Reiss RA, Schwert DP, Ashworth AC. 1995. Field preservation of Coleoptera for molecular genetic analyses. Environmental Entomology 24: 716-719.
These authors tested five preservatives for nuclear and mitochondrial DNA preservation. Carabid beetles collected at Kuujjuarapik, Quebec, from shore-line debris on Hudson Bay were placed into either 95% ethanol, Carnoy fixative (3:1 methanol:acetic acid), DNA isolation buffer (half whole, half homogenized with a pestle), cryotubes immersed in liquid nitrogen followed by storage at -80°C, or glass vials containing tissue paper soaked in ethyl acetate; all treatments except liquid nitrogen were at room temperature. Ethanol is widely used as both a fixative and a preservative, though it does often distort or remove body colouration, and is considered a hazardous material for transport. Ethyl acetate is sometimes used by entomologists to preserve morphology, as it causes less distortion than other techniques such as drying (pinned) or ethanol.
Overall, cryopreservation performed best, producing excellent results when the specimens were subjected to DNA extraction and basic molecular techniques examining both nuclear and mitochondrial DNA. Ethanol also performed well, though specimens maintained in ethanol longer than about 6 weeks showed significant degradation. DNA isolation buffer also performed well, as long as specimens were very thoroughly ground and homogenized; intact specimens did not yield good results.
These authors recommend ethanol for remote field studies where the equipment associated with liquid nitrogen would be very difficult to transport and maintain, and DNA isolation buffer combined with thorough grinding where ethanol cannot be carried due to its hazardous nature.
Wednesday, August 13, 2008
Tuesday, August 12, 2008
Rogers 2001
Rogers DC. 2001. Revision of the nearctic Lepidurus (Notostraca). Journal of Crustacean Biology 21: 991-1006.
This author revised the tadpole shrimp (Crustacea: Branchiopoda: Notostraca) occurring in North America, with particular attention to the Western USA, and the description of a new species from northern California identified with the aid of DNA sequence data. The genus had previously been rather confused, with species descriptions and type locality often unclear or poorly described. Many of the morphological traits used in species identification in this group of crustaceans are known to be highly polymorphic within populations, and some are known to be frequently damaged or destroyed by predators or other factors, with subsequent regeneration if the animal survives.
In addition to describing the new species and providing a list of diagnostic traits for each of the six North American species, this author constructed a dichotomous tree for species identification, and tested the morphological effects of variation in diet and amputation / regeneration, removing some hypervariable traits from the diagnostic criteria. Biogeographic distributions focus on California and adjacent areas, with only a few collections from Canada.
This author revised the tadpole shrimp (Crustacea: Branchiopoda: Notostraca) occurring in North America, with particular attention to the Western USA, and the description of a new species from northern California identified with the aid of DNA sequence data. The genus had previously been rather confused, with species descriptions and type locality often unclear or poorly described. Many of the morphological traits used in species identification in this group of crustaceans are known to be highly polymorphic within populations, and some are known to be frequently damaged or destroyed by predators or other factors, with subsequent regeneration if the animal survives.
In addition to describing the new species and providing a list of diagnostic traits for each of the six North American species, this author constructed a dichotomous tree for species identification, and tested the morphological effects of variation in diet and amputation / regeneration, removing some hypervariable traits from the diagnostic criteria. Biogeographic distributions focus on California and adjacent areas, with only a few collections from Canada.
Michelutti et al. 2007
Michelutti N, Douglas MSV, Smol JP. 2007. Evaluating diatom community composition in the absence of marked limnological gradients in the high Arctic: a surface sediment calibration set from Cornwallis Island (Nunavut, Canada). Polar Biology 30: 1459-1473.
These authors measured a range of water chemistry and climatological variables in a large number of lakes and ponds on and near Cornwallis Island. This island is remarkably boring in its geology, with little in the way of relief or patterns of geological variation, and provides a sort of negative control for studies of Arctic limnology and the variables exerting the strongest control on diatom species assemblages.
Overall, this study supports the hypothesis that climate and water chemistry variables are the major determinants of diatom diversity in Arctic ponds and lakes. Cornwallis’ ponds and lakes varied little in altitude, latitude, temperature, or a large number of water chemistry variables, and varied little in diatom communities, too, when compared to the existing database of Arctic limnology and diatoms.
These authors measured a range of water chemistry and climatological variables in a large number of lakes and ponds on and near Cornwallis Island. This island is remarkably boring in its geology, with little in the way of relief or patterns of geological variation, and provides a sort of negative control for studies of Arctic limnology and the variables exerting the strongest control on diatom species assemblages.
