Cited by J.N. Langley and H. Sewall, 'On the Changes in Pepsin-forming Glands During Secretion', Journal of Physiology 2 (4) (1879), pp. 281-322.
Description:

'Nussbaum [note: 'Nussbaum. Arch. f. Mik. Anat. Bd. XIII. p. 746, 1877.'] has described the appearance of the oesophageal glands after treatment with osmic acid. It will have been seen that we differ from him in many points.

1. He finds that after "several days' hunger" the alveoli are like the alveoli of the pancreas and present two zones nearly equal. We find that frogs killed four days after feeding present alveoli granular throughout. In two or three days after feeding some frogs do not completely recover their granules, possibly Nussbaum may have taken some such slowly reacting frogs, as the typically hungry. Possibly in some frogs the granular zone does not in the ordinary hungry condition stretch quite to the periphery; in the healthy frogs which we have observed it did, nevertheless the matter does not seeim to us very important.

2. On feeding, he finds the granules increase so that in three hours the alveoli are granular throughout, an appearance which they preserve until about five hours after feeding, and only at about the fifteenth hour has the outer clear zone again become obvious. We find that from the beginning of digestion onwards there is a using up of granules.

3. According to him, if small pieces of cork be given to a frog and the cardia ligatured so that the cork continues to stimulate the oesophageal mucous membrane, the granules disappear entirely in three to four hours from the alveolar cells, whilst the nuclei become shrunken and angular.

This we have been quite unable to observe.

In the granular throughout stage of Nussbaumn after five hours feeding, he describes coarser particles staining deeply black with osmic acid, immediately underneath the membrana propria. We have described in a similar position collections of small highly refracting granuiles which stain very deeply with osmic acid; these "border" granules perhaps represent the coarser particles of Nussbaum.

That which we regard as the most important point of difference is the effect of feeding: Nussbaum finds that the granules increase at first, we find only a decrease.' (285)

 

'Nussbaum connected the presence of granules in the oesophageal glands with the presence of ferment. His starting point was, however, not so much the actual presence of granules as their staining with osmic acid; according, to him, when the granules disappeared, the blackening from osmic acid ceased. This seems to us untenable, for if the gland be treated with osmic acid alone for two hours, the granules are not more deeply stained than the rest of the protoplasmic cells; whilst the border-granules are deep black, so that on Nussbaum's view, it should be these and not the central granules which would represent the ferment. We may remark too that Nussbaum's observations on the granularity of the cells do not with any accuracy correspond with the results of Swieçicki on the amount of ferment. The former finds the granules in the cells increase for three hours after feeding, remain stationary for two hours and then diminish. The latter found the ferment increase up to 6-10 hours and only then diminish.

From general reasoning we are inclined to connect the number of central granules with the amount of ferment, and we rnay point out that Grützner's [note: 'Grützner. Pflüger's Arch. Bd. XVI. p. 122, 1877.']  results agree thoroughly with this view. He found that the ferment was greatest during hunger, and diminished during digestion, at first rapidly, then more slowly.' (286)

 

'The greater curvature contains more border-cells than any other portion of the stomach, the pyloric glands of the smaller curvature contain at most an occasional border-cell here and there, yet the amount of pepsin produced by the two gland-forms is scarcely different. The conclusion is irresistible that the border-cells do not form the ferment.' [re: Nussbaum's assertion to the contrary. No direct reference to Nussbaum here.] (295)

 

'The granules we consider as stored up cell-products, which, on suffering molecular re-arrangement during the secretion, give rise amongst other substances to the proteolytic ferment.

We cannot agree with Nussbaum's view that the depth of staining with osmic acid is a trustworthy index of the amount of ferment present in the cells. On his view, it appears to us, the border, rather than the central, granules should be connected with the ferment.' (297-298)

 

Cited by J.N. Langley, 'On the Changes in Serous Glands during Secretion', Journal of Physiology 2 (4) (1879), pp. 261-322.
Description:

'Nussbaum [note: 'Arch. f. Mik. Anat., Bd. XIII. s. 721, 1877.'] found the transition cells to stain with osmic acid a deep black, compared with the alveolar-cells.

