Lacelle et al. 2010

Lacelle D, Radtke K, Clark ID, Fisher D, Lauriol B, Utting N, Whyte LG. 2011. Geomicrobiology and occluded O2-CO2-Ar gas analyses provide evidence of microbial respiration in ancient terrestrial ground ice. Earth and Planetary Science Letters 306: 46-54.

These authors compared the gas composition of ice from massive ground ice bodies (e.g. 30 000-year-old buried snowbanks) to atmospheric gas concentrations and gas contents from glacial ice. They also cultivated some microorganisms collected from within the ice, and used some non-culture-dependent methods to examine the diversity of those organisms.

Loss of O2, as determined by comparison of the ratio of O2 to Ar in samples, as well as changes to the 13C-CO2 contents indicate heterotrophic microbial activity in the ice, most likely by organisms living in high-salt brine channels in cracks in the ice, using dissolved organic carbon as both energy and C source.

This paper provides an overview of methods for extracting and measuring the gases trapped in microscopic bubbles in permafrost and ground ice, as well as interesting findings about microbial activities.

Palmer et al. 2010

Palmer K, Drake HL, Horn MA. 2010. Association of novel and highly diverse acid-tolerant denitrifiers with N2O fluxes of an acidic fen. Applied and Enironmental Microbiology 76: 1125-1134.

These authors examined soils from an acidic fen in southern Germany, and discovered novel denitrifiers that are apparently adapted to local conditions and contribute to the cycling of nitrogen within the fen. Methods employed included measurement of soil parameters, microcosms to examine denitrification rates (both total denitrification and net production / consumption of N2O), cell counts of cultured organisms, and phylogenetic analysis using narG and nosZ sequences and RFLP.

Tuomivirta et al. 2009

Tuomivirta TT, Yrjälä K, Frize H. 2009. Quantitative PCR of pmoA using a novel reverse primer correlates with potential methane oxidation in Finnish Fen. Research in Microbiology 160: 751-756.

These authors developed novel primers for studying the methanotrophs of Finnish peatland fens. The existing primer pairs widely used to study methanotrophs, based on the gene pmoA, did not consistently amplify useful products in PCR using template DNA from these fens.

The novel primer pair was tested on 114 samples from two Finnish fens, representing the northern and southern peatlands of Finland. While A189f/A682r failed to provide useful products, the new primer A6821r in conjunction with A189f did produce strong bands. In qPCR, these primers produced results correlated with measured methane oxidation potential, further supporting their utility in these systems.

Schmidt et al. 2008

Schmidt SK, Reed SC, Nemergut DR, Grandy AS, Cleveland CC, Weintraub MN, Hill AW, Costellow EK, Meyer AF, Neff JC, Martin AM. 2008. The earliest stages of ecosystem succession in high-elevation (5000 metres above sea level), recently deglaciated soils. Proceedings of the Royal Society of London, Series B 275: 2793-2802.

These authors describe the microbial community and soil parameters of a chronosequence at the foreground of a receding glacier high in the Peruvian Andes. From a combination of aerial photography and previous work at this site, a series of sites of soils of increasing ages from zero to 79 years old was established. No surface plants, even lichens, are present on any of these new soils, and soil nutrient levels (carbon, nitrogen) are very low; the only organisms present are microorganisms.

Two previous hypotheses had been proposed to explain the dynamics of very early primary succession on new soil. Organic matter has been observed to accumulate slowly in new soils; the source of this material is either aeolian deposits (i.e. wind-borne plant detritus and pollen) or in-situ fixation of CO2 and N2. These are not mutually exclusive hypotheses, but the relative contributions of each are explored in this study.

The methods used here cover an extensive list of soil parameters. Three sets of soil samples were collected: for microbiological analysis, N-fixation measurement, and all other chemical analyses. The other chemical analyses include photosynthetic pigment extraction, soil total and mineral nitrogen, pyrolysis for identifying sources of carbon compounds (i.e. microbial-autotroph, microbial-heterotroph, plant), enzyme assays for common and informative microbial enzymes, and soil stability analysis of the resistance of these new soils to erosion forces such as water runoff.

