Sealed Capillary Tube Research Articles

Crystalline ices – Densities and comparisons for planetary and interstellar applications

Results are presented from recent density measurements on twenty-nine crystalline ices at temperatures applicable to the outer Solar System and the interstellar medium. For each ice we report both a density and a refractive index at 670 nm, quantities needed to calculate infrared band strengths, complex indices of refraction, and, ultimately, molecular abundances. Comparisons are made between densities obtained from micrometer-size crystalline ice samples prepared by vapor-phase deposition and the larger crystals (mm scale) used in diffraction work, and often made by the slow cooling of a liquid or a vapor in a sealed capillary tube. A strong positive correlation is found between the two sets of crystalline-ice densities.

Investigating the performance of in situ quantitative nuclear magnetic resonance analysis and applying the method to determine the distribution of saccharides in various parts of carrot roots (Daucus carota L.)

In order to explore the performance of the analytical method called in situ quantitative nuclear magnetic resonance spectroscopy – is q NMR – the distribution of glucose, fructose and sucrose in various parts of a carrot root (Daucus carota L.) – primary xylem, secondary xylem, phloem, cortex; top part and lower part – was determined. The influence on the quality of spectra of drying samples before analysis was studied, as well as the influence of the length of strips of tissue used in analysis. Finally samples as small as 240mm3 could be studied directly, with minimum prior treatment (only drying), along with deuterated water for locking and a sealed capillary tube containing a solution of 0.5% of the sodium salt of (trimethylsilyl)propionic-2,2,3,3-d4 acid, used both as an internal reference and for quantification. With optimized parameters, the coefficients of variation for measurements were observed to have an average value of 0.038, with a standard deviation of 0.047.

Self‐pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans, could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.

Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions.

The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures.

Inactivation Kinetics of Avirulent Bacillus anthracis Spores in Milk with a Combination of Heat and Hydrogen Peroxide

The combined effect of heat and hydrogen peroxide (HP) on the inactivation of avirulent Bacillus anthracis spores (Sterne strain 7702; strain ANR-1, an avirulent Ames derivative lacking the pXO2 plasmid; and strain 9131, a plasmid-less Sterne strain) was evaluated in milk. The study temperature ranged from 90 to 95°C, and the concentration of added HP varied from 0.05 to 0.5%. Decimal reduction times (D-values) were determined using a sealed capillary tube technique. The mean D- and z-values of hydrated freeze-dried spores of all three strains in milk ranged from 550 s at 90C to180s at 94°C and from 8.6 to 9.0°C, respectively. When 0.05% HP was added to the milk, the D-values were decreased at least threefold, and at 0.5% HP the D-values ranged from 1 to 10 s. At 90°C, all three strains had similar D-values when 0.05% HP was added. Increasing the concentration of HP to 0.5% had a greater reducing effect on the D-value for strain 7702 than on the values for strains ANR-1 and 9131. The rate of inactivation of each strain followed first-order reaction kinetics at each temperature-peroxide combination. Equations in the form of D = Constant × (HP concentration)n had R2 values greater than 0.97 for strains ANR-1 and 7702 and of at least 0.7 for strain 9131. This study suggests that a combination of high temperature (from 90 to 95°C) and HP could be used for inactivation of B. anthracis spores in the event of deliberate contamination of milk such that the contaminated milk could be disposed of safely.

Transient Responses of a Wetting Film to Mechanical and Electrical Perturbations

This article reports real-time observations and detailed modeling of the transient response of thin aqueous films bounded by a deformable surface to external mechanical and electrical perturbations. Such films, tens to hundreds of nanometers thick, are confined between a molecularly smooth mica plate and a deformable mercury/electrolyte interface on a protuberant drop at a sealed capillary tube. When the mercury is negatively charged, the water forms a wetting film on mica, stabilized by electrical double layer forces. Mechanical perturbations are produced by driving the mica plate toward or by retracting the mica plate from the mercury surface. Electrical perturbations are applied to change the electrical double layer interaction between the mica and the mercury by imposing a step change of the bias voltage between the mercury and the bulk electrolyte. A theoretical model has been developed that can account for these observations quantitatively. Comparison between experiments and theory indicates that a no-slip hydrodynamic boundary condition holds at the molecularly smooth mica/electrolyte surface and at the deformable mercury/electrolyte interface. An analysis of the transient response based on the model elucidates the complex interplay between disjoining pressure, hydrodynamic forces, and surface deformations. This study also provides insight into the mechanism and process of droplet coalescence and reveals a novel, counterintuitive mechanism that can lead to film instability and collapse when an attempt is made to thicken the film by pulling the bounding mercury and mica phases apart.