Overall, this study supports the hypothesis that climate and water chemistry variables are the major determinants of diatom diversity in Arctic ponds and lakes. Cornwallis’ ponds and lakes varied little in altitude, latitude, temperature, or a large number of water chemistry variables, and varied little in diatom communities, too, when compared to the existing database of Arctic limnology and diatoms.
Monday, August 11, 2008
Maciorowski et al. 1997
Maciorowski Z, Veilleux C, Gibaud A, Bourgeois CA, Klijanienko J, Boenders J, Vielh P. 1997. Comparison of fixation procedures for fluorescent quantitation of DNA content using image cytometry. Cytometry 28: 123-129.
These authors evaluated four different fixatives for nuclear DNA quantitation of peripheral blood and breast cancer tumour cells, in the context of optimization of procedures for multi-colour, multi-parameter analyses, for example simultaneous DNA quantitation and FISH probe examination. The four treatments were “no fixative” (air-dried only), ethanol, ethanol / acetic acid, and paraformaldehyde / ethanol.
The fixation procedures were performed on cells already isolated from tissues, and already separated from other cells and substrates. Staining was with Propidium iodide, at concentrations comparable to current best practices recommendations, but the temperature and time of incubation seems to have varied for fixed vs. “no fixative” cells, and was never performed on ice.
Histograms of the relative DNA contents of various cells and treatments are presented, and none look completely useless. However, the authors state that the best treatment was with either paraformaldehyde or acetic acid with ethanol, depending on the other cellular parameters of interest.
These authors evaluated four different fixatives for nuclear DNA quantitation of peripheral blood and breast cancer tumour cells, in the context of optimization of procedures for multi-colour, multi-parameter analyses, for example simultaneous DNA quantitation and FISH probe examination. The four treatments were “no fixative” (air-dried only), ethanol, ethanol / acetic acid, and paraformaldehyde / ethanol.
The fixation procedures were performed on cells already isolated from tissues, and already separated from other cells and substrates. Staining was with Propidium iodide, at concentrations comparable to current best practices recommendations, but the temperature and time of incubation seems to have varied for fixed vs. “no fixative” cells, and was never performed on ice.
Histograms of the relative DNA contents of various cells and treatments are presented, and none look completely useless. However, the authors state that the best treatment was with either paraformaldehyde or acetic acid with ethanol, depending on the other cellular parameters of interest.
Carbonari et al. 2008
Carbonari M, Mancaniello D, Tedesco T, Fiorilli M. 2008. Flow acetone-staining technique: a highly efficient procedure for the simultaneous analysis of DNA content, cell morphology, and immunophenotype by flow cytometry. Cytometry Part A 73A: 168-174.
These authors have developed a cell-handling procedure that allows simultaneous examination of nuclear DNA content, cell morphology, and some aspects of membrane-associated immunoproteins in mammalian cells from culture. The foundation of this technique is the use of 80% acetone at 8°C as a fixative during the initial stages of cell processing for flow cytometry.
These authors tested a range of fixative agents and conditions, and found that while previous work had rejected acetone as a useful fixative, careful control of temperature at each stage of fixation and preparation allows the useful properties of acetone to outweigh its negative qualities for this type of work.
The application of this technique to my own work seems very limited, as I am not interested in immunochemistry, only in nuclear DNA content.
These authors have developed a cell-handling procedure that allows simultaneous examination of nuclear DNA content, cell morphology, and some aspects of membrane-associated immunoproteins in mammalian cells from culture. The foundation of this technique is the use of 80% acetone at 8°C as a fixative during the initial stages of cell processing for flow cytometry.
These authors tested a range of fixative agents and conditions, and found that while previous work had rejected acetone as a useful fixative, careful control of temperature at each stage of fixation and preparation allows the useful properties of acetone to outweigh its negative qualities for this type of work.
The application of this technique to my own work seems very limited, as I am not interested in immunochemistry, only in nuclear DNA content.
Rousselle et al. 1998
Rousselle C, Robert-Nicoud M, Ronot X. 1998. Flow cytometric analysis of DNA content of living and fixed cells: a comparative study using various fixatives. Histochemical Journal 30: 773-781.
These authors tested a range of fixatives for their effects on cell parameters measured in flow cytometry. Two different fluorochromes, Hoechst 33342 and Propidium iodide were tested, with a standard fixation procedure that varied little between treatments. Fixatives were derived from the relevant literature, including static rather than flow cytometric experiments.