These authors focused on the cyanobacterial fraction of the microbial community in this study; some details of other components of the biota are described in an earlier paper, Nemergut et al. (2007). Cyanobacteria are autotrophs also capable of fixing atmospheric nitrogen, thus they are ideal primary colonizers of new soil as they require little more than a source of moisture and air. Analysis of the community included the use of the P-test (Martin 2002); note that as in Nemergut et al. (2007), he is one of the authors of this study. The analytical approach is very similar to that employed in the earlier study, with a comparison of discovered sequences to published sequences from around the world. In this study, cyanobacterial sequences from zero and 4-year-old soils were similar to sequences from an extremely broad sample of habitats, including Antarctic lake ice, marine subseafloor sediments, urban aerosols, forest soils, and oil-polluted soils.

The soil chronosequence showed a clear pattern of stages of primary succession at every level of analysis. The soil microbial community became both more abundant and more diverse through time, soil nutrients increased, the chemical environment included increasing amounts and diversity of complex organic molecules, key enzyme pathways became established, and soil stability increased as soils aged. N-fixation showed a peak, with increasing N-fixation activity from the zero to 4-year-old soils (by two orders of magnitude), then declining by about half in the 79-year-old soils. This mirrors and precedes a widely-observed pattern in plant primary succession, in which nitrogen-fixing plants are among the first colonizers, but decline in abundance at later stages of succession. Enzyme and organic molecule patterns were consistent with a total absence of heterotrophs in the extremely young soils, increasing occurrence of organisms capable of decomposing plant matter in the 4-year-old soils, and a molecular ecology qualitatively similar to a mature plant-associated soil in the 79-year-old soil.

The list of procedures and level of detail of analysis in this paper is impressive. Many, though certainly not all, of these techniques will be models for my own work, especially in the summer of 2010. The molecular-diversity techniques pioneered by Martin (Martin 2002, Nemergut et al. 2007, this paper) as well as the techniques of analyzing soil pigments and soil nutrients are all very interesting.

Martin and Rygiewicz 2005

Martin KJ, Rygiewicz PT. 2005. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology 5:28.

These authors designed new primers for PCR and related molecular biology investigations of soil fungi, especially mycorrhizae. These are very diverse organisms, and, because of the commonalities between fungal and plant DNA, studies of fungal samples closely entwined with plant tissue as in root-associated mycorrhizae can be very complex. The new primers were designed to produce a range of PCR products suitable for techniques such as qPCR, Length-Heterogeneity PCR (LH-PCR) and T-RFLP analyses.

The primers as a suite were designed around a nested approach, with new outer primers amplifying a long DNA sequence of approximately 1000bp, and later primer pairs amplifying regions within that long sequence. Most inner primer pairs generate products of approximately 500bp length.

These authors also used a different DNA extraction method, based on xanthogenate and Tween (X/T) that involves little to no tissue grinding, compared to the standard method based on CTAB. The X/T method preferentially extracts fungal DNA from cells on the outside of particles, for example fungal cells not penetrating plant roots. This reduces the amount of plant DNA and associated plant-derived compounds in resulting extracts. Combining the two techniques may allow for some interesting studies of fungal micro-ecology.

This paper’s novel primers should be more specific and more useful to my own qPCR studies of Arctic soil microbes, including soil fungi. The methods section detailing some of the decisions made and considerations involved in primer design will also be useful.

Dandie et al. 2007

Dandie CE, Miller MN, Burton DL, Zebarth BJ, Trevors JT, Goyer C. 2007. Nitric oxide reductase-targeted real-time PCR quantification of denitrifier populations in soil. Applied and Environmental Microbiology 73: 4250-4258.

