounts of time (10, 20, 40 and 80 seconds). After removing the filter paper 100 ul of sterile water was applied to the slide, and immediately 50 ul was removed and serially diluted to determine the number of bacteria recovered from the slide. As expected majority of the results showed that the eighty-second ethanol exposure time had the lowest number of bacterial colonies recovered. Unexpectedly, the water control (sterile water on filter paper) had an even lower number bacterial of colonies recovered when compared to the ethanol treatments. Overall, the trials performed with water were the most effective at reducing the number of bacteria recovered.

59 LIPID DYNAMICS IN THE EARLY EMBRYONIC DEVELOPMENT OF ZEBRAFISH BY DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY IMAGING AND NANOELECTROSPRAY–MASS SPECTROMETRY

This study introduces a combination of mass spectrometry-based analytical approaches with little or no sample preparation for the study of zebrafish (Danio rerio) lipid metabolism during early development (0 through 96 h post-fertilization, hpf). Desorption electrospray ionization mass spectrometry (DESI-MS) imaging and nanoelectrospray (nESI) MS were used. Embryos (N = 107) were placed onto filter paper squares and analysed by DESI-MS imaging. Embryos were arranged in rows with each one corresponding to a different developmental time, so that each DESI-MS image contained arrays of samples at different hpf. After completion of DESI-MS acquisition, the arrays were cut into strips of paper, isolating each embryo, which was subsequently inserted into a nESI tip and analysed. All experiments were performed using a linear ion trap mass spectrometer. DESI-MS images were acquired in the positive ion mode using acetonitrile doped with 10 ppm AgNO3, which allowed for the detection of cytosolic neutral lipids, such as cholesteryl esters (CE), diacylglicerols (DAG), triacylglycerols (TAG), squalene, and ubiquinone. For the nESI, experiments were run in full mass scan and MS/MS, in both negative and positive ion mode, using a mixture of dimethylformamide and acetonitrile to detect membrane phospholipids (e.g. phosphatidylcholines and phosphatidylglycerols). Principal component analysis was used to explore DESI-MS images and nESI data in an unsupervised fashion. The combination of DESI-MS imaging, providing chemical and spatial location, and nanoESI, providing a broader and more extensive structural information, showed that the lipid content changes dramatically over the first days of development. Accumulation of DAG and TAG, which are usually concentrated in the embryos yolk sac as an important storage for early development, were observed between 0 and 48 hpf. Embryos at the intermediate phase of development (24 hpf) were distributed between those of 0 and 48 hpf, reflecting the dynamics associated with development and might be correlated with TAG consumption and de novo synthesis. After 72 and 96 hpf, samples differed from prior developmental stages by the content of squalene, ubiquinone, and CE 22 : 5 (overexpressed), while the TAG content decreased. Further decrease of TAGs and ubiquinone occurred between 72 and 96 hpf. These observations indicated that zebrafish embryos rely entirely on the yolk sac for the nutrients needed to sustain growth and survival during the first four days of development. Yolk lipids are the likely source of TAGs, as well as cholesterol, a required component of cell membranes and a precursor for bile acids. The abundance of ubiquinone can be tied to zebrafish embryo growth, differentiation and organogenesis, and activation of mitochondria.

First Report of Alternaria Black Spot of Pomegranate Caused by Alternaria alternata in Spain.