This he found, after two hours treatment with osmic acid, 1 per cent. I imagine that he must have hardened the osmic-treated gland in alcohol before making sections, since osmic acid alone only stains the transition-cells a yellowish-brown.

With regard to this point the following details may be. given. A piece of the gland is taken from a freshly-killed one-day-hungry rabbit, and placed in osmic acid 1 per cent. for two hours. Sections are then made at once, without the gland having been placed in alcohol, and mounted in dilute glycerine or in osmic acid. In such preparations the staining is yellowish-brown, no parts are stained very deeply; there are none which can be described as staining black. The difference of tint is not very great, but the ducts strike one at once as the darker parts; the ductules and transition-cells are not conspicuous, they differ from the alveolar-cells in being stained evenly throughout, whereas the latter are finely dotted; amongst these fine dots there is seen towards the periphery an evenly stained usually irregular nucleus, lightly stained. The nuclei of the transition-cells are stained to much the same extent as the cell-substance, and are thus with difficulty, or not at all, seen.

lf sections are made after twenty-four hours treatment with osmic acid, instead of after two, the appearances presented are very similar; the ducts are still the most deeply stained parts. The tint may have changed from a yellowish-brown to a brownish-yellow: still however the ductule- and transition-cells are not striking to the eye, and not stained much more deeply than the alveolar-cells.

If the osmic acid be renewed and, later on, sections cut, it is seen that only after several days do the transition-cells attain a colour to which Nussbaum's description can be fairly applied. 

...

It is... the alcohol treatment after osmic acid, which brings out clearly the difference of the staining power of the alveolar compared with the remaining gland-cells.

In a previous paper [note: 'Journal of Physiology, Vol. I. p. 69, 1878. At that time I could not obtain any appreciable amount of ferment from the sub-maxillary gland of the rabbit; since I have made no further experiments in that direction, I treat the subject here purely from a histological standpoint.'] I contested the justice of Nussbaum's conclusion that ferment is formed in, and only in, transition-cells. I there pointed out that after two hours osmic acid the ducts stain darkest, and the ductules equally with the transition-cells; consequently there is no reason for limiting the ferment to the transition-cells if we are to judge of the presence of ferment by the depth of staining with osmic acid. Nussbaum in his earlier paper [note: 'Arch. f. Mik. Anat., Bd. XIII. s. 721, 1877.'] describes the ducts as being stainied brown, and the transition-cells deep black (tiefschwarz); in his later paper [note: 'Arch. f. Mik. Anat., Bd. XVI. s. 543, 1879'] he says that in ferment-holding glands the transition-cells are always more deeply stained than the ducts. In neither does he mention the colouration of the ductules. In glands treated with alcohol after osmic acid it is true that the transition-cells may be slightly darker than the ducts, but this is not the case if the alcohol treatment is omitted. The striation of the outer portion of the duct-cells gives a different character to the staining here and in the transition-cells. The staining of the ductule-cells is however quite comparable to the staining of the transition-cells; the tint is in all cases similar, whether yellow-brown, brown o rblack, according to different modes of treatment; the cells are, in both, equally stained throughout. The depth of staining may be somewhat less in the ductules than in the transition-cells, frequently however it is not; it must be remembered that the transition-cells are larger than the ductule-cells, and on that account alone in most sections look darker. Even however granting that the ductule-cells are less darkly stained than the transition-cells, still there is sufficient similarity between them, and a sufficient difference of both from the alveolar-cells, to make it impossible on simple colouration, to say that the transition-cells owe their colour to ferment, and at the same time deny the presence of ferment in the ductules.' (270-272)

Cited by J.N. Langley, 'On the Histology and Physiology of Pepsin-forming Glands', Philosophical Transactions of the Royal Society of London 171 (1881), pp. 663-711.
Description:

