Recent Advances in Microtechnic. I. Methods of Studying the Development of the Male Gametophyte in Angiosperms

Among methods used for a study of nuclear details in the development of pollen grains, the following were found to be very satisfactory: (1) warming the entire grains in aceto-carmine and then clearing with chloral hydrate; (2) making smear preparations stained with crystal-violet-iodine or iron alum hematoxylin. For paraffin sections, a counterstain with dilute alcoholic erythrosin is often very useful after the usual iron hematoxylin technic.

A method of making cultures of pollen tubes on slides coated with thin films of sugar agar is described in detail. The tubes can be fixed by immersing the slide in formol-acetic-alcohol and then stained by any desired schedule. Iron alum hematoxylin was found to be the most satisfactory, but the Feulgen reaction is very valuable in such cases where the nuclei are obscured by the density of the pollen tube cytoplasm. Living pollen tubes can be kept under observation by dissolving a small quantity of neutral red or other vital stain in the sugar agar before it is spread on the slide.

For studying stages in fertilization or gametogenesis, styles should be fixed and sectioned only after a preliminary study with iodine-chloral-hydrate or safranin-anilin-blue or aceto-carmine. Once the extent to which pollen tubes grow in a given time in the stylar tissues has been determined, it is possible to fix material with some knowledge of what it is going to show.

Some other methods, that have not been tried by the authors but appear to be valuable, are also briefly described.     

Tannic Acid and Iron Alum with Safranin and Orange G in Studies of the Shoot Apex

A schedule is given for staining the cell walls of young plant tissues in tannic acid and iron alum after the protoplasts have been stained in safranin and orange G. Sections are placed for one minute in 2% aqueous ZnCl₂, and are then stained in a 1/25,000 aqueous solution of safranin O. From this they are placed for five minutes in a bath consisting of orange G (2 g.), tannic acid (5 g.). water (up to 100 cc.) and HC1 (4 drops). This is followed by five minutes in 5% aqueous tannic acid and two minutes in a 1% solution of iron alum. A brief rinse in tap water is given between each stage; the slides are raised and lowered about a dozen times at each change to ensure that the new solution reaches the material quickly. The method was originated for shoot apices but it also works excellently on more mature tissues and on adult material. It has the advantage of allowing extremely easy detection of protophloem in the strands even at the very onset of vascular differentiation.     

Iron and temperature as sporangiospore germination factors of Pilobolus longipes

Sporangiospores of Pilobclus longipes germinated on a medium containing ascorbate and FeSO₄, but neither ascorbate nor FeSO₄ alone caused spores to germinate. The iron chelates (hemin, coprogen, and ferrichrome) that are known to promote mycelial growth of this and other species of Pilobolus had little or no effect on spore germination, suggesting that under these conditions dormant spores are unable to reduce iron III.Regardless of the medium used, maximum germination required treatment at two temperatures. The early stage of germination, spherical growth, was favored by treatment for several hours at about 38°C while optimum germ tube formation required incubation at lower temperatures (25°C). Under most conditions the requirement for a heat treatment was nearly absolute.When the iron-ascorbate and the heat treatments were separated it was found that they need not be applied simultaneously provided that iron and ascorbate are given first. Spores that were heated first and then given iron and ascorbate at lower temperatures did not germinate. Apparently dormancy of these spores is broken by available iron but a heat treatment is usually required to complete the germination process.     

The Smear Technic in Plant Cytology

The following method of making permanent smears of pollen mother cells is in general use and gives excellent results. Determine the stage of meiosis from aceto-carmin mounts. Smear the pollen mother cells on a dry slide. Fix in Navaschin's or a modified Flemming's solution from 1 to 2 hours. Wash in 10 to 20% alcohol from 15 to 30 minutes. Stain in 1% aqueous crystal violet from 1 to 5 minutes. Rinse in water and pass thru 30 to 50% alcohol, about 15 to 20 seconds in each. Transfer to 80% alcohol containing 1% iodine and 1% potassium iodide for 30 seconds. Destain with absolute alcohol, followed by clove oil. xylol, balsam and cover.

Permanent smears for chromosome counts can be quickly made by smearing pollen mother cells on a dry slide, fix and stain with aceto-carmin, dehydrate with mixtures of absolute alcohol and acetic acid, follow with xylol, balsam, and cover.     