Since 2010, a new foliar and fruit disease was observed in pomegranate (Punica granatum L.) orchards in Alicante Province (eastern Spain). Symptoms included black spots on leaves and fruits, as well as chlorosis and premature abscission of leaves. Fungal isolates were obtained by surface-disinfecting small fragments of symptomatic leaf and fruit tissues in 0.5% NaOCl, double-rinsing in sterile water, and plating them onto potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. Gray-to-black colonies were obtained, which were identified as Alternaria sp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (4). Conidia (n = 100) measured (12.2-) 20.2 (-27.6) × (5.7-) 9.2 (-12.0) μm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures were obtained and their genomic DNA was extracted. The internal transcribed spacer (ITS) region of rDNA and partial sequences of the beta tubulin gene were amplified and sequenced with primers ITS1-ITS4 and Bt1a-Bt1b, respectively (3). BLAST analysis of the sequences showed that they were 100% identical to a pathogenic A. alternata (Fr.) Keissl. isolate obtained from black spot disease of pomegranate in Israel (Accession No. JN247826.1, ITS and Accession No. JN247836.1, beta tubulin) (2). As all the sequences obtained showed 100% homology, ITS and beta tubulin sequences of a representative isolate (1516B) were submitted to GenBank (KF199871 and KF199872, respectively). In addition, a PCR reaction with specific primers (C_for/C_rev) designed to recognize highly virulent isolates of A. alternata causing black spot of pomegranate was used with all isolates (2). A characteristic fragment of ~950 bp was amplified in two isolates: 1552B and 1707B. Pathogenicity was assessed on plants and detached fruit of pomegranate cv. Mollar (1). Two-year-old pomegranate trees were inoculated with isolates 1552B and 1707B by spraying a conidial suspension (106 conidia/ml) onto the upper and lower leaf surfaces. Five plants per fungal isolate were used and five control plants were sprayed with sterile water. Plants were covered with transparent plastic bags and incubated in a growth chamber for 1 month at 25°C, with a 12-h photoperiod. One-month-old fruits were surface sterilized in 1.5% sodium hypochlorite solution for 1 min and rinsed twice in water. Two filter paper squares (5 × 5 mm) were dipped in the conidial suspensions and placed on the fruit surface. Inoculated fruit were incubated in a humid chamber in the dark at 25°C. Ten fruit per fungal isolate were used and 10 control fruit were inoculated with sterile water. Black spots were visible on inoculated leaves and fruit, 10 and 3 days after inoculation, respectively. Symptoms were not observed on controls. The fungus was re-isolated from leaf and fruit lesions, confirming Koch's postulates. Leaf black spot of pomegranate caused by A. alternata was first described in India in 1988, and later in Israel in 2010 affecting both fruit and leaves (1). To our knowledge, this is the first report of the disease in Spain, where it could represent a threat for pomegranate cultivation due to the increasing amount of area dedicated to this crop.

A Plant Stem Inoculation Assay for Assessing Transmission of Phytophthora infestans from Potato Seed Tubers to Emerged Shoots.

A stem inoculation assay was developed to assess transmission of Phytophthora infestans from the base of belowground potato stems to emerged aboveground shoots. Sporangia of P. infestans subsequently developed on aboveground stems after belowground stems were inoculated with isolates BF05 (US 8 genotype), WA10.1 (US 24), and 110B (US 11) by (i) placing a filter paper square saturated with a spore suspension onto nonwounded belowground stem tissue, (ii) placing a filter paper square saturated with a spore suspension onto wounded belowground stem tissue, and (iii) dipping the base of the cut belowground stem into a spore suspension. Plant stems were then incubated in a humidity chamber at 10 to 21.5°C for 7 to 28 days. Incidence of aboveground stems with sporulation was significantly less when stems were inoculated with isolate 110B than with isolates BF05 and WA10.1 but length of latent period did not differ among the three isolates. Incidence of aboveground stems with sporangia was significantly (P < 0.05) greater for 'Russet Norkotah' than for 'Umatilla Russet' when inoculated near the point of seed piece attachment. Incidence of aboveground stems with sporulation did not differ among incubation temperatures of 10, 15, and 21.5°C. Latent period significantly increased as temperature decreased. Age of stem did not appear to affect incidence of transmission. Incidence of transmission of P. infestans to aboveground stems from the base of belowground stems was significantly lowered when aboveground stems were treated with selected fungicides 1 h before inoculation.

