Seasonality of vesicular-arbuscular mycorrhizae in sedges in a semi-arid tropical grassland

Vesicular-arbuscular mycorrhizal (VAM) colonization and spore numbers in the rhizosphere of Cyperus iria L. and C. rotundus L., growing in a semi-arid tropical grassland, was studied during the 1993 and 1994 monsoons. In addition, climatic and chemical properties of the soils were determined in order to investigate their influence on mycorrhizal variables. VAM fungal association in the sedges was confirmed by plant- and root-trap culture techniques. The soil nutrients exhibited seasonal variations, but were highly variable between years. Intercellular hyphae and vesicles with occasional intraradical spores characterized mycorrhizal association in sedges. Dark septate fungi also colonized roots of sedges. Temporal variations in mycorrhizal colonization and spore numbers occurred, indicating seasonality. However, the patterns of mycorrhizal colonization and spore numbers were different during both the years. The VAM fungal structures observed were intercellular hyphae and vesicles. Changes in the proportion of root length with VAM structures, total colonization levels and spore numbers were related to climatic and edaphic factors. However, the intensity of influence of climatic and soil factors on VAM tended to vary with sedge species.     

Laboratory Hints from the Literature: A Department Devoted to Abstracts of Books and Papers from Other Journals Dealing with Stains and Microscopic Technic in General

A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy.     

Transformed soybean (Glycine max) roots as a tool for the study of the arbuscular mycorrhizal symbiosis

Ri T-DNA transformed roots have been used effectively in studying the interaction between various plant hosts and arbuscular mycorrhizal (AM) fungi. We investigated the in vitro monoxenic symbiosis between the AM fungus Glomus intraradices and transformed soybean roots (TSRs). Comparisons were made between TSR system and plants of the same genotype. The extraradical fungal structures generated in vitro culture showed normal development. Straight runner hyphae branched into short simple branched absorbing structures and spores were initiated. AM symbiosis was confirmed by the presence of arbuscules and vesicles in cortical cells of the TSRs. The frequency of intraradical colonization in TSRs was higher than in plants grown in soil, whereas the intensity values of intraradical colonization in TSR cultures were similar to those in whole plants. These results show that TSR cultures were able to support the growth and characteristic development of G. intraradices.     

Parasitic and mutualistic associations between a mycorrhizal fungus and soybean: The effect of phosphorus on host plant-endophyte interactions

Soybean [ Glycine max (L.) Merr. cv. Kent] plants were colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe in pot cultures using an inert medium and a nutrient solution. Phosphorus was provided initially as 0, 25,50, 100 or 200 mg hydroxyapatite [HAP, Ca₁₀(PO₄)₆(OH)₂] per pot. Under the low (0 mg HAP) and high (100 and 200 mg HAP) P regimes, VAM plants showed 20, 25 and 38% growth retardation, respectively, relative to non-colonized controls. At 50 mg HAP, VAM plant growth was significantly enhanced (14%). Dry weight and P content of both VAM and control plants increased with increased P availability throughout the HAP gradient. Intraradical VAM fungal biomass increased linearly with increasing P availability. Extraradical VAM fungal biomass was smaller than the intraradical component of the fungus at the lowest and highest levels of P addition in the growth medium. The ratio of extra- to intraradical mycelium, a suggested index of VAM fungal effectiveness, was greatest for the 50 mg HAP treatment, coinciding with growth enhancement of the host plant. This enhanced growth of the host at an intermediate P level was apparently a result of increased P uptake by the endophyte.     

An improved method for clearing and staining free-hand sections and whole-mount samples

BACKGROUND AND AIMS: Free-hand sectioning of living plant tissues allows fast microscopic observation of internal structures. The aim of this study was to improve the quality of preparations from roots with suberized cell walls. A whole-mount procedure that enables visualization of exo- and endodermal cells along the root axis was also established. METHODS: Free-hand sections were cleared with lactic acid saturated with chloral hydrate, and observed with or without post-staining in toluidine blue O or aniline blue. Both white light and UV light were used for observation. Lactic acid was also used as a solvent for berberine, and fluorol yellow for clearing and staining the samples used for suberin observation. This procedure was also applied to whole-mount roots with suberized celllayers. KEY RESULTS: Clearing of sections results in good image quality to observe the tissue structure and cell walls compared with non-cleared sections. The use of lactic acid as a solvent for fluorol yellow proved superior to previously used solvents such as polyethylene glycol-glycerol. Clearing and fluorescence staining of thin roots such as those of Arabidopsis thaliana were successful for suberin visualization in endodermal cells within whole-mount roots. For thicker roots, such as those of maize, sorghum or tea, this procedure could be used for visualizing the exodermis in a longitudinal view. Clearing and staining of peeled maize root segments enabled observation of endodermal cell walls. CONCLUSIONS: The clearing procedure using lactic acid improves the quality of images from free-hand sections and clearings. This method enhances the study of plant root anatomy, in particular the histological development and changes of cell walls, when used in combination with fluorescence microscopy.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

