cytological study on the salivary corpuscles as well as

Cytological Study on the Salivary Corpuscles

as well as the Oral Epithelial Cells with

Particular Reference to the Glycogen

and Enzyme Reactions.

The 17th Report of Histochemical Study of Peroxidase

By

Shoji Maruyama

Department of Anatomy, School of Dentistry, Nihon University, Surugadai, Tokyo.

(Director: Prof. T. Mitsui, M. D.)

Almost invariably, the leucocytes are discharged into the human oral cavity in varying amounts. These leucocytes are predominantly

neutrophil and called salivary corpuscles. The salivary corpuscles

possess much less activity than the neutrophils in tissue fluids, because the saliva is one of the unsuitable conditions for the blood cells due to its hypotonia, mucin content and increase in acidity etc. Therefore, the leucocytes in saliva gradually lose their active motility with the

lapse of time, changing into degenerative cells. In order to examine the degree of degeneration of these salivary corpuscles, several special

stainings of blood cells were employed here,, because the R om an o wsk y staining such as Wright's or Ma y-G iems a's staining is insufficient for differentiation of the degenerating leucocytes.

This study was undertaken in order to make it clear how the leucocytes are cytologically influenced by saliva, as part of a series of investigations concerning the peroxidase staining of the blood cells.

Materials and Methods.

A large number of saliva smears were obtained from 114 human subjects aged from 6 to 56 years. The saliva was taken from the sites of the gingival margin, the palatine tonsils or the hard palate by means

of cotton applicator. Epithelial cells of the oral mucosa as well as

peripheral blood specimens of the same human subjects were simulta- neously examined.

250 S. Maruyama

Staining methods were as follows : 1) Glycogen staining.

The saliva smear was fixed with the S c h a u d i n n's mercuric chlo-ride alcohol (sublimate -alcohol) for thirty minutes'''. This fixative is a mixture of 2 cc. saturated aqueous mercuric chloride, 1 cc. absolute alcohol and several drops of glacial acetic 'acid. After this fixation, the smear was washed in distilled water for 1 minute, treated with about 0.5% iodine in 70% alcohol for 10 minutes, immersed in 70% alcohol for 10 minutes, next washed again in distilled water for 10 minutes. After -this iodization, Ito's modificatioe of the periodic acid leuco-fuchsin method was employed as follows :

a) Oxidize for 30 minutes in- the potassium periodate (K1.04) solution (a mixture of 0.5 gr. K104, 10 cc. distilled water and 24 cc.

0.1 N hydrochloric acid ; this remains active for some months). In summer, this oxidizing time may be 10-20 minutes.

b) Wash in distilled water for 5 minutes. c) Treat with the Schif f's leuco•fuchsin reagent for 30 minutes.

In summer, this treating time may be 15-20 minutes. The Schif f's reagent was prepared by the following method. Dis- solve 1.0 gr. basic fuchsin (parafuchsin) in 200 cc. hot distilled

water -(90°-95°C.). Filter at 50°C. as it cools, add 20 cc. N hydrochloric acid. After cooling below 25°C., add 1.0 gr.

sodium bisulfite (NaHS03) and shake sufficiently. Stopper tightly and store in the dark overnight at room temperature.

The solution should now be clear light yellow. When kept at 5°C., it remains active for some months, but it becomes useless

when any pink tint appears. d) Pass through 3 changes, 5 minutes each, of the sodium bisulfite.

This is made extempore by adding 10 cc. N hydrochloric acid to 200 cc. 0.5% sodium bisulfite solution. This mixture should

always be prepared anew. e) Wash in running water for 10 minutes.

0 Stain with the De la f ield's hematoxylin solution for 2 minutes, next wash with tap water, dry and examine.

Both the above Sc ha u d inn's solution and gasecus formaldehyde were better fixatives for the glycogen staining than formol-ethanol

: 2), methanol, ethanol or 10% formalin. 2) Iodine reaction.

Unfixed saliva smear was previously stained with either hemato-xylin or safranine red for 10 minutes in order to differentiate leuco-

Cytological Study on the Salivary Corpuscles. 251

cytes ; fuchsin and methylenblue were entirely improper for it. Next,

about 0.4 gr. iodine crystals were slowly heated in a covered heat-resisting Petri dish, which was soon saturated with the violet colored iodine vapors, then the smear was put into the above dish, and let stand about 10 to 15 minutes. It is necessary in this reaction that the iodine vapors are always slowly volatilizing in the dish, and that the result is examined. promptly after the reaction is finished.

