Promotion of stipe elongation in Flammulina velutipes by a diffusate from excised lamellae supplied with nutrients

Flammulina velutipes fruit bodies were grown on partly decayed Populus tremuloides sawdust supplemented with wheat bran and malt extract. In each culture there was a gradation in fruit body size, which served to select test specimens at an early stage of growth. Diffusates collected in agar blocks were applied on the apex of decapitated stipes. Plain agar and dilute potato dextrose agar (PDA/2) alone had the same slight effect on growth. Lamellae placed on plain agar caused limited growth promotion. Lamellae on PDA/2 gave 100–150% more growth promotion than on plain agar during early development, but the activity decreased to zero during the middle of the stage of rapid elongation. Lamellae of that age had no effect on young stipes and the older stipes were insensitive to diffusate from young lamellae. Very small amounts of lamellae promoted stipe elongation. Potato extract alone did not stimulate production of the lamellar diffusate and glucose was less effective than the two nutrients combined. A delay of 2 h in applying lamellar diffusate reduced stipe elongation, and there was no response after 12 h delay. Pilear trama did not produce growth-promoting diffusate.

The negative staining of "difficult" bacteria like Arthrobacter globiformis for electron microscopy

A method is suggested for the negative staining of "difficult" bacteria like Arthrobacter globiformis for electron microscopy when the usual procedures do not give satisfactory results. It involves glutaraldehyde fixation, and thorough washing and drying of the cell suspension on a grid sitting on a bed of plain agar, followed by a rapid flushing with 2% phosphotungstic acid.

Regeneration on Phycomyces sporangiophores. II. Regeneration on isolated segments from different positions along the sporangiophore axis

Apical, middle, and basal 5-mm segments from decapitated Phycomyces blakesleeanus sporangiophores cultured on plain agar blocks regenerated sporangiophores after wound closure. Hyphae formed very rarely. Maximum regeneration frequency decreased between apex and base from 100% to close to 70% in stages 1 and 3, and from 80% to 23% in stage 4. The regeneration speed declined along the axis. The frequency of two or more initials per segment decreased basipetally in stages 1 and 3, but not in stage 4. Most segments formed sporangiophores at the apical end but regeneration from the basal end alone increased towards the base in stages 1 and 3, and was found on up to 30% of the stage 4 segments without gradient along the axis. A few segments regenerated at both ends at all positions. Initials emerged laterally at the apical end of most top segments and from the wound closure wall on almost all others. The diameter of single sporangiophores on apical segments was larger and their maximum stage 1 length shorter than on the rest. Both were less on stage 4 than on 1 and 3. The final length on either stage 1 or 4 was the same at all positions but the former was more than twice as great as the latter. One-millimeter stage 1 segments regenerated very thin sporangiophores but polarity was much weaker than in 5-mm pieces.Almost 90% of stage 1 segments with whole apices and open basal ends continued growth without branching. Most of those which stopped regenerated near the tip. Stage 3 and 4 segments tied off basally continued growth without regenerating above the tie, but if the end was open the sporangia stuck to the agar because of turgor loss, elongation stopped, and regeneration occurred close to the sporangia at slower speed than on decapitated apical segments.Cytoplasmic extrusion and formation of exudation drops were studied, wound closure was followed on middle stage 1 segments, and preliminary observations were made on movement of cell contents during regeneration.

Regeneration on Phycomyces sporangiophores. I. Induction by decapitation and basal cutting on sporangiophores attached to mycelium and after isolation

Phycomyces blakesleeanus sporangiophores attached to mycelium and isolated sporangiophores cultured on plain agar blocks were decapitated at 1–2 mm below the apex (stage 1) or the top of the sporangium (stages 3, 4). One or more sporangiophore initials regenerated at or very near the wound closure after formation of a cross wall. They emerged only from the side on 82–85% of all parents and on most of the rest from the wound closure wall. Lateral initials frequently perforated the original wall. Regeneration on isolated and attached sporangiophores reached 100% at 12 h on stage 1 and 18 h on stage 3, but only 80% after 24 h on stage 4. The frequency of two or more initials per parent decreased from stage 1 to 4 and was greater on attached than on isolated specimens. Fewer than half of all parents with two initials showed maturation of both. Single sporangiophores elongating on isolated stage 1 parents had a larger diameter, greater stage 1 length, and 66% greater final length than on stage 4. Stage 1 was always very short (average maximum, 5.1 mm). Sporangium formation and maturation were most synchronized after regeneration on stage 1 and least on stage 4.Sporangiophores cut at 5 mm above the base regenerated less frequently than after decapitation and the decrease was larger in stage 4 than in stage 1. Hyphae and sporangiophores regenerated alone or together, but hyphae were most common on attached stage 1 stumps. Regeneration was always from the wound closure wall, and two or more sporangiophore initials per parent were less frequent on stumps than near the apex.Observations on outflowing of drops of cell contents and on wound closure are discussed in relation to regeneration.

Variation of Bacillus anthracis with special reference to the non-capsulated avirulent variant

Normal virulent B. anthracis all possess the genetical potentiality of producing capsules, but the capsule is only produced in vivo or under special cultural conditions suitable for capsule formation, especially in the presence of CO2.During multiplication the potentially capsulated cells regularly produce a small number of a stable non-capsulated avirulent variant. All laboratory stock cultures of B. anthracis examined were found to contain from 0·14 to 32·4% of this avirulent variant. The variant has also been demonstrated in hair and wool from anthrax endemic areas, which suggests that the dissociation may also occur in nature.Both the normal potentially capsulated cell and the variant incapable of producing capsules grow on plain agar in air as rough colonies with apparently non-capsulated cells. But when they are grown on serum agar in the presence of CO2, the normal potentially capsulated cell gives rise to a smooth mucoid colony with fully capsulated cells, while the variant still produces a rough colony with non-capsulated cells. If we regard the smooth mucoid colony produced by normal virulent B. anthracis on serum agar in CO2 as the normal colony form of B. anthracis, then the dissociation of the avirulent non-capsulated variant with the rough colony form falls in line with the usual S-R dissociation of other bacteria.The more widely known variation of B. anthracis, which had long been held as an exception to the usual S-R variation of other bacteria, refers to colonies grown on plain agar in air. This variation is different from the one just described and is not so clear-cut and significant. Its underlying cause is not quite clear except that the chain length of the growth appears to play some part.A summarized picture showing the whole range of variation in colony appearance and cell structure of B. anthracis is presented.

THE MECHANISM OF BACTERIOSTASIS

1. Between Gram-positive and Gram-negative organisms gentian violet exhibits the same type of selective activity whether the dye be added to the media on which the bacteria are planted unstained (extrinsic bacteriostasis), or the organisms be stained with it before being planted on plain agar (intrinsic bacteriostasis). In both instances the Gram-positives are inhibited and the Gram-negatives unaffected. 2. Between Gram-positive spore-bearing aerobes and the commoner Gram-negative bacteria, acid fuchsin, related sulfonic substances, and the flavines exhibit one type of selective activity when the dye is added to the media (extrinsic bacteriostasis) and the opposite type when it is added directly to the bacteria (intrinsic bacteriostasis). In the former case, the Gram-positive spore bearers are inhibited and the Gram-negatives unaffected; in the latter case the Gram-negatives are inhibited and the Gram-positive spore bearers unaffected. 3. Selective bacteriostasis is not necessarily conditioned by selective penetrability. Stained organism may grow, and dyes which do not stain well may inhibit reproduction. 4. There is evidence that the phenomena of bacteriostasis may be due to changes effected by the dye at the surface of the organisms.