Effect of the VAM fungus Glomus sp. on the growth and yield of soybean inoculated with Bradyrhizobium japonicum

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N. During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did not differ in the presence of either strain. VAM positively affected biomass production, foliar concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass) and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of rhizobia.     

Interactions of Vesicular-Arbuscular Mycorrhizal Fungi,Phosphorus, and Heterodera glycines on Soybean

Effects of vesicular-arbuscular mycorrhizal (VAM) fungi and soil phosphorus (P) fertility on parasitism of soybean cultivars Bragg and Wright by soybean cyst nematode (SCN) were investigated in field micropiot and greenhouse experiments. VAM fungi increased height of both cultivars and yield of Wright in microplot studies in 1986 and 1987. Conversely, yield of mycorrhizal and nonmycorrhizal plants of both cultivars was suppressed by SCN. Soil population densities of SCN were unaffected by VAM fungi in 1986 but were greater in microplots infested with VAM fungi than in control microplots in 1987. Growth of Wright soybean was stimulated by VAM fungi and suppressed by SCN in greenhouse experiments. The effect of VAM fungi on SCN varied with time. Numbers of SCN in roots and soil were decreased by VAM fungi by as much as 73% at the highest SCN inoculum level through 49 days after planting. Later, however, SCN numbers were usually comparable on mycorrhizal and nonmycorrhizal plants. Soil P fertility generally had no effect on SCN. Results of a split-root experiment indicated that VAM fungal suppression of SCN was not systemic.     

Analysis of the kinetics of natural killer cell activity in mice during an active infection with Pseudomonas aeruginosa

The present study was designed to determine the effect of Pseudomonas aeruginosa (PA) infection on the activity of natural killer (NK) cells in mice. Following a sublethal injection of the bacterium, increased NK cell activity is evident as early as 24 h and peaks within 72 h, returning to normal levels by 168 h. Interestingly, the route by which PA was administered was very important with respect to increased NK activity. For example, the greatest augmentation of activity was seen in the peritoneal cavity when mice were injected intraperitoneally and in the spleen when injected intravenously. Peripheral blood leukocytes expressed the greatest augmentation in animals which received an intravenous injection of viable PA. In addition, a nonviable preparation of PA was used and found to significantly augment NK cell activity in a dose-dependent manner. To determine whether the presence of the organism is required for augmentation of NK activity, the rate at which PA is cleared from the animals was evaluated. Regardless of the route of injection, PA is effectively cleared within 24 h, thus eliminating the possibility that viable PA is required for augmentation of NK activity. This augmentation is proximal to the route of injection with little systemic effect seen. The data presented in this report illustrate that both viable and nonviable preparations of PA produce a significant increase in NK cell activity. This augmentation may suggest a role for these cells as effectors in natural resistance to infectious disease.     

Seasonal carbon allocation to arbuscular mycorrhizal fungi assessed by microscopic examination, stable isotope probing and fatty acid analysis

Background and Aim

Climate change models are limited by lack of baseline data, in particular carbon (C) allocation to – and dynamics within – soil microbial communities. We quantified seasonal C-assimilation and allocation by plants, and assessed how well this corresponds with intraradical arbuscular mycorrhizal fungal (AMF) storage and structural lipids (16:1ω5 NLFA and PLFA, respectively), as well as microscopic assessments of AMF root colonization.

Methods

Coastal Hypochoeris radicata plants were labeled with ¹³CO₂ in February, July and October, and ¹³C-allocation to fine roots and NLFA 16:1ω5, as well as overall lipid contents and AM colonization were quantified.

Results

C-allocation to fine roots and AMF storage lipids differed seasonally and mirrored plant C-assimilation, whereas AMF structural lipids and AM colonization showed no seasonal variation, and root colonization exceeded 80 % throughout the year. Molecular analyzes of the large subunit rDNA gene indicated no seasonal AMF community shifts.

Conclusions

Plants allocated C to AMF even at temperatures close to freezing, and fungal structures persisted in roots during times of low C-allocation. The lack of seasonal differences in PLFA and AM colonization indicates that NLFA analyses should be used to estimate fungal C-status. The implication of our findings for AM function is discussed.     