Thermal Resistance of Salmonella enterica Serotypes, Listeria monocytogenes, and Staphylococcus aureus in High Solids Liquid Egg Mixes

Decimal reduction times (D-values) were determined for Salmonella enterica serotypes, Listeria monocytogenes, and Staphylococcus aureus in two high solids egg mixes designated A and B (water activity [aw] = 0.76 and 0.82; solids = 53.12 and 52.63%; pH = 5.09 and 5.29; viscosity = 183 and 119 centipoise/s, respectively) using a low-volume (0.06 ml) sealed glass capillary tube procedure. For Salmonella, D-values ranged from 0.035 (70°C) to 0.193 min (64°C) in product A and from 0.048 to 0.193 min in product B. For Listeria, D-values ranged from 0.133 (70°C) to 0.440 min (64°C) in product A and from 0.074 to 0.364 min in product B. For Staphylococcus, D-values ranged from 0.332 (70°C) to 1.304 min (64°C) in product A and from 0.428 to 1.768 min in product B. For Listeria, the D-values of all heating temperatures were significantly higher (P < 0.01) in product A than in product B. The similar trend was also observed for Salmonella and Staphylococcus but only at 66°C for Salmonella and 64°C for Staphylococcus. Greater temperature dependence was observed for Salmonella inactivation in the low aw and low pH product (A), while the product (B) with the higher aw and pH had greater temperature dependence for Listeria. Compared across both egg mixes and all heating temperatures, the Staphylococcus strains were from 6.2 to 11.7 times more heat resistant than S. enterica serotypes and from 2.2 to 7.5 times more heat resistant than L. monocytogenes.

Survival of Honey Bee (Hymenoptera: Apidae) Spermatozoa Stored at Above-Freezing Temperatures

The development of practical techniques for the storage of honey bee, Apis mellifera L., semen would significantly improve our ability to breed for desirable genotypes and maintain genetic diversity in populations. Artificial insemination of queens has been possible for some time, but the semen used is usually freshly collected, or held for < 1 wk at room temperature. I examined the limitations of spermatozoal survival at nonfrozen temperatures. Pooled, diluted semen was stored in sealed capillary tubes at room temperature (25 degrees C) or in a refrigerator set to 12 degrees C, for periods up to 1 yr. Survival of spermatozoa was assayed by a dual fluorescent staining technique using SYBR-14 and propidium iodide stains, which readily distinguishes live and dead cells. No significant loss of viable spermatozoa occurred within the first 6 wk. Between weeks 6 and 9, the percent live spermatozoa fell from 80 to 58%, and remained at that level until after 39 wk. By week 52, samples at room temperature, but not at 12 degrees C, fell to 18.9% live spermatozoa. Nonfrozen storage of honey bee semen has potential for short-term preservation of germplasm, however several factors need to be studied further to optimize survival rates.

Nisin in Milk Sensitizes Bacillus Spores to Heat and Prevents Recovery of Survivors

Decimal reduction times (D values) were determined for Bacillus cereus T spores and B. stearothermophilus ATCC 12980 spores in skim milk supplemented with various concentrations (0, 2,000, and 4,000 IU/ml) of the bacteriocin nisin by using an immersed, sealed capillary tube procedure. For both organisms, the addition of nisin lowered the apparent D values. For B. cereus, the addition of 2,000 IU of nisin per ml to skim milk before heating significantly (P ≤ 0.05) lowered the apparent D value compared to the control treatment. The D values at 97°C were 7.0, 4.8, and 4.7 min for the control and 2,000- and 4,000-IU/ml nisin treatments, respectively. At 103°C, the D values were 1.5, 0.85, and 0.88 min for the control and 2,000-and 4,000-IU/ml nisin treatments. When calculated across both nisin treatments, the mean reductions in apparent D values at 97, 100, and 103°C due to addition of nisin in comparison to the controls were 32, 20, and 42%, respectively. The zD values for B. cereus ranged from 8.0 to 8.9°C. With B. stearothermophilus, the apparent D values at 130°C were reduced by 13 and 21% respectively, because of the presence of 2,000 or 4,000 IU of nisin per ml. The D values were 16.0, 13.8, and 12.5 s for the control and 2,000- and 4,000-IU/ml nisin treatments, respectively. There was a significant (P ≤ 0.05) decrease in the apparent D value between the control and 4,000-IU/ml treatment. Overall, log populations of survivors for B. stearothermophilus compared to the control were lower at any given sampling time due to the presence of nisin. The results of these studies suggest that spore control is likely due to enhanced sensitivity of spores to heat and the presence of residual nisin in the recovery medium that could prevent outgrowth of survivors.