Colchicine-Feulgen Leaf Smears

The colchicine-Feulgen leaf smear has many advantages. It (1) prevents spindle formation, (2) allows the chromosomes to be widely spread in the cell, (3) straightens the chromatids, (4) allows the constrictions to become very noticeable, (5) increases the number of chromosome plates by preventing anaphase, (6) facilitates smearing, and (7) stains only the chromosomes. (8) Young leaves are generally easily obtainable, while roots of the proper sort are to be had only under very special conditions. (9) Mitoses are frequently more numerous in young leaves than in roots. The schedule for specimens of Phlox involves a 1 to 2 hour pretreat-ment of young leaves in a 0.2% colchicine solution, fixation in Sem-men's Carnoy (3 vol. absolute alcohol, 1 vol. glacial acetic, 1 vol. chloroform), hydrolysis for 25 minutes in 10% HC1 at 58° C., staining in decolorized fuchsia, smearing in 45% acetic acid, and running the material on slide and cover glass thru acetic alcohols (1:1, 1:3, 1:9), absolute alcohol, xylol, and balsam.     

Controlled Differentiation of Cells and Connective Tissue Fibers in Silver Staining

Controlled silver staining of connective tissue fibers and sometimes of these fibers and cells simultaneously can be obtained. 1. Fix in 10% formalin. Embed in paraffin and cut sections as usual, but do not mount them on slides. Deparaffinize and hydrate through xylene, alcohols and distilled water and henceforth treat them the same as frozen sections. Real frozen sections can also be used. 2. Treat with a freshly prepared 1% solution of KMnO₄, usually 15-60 sec, sometimes up to 10 min. 3. Wash in distilled water, 5-10 sec. 4. Decolorize in 2% potassium metabisulfite, 10-20 sec. 5. Place in distilled water, 1 min. 6. Sensitize with 2% iron alum, 1 min. 7. Place in distilled water, 1 min. 8. Impregnate in Gomori's silver oxide solution, 2 min. 9. Wash in a 1.5% aqueous solution of pyridine, about 15 sec. 10. Reduce in a mixture containing 0.25% gelatin and 2% formalin 1 min. 11. Repeat steps 7 to 10 once or several times until the connective tissue fibers are completely stained. For cell staining (which may fail) proceed as follows: After the first insufficient staining of the connective tissue fibers, rinse in distilled water, dip for 1 sec in Gomori's solution and reduce immediately in gelatin-formalin without previous washing in pyridined water. This step can be repeated. 12. If the staining is too strong, decolorize as needed in 2% iron alum. 13. Toning in 0.2% gold chloride, 5 min or more, followed by fixation in 5% sodium thiosulfate, 1 min, is optional. Counterstain as desired. 14. Wash in tap water, dehydrate, clear in xylene and mount in balsam. The same technique applied to sections attached to slides gives good results but inferior to that obtained in paraffin sections processed in the loose, unmounted condition.     

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.     

Frozen Section Micro-Incineration

A method for micro-incineration of frozen sections is described. Material containing diffusible or soluble salts is cut on the freezing microtome and the sections are placed into xylol and mounted out of xylol onto Corex D slides previously filmed with glycerin-gelatin medium. Material containing non-diffusible or insoluble salts can be fixed in 10% formalin before sectioning. Sections of the fixed material are dehydrated thru 50, 70, and 95% ethyl alcohol and mounted out of absolute alcohol onto Corex D slides previously fumed with glycerin-gelatin medium. After mounting by either procedure the sections are incinerated in an electric furnace and the temperature of incineration is dependent on the type of tissues to be incinerated and the character of the salts present. The method is time saving and when no fixation is required the whole procedure can be carried out in one hour.     

The Dissection Staining and Mounting of Styles in the Study of Pollen-Tube Distribution

The methods here outlined have been given in general in previous publications; but they are now summarized, with special reference to Datura styles, for the benefit of those desiring further details.

Pistils, artificially pollinated and kept at 18 to 22°C, are slit along two sides, scalded 30 seconds to 2 minutes in water at 70 to 75°C, and killed by immersing for several hours in 50% alcohol containing 6% formalin. Within 12 hours of killing, they are dissected with needles and forceps under a wide field binocular microscope, removing the cortex from style and stigma. The strand of conducting tissue thus separated from the cortex is stained within 24 hours of dissection in a watch glass containing a mixture of 8 parts 1% aqu. acid fuchsin and 2 parts of 1% aqu. or alc. light green. Staining requires 3 to 6 hours, and may be continued over night if stain is not too concentrated. The strand of tissue is cleared several hours or over night in 80% lactic acid, carefully spread out on a slide, and mounted in lactic acid. After a few days the preparations are sealed around the edges of the cover glasses with damar in xylol or paraffin to which gum mastic has been added.