All fixative treatments were applied after initial cell processing steps, including isolation of individual cells using a series of Phosphate-buffered-saline (PBS) washes and Trypsin. No detergent was used to permeabilize or remove cell membranes, though there is some discussion of the use of saponin as a permeabilizing agent in other studies.
Hoechst 33342 will penetrate living cells and stain the DNA in a stoichiometric fashion, allowing these authors to compare fixatives to living cells with this stain. Under these treatments, 68% or 70% ethanol (the figure disagrees with the text on this detail) provides excellent resolution of nuclear DNA content and cell cycle (G0/1 vs. S vs. G2 phases, ratio of G2 to G0/1). Other fixatives such as 85% methanol and Acetone also provide good results, with poor and inconsistent results from Carnoy and Boehm-Sprenger treatments.
The results for Propidium iodide were similar, though because Propidium iodide does not penetrate living cells, ethanol was used as the standard for comparison.
Cell size and cell granularity were measured under all treatments, using forward scatter for size and orthogonal (presumably equivalent to side-scatter) scatter for granularity. It is not clear in this paper what the precise definition of “granularity” might be, but it seems to be related to membrane permeabilization and DNA staining stoichiometry. In any case, all fixatives except 1% formaldehyde caused significant cell shrinkage, and all fixatives caused increased granularity. Though they do not discuss it, ethanol fixation produced relatively non-variable cell shrinkage compared to the other fixatives, shrinking cells to 0.74 +- 0.03 of living cell volume vs. 0.81 +- 0.05 for methanol.
This study did not find that alcohols increased cell aggregations, in contrast to an earlier study by Schimenti and Jacobberger (1992) in which 67%, 81% and 90% ethanol fixation caused cell clumping and increased debris. Alcohols increase cell membrane permeabilization, presumably allowing greater access to the DNA by stains. In this study, acetone treatment was basically not good compared to other fixatives.
This study did not examine the effects of long-term storage in fixatives such as ethanol. Fixatives were applied for 30 minutes, then washed away by centrifugation of cells to a pellet, removal of fixative supernatant, and resuspension in PBS. PBS seems to be the fluid of choice for many flow cytometry studies, and these authors note that separated cells can be stored for months in cold ethanol before flow cytometry, though they do not describe a procedure to replace ethanol with PBS or other solutions.
These authors tested a range of fixatives for their effects on cell parameters measured in flow cytometry. Two different fluorochromes, Hoechst 33342 and Propidium iodide were tested, with a standard fixation procedure that varied little between treatments. Fixatives were derived from the relevant literature, including static rather than flow cytometric experiments.
All fixative treatments were applied after initial cell processing steps, including isolation of individual cells using a series of Phosphate-buffered-saline (PBS) washes and Trypsin. No detergent was used to permeabilize or remove cell membranes, though there is some discussion of the use of saponin as a permeabilizing agent in other studies.
Hoechst 33342 will penetrate living cells and stain the DNA in a stoichiometric fashion, allowing these authors to compare fixatives to living cells with this stain. Under these treatments, 68% or 70% ethanol (the figure disagrees with the text on this detail) provides excellent resolution of nuclear DNA content and cell cycle (G0/1 vs. S vs. G2 phases, ratio of G2 to G0/1). Other fixatives such as 85% methanol and Acetone also provide good results, with poor and inconsistent results from Carnoy and Boehm-Sprenger treatments.
The results for Propidium iodide were similar, though because Propidium iodide does not penetrate living cells, ethanol was used as the standard for comparison.
Cell size and cell granularity were measured under all treatments, using forward scatter for size and orthogonal (presumably equivalent to side-scatter) scatter for granularity. It is not clear in this paper what the precise definition of “granularity” might be, but it seems to be related to membrane permeabilization and DNA staining stoichiometry. In any case, all fixatives except 1% formaldehyde caused significant cell shrinkage, and all fixatives caused increased granularity. Though they do not discuss it, ethanol fixation produced relatively non-variable cell shrinkage compared to the other fixatives, shrinking cells to 0.74 +- 0.03 of living cell volume vs. 0.81 +- 0.05 for methanol.
This study did not find that alcohols increased cell aggregations, in contrast to an earlier study by Schimenti and Jacobberger (1992) in which 67%, 81% and 90% ethanol fixation caused cell clumping and increased debris. Alcohols increase cell membrane permeabilization, presumably allowing greater access to the DNA by stains. In this study, acetone treatment was basically not good compared to other fixatives.