Solution and Crystal Structures of a Sperm Whale Myoglobin Triple Mutant That Mimics the Sulfide-binding Hemoglobin fromLucina pectinata

The bivalve mollusc Lucina pectinata harbors sulfide-oxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O2, but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86-89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with approximately 700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln64(E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe68(E11) is rotated approximately 90 degrees about chi2 and located approximately 1-2 A closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.

Thermal Resistance of Aeromonas hydrophila in Liquid Whole Egg

Aeromonas hydrophila (AH) is a psychrotrophic spoilage bacterium and potential pathogen which has been isolated from a variety of refrigerated foods of animal origin, including raw milk, red meat, poultry, and commercially broken raw liquid whole egg (LWE). Decimal reduction times (D values) of 4 strains of AH (1 egg isolate, 2 egg processing plant isolates, 1 ATCC type strain) were determined in LWE using an immersed sealed capillary tube (ISCT) procedure. Initial populations (7.0 to 8.3 log CFU/tube in 0.05 ml LWE) were heated at 48, 51, 54, 57, and 60°C, and survivors were plated onto starch ampicillin agar (48 h at 28°C). D values ranged from 3.62 to 9.43 min (at 48°C) to 0.026 to 0.040 min (at 60°C). Both processing plant isolates were more heat resistant than the ATCC strain. Decimal reduction time curves (r2 ≤ 0.98) yielded ZD values of 5.02 to 5.59°C, similar to those for other non-spore-forming bacteria. D values of the most heat resistant AH strain were also determined in LWE at 48, 51, and 54°C using a conventional capped test tube procedure (10 ml/tube). Cells heated in test tubes yielded nonlinear (tailing) survivor curves and larger (P ≤ 0.05) apparent D values at each temperature than those obtained using the ISCT method. This study provides the first thermal resistance data for AH in LWE and the first evidence that straight-line semilogarithmic thermal inactivation kinetics may be demonstrated for Aeromonas using the ISCT procedure.

Thermal and Biological Treatments to Control Psychrotrophic Pathogens

Over the past decade, advances in egg processing technologies have permitted commercial production of ultrapasteurized liquid whole egg (LWE) products with a shelf-life of greater than 10 wk at 4 C. The inactivation and control of psychrotrophic pathogens such as Listeria monocytogenes and Aeromonas hydrophila in extended shelf-life LWE and conventionally pasteurized egg products is an ongoing food safety concern. This manuscript reports on the common features of these two psychrotrophic pathogens, their incidence in egg products, and their survival, growth potential, and heat resistance in liquid egg. Furthermore, this manuscript reports in detail on the results of two specific studies conducted in our laboratory whose objectives were: 1) to determine the heat resistance (D-values) of A. hydrophila in LWE using a low-volume immersed sealed glass capillary tube (ISCT) procedure; 2) to assess the impact of methodology (i.e., ISCT procedure vs a conventional capped test tube procedure) on the apparent thermal resistance of A. hydrophila; and 3) to report on the use of the bacteriocin nisin to restrict the survival of L. monocytogenes in ultrapasteurized LWE stored at refrigeration temperatures.

An enclosed Curie point pyrolysis-GC technique for studying the oxidative or thermal degradation of polymers and volatile oligomeric oils

The technique of pyrolysis-GC has been extended to the study of oxidative degradation by a method that can also deal with volatile oil samples and oligomers. This has been done by pre-degrading the sample in a sealed capillary tube containing either air or inert gas, and then breaking open this tube within the carrier gas flow at the injection port of a GC apparatus. This approach is possible because the sample is deposited on a Curie-point filament wire within the sealed tube, and this wire can be rapidly heated in the usual way by an external RF field. The new technique can therefore be described as enclosed Curie-point pyrolysis (ECP). Although there is a need for improving the reproducibility of the absolute product yields obtained by ECP, the relative yields of products display very good reproducibility. Examples of the use of the approach to study the oxidation of polyisobutylene oils are provided. The results show distinct oxidation products which are not present in the thermolysis pyrograms. A comparison of results from conventional resistive filament pyrolysis with those from the ECP method provides a procedure for distinguishing gas-phase versus melt-phase secondary reactions of the pyrolysis and oxidation products.