Biocide-Containing Varnish for the Protection of Sandstone: Comparison of Formulations and Laboratory Test Methods

Two formulations of acrylic varnish, with and without either of two dry film biocides--one a mixture of isothiazolinones and benzimidazole derivatives, and the other a carbamate--were tested in vitro for their activity against mixtures of filamentous fungi and cyanobacteria found on sandstone buildings. Growth on filter-paper squares coated with the varnishes was assessed semi-quantitatively by naked eye, quantitatively by image analysis and chemically by measurement of ergosterol and chlorophyll a. The lower solvent content (higher resin) varnish was more inhibitory to cyanobacteria than the higher varnish content, whilst the opposite was true for the fungal inoculum. The carbamate biocide was effective against cyanobacteria, unlike the isothiazolinone mixture, but the latter produced more inhibition of fungal growth. The three assay methods produced generally similar results, although visual observation was obviously the most imprecise. There was an anomaly in the ergosterol measurements, which was considered to be caused by the varying ergosterol content and unequal inhibition of the three fungal genera used in the inoculum. Fusarium sp. was shown to contain higher levels of this membrane component than Cladosporium sp. and Penicillium sp. For this reason, the most appropriate method overall, giving reliable quantitative results, was deemed to be the image analysis.

Chemosensory cues allow male Tenebrio molitor beetles to assess the reproductive status of potential mates

Males of many insect species, including beetles, choose their mates according to their reproductive status. However, the ways in which male beetles evaluate female reproductive status have received little attention. We tested the existence of male mate choice in the mealworm beetle, Tenebrio molitor, by observing mating and courtship behaviour of males given simultaneous access to pairs of females differing in their reproductive status: (1) mature versus immature; (2) virgin versus previously mated; (3) familiar (mated with the experimental male) versus unfamiliar (mated with a different male). Males courted and mated preferentially with mature and virgin females. To determine whether chemical cues played a role in these discriminations, we exposed males to filter paper squares bearing chemical cues from different types of females: (1) virgin versus mated; (2) mature versus immature. Males were significantly more attracted to those squares bearing chemical cues from virgin and mature females, suggesting that males can assess female reproductive status on the basis of chemical cues alone.

Chemosensory cues allow male Tenebrio molitor beetles to assess the reproductive status of potential mates

Males of many insect species, including beetles, choose their mates according to their reproductive status. However, the ways in which male beetles evaluate female reproductive status have received little attention. We tested the existence of male mate choice in the mealworm beetle, Tenebrio molitor , by observing mating and courtship behaviour of males given simultaneous access to pairs of females differing in their reproductive status: (1) mature versus immature; (2) virgin versus previously mated; (3) familiar (mated with the experimental male) versus unfamiliar (mated with a different male). Males courted and mated preferentially with mature and virgin females. To determine whether chemical cues played a role in these discriminations, we exposed males to filter paper squares bearing chemical cues from different types of females: (1) virgin versus mated; (2) mature versus immature. Males were significantly more attracted to those squares bearing chemical cues from virgin and mature females, suggesting that males can assess female reproductive status on the basis of chemical cues alone.

Derivatization of F 2-isoprostanes with 1-pyrenyldiazomethane and their subsequent determination by fluorescence high-performance liquid chromatography

F 2-isoprostanes are produced during free radical oxidation of cell phospholipids and are considered reliable biomarkers of oxidative stress. Currently, mass spectroscopy is the method of choice to detect F 2-isoprostanes. However, due to numerous isomeric forms, analysis of F 2-isoprostanes using MS detectors requires preliminary chromatographic separation. The current study was undertaken to develop a method of HPLC separation and quantification of different isomers of prostaglandin F 2α, including 8-iso-PGF 2α, following derivatization with 1-pyrenyldiazomethane (PDAM) to a highly fluorescent 1-pyrenylmethyl ester. HPLC separation and quantification of 1-pyrenylmethyl esters of PGF 2α isomers at picogram level are complicated by numerous interfering products of the degradation of 1-pyrenyldiazomethane. A procedure of derivatization and purification was developed to reduce these interfering contaminants. The procedure of derivatization includes sorption of PGF 2α isomers from solution in a hexane:ethyl acetate mixture (10:1) on a cellulose support prepared in the form of small (4×4 mm) filter paper squares. Bound PGF 2α isomers are derivatized by 1-pyrenyldiazomethane dissolved in the same hexane:ethyl acetate mixture. During subsequent washing of the cellulose squares by the hexane:ethyl acetate mixture (10:1), fluorescent derivatives of PGF 2α remain bound to cellulose while a significant portion of the contaminants are washed out. The 1-pyrenylmethyl esters of PGF 2α can be quantitatively extracted from cellulose by an ethyl acetate:methanol (1:1) mixture. The next step in eliminating interference is a solid-phase extraction on silica cartridges using ethyl acetate for application of the sample and an ethyl acetate:methanol (1:1) mixture for elution. Final purification is achieved by normal-phase HPLC with wet ethyl acetate as the mobile phase. This chromatographic method displays remarkable resolution in the separation of different PGF 2α isomers and can be used not only for sample purification but also for pre-MS separation. The purified 1-pyrenylmethyl esters of PGF 2α were quantitatively analyzed by reverse-phase HPLC with fluorescent detection, with a detection limit of 5–10 pg.