Directions are given for counting the ends of pollen tubes and for keeping the records when genetic studies are being made.     

INHIBITION OF FERN SPORE GERMINATION BY LIPOPHILIC SOLVENTS

Several alcohols and other solvents inhibit germination of spores of the fern, Onoclea sensibilis L. The inhibition is reversible when spores are transferred to solvent-free media. The effectiveness of different solvents, measured by the concentration needed to inhibit germination by 50%, increases with their lipid solubility. The activity of straight-chain alcohols from methanol through n-heptanol is highly correlated with lipid solubility, whereas the correlation is weaker for several other solvents. The results indicate that some lipophilic site in the spore is important in germination.     

Bacillus megaterium spore germination is influenced by inoculum size

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.     

The Fixation of Tissues Vitally Stained with Trypan Blue

The following fixative is recommended for tissues vitally stained with trypan blue: Chloroform, 2 parts; absolute ethyl alcohol, 2 parts; glacial acetic acid, 1 part; mercuric chloride to the point of saturation.

The tissue should be fixed 1 to 2 hours; transferred to 95% ethyl alcohol for 12 hours; to absolute alcohol for 12 to 24 hours; to a mixture of absolute alcohol and xylol for 1/2 hour, and finally to xylol, before embedding in paraffin. Cedar oil may be used for clearing in the place of xylol; in that case the tissues should be transferred from absolute alcohol to a mixture of absolute alcohol and cedar oil for 24 hours before placing in cedar oil alone.

Various counterstains can be used; Mayer's carmalum is excellent.     

Temperature and photocontrol of onoclea spore germination

Germination of Onoclea sensibilis L. spores is controlled by light and temperature. Temperatures of 30 C can induce maximal germination in the dark to a level of 60 to 95% of that induced by a saturating dose of red light (0.38 joules/square meter) providing the spores are placed at the elevated temperature immediately after being sown. Maximum dark germination occurs with a minimum exposure of 16 to 24 hours at 30 C, suggesting that the temperature treatment is required for the induction of germination rather than for the germination process per se. Interaction of temperature and light for induction of germination shows nonadditive behavior. Germination induced by light and temperature applied consecutively never exceeded that which could be induced by a saturating dose of red light alone. Imbibition of the spores at 25 C in the dark for 12 or more hours prior to incubation at 30 C results in a loss of thermosensitivity. Dose response curves for red light induction of germination after varying times of imbibition at 25 C show no concomitant loss of sensitivity of the spores to red irradiation. This suggests that the mechanism and/or pathway of thermoinduction of germination differs from that of photoinduction. The loss of thermosensitivity as a result of presoaking at 25 C can be prevented if the spores are imbibed at 25 C in osmotic agents such as 0.3 molar mannitol or 0.1 gram per liter of polyethylene glycol 400 or in 0.08% dimethylsulfoxide or 10 micrograms per milliliter of herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3-(2H)pyridazinone). The latter two substances are hypothesized to act upon membranes. These results suggest that the degree of hydration and possibly changes in membrane properties play a role in the change in sensitivity of Onoclea spores to temperature.     

A Modified Bradley's Squash Technique for Studying Embryo Sacs in Gramineae

This procedure is especially suited for studying the embroyology of sexual and apomictic grasses. Material is fixed in a 2:2:1 alcohol-chloroform-propionic acid mixture for a minimum period of 2 days, soaked in 4% iron alum at 75 C for 7 min, and 2 min each in 2 changes of distilled water, also at 75 C. After 2-3 min in cold water, it is macerated in 50% HCI for 10 min at about 22-25 C, washed and mordanted for 12-16 hr in 50% alcohol saturated with ferric acetate. Ovules are then dissected out and squashed in 1% carmine in 45% propionic acid. Squashing should be firm enough to separate and flatten the embryo sacs but not to burst them. The slides are set aside for 12-24 hr for intensification of the stain.     

A simple stain for myelin in frozen sections: a modification of Mahon's method.

A simple and rapid method for demonstrating myelinated nerve fibers in frozen sections of the central and peripheral nervous system is described. Material fixed by perfusion with mixed aldehydes gives the best results but the method also works on specimens fixed by immersion in formaldehyde. Frozen sections varying in thickness from 15-50 micron are mounted on slides subbed with chrome alum-gelatin. After hydration (60-120 min), sections are mordanted (20-40 min) in 2.5% iron alum and rinsed briefly in three changes of distilled H2O (total 2 min). Staining is for 60-180 min in 20 cc freshly made 10% alcoholic hematoxylin diluted with 165 cc distilled H2O to which 15 cc saturated Li2CO3 is added. The sections are washed in distilled H2O (5-15 min) and dehydrated in graded alcohols without differentiation in mordant, and covered. Myelin stains a dark blue-purple against a light grey background. Fiber tracts, as well as individual myelinated fibers, are clearly demonstrated.     