3) Dopa reaction. Smears were fixed with the formol-ethanol (2 :8) for 5 minutes,

washed in distilled water, next stained with the Dopa solution (pH 7.4) for 20 minutes at 37°C., then counterstained with the Pfeiff er's fuchsin solution for 30 seconds. The Dopa solution is a mixture of 10 cc. 0.1 % 3, 4-dihydroxyphenylalanine and 3 drops 0.1 N caustic

potash. 4) Peroxidase reaction.

The NAS-Benzidine method" was utilized. 5) Staining of the plasma membrane (Tannin fuchsin method)..

a) Fix the smear with methanol for. 1 minute, and wash in distilled water, As the fixative for the staining, methanol is

preferable to the Zenker's solution. b) Immerse in 5.0% tannic acid solution for 5 minutes, and wash

in distilled water. c) Stain with the Z i e h l's carbol fuchsin diluted twice, for 1

minute. d) Wash in distilled water, and dry.

e) Immerse in acetone for 1 minute. f) Clear with xylol sufficiently ; • pass through 3 changes of xylol.

6) Iron reaction (Turnbull blue reaction). a) Fix smear with ethanol-formol (14 : 1) for 30 minutes, and wash

in distilled water. b) Impregnate for 2 hours in saturated aqueous ammonium sulfide

((NH4)2S) solution, and wash thoroughly in distilled water. c) Soak for 1 hour in equal volumes of 1.0% hydrochloric acid

and 20.0% potassium ferricyanide, freshly mixed. d) Wash in distilled water, and counterstain with 0.5% basic

fuchsin in 50% alcohol for 20 minutes.

252 S. Maruyama

Results obtained.

1) Glycogen reaction. (Fig. 2-5). Among leucocytes in the peripheral blood, the neutrophil shows

the strongest reaction diffusely in the protoplasma, and the eosinophil shows a weaker reaction around its granules alone, while others show merely few reacting granules in the protoplasma, namely the order of the intensity of the glycogen reaction is as follows : Neutrophils, eosino-

phils, basophils, monocytes, lymphocytes. According to Yokoi (1949)" the polymorphonuclear neutrophils show a stronger glycogen reaction than in their immature stages in bone marrow. These glycogen

granules stain dark violet in a strong reaction, while red purple in a weak. However, it is questionable whether the glycogen reacting sub-stances of the leucocytes are essentially present in the localized, granular states or present homogeneously within the protoplasma". The salivary corpuscles being predominantly neutrophils, their glycogen reactions are expected , to be intensely positive. However, the reaction intensity was varied and generally weaker than expected due to the inhibiting influence of saliva. These findings can be summ?rized as follows :

a) Group demonstrating much the same intensity as neutrophils in the peripheral blood. This group is very limited and thought

to be fresh salivary corpuscles. . b) Group demonstrating fewer and finer glycogen granules than

in normal neutrophils. c) Group demonstrating glycogen granules in the ectoplasmatic

zone alone. d) Group demonstrating a conglomeration of glycogen granules in

the endoplasma. e) Group demonstrating purple, linear substances in the proto-

plasma. 1) Group demonstrating abnormally dark purple, coarse granules

diffusely in the protoplasma (Fig. 2, 4) ; the reaction is clearly stronger than that of neutrophils in the peripheral blood. These positive granules are so coarse and numerous that they

may be confused with the eosinophil granules, but the glycogen reaction of the eosinophil leucocyte, as stated already, is much

weaker than neutrophil leucocyte, and occurs only around the

granules, but not within the granules. This differentiation becomes more simple when the smear stained with the glycogen

stain, is decolorized with a mixture of hydrochloric acid and


ethanol, next stained with hematoxylin eosin solution, in which case the eosinophil granules appear red, bacterium blue, while neutrophil granules remain unstained.

g) Glycogen negative group. These are degenerating cells, which frequently possess intracellular vacuoles and faintly stained

nuclei with obscure contours. Of these seven different groups, the reaction negative or weak

positive group was most numerous, while reaction intense group, es-pecially the group of f) was rare.