'The oesophageal glands. - These glands have been described by Swiecicki, [note: 'Swiecicki, Pflüger's Archiv., Bd. xiii., s. 444, 1876'] Nussbaum, [note: 'Nussbaum, Max Schultze's Arch., Bd. xiii., 1877.'] and Partsch. [note: 'Partsch, Max Schultze's Arch. Bd. xiv. s. 179, 1877.'] The glands are of the complex tubular type; [note: 'I apply the term "simple tubular" to such glands as consist of one tube; when several tubes are given off by one duct, I call the glands "compound" tubular; when the tube or tubes arising from a duct divide, I call the gland a "complex" tubular gland. Klein describes the oesophageal glands as acinous glands (Stricker's 'Handbook,' vol. i., p. 538)']  amongst the proper secreting cells are mucous cells, these occur in smallest number in the final dilatations of the ducts. In the ducts ciliated cells are sometimes, though rarely, to be seen. The secretory cells are cylindrical or conical and are smaller than the gastric gland-cells. Nussbaum [note: 'Op. cit., s. 748.'] has shown that, they contain in the fresh state conspicuous granules; in a teased-out fresh preparation many of these granules are seen floating in the fluid: they are three to five times as large as the granules seen on teasing out similarly the gastric glands; they are even larger than the granules of the pancreas.' (664)

 

'On adding alcohol the granules sometimes run together before the partial solution takes place. Thus, in one instance, I watched three granules lying close together; first one ran into its neighbour, then this into the remaining granule, the whole forming one large granule; in it several brighter spots appeared; later, the greater part suddenly vanished leaving four or five rather bright particles arranged so as to produce the appearance of a fragment of a small-meshed network, I have little doubt that the apparent network seen in the cells in alcohol specimens has its origin from these residual particles.' [No explicit citation of Nussbaum here. cf. Langley's earlier criticism of Nussbaum's use of alcohol to fix specimens.]

 

'It was shown by Nussbaum [note: 'Op. cit.'] that the cesopha'eal gland granules are preserved by osmic acid. In treating glands with this reagent I usually use the following method. The tissue is placed in a 1 per cent. solution for twenty-four hours, removed to 50 per cent. alcohol for fifteen minutes and then transferred to 75 per cent. alcohol. Sections are cut on the following day. In sections so prepared the granules are stained not very deeply and have a yellow-brown tint. The sections as a whole are less stained than similarly prepared sections of the stomach. The tint of staining of the gastric gland granules tends to be brown-black rather than yellow-brown.' (665)

 

'The extent to which the disappearance of granules proceeds varies in different cases: in many cases two days after feeding [note: 'Nussbaum (op. cit., s. 749) made some observations upon the direct stimulation of the oesophageal mucous membrane, the cardia being ligatured before the animal was fed with sponge. He found under such circumstances that the granules entirely disappeared from the cells in three to five hours. Sewall and myself (op. cit., p. 285) were unable to observe any such rapid action.'] with a rather large piece of sponge, occasionally in a less time, scarcely any granules are left; and in some glands not a granule is to be seen (Plate 77, fig. 6 (a), 6 (b)).' (671)

 

'In the account of the changes in the oesophageal glands given by Sewall and myself there were two points which clashed with the earlier observations which Nussbaum made on osmic acid specimens. He described the gland-cells of the normal hungry Frog as having a large clear zone, and found that on feeding the animal the granules increased so that in three to five hours a clear zone was no longer to be seen.

Grutzner's [note: 'Grützner, Pflüger's Arch., Bd. xx. s. 395, 1879.'] results suggest an explanation of the divergence between the account of Nussbaum and that of Sewall and myself. He finds that in the normally hungry Frog the oesophageal glands are granular throughout and diminish in granularity during digestion; but finds also that if a Frog is kept longer than usual without food a clear zone is then formed in the oesophageal glands and that on feeding there is at first an increase of granules. Thus according to Grützner, Nussbaum's results would represent what occurs in a pathological and not what occurs in a normal condition,

During the last year and a half I have made a considerable number of observations with the view of determining the points at issue. As regards the state of the glands in the normal hungry Frog I have seen no reason to alter my first-formed opinion.