This study did not examine the effects of long-term storage in fixatives such as ethanol. Fixatives were applied for 30 minutes, then washed away by centrifugation of cells to a pellet, removal of fixative supernatant, and resuspension in PBS. PBS seems to be the fluid of choice for many flow cytometry studies, and these authors note that separated cells can be stored for months in cold ethanol before flow cytometry, though they do not describe a procedure to replace ethanol with PBS or other solutions.
Saturday, August 9, 2008
Martin et al. 2007
Martin GG, Oakes CT, Tousignant HR, Crabtree H, Yamakawa R. 2007. Structure and function of haemocytes in two marine gastropods, Megathura crenulata and Aplysia californica. Journal of Molluscan Studies 73: 355-365.
These authors characterised the blood cells of two species of gastropods chosen for their importance to other areas of research. Other studies have reported either one or two types of cells in mollusc blood, either with or without granules visible as electron-dense patches in electron microscopy.
This paper found only a single, generally granule-lacking type of cell in the blood of each species. These cells show phagocytic and aggregation abilities, but the blood does not clot. These cells appear to be general-purpose circulating amoeboid cells, capable of responding to stress, wounds, shell damage, infection, and other insults to the organism. This contributes to the circumstantial and weak evidence that molluscan haemocytes are probably not endopolyploid, as they apparently must be produced rapidly in large numbers by stem cell lineages to respond to changes to both the external and internal environments.
These authors characterised the blood cells of two species of gastropods chosen for their importance to other areas of research. Other studies have reported either one or two types of cells in mollusc blood, either with or without granules visible as electron-dense patches in electron microscopy.
This paper found only a single, generally granule-lacking type of cell in the blood of each species. These cells show phagocytic and aggregation abilities, but the blood does not clot. These cells appear to be general-purpose circulating amoeboid cells, capable of responding to stress, wounds, shell damage, infection, and other insults to the organism. This contributes to the circumstantial and weak evidence that molluscan haemocytes are probably not endopolyploid, as they apparently must be produced rapidly in large numbers by stem cell lineages to respond to changes to both the external and internal environments.
Erenpreisa et al. 2002
Erenpreisa J, Ivanov A, Cragg M, Selivanova G, Illidge T. 2002. Nuclear envelope-limited chromatin sheets are part of mitotic death. Histochemistry & Cell Biology 117: 243-255.
These authors investigated the structure and formation of nuclear envelope-limited chromatin sheets (ELCS) in two lines of mutant human cells commonly used in cancer studies. ELCS are membranous structures of unknown function; they are flat folds of inner nuclear envelope that project outwards from the nucleus, sometimes as far as the cytoplasm, and enfold chromatin. Larger projections containing chromatin are termed nuclear pockets (NP), and are strongly associated with some types of cancer such as leukaemia in mammals, but are also found less commonly in healthy tissues including spermatogonia.
Cells were treated with irradiation to induce double-stranded breaks and with a microtubule inhibitor to inhibit mitosis. Earlier investigations by these and other authors have developed the concept of “mitotic death”, which is a syndrome of cells including failed mitosis, the uncoupling of mitosis from DNA replication, delayed (but inevitable) apoptosis, and the formation of giant cells with high levels of aneuploidy and often endopolyploidy.
This paper demonstrates that mitotic death can be induced by quite different mechanisms, in this case by the failure of mitosis driven by microtubule depolymerisation and by a large number of double-stranded DNA breaks induced by radiation. In both cases, DNA repair was effected over considerable time, though apparently cells were unable to “repair” their aneuploidy.
Of particular interest to my work in this paper is their investigations of nuclear morphology. Cells were suspended in solution, then treated to prepare them for either nuclear DNA contents measures (by PI-flow cytometry and by Feulgen staining), light microscopy, or electron microscopy. For light microscopy cell- and nuclear-morphology investigation, these authors fixed cells in a 1:1 mixture of ethanol and acetone, hydrolysed the cells (and presumably freed the nuclei) in 0.1N HCl, and washed them in a poorly-described solution that may be either MacIlvain buffer (pH 5) or Toluidene blue stain in MacIlvain buffer. All of this occurred at 4°C. This was followed by a rinse in distilled water, and dehydration of slides carrying mounted cells in “warm tertiary butanol”. I have seen ethanol used as a fixative and preservative agent in histology studies before, but this represents the clearest description I have encountered of the process as applied specifically for investigations of cell nuclear morphology. There are some obvious similarities with our procedure for Feulgen staining, which helps to confirm that ethanol preservation may be reversible for whole-nuclear investigations.