These authors examined the responses of two major components of the denitrifying bacteria fraction of soil bacteria to the addition of labile carbon (glucose) under denitrifying conditions. Denitrification is presented as a four-step process, with enzymes responsible for shuttling nitrate to N2 via nitrite, nitric oxide, and nitrous oxide. In this study, one of the enzymes responsible for the reduction of NO to N2O, cNOR, was examined using primers optimized for two different groups of denitrifying bacteria. This gene is found only in denitrifiers, unlike another enzyme, qNOR, found in many microorganisms and associated with detoxification, rather than utilization, of dangerous nitric oxide.

Primers for qPCR are presented in a table. Specific primers for the two variants of cNOR were developed in this study for use with SYBR green-based qPCR. 16s rRNA sequences were also studied, to examine the total population of soil bacteria; for these qPCR reactions, the TaqMan primers-plus-probe system was used, based on oligonucleotides published by Suzuki et al. 2000.

Two experiments were carried out. In the first, a preliminary experiment to establish the utility of qPCR in this area was based on inoculating soils with cultures of bacteria of known cell density, followed by qPCR evaluation of those soils. Under most conditions qPCR performed well, though at low cell densities of some genera of bacteria the signal was not distinguishable from the background noise also associated with sterilized soil. The second experiment forms the main body of work of this paper, and is an examination of the population dynamics of soil bacteria, divided into the hierarchical categories “denitrifiers” and “all bacteria”, under denitrifying conditions and with varying levels of added labile carbon in the form of glucose solutions in distilled water. In the second experiment, soil nitrate was maintained at a high level, to ensure sufficient raw material for detectable denitrification activity. As N2O accumulation was one of the measures of activity, nitrous oxide reductase activity that would reduce N2O to N2 was inhibited by maintaining an atmosphere of 10% acetylene in culture jars. Soils were maintained at 70% WFPS to encourage denitrification.

Total microbial biomass was also measured, using the CHCl3 fumigation-extraction technique. While cNOR sequences are almost certainly restricted to one copy per genome, 16s rRNA sequences may range in copy number up to 15 per genome, thus estimates of bacterial populations by qPCR of 16s may have a large error associated with it. Fumigation-extraction captures all carbon associated with cells, thus contributions by archaea and fungi will not be found by molecular methods such as 16s qPCR that are specific to bacteria. However, in this study, estimates of total bacterial population by the two methods were well correlated, with r2 = 0.69.

Denitrification occurred in this study. Soils treated with additional glucose showed greater depletion of nitrate, as expected when denitrifiers increase their activity in response to a food supply and conditions already favour denitrification. These authors provide two possible mechanisms, non-mutually-exclusive, that could lead to increased denitrification activity under added glucose. First, the population of denitrifiers could expand, through both additional cell replication and activation of dormant cells. This would increase the proportion of the bacterial population composed of denitrifiers. Second, the total population of soil organisms could increase, leading to increased respiration, a decrease in oxygenation, and establishment of anaerobic conditions more favourable for denitrification. This would not necessarily change the proportion of the population composed of denitrifiers. In this study, denitrifiers increased their proportion of the population as measured by comparative qPCR from less than 1% to about 2.4% of cell numbers.

This change in population components is central to the approach using qPCR advocated in this paper. As these authors state:

“Although absolute numbers may not be achievable, gross differences and changes in population size are still detectable. The differences observed between the two denitrifier populations studied are then real differences in the responses of these populations to the conditions tested.”

This general approach of examining relative changes in populations is applicable to a very wide array of studies of environmental microbiology, including my own planned studies in which the environmental factor under examination is biogeographical (i.e. latitude) and the functional diversity response is in terms of greenhouse gas cycling."

This paper is of great value to my studies. The qPCR methods are directly applicable, for example the primers presented here will be useful if I decide to examine multiple components of the denitrification pathway. The approach, as described above, is also useful. And the reference list is composed almost entirely of papers I am surprised I have not yet found in my literature searches.

Klotz and Stein 2008

Klotz MG, Stein LY. 2008. Nitrifier genomics and evolution of the nitrogen cycle. FEMS Microbiology Letters 278: 146-156.