In some Frogs a large clear zone in the oesophageal glands does occur, namely, in those in which there are signs of general inflammation. In nearly all cases in which I have found a marked clear zone in the gland-cells of a hungry Frog, the animal had some mark or other of an ill state of health. I have frequently selected lively, active Frogs, and sluggish, unhealthy ones from a batch brought to the laboratory, and in a few days examined the oesophageal glands. The gland-cells in the former were granular throughout; those of the latter had almost always a clear zone.' (672)

 

'Swiecicki concluded from his observations that the oesophageal glands are at any rate the chief source of pepsin, and that the gastric glands produce little, perhaps indeed none. [note: 'He says: "Alle diese Thatsachen spreben hiernach dafür dass bei den Frischen die Pepsinbildung vorzugsweise, ja vielleicht nur allein in dem Oesophagus von statten geht, während der die Belegzellen führende Magen die Saüre bildet" (s. 452).']

Nussbaum suggested that the pepsin arose from the oesophageal gland granules. He found a correspondence between the number of granules in the gland-cells and the amount of ferment contained by the glands. Sewall and myself, whilst differing from Nussbaum as to the times of increase and decrease of granules, came nevertheless to the conclusion that the granules were connected with the formation of ferment. To this conclusion we came partly on general grounds, partly by comparing Grützner's results on the times of increase and decrease of pepsin with our own on the times of increase and decrease of granules.

... I think we can fairly conclude that the granules give rise to the ferment.' (678-679)

 

'In the cell-protoplasm there is during secretion an increase of substance capable of reducing osmic acid.

In all the cases investigated above we have seen that the protoplasm of the "quiescent" gland-cells stains very slightly with osmic acid, whilst the protoplasm of the active cells stains more or less deeply.

This is opposed to the observations of Nussbaum, but Nussbaum, I think, directed his attention so much to the granules contained by the cells that he overlooked the cell-protoplasm.

I have not been able to satisfy myself of the meaning of this increase in stainingpower of the protoplasm. Since the granules split up during activity, we might imagine that the cells do not at once cast out the whole of the substances formed, but retain a certain proportion, and that this diffused throughout the cell causes it to stain more than normally with osmic acid.

There is, however, another way of looking at the fact. Generally speaking, the formation of granules goes hand-in-hand with a diminution in the power of staining of the cell-substance. In the latter period of digestion, when the granules are increasing the cell-substance stains less and less with osmic acid. In Triton taeniatus during the rapid increase in the granules which takes place from the fourth to the eighth hour of digestion there is a rapid diminution in the staining power of the cell.

This would indicate that the protoplasm of the cells, in passing through those changes which result in the formation of granules, uses up the substance stainable with osmic acid. This substance might be either taken up by the cell during secretion to be further assimilated, or it might be an integral part of the cell-protoplasm.

The facts we have at present are, I think, insufficient to allow any satisfactory conlclusion to be drawn on this point; but whatever the cause of it may be, it is, I think, a very general phenomena of cells to stain more deeply with osmic acid during active secretion than duiing rest. I have on a former occasion [note: 'Proc. Royal Soc., vol. xxix., p. 377, 1879; Jour. of Physiol., vol. ii., p. 261, 1879.'] pointed out that this is the case in the parotid, sub-maxillary, lachrymal, and infra-orbital glands of the Rabbit.

In parotid and sub-maxillary glands the resting cells treated with osmic acid show lightly-stained cell-substance with darker-stained granules; when the cells are similarly treated after a period of secretion the cell-substance is distinctly more deeply stained, the depth of staining increasing within certain limits with the length of time during which the cells have secreted.