ベクロメタゾン点鼻剤の鼻腔内分布の検討 呼吸状態，鼻腔内形態の影響

Although betamethasone (BTM) nose drops have been the most common treatment for olfactory dysfunction, only a few reports are available in regard to the distribution of this drug by this mode. From the point of view of delivery to the olfactory cleft, the spray form may be a feasible alternative. In order to establish the treatment of olfactory dysfunction by beclomethasone (BCM) spray, the authors examined its distribution in a silicon replica prepared from a mold of a cadaver nasal cavity, available from Koken Co. Ltd., which was used as such and also modified by dental paste to simulate septal deformity or nasal polyp. The effects of respiration on drug delivery were also examined. Nine pieces of 8 mm square filter paper were placed on different sites of the replica. After the application of BCM spray or BTM nose drops, the concentrations of each drug on the filter paper squares were determined by HPLC. A larger concentration of BCM spray than of BTM drops was present even in models with deviation or nasal polyp, if the olfactory cleft remained open. In addition, there was no difference in amount of BCM delivered into the holding olfactory cleft between conditions of simulated inspiration and of breath. During inspiration, more BCM spray entered the upper pharynx, although the instructions for the use of BCM suggest that the spray should be inhaled. The amounts of BCM in the anterior nasal cavity and nasal fissures were increased when applied in the upward direction. Our results suggest that BCM spray can be used in the treatment of olfactory dysfunction.

Patterns of deposition of urine containing chemosignals that affect puberty and reproduction by wild stock male and female house mice (Mus domesticus)

House mice release chemosignals in their urine that influence the timing of puberty and reproductive condition in conspecific females. These experiments tested the possibility that mice depositing urine containing chemosignals that affect puberty and reproduction do so differentially with respect to urine cues from conspecifics. Mice were tested in cages containing samples of urine or water on cotton in wire mesh capsules protruding from the cage floor. Their urine deposition patterns were recorded on squares of filter paper positioned below the cages. Males deposited more urine than females housed in groups, estrous females, diestrous females, or prepubertal females. All groups of mice deposited urine in a nonrandom fashion with regard to urine cues from conspecifics. Male mice deposited more urine near locations previously soiled by females than near water or other males. Grouped females deposited more urine near male urine cues and avoided depositing urine near urine from other group-housed females. Both estrous and diestrous female mice deposited more urine near males than near other urine cues or water, possibly to attract mates. Prepubertal females avoided depositing their urine near male urine and urinated more near urine from grouped females than near other urine types or water. Young females may be avoiding possible male mates until they have attained puberty. This avoidance behavior may enhance the long-term reproductive success of the females that otherwise might mature and mate at too young an age.

An improved cellophane method for in vitro germination of recalcitrant pollen.

The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper. The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min. This arrangement keeps the cellophane flat as it absorbs medium. The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish. The lower leaves of the booklet keep the cellophane moist for up to 24 hr. Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique. Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.