These authors review the role of nitrifying microorganisms in the current nitrogen cycle, and their evolution and the emergence of biological nitrogen cycles in early Earth history. The current global nitrogen cycle has changed considerably in the past decades, due to the large increase in nitrogen in the cycle due to human activities. The early-Earth nitrogen cycle was probably mostly driven by abiotic processes. After the development of an oxygen-rich atmosphere, nitrogen cycling was almost entirely biotic, with most key processes driven largely or entirely by bacteria and archaea. In the last few decades, the anthropogenic abiotic processes of fertilizer production and fossil-fuel combustion combined with increased cultivation of N2-fixing crops, has transformed the global nitrogen cycle.

As presented in this paper, there are two lobes to the global nitrogen cycle. N2 gas in the atmosphere is fixed to NH3, by nitrogenase in bacteria and archaea, by the Haber-Bosch industrial process, and (in small quantities) by hydrothermal vents. The process of nitrification converts this ammonia to nitrite/nitrate. Nitrite/nitrate are returned to the atmosphere as N2 through denitrification, with production of N2O under weakly anaerobic conditions. The other lobe of the cycle is a “short circuit” that avoids the atmospheric N2 pool and cycles nitrite/nitrate back to ammonia through the processes of ammonification and through production and decomposition of organic matter containing nitrogen. There is another, minor short circuit, as “anammox”, anaerobic ammonia oxidation, returns ammonia to N2 directly.

This “mini review” focuses on the nitrification portion of the cycle. The first step, oxidation of ammonia to hydroxylamine (NH2OH) is carried out by ammonia oxidizing bacteria, abbreviated AOB. There are many acronyms in this paper, reflecting the many acronyms in the existing literature regarding global biogeochemical cycles. NOB are nitrite oxidizing bacteria, and they take the oxidized products of AOB, especially nitrite through to nitrate. Anammox bacteria, on the other hand, may run the same net process of NH3 to NO3- directly, without collaboration with other cells.

The discussion of the plausible evolutionary scenarios in this paper is interesting but not particularly relevant to my current research. This discussion focuses on the relative timing of major events, such as the emergence of nitrification, complete and incomplete denitrification, an oxygenated atmosphere, and nitrogen fixation. These factors interact with each other, creating conditions favourable or not to the evolution of each other and of possible detail shifts within.

The description of the role of hydroxylamine produced by early nitrifiers in stimulating evolution of metabolic pathways responsible for its detoxification initially reads as speculation, but a long and detailed description of the ways in which the various components of those metabolic detox enzymes and pathways function provides plenty of support for the arguments. One aspect of this discussion is that some enzymes are currently misclassified, and that very similar enzymes in different organisms have different names reflecting different ultimate functions rather than the usual (and preferred) enzyme naming scheme that reflects proximate function.

In the discussion concerned with anthropogenic climate change and nitrogen dynamics, especially in soils and ocean waters, interactions with methane are briefly considered. This is based on the observation that methanotrophs are often also ammonia-oxidizers, operating under a budget of consumption of both molecules that shifts as ammonia from fertilizer is added to the system.
Of greatest relevance to my current work is the section describing the gene ncyA. This encodes an enzyme (nitrosocyanin) involved in the pathway from ammonia to nitrite, and has only been found in AOB to date, as opposed to NOB, anammox, or heterotrophs; it seems to be involved in the chemistry of obligate chemolithotrophy as expressed by AOB. It seems likely the enzyme binds and reduces NO, a highly toxic intermediate in ammonia oxidation. The regulatory region adjacent to the gene is also suggestive of roles in this metabolic pathway, and regulation is linked to concentration of various nitrogen-with-oxygen compounds.

This review is very useful to my current research. This paper and the major references in it will be key to constructing a diagram of the complex nitrogen transformations occurring in soils, which will allow targeted hypothesis generation and testing regarding the communities and processes in the soils I am studying.