In the lachrymal and infra-orbital glands the resting cells treated with osmic acid show a networked appearance, from the presence of a lightly-stained protoplasmic with a darker interprotoplasmic portion. When the cells are similarly treated after a period of secretion the networked appearance disappears from the outer portion of the cells; it now is homogeneous - the protoplasm stains deeper than before. After prolonged secretion the cells stain equal and fairly darkly throughout.

In all these glands, however, the increase of substance in the cell-protoplasm capable of staining with osmic acid, although distinct, is not so marked as it is in the gastric glands. Grützner [note: 'Grützner, Pflüger's Arch., Bd. xx., s. 399, 1879.'] has independently come to a somewhat similar conclusion. He found in all the salivary and gastric glands investigated by him a difference of tint in osmic acid specimens of the resting and of the active glands. The former he found to be grey-green, the latter a dirty (schmutzig) brown. This, indeed, represents not unfairly the general difference in tint of the glands in the respective states when examined with not too high a power. I should prefer to call the tint of the one yellow-brown and that of the other brown-black. Grützner applied his description to the cells as a whole; the yellow-brown tint is, however, in the main due to the staining of the cell-granules, the brown-black tint in the main to the staining of the cell-protoplasm.' (707)

Cited by J.N. Langley, 'Some Remarks on the Formation of Ferment in the Sub-maxillary Gland of the Rabbit', Journal of Physiology 1 (1) (1878), pp. 68-71.
Description:

'Early in the past year (1877), I commenced to make some experiments to test the conclusions arrived at by Nussbaum [note: ' "Die Fermentbildung in den Druisen," Archiv für microsc. Anat. XIII. (1876 [sic]), p. 721.'], concerning the recognition under the microscope of the amylolytic ferment of the salivary glands. Circumstances prevented me from carrying them out as I had purposed, and in the meanwhile, Grützner [note: 'Pflüger's Archiv, xvi. (1877), p. 105'] has been working at the subject, and arrived at results which invalidate Nussbaum's conclusions. Incomplete as my experiments are, still they may serve to fill up, to a certain extent, a gap in Grützner's observations so far as the sub-maxillary gland of the rabbit is concerned.

The starting-point of Nussbaum's work was the fact that the amylolytic ferment as prepared by von Wittich's method, reduced osmic acid; from this he argued that those parts of a gland wlhich contain ferment, must be stained black by this reagent, and that if the ferment be extracted from the gland before the latter is treated with osmic acid, the parts which are no longer deeply stained must represent the parts which are concerned in ferment-production. His method of preparation in order to observe the difference of colouration before and after the removal of the ferment, is the following: he places one piece of the gland warm from the body, in a 1 per cent. aqueous solution of osmic acid for a definite time, and another piece in osmic acid of a like strength for a like time, after it has been extracted with glycerine for two or three days, with or without previous hardening in alcohol. Treating in this way the sub-maxillary gland of the rabbit, he obtained from the piece of the gland laid fresh in osmic acid, certain cells of the alveoli, very deeply stained; these were the more centrally lying cells coming immediately after the ductules [note: 'I use the word ductules for those small terminal ducts which have lost the characteristic appearance of the larger ducts, in no longer possessing a cylindrical epithelium with a striated outer border; they were called by their discoverer, Ebner, " Schaltstücke."'], which for convenience, and for reasons to be given presently, I will call "transition" cells; in the piece however that had been treated with glycerine for the extraction of ferment, he found the transition-cells to be stained not more deeply than those in the peripheral parts of the alveoli. He concludes then from this, that the amylolytic ferment is formed in the transition-cells; the more peripheral alveolar cells not being concerned in ferment-production.

Leaving for the moment the consideration as to whether this deduction is logical or no, I would first discuss the facts on which the deduction is founded.

The appearances described by Nussbaum after the gland has been treated in the above-mentioned ways, do not, I think, represent the actual state of things.' (68-69)