Relationship between Heat Activation and Percentage Colony Formation for Bacillus stearothermophilus Spores: Effects of Storage and pH of the Recovery Medium

Colony counts of unheated spores were higher in a medium at pH 5·9 than in media of higher pH. The pattern was reversed as the spores were heated. Slide germination studies showed that about 8% or less of unheated spores germinated in slide culture. Optimal heat activation resulted in about 50% germination. The colony counts of heat activated spores dropped significantly upon storage for 9 months, but those of unheated spores did not.     

Feulgen's Reaction Applied to Protozoa and Small Worms, Mounted In Toto in Venetian Turpentine

Permanent mounts of certain protozoa and small worms are obtained as follows: kill suspensions of the organisms with Feulgen's fixative (6% HgCl₂ in 2% aqu. acetic acid) for 3 to 24 hours. After pipetting off the fixative, add successively: 70% iodized alcohol; ditto, 30 minutes later; 50%, then 35% alcohol; 2 baths distilled water; normal HCl. Transfer to cold water and heat to 60°C for 4 to 5 minutes or longer. Cool under running water; and wash in distilled water.

Stain 1 to 3 hours in Feulgen's fuchsin sulfurous acid (1 g. of a suitable basic fuchsin, e. g. rosanilin hydrochloride, boiled in 200 cc. water, cooled, and allowed to stand 24 hours after adding 20 cc. normal HCl and 1 g. sodium bisulfite). Pass thru 3 baths of 200 cc. distilled water with 10 cc. normal HCl and 1 g. sodium bisulfite. Transfer to water and then to 35%, 70%, and 95% alcohols successively. Counterstain with fast green FCF, orange G or eosin Y in 95% alcohol. Pass thru two changes of absolute alcohol.

Transfer to 10% Venetian turpentine and place in a dessicator; mount after the turpentine has become concentrated.

If sections instead of total mounts are desired, the material should go from absolute alcohol, thru alcohol-xylol and xylol to paraffin (or preferably paraffin of M. P. 56°C with 3% bees-wax). The paraffin may be added to the material in the test tube, and cooled after the organisms have settled. Then break the tube, trim a block, and cut.     

Iron-Alum Aceto-Carmine Staining for Chromosomes and Other Anatomical Features of Rhodophyceae

The chromosomes, certain intracellular structures and gross anatomical details of many red algae, which, as a class, have proved technically difficult material, can be demonstrated by staining with aceto-carmine after a mordant bath of iron alum. Acetic-alcohol mixtures are used as nuclear fixatives and formalin-acetic-alcohol and other similar fluids for preservation of anatomical features. The tougher more cartilaginous thalli of some species can be softened, if squashes are desired, by prolonging fixation (24-48 hr.) in acetic alcohol and subsequent washing. The fixatives are washed out of the material before the latter is transferred to 0.5-5.0% ferric ammonium sulphate, the concentration of which may be altered according to the material. Excess mordant is removed by washing and the material stained in Belling's aceto-carmine containing a trace of ferric acetate as a “ripener”. The degree of heating before covering is critical as it controls the quality of the staining. Squashing must be very thorough to spread the chromosomes which are usually very small but only slight controlled pressure is necessary when diffuse structures such as carposporophytes, nemathecia or medullary filaments are being demonstrated. Paraffin sections mounted on slides can also be stained by this method.     

Mallory's Connective Tissue Stain Following Hematoxylin

The following combination of hematoxylin with Mallory's connective tissue stain is useful in bringing out nuclei as well as in differentiating tissue:

Slightly overstain in Mayer's hematoxylin (50 g. potassium alum and 0.2 g. sodium iodate added to 1 liter 0.1% aqueous hematoxylin). Wash; and stain 30 seconds to 1 minute in 0.04% aqueous acid fuchsin-Stain 4 minutes in: 0.5 g. anilin blue and 2 g. orange G dissolved hi 100 cc. of 1% aqueous phosphomolybdic acid. Pass thru 95% alcohol to absolute; clear in xylol and mount in balsam.     

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. CONCLUSIONS: This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.