Li et al. 2009

Li X-R, Du B, Fu H-X, Wang R-F, Shi J-H, Wang Y, Jetten MSM, Quan ZX. 2009. The bacterial diversity in an anaerobic ammonium-oxidizing (anammox) reactor community. Systematic and Applied Microbiology 32: 278-289.

These authors studied the bacterial community that developed inside a bioreactor running on sewage sludge under anaerobic conditions. Like Lim et al. (2008), the main focus of this study was in the applications of ammonia-oxidizing bacteria (AOB) to water treatment facilities. The expected chemistry of anaerobic ammonia oxidation catalyzed by microorganisms (“anammox”) includes the use of nitrite as the electron acceptor in a near-one-to-one ratio with the consumption of ammonia or ammonium. The energy derived from this process is used by the cell to fix CO2, thus making these organisms autotrophs. This alters the underlying stoichiometry slightly, as some nitrite is diverted to CO2 fixation rather than ammonia oxidation.

The study of anaerobic AOB is still quite new, with the five described genera of such organisms all named with “Candidatus” prefixes, indicating recent species descriptions. All are in one group (taxonomic level unknown), the Brocadiales, within the phylum Planctomycetes. Aerobic AOB are in other groups, and include some species within the genus Nitrosomonas in the Beta-Proteobacteria that are capable of limited ammonia-oxidizing activity under anaerobic conditions, and can apparently survive long periods without oxygen.

These authors did not develop novel primers for PCR or qPCR in this study. Instead, they used published primer sets; I gather they did not use the TaqMan double-dye system for qPCR, as no mention of probes is made. The target genomic sequences were portions of the 16s rRNA gene, using E. coli as a standard. Oddly, the overall procedure included normal PCR, followed by cloning and insertion into plasmids, followed by qPCR of plasmid DNA containing the 16s sequences. It is unclear to me exactly why this was done, though later in the paper there are a few sequence-based phylogenetic trees that might have been based on sequences derived from this cloning procedure. In any case, the qPCR did provide informative results regarding the composition of bacterial groups within the reactor.

Of the sequences identified, the great majority were unlike cultivated organisms, highlighting the utility of these techniques in studying environmental samples. AnAOB produced approximately 16% of sequences, with aerobic AOB less than 1%. Non-AOB in three phyla constituted the majority of sequences, including 38% Chlorobi, 21% Chloroflexi, and 7% Bacteriodetes. These are filamentous heterotrophic bacteria, and appear to be closely associated with the granules that formed in the reactor solution after a few months. These authors suggest further research on the ecophysiology of these groups to answer questions regarding energy and material cycles within these systems.

In addition to 16s sequences, the hzo locus was also studied. This is a gene that produces an enzyme that catalyzes the oxidation of hydrazine (rocket fuel; N2H4) to N2 gas. No mention is made of the possibilities for N2O production or consumption in this process. The gene is restricted to AnAOB only, or at least that is the inference based on the observation that hydrazine is a unique intermediate molecule of the anammox process.

This study provides a useful example of the combination of qPCR and molecular-phylogenetic approaches in studying a microbiological system. Applied together, the two approaches allow the extraction of useful information regarding taxonomic diversity, both richness and evenness, among functional groups of organisms.

Kostina et al. 1994

Kostina NV, Stepanov AL, Umarov MM. 1994. Study of the complex of nitrous oxide-reducing microorganisms in the soil. Eurasian Soil Science 26: 81-87.

These authors extracted microorganisms from a range of soils apparently collected from various places in Russia and produced cultures of organisms capable of reducing nitrous oxide. The only nitrogen source in culture vials was N2O, and conditions were rendered anaerobic by flushing with argon gas.

Most cultures gradually lost their ability to reduce N2O, especially mixed-species cultures. A few pure strains were isolated that did not show this loss, and maintained high levels of activity in storage.