Microscale Analysis of Glycosphingolipids by Methanolysis, Peracetylation, and Gas Chromatography

A method for the analysis of pure samples of individual glycosphingolipids by microscale methanolysis, peracetylation, and gas chromatography is described. Solvolysis of glycosphingolipids in dry methanolic HCl and peracetylation were conducted in a single 4.5-cm sealed capillary tube (2 mm i.d.), after which the products were directly injected into a gas chromatograph. Total-component analysis (i.e., analysis of the sugar, fatty acid, and sphingosine moieties) was possible after a 45-min chromatographic run. Time-course studies of the acid-catalyzed methanolysis of Galβ1-4GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-1Cer ganglioside at 80, 110, and 150°C showed that methanolysis was complete after 2 h at 110°C. Rates of methanolysis of individual components were compared and the release of the fatty acid moiety from the long-chain base was shown to be the slowest reaction. The methanolysis of all glycosidic bonds was complete in 0.5 h. Peracetylated methanolysis products were very stable over time and provided for good gas chromatographic detection of subnanomolar amounts of hexose, hexosamine, fatty acid, sialic acid, and long-chain sphingoid base components. Recoveries of fucose and N-acetylglucosamine were determined with reference samples of Fucα1-2Lac and lacto-N-fucosylpentaose II. Applications of the method are presented for the component analysis of a gift mixture of NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-1Cer ganglioside and NeuAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-Cer ganglioside and analysis of NeuAcα2-3Galβ1-4Glcβ1-1Cer isolated from human plasma.

Crystallisation of zeolitic molecular sieves: direct measurements of the growth behaviour of single crystals as a function of synthesis conditions

A technique is described for the milligram-scale synthesis of zeolitic molecular sieves in sealed capillary tubes. The method is ideally suited to the measurement of the linear growth rates of individual crystals. Spectroscopic characterisation of individual crystals within the sealed capillaries has been demonstrated. Detailed results for growth rates as a function of synthesis temperature and composition are presented for the isostructural zeolite ZSM-5 and silica molecular sieve silicalite (MFI framework). Length growth rates follow the Arrhenius law over a wide temperature range and have an apparent activation energy of 80 ± 3 kJ mol–1. Width growth rates show more complex behaviour and yield apparent activation energies in the range 62–81 kJ mol–1, depending upon the temperature range chosen and the synthesis conditions. The crystal aspect ratio is found to increase with increasing temperature and also when the crystals are growing under chemical constraint. The maximum value observed for the length growth rate {0.5 dl/dt=(1.9–2.0)× 10–2µm h–1} at 368 K agrees well with that found in earlier studies, and it is suggested that this represents a rate limit dependent upon the integration of growth species into the crystal surface. Addition of aluminium compounds, tetramethylammonium salts or ethanol reduces the growth rate, and a number of possible mechanisms are suggested. Some observations have also been made on zeolites EU-1 (EUO), ZSM-39 (MTN) and ZSM-48.

The micro-scale simulation of maturation: outline of a new technique and its potential applications

A micro-analytical technique has been developed for artifically maturing sedimentary organic matter and then quantifying the major components generated during this process in a single analytical step. Such a capability is well-suited for examining the compositional relationships between kerogens and petroleums, determining reaction kinetics and making precursor-product mass balances. According to this technique, simulations are made using sealed glass capillary tubes (heated here for three days at 300 °C, 330 °C and 350 °C). Pyrolysis products are then released directly into a combined thermovaporisation/pyrolysis-GC instrument and the major components of the entire C1 yto C35 range can be quantified in a single step using gas chromatography. Alkene yields are very low and pyrolysates are oil-like. This is exemplified by the fact that then−C9−C19 alkane distribution range of simulated whole petroleum chromatograms, from originally immatureGloeocapsamorpha-typz alginite A, resembles that seen in crude oils generated from this same kerogen type in nature. Sealed tube experiments usingBotryococcus type alginite A generated a »high wax« pyrolysate. The relative abundance ofn-alkanes in the C2−C32 range of many kerogen pyrolysates does not appear to change significantly despite an approximately fifteen-fold difference inn-alkane yield between the 300 °C and 350 °C experiments. Kerogens which are »paraffinic« oil-prone, and whose pyrolysates are very rich inn-alkanes might therefore generate petroleums in nature with a fixed wet gas (C2-C4) to oil (C5+) ratio. Alginite B in a Greenland shale is much more thermally labile than eitherBotryococcus-type alginite A orGloeocapsamorpha-type alginite A. The mass balance capabilities of the technique have been tested using Alum Shale kerogen. Two gas chromatograms were obtained, one for the free compounds generated during simulation, and one for the high temperature pyrolysate of the kerogen residue, for each heating experiment. Precursor-product relationships were qualitatively assessed, and dead carbon formation was quantified in this exercise.