Fungal lipid accumulation and development of mycelial structures by two arbuscular mycorrhizal fungi

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1omega5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1omega5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1omega5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.     

Fungal Lipid Accumulation and Development of Mycelial Structures by Two Arbuscular Mycorrhizal Fungi

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1ω5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1ω5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1ω5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.     

A Note on the Staining of the Skeleton of Cleared Specimens with Alizarin Red S

A selective, progressive method for staining the skeleton in cleared specimens, developed with rat material.

Fix in 95% alcohol for at least 48 to 96 hrs. Even longer fixation is desirable. Then place in a 1% solution of KOH until the bones are clearly visible through the surrounding tissues. Transfer directly to a dilute solution of alizarin in KOH, one part alizarin to 10,000 parts of 1% KOH. Allow the stain to act until the desired intensity is attained. Fresh stain may be added if necessary.

Complete the clearing process, (1) in Mall's solution, water 79 parts, glycerine 20 parts and KOH 1 part; (2) in increased concentrations of glycerine. Store in pure glycerine.

The success of the method depends on obtaining the proper degree of clearing before staining. If the specimen is insufficiently cleared, a general staining of all tissues usually occurs.     

Interactions between Nitrogen Fixation, Mycorrhizal Colonization, and Host-Plant Growth in the Phaseolus-Rhizobium-Glomus Symbiosis

Bean (Phaseolus vulgaris L. cv. Dwarf) roots were inoculated with Rhizobium phaseoli and colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum Gerd. and Trappe or left uncolonized as controls. The symbiotic associations were grown in an inert substrate using 0, 25, 50, 100, or 200 milligrams hydroxyapatite (HAP) (Ca₁₀[PO₄]₆[OH]₂) per pot as a P amendment. Plant and nodule dry weights and nodule activity increased for both VAM and control plants with increasing P availability, but values for VAM plants were significantly lower in all parameters than for controls. Inhibition of growth and of N₂ fixation in VAM plants was greatest at the lowest and highest P regimes. It was smallest at 50 milligrams HAP, where available P at harvest (7 weeks after planting) was 5 micrograms P per gram substrate. At this level of P availability, the association apparently benefited from increased P uptake by the fungal endophyte. Percent P values for shoots, roots, and nodules did not differ significantly (p > 0.05) between VAM and control plants. The extent of colonization, fungal biomass, and the fungus/association dry weight ratio increased several fold as HAP was increased from 0 to 200 milligrams. It is concluded that intersymbiont competition for P and photosynthate was the primary cause for the inhibition of growth, nodulation, and nodule activity in VAM plants. Impaired N₂ fixation resulted in N stress which contributed to inhibition of host plant growth at all levels of P availability.     

Rapid methods for quantifying VAM fungal infections in maize roots

Roots of maize (Zea mays cv W64A × W182E) infected by vesicular arbuscular mycorrhizal (VAM) fungi (Glomus versiforme (Karst) Berch or a Glomus species isolated from an alfalfa soil) exhibit a bright yellow pigmentation. The percentage of pigmented roots can be quantified by a rapid visual estimate or by a grid intersect method. Both methods gave similar estimates of VAM infection to those obtained using a grid intersect count on cleared roots stained with chlorazol black E. Thus for experimental or field evaluation where speed and quantity are important, the rapid visual estimate (less than one minute for each washed root system) yields reliable results. The yellow root intersect method takes longer (5–15 minutes per root system) but gives more reproducible results. The yellow root pigmentation is light sensitive However, root systems can be reliably assayed after 1 week when stored at 5°C in the dark or after 1 year if dried.     

A Method for Staining Pollen Tubes in Pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsia, 6 ml; 1% aqueous aniline blue, 4 ml; 1 % orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45±2 C They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45±2 C, hydrolyzed in the clearing and softening fluid at 58±1 C for SO min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

Early events in the life of apple roots: variation in root growth rate is linked to mycorrhizal and nonmycorrhizal fungal colonization