Pseudomonas spp. and Bacillus spp. contributed the vast majority of N2O-reducing activity in all soils, with other groups including Aeromonas spp., Micrococcus spp., Flavobacterium spp., Erwinia spp., and an organism identified as “similar to Corynebacterium” also showing some activity. Neither actinomycetes nor eukaryotes were found in any of the cultures capable of N2O-reduction, indicating this is a physiological process not possessed by these organisms.

This is an English translation of a paper that was probably originally in Russian: Pochvovedeniye 1993 25: 72-76.

Wagner et al. 2009

Wagner D, Kobabe S, Liebner S. 2009. Bacterial community structure and carbon turnover in permafrost-affected soils of the Lena Delta, northeastern Siberia. Canadian Journal of Microbiology 55: 73-83.

These authors examined the microbial communities at two depth bands (near-surface and near-permafrost) in low-centred tundra polygons at the vast permafrost wetland of the delta of the Lena River. The delta covers more than 60 000 km^2, and much of it appears to be a reserve or national park of Russia. The CAVM (Walker et al. 2002) describes most of the delta as vegetation type W2, sedge, moss, dwarf-shrub wetland, and satellite images from Google maps shows very extensive lake and pond coverage of the landscape. In short, it’s pretty wet, and generally cold.

The general finding of this paper is that while near-surface communities include a wide diversity of aerobic and facultatively-anaerobic bacteria, the deeper, colder, anaerobic portions of the soil contain almost no aerobes, and are instead dominated by “fermenting” species capable of decomposing recalcitrant organic carbon molecules under negative-redox conditions. There is a sharp temperature gradient, which combined with the poorer quality of carbon, the lack of oxygen and negative redox conditions, and the general water saturation at depth creates conditions near the permafrost suitable only for the slow microbial metabolisms. None of this is particularly surprising, but the observation of decreased biodiversity with water saturation does suggest the worrying possibility that increased water in this system, driven by melting permafrost and climate change (particularly upstream in the long and North-flowing Lena) could drive these microbial communities to lose some “physiological skills” such as the ability to oxidize methane, a metabolic pathway possessed only by some aerobic prokaryotes.

This paper is quite important to my own work, I think. Besides emphasizing the role of water content in structuring soil chemical and especially biological conditions, the description of the methods used to measure microbial biodiversity should be useful. However, while the BIOLOG plates seem interesting, the results of this technique are not at all well explained in this paper. I do not know what is indicated by the relationship shown in Figure 3, for example, of changes in colour development associated with carbon turnover of various categories of organic substrates. Several of the figures are simple plots of principal component analysis (PCA), literally just PC1 vs. PC2 with some outlines drawn around some clusters. I’m sure there is more of interest in this paper besides the coarse outline of biodiversity differences in communities, but without a more thorough explanation of the nearly-raw data I cannot see it.

Wrage et al. 2004

Wrage N, Lauf J, del Prado A, Pinto M, Pietrzak S, Yamulki S, Oenema O, Gebauer G. 2004. Distinguishing sources of N2O in European grasslands by stable isotope analysis. Rapid Communications in Mass Spectrometry 18: 1201-1207.

These authors used the signature ratios of stable isotopes of oxygen and nitrogen in a range of soil chemicals and microbial metabolic pathways to identify the source of N2O produced in grasslands monitored as part of a long-term greenhouse gas international experiment. There are 3 known pathways to N2O production: nitrification, in which N2O is produced as a by-product of ammonium oxidation to nitrate, nitrifier denitrification, in which nitrifying organisms reduce nitrite to dinitrogen gas via N2O, especially under anaerobic conditions, and denitrification, in which nitrate is reduced to dinitrogen gas via N2O by denitrifying organisms.

Previous studies had often used acetylene to inhibit N2O production, but this has been found to be unreliable. In contrast, the stable isotope approach used here was able to detect both N2O production and consumption even when reservoirs of N2O were very low. Nitrification was the most important N-transforming process found in these systems, with most N2O produced probably from reduction pathways.

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.

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.

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.

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.

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.