Automated polymerase chain reaction in capillary tubes with hot air.

We describe a simple, compact, inexpensive thermal cycler that can be used for the polymerase chain reaction. Based on heat transfer with air to samples in sealed capillary tubes, the apparatus resembles a recirculating hair dryer. The temperature is regulated via thermocouple input to a programmable set-point process controller that provides proportional output to a solid state relay controlling a heating coil. For efficient cooling after the denaturation step, the controller activates a solenoid that opens a door to vent hot air and allows cool air to enter. Temperature-time profiles and amplification results approximate those obtained using water baths and microfuge tubes.

Luminescence and Raman Spectroscopic Characterization of Tyrosine Oxidized by Persulfate

To gain insight into the photochemistry of oxidized amino acids, we have measured chemiluminescence and Raman spectra of persulfate oxidized tyrosine (POT). Chemiluminescence kinetics were obtained from a sealed capillary tube containing a basic POT solution. Excitation at 488.0 nm and monitoring emission at 633 nm produced a first-order rise and a second-order decay in intensity to steady-state levels. We collected steady-state emission spectra from a flow system excited by argon-ion and helium-neon laser radiation. The full-width at half-maximum, position, and intensity of fluorescence were measured. The full-width at half-maximum (3600 cm−1) was independent of excitation wavelength. At higher energy excitation, the emission maximum was independent of excitation line. At lower energy excitation, the emission maximum varied with the excitation line. The emission intensity dropped by a factor of 6 as the excitation wavelength was varied from 465.8 nm to 514.5 nm. Luminescence was also observed upon excitation at 632.8 nm of this compound dissolved in DMSO. Raman data, obtained from solid polymer suspended in a KBr pellet (rotating sample cell, 488.0 nm excitation, S cm−1 bandpass), revealed broad bands at 1385 cm−1, 1590–1615 cm−1, and 2930 cm−1 superimposed upon a weak fluorescent background. Upon ultraviolet laser excitation (363.8 nm, 5 mW power on the sample, 5 cm−1 bandpass), a single broad band appeared, centered at 1300 cm−1. The difference from visible excitation implies that resonance enhancement from an ultraviolet absorbing chromophore occurred.

Potential sources of error during virus thermal inactivation

A review of virus thermal inactivation data published in the literature demonstrated variations in reported virus resistance. Examination of the methods used indicated that numerous studies were made by heat processing virus suspensions in test tubes. Duplication of some of the methods using milk suspensions of poliovirus 1 showed virus persistence after heating as a result of uneven temperature distribution inside the test tubes. Unless the containers (preferably sealed ampoules or capillary tubes) are completely submerged in the water bath and agitated vigorously, apparent virus persistence may be encountered.

The sealed capillary migration technique and thymocyte migration in vitro

The in vitro migration of thymocytes in sealed capillary tubes was influenced by cell number, temperature, cell viability and oxygen supply. Migration was successively slower over a 24 h period. There was no major influence of gravity or the degree of initial packing of the cells by centrifugation. Migration was inhibited by cytochalasin B, although the cells escaped from this effect after 8 h. A transient stimulatory effect on migration was seen after addition of serum or supernatants from cultured thymocytes. There was no effect of isoproterenol, theophylline or carbacholine. A paradoxic effect was obtained with high doese of some metabolic inhibitors, which produced increased migration in spite of cell death. The technique may be used for studies on lymphocyte migration in vitro, but care must be taken to exclude a toxic influence of added substances, and expected effects should preferably be looked for early after onset of migration, since cell viability is gradually decreased.