Early events of mycorrhizal and nonmycorrhizal fungal colonization in newly-emerging roots of mature apple (Malus domestica Borkh) trees were characterized to determine the relationship of these events to fine root growth rate and development. New roots were traced on root windows to measure growth and then collected and stained to quantify microscopically the presence of mycorrhizal and nonmycorrhizal fungal structures. Most new roots were colonized by either mycorrhizal or nonmycorrhizal fungi but none less 25 days old were ever internally colonized by both. Compared to nonmycorrhizal colonization, mycorrhizal colonization was associated with faster growing roots and roots that grew for a longer duration, leading to longer roots. While either type of fungi was observed in roots as soon as 3 days after root emergence, intraradical colonization by mycorrhizal fungi was generally faster (peaking at 7 to 15 days) than that by nonmycorrhizal fungi and often occurred more frequently in younger roots. Only 15 to 35% of the roots had no fungal colonization by 30 days after emergence. This study provides the first detailed examination of the early daily events of mycorrhizal and nonmycorrhizal fungal colonization in newly emerging roots under field conditions. We observed marked discrimination of roots between mycorrhizal and nonmycorrhizal fungi and provide evidence that mycorrhizal fungi may select for faster growing roots and possibly influence the duration of root growth by non-nutritional means.     

A method for staining pollen tubes in pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsin, 6 ml; 1% aqueous aniline blue, 4 ml; 1% orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45 +/- 2 C. They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45 +/- 2 C, hydrolyzed in the clearing and softening fluid at 58 +/- 1 C for 30 min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

Occurrence and importance of mycorrhizae in aquatic trees of New South Wales,Australia

Vesicular arbuscular mycorrhizal (VAM) infection was found in KOH-cleared and lactophenolblue-stained roots of Salix babylonica, Melaleuca quinquenervia and Casuarina cunninghamiana. These are all trees growing on creeks and river banks, in stationary or slowly flowing fresh or brackish waters in swamps, creeks, drains and channels, and in seepage areas of New South Wales, Australia. Larger and older roots lacked VAM infection in the inner cortex, probably due to suberisation of cells, and the endophyte was restricted to the epidermal layers. Spores and sporocarps of the VAM fungi Glomus fasciculatus, G. mosseae, Sclerocystis rubiformis, Gigaspora margarita and an unidentified Scutellospora sp. were wet sieved and decanted from aquatic sediments and soils. The presence of similar VAM fungal spores in the aquatic sediments and terrestrial soil suggests that they probably enter the aquatic sediments through run off from the land ecosystem. All three plants formed vesicular arbuscular (VA) mycorrhizae almost exclusively in the marshy, periodically inundated soils, but the same plant species formed endo-/ ectomycorrhizae when growing in soil with higher redox potentials (E _h). Salix and Melaleuca tree roots possessed both VAmycorrhizae and ectomycorrhizae. VAM roots of Casuarina were equipped with both N-fixing Frankia nodules and proteoid roots. VAM endophytes did not invade nodular cortical tissues, suggesting the presence of an exclusion mechanism which needs further study. The highest VAM infection was found in nodulated specimens. Free-floating roots growing in water close to the banks were non-mycorrhizal but were mycorrhizal in the bottom-rooting state. VAM spore number and mycorrhizal infection seem to be associated with redox-potential, i.e. lower at sites such as swamps, water or sediments with lower E _h values than in terrestrial soils with higher E _h values. A relationship between soil moisture gradient and VAM infection pattern became apparent from the study of a C. cunninghamiana transect on a creek embankment, i.e. typical vesicles and arbuscules were found in roots from drier soils, there was a lack of arbuscules in relatively wet soils but large lipid-filled intracellular vesicles were present, and typical vesicles and arbuscules were absent in flooded creek beds where roots were associated with coenocytic intercellular hyphae with abundant lipid droplets. The importance of VA mycorrhiza, ectomycorrhizae, N-fixing root nodules and proteoid roots at the land-water interface is discussed with reference to the use of these trees as pioneering species for stabilising river and stream banks, reducing erosion, windbreaking, and as a long-term and inexpensive means of achieving biological control of aquatic weeds by shading waterways.     

Aceto-Iron-Haematoxylin-Chloral Hydrate for Chromosome Staining

Aceto-iron-haematoxylin can be used combined with the clearing agent chloral hydrate for the squash method. The stain is prepared by dissolving 2 gm of chloral hydrate in 5 ml of a stock solution of 4% haematoxylin and 1% iron alum in 45% acetic acid, which has been allowed to ripen for 24 hr to 1 wk. Heat must not be used to hasten solution. The material (fixed in 1:3 acetic-alcohol) is put on a slide, the fixative removed and a drop of stain added; if necessary the material is crushed before the cover slip is placed in position. The preparations are now carefully heated until a slight colour change occurs. Squashing needs more pressure than in other techniques. This stain gives best results in zoological and botanical material not requiring hydrolysis, e.g., leucocytes, ascites cells, and cells undergoing spermatogenesis and microsporogenesis. Well-spread and selectively stained mitotic and meiotic figures can be obtained.