Behavioral Responses of Male Eoreuma loftini (Lepidoptera: Pyralidae) to Ovipositor Extracts1

Eoreuma loftini (Dyar) adults were observed in the laboratory to study their behavior before and during the mating process. Characteristic female calling behavior and male response to females and crude sex pheromone extracts made from excised ovipositors were noted. Bioassays were performed on ovipositor extracts, and levels of male activity at various hours after darkness were determined. Males began responding to extracts of ovipositors at darkness, and activity peaked between 5 and 9 h after darkness. Males exposed to 0.5-1.0 female equivalents of extract exhibited responses indistinguishable from those to calling females. In the presence of pheromone extracts, males often attempted to mate with other males, the nylon mesh covering, and the filter-paper square used to dispense the pheromone.

A non-specific aggregation pheromone in two Australian reptile ticks

Larvae, nymphs, adult males and adult females of each of the Australian reptile tick species Aponomma hydrosauri and Amblyomma limbatum took up significantly aggregated distributions when left for 12 h in the dark in petri dishes. Nymphs of the two species placed together formed co-aggregations. Pheromonal communication was implicated by the attraction of nymphs to filter paper squares which had been in contact with other nymphs. Each species was attracted to pheromone from the other species and no conspecific preference was detected. A common ancestral pheromone is suggested, which may have been retained after speciation by stabilizing selection for a behaviour of major importance to tick survival.

Contamination of Electronic Equipment after an Extended Urban Exposure

Water soluble contaminants on electromechanical telephone switching equipment exposed for up to 40 years to the New York City environment have been chemically analyzed by a combination of methods, and their moisture pickup characteristics have been monitored by instrumental gravimetric techniques. Samples were collected from structural surfaces by extraction with filter paper squares moistened with distilled water. Elements with atomic numbers greater than 11 were identified by energy dispersive x‐ray analysis. Selected anions were identified by microchemical tests and infrared spectroscopy. Nitrate and sulfate concentrations of 46 samples from one location were measured using nitrate and lead ion selective electrodes (the latter for titration of sulfate). Several hundred samples from this and four other locations were analyzed using a chloride ion selective electrode. On zinc surfaces, chloride concentrations averaged 27 μg/cm2 for typical locations, while sulfate and nitrate concentrations averaged 48 and 6 μg/cm2, respectively. On aluminum surfaces, chloride concentrations averaged <2 μg/cm2, while sulfate and nitrate concentrations averaged 25 and 3 μg/cm2, respectively. Moisture pickup by contaminants on zinc and aluminum surfaces was found to occur above 26 and 46% RH, respectively.

Isoelectric focusing in thin layer polyacrylamide gel combined with a zymogram method for detecting enzyme microheterogeneity: Sample application

In the study of the microheterogeneity of the phospholipase C of Clostridium perfringens using combined zymogram gel isoelectric focusing, different methods of sample application were tested. Of fifteen filter papers tried Whatman 3MM, Whatman No. 1 and Schleicher & Schull 2043B were least adsorptive and had sufficient absorptive capacity. Their absorptive capacity was about 1 μl 3 mm 2 . However, with the sensitive egg yolk zymogram procedure used even these filter papers were shown to adsorb 1 μg protein/12 mm 2, significant levels in relation to the sensitivity of the detection technique. Application of small samples directly onto gel surface increased the sensitivity of the system 200–400 times over loading on filter paper squares. It was observed that enzymic components were selectively adsorbed to filter paper when applied below their pI values on gels with preformed pH gradients, thus reducing the apparent microheterogeneity observed. Problems associated with other application procedures were noted.

A new assay of phosphorylase based on the filter paper technique

A rapid method for the assay of phosphorylase has been developed, similar in technique to the method described by Thomas et al. (8) for glycogen synthetase. It utilizes the ability of phosphorylase to incorporate 14C-labeled glucose from 14C-glucose 1-phosphate into glycogen, which is then precipitated on filter paper squares by immersion in ice cold 66% ethanol. After removal of surplus 14C-glucose 1-phosphate, the papers are counted directly in a vial containing scintillation fluid. Several buffers commonly used in phosphorylase assays were compared and it was found that measuring in a 100 m M maleate buffer gave approximately 15% lower activity than assaying in any of the other seven buffer systems tested.