These authors investigated the structure and formation of nuclear envelope-limited chromatin sheets (ELCS) in two lines of mutant human cells commonly used in cancer studies. ELCS are membranous structures of unknown function; they are flat folds of inner nuclear envelope that project outwards from the nucleus, sometimes as far as the cytoplasm, and enfold chromatin. Larger projections containing chromatin are termed nuclear pockets (NP), and are strongly associated with some types of cancer such as leukaemia in mammals, but are also found less commonly in healthy tissues including spermatogonia.
Cells were treated with irradiation to induce double-stranded breaks and with a microtubule inhibitor to inhibit mitosis. Earlier investigations by these and other authors have developed the concept of “mitotic death”, which is a syndrome of cells including failed mitosis, the uncoupling of mitosis from DNA replication, delayed (but inevitable) apoptosis, and the formation of giant cells with high levels of aneuploidy and often endopolyploidy.
This paper demonstrates that mitotic death can be induced by quite different mechanisms, in this case by the failure of mitosis driven by microtubule depolymerisation and by a large number of double-stranded DNA breaks induced by radiation. In both cases, DNA repair was effected over considerable time, though apparently cells were unable to “repair” their aneuploidy.
Of particular interest to my work in this paper is their investigations of nuclear morphology. Cells were suspended in solution, then treated to prepare them for either nuclear DNA contents measures (by PI-flow cytometry and by Feulgen staining), light microscopy, or electron microscopy. For light microscopy cell- and nuclear-morphology investigation, these authors fixed cells in a 1:1 mixture of ethanol and acetone, hydrolysed the cells (and presumably freed the nuclei) in 0.1N HCl, and washed them in a poorly-described solution that may be either MacIlvain buffer (pH 5) or Toluidene blue stain in MacIlvain buffer. All of this occurred at 4°C. This was followed by a rinse in distilled water, and dehydration of slides carrying mounted cells in “warm tertiary butanol”. I have seen ethanol used as a fixative and preservative agent in histology studies before, but this represents the clearest description I have encountered of the process as applied specifically for investigations of cell nuclear morphology. There are some obvious similarities with our procedure for Feulgen staining, which helps to confirm that ethanol preservation may be reversible for whole-nuclear investigations.
Friday, August 8, 2008
Vermeij and Roopnarine 2008
Vermeij GJ, Roopnarine PD. 2008. The coming arctic invasion. Science 321: 780-781.
In this short “perspectives” article, these authors describe the historical biogeography of the North Pacific, near shore Arctic, and North Atlantic oceans, in the context of predicted patterns of climate warming over the next fifty years. In general, the climate of these areas is likely to become similar to that during the mid-Pliocene, about 3.5 million years ago. During the mid-Pliocene, large numbers of Pacific lineages of marine animals, especially molluscs, successfully colonized the Arctic ocean and established populations in the North Atlantic. While cores from the Arctic Ocean seabed suggest permanent ice-cover at the highest latitudes, there is some evidence to suggest the near shore Arctic ocean included regions that were largely ice-free. This probably resulted in much higher productivity at these locations, similar to the high productivity of the Bering Sea, and allowing large-bodied, planktotrophic animals to disperse northwards and eastwards in the generally north-east flowing currents. This pattern is expected to repeat under global warming, and because Pacific lineages are generally ecologically quite distinct from extant Atlantic species, the North Atlantic should see increased biodiversity overall. Colonization in the opposite direction, of Atlantic lineages into the North Pacific, is considered unlikely due to generally unfavourable water currents and the intensely competitive and predatory biotic environment of the Bering Sea.
In this short “perspectives” article, these authors describe the historical biogeography of the North Pacific, near shore Arctic, and North Atlantic oceans, in the context of predicted patterns of climate warming over the next fifty years. In general, the climate of these areas is likely to become similar to that during the mid-Pliocene, about 3.5 million years ago. During the mid-Pliocene, large numbers of Pacific lineages of marine animals, especially molluscs, successfully colonized the Arctic ocean and established populations in the North Atlantic. While cores from the Arctic Ocean seabed suggest permanent ice-cover at the highest latitudes, there is some evidence to suggest the near shore Arctic ocean included regions that were largely ice-free. This probably resulted in much higher productivity at these locations, similar to the high productivity of the Bering Sea, and allowing large-bodied, planktotrophic animals to disperse northwards and eastwards in the generally north-east flowing currents. This pattern is expected to repeat under global warming, and because Pacific lineages are generally ecologically quite distinct from extant Atlantic species, the North Atlantic should see increased biodiversity overall. Colonization in the opposite direction, of Atlantic lineages into the North Pacific, is considered unlikely due to generally unfavourable water currents and the intensely competitive and predatory biotic environment of the Bering Sea.
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