On the other hand, lymphocytes as well as eosinophils were also demonstrated from the saliva in far smaller numbers than neutrophils. But the glycogen reactions of the former two were always weak and their reaction positive granules lost sharp contours as compared with the same leucocytes in the peripheral blood. Furthermore, it should be noted that the neutrophils in the peripheral blood as well as in the saliva, frequently indicated a strong glycogen reaction in the extreme margins of the cell bodies, and that the halo of the saliva surrounding salivary corpuscles stained faint purple indicating weak positive glycogen reaction. The glycogen reaction of the oral epithelial cells was varied. A small fraction of the cells were unstained with the periodic acid leuco-fuchsin solution, while most of them were stained in various degrees. Namely, some of them possessed dark purple granules diffusely in the protoplasma, some possessed purple granules localized in either ecto-plasma or endoplasma, while some possessed faint purple granules dispersed in the protoplasma, and others possessed homogeneously purple stained protoplasma. The shape of the glycogen reaction granules is varied, for example, coarse and spherical just like the eosinophil granules, rod-shaped, needle-shaped, fine granular, flocculent or irregular form.

There was no relationship between intensities of the glycogen reaction and shapes of the epithelial cells. Vacuoles unstainable with the glycogen stain, were rarely seen within their protoplasma.

The nuclei of the salivary corpuscles and of the oral epithelial cells were glycogen negative".

2) Iodine reaction. (Fig. 1).

As already stated by Ikeda (1951)" at our laboratory, protoplasma of the blood neutrophils shows a homogenous yellow or light brownish tinge by the iodine reaction. The salivary corpuscles, due to the in-fluence of saliva, indicated somewhat different reactions from the blood

254 S. Maruya ma

neutrophils. The detailed results were as follows : a) Leucocytes demonstrating homogeneous faint yellow proto-

plasma. b) Leucocytes demonstrating brown -fine or coarse granules within

the protoplasma. c) Leucocytes demonstrating large conglomerate iodophil granules

in the endoplasma. d) Leucocyte demonstrating intracellular vacuoles which are sur-

rounded with iodophil substance. e) Leucocyte demonstrating a weak iodophil nucleus.

f) Entirely unstained leucocytes with iodine these are highly degenerating cells.

Of these, group a) and f) were most numerous, while group e) was very rare. On the other hand , iodine reaction of the oral epithelial cells were generally more intense than in the salivary corpuscles. Reac-fion positive granules were of various sizes and of various forms ; some were coarse spherical, while some were fine granular , and others were conglomerate in the endoplasma . The epithelial cells with homogeneous-ly dark brown protoplasma were not unusual , and nuclei of the epithelial cells were always intense iodophil, so that the previous staining of these nuclei with hematoxylin solution before the iodine reaction, was not necessary. Of these, the cells with homogenously brownish stained

protoplasma were most numerous, while those with coarse dark brown granules were less in number. The epithelial cells rarely possessed vacuoles, which did not stain with the iodine vapor. Linear folds of the epithelial cell which stained most intensely brown, were often demonstrable, especially from the cells of the hard palate.

It is of interest that both nucleus and protoplasma of the oral epithelial cells were more intense iodophil than those of the salivary

corpuscles.

3) Peroxidase and Dopa oxidase (melanase) reactions. (Fig. 1). Most of salivary corpuscles could be stained with the peroxidase

stain, since they were myelogenous leucocytes. In general, the neutro-

phils can also be identified by Wright's stain unless they are degenerated in the saliva, while it becomes very difficult to distinguish them from the lymphocytic series when the neutrophils are degenerated, for example, their nuclei become mononuclear or the vacuoles occur in the

protoplasma. As has already been reported by Ik ed a (1951), with the NAS-Benzidine solution,- the protoplascna , of fresh neutrophils in saliva is full of fine or large deep blue granules, and in propoition to


the degree of degeneration by saliva, the granules become sparse, not

uniform in size, fewer in number, not well.-defined, mixed with brown

granules (so-called double phase"), until no reacting granules appear in the protoplasma although they are myelogenous. It is not unusual that

intensely degenerated salivary corpuscles possessing several large vacu-oles, demonstrate much the same distinct numerous peroxidase granules in the protoplasma as in fresh neutrophil leucocytes. The Dopa reaction

positive granules in the myelogenous leucocytes appear dark brown. Both the Dopa melanase and peroxidase reaction being a similar oxidase reaction in the broad sense, there is no doubt that both reactions demonstrate almost similar results about blood cells. However, when

precisely examined, the following characteristic findings of salivary corpuscles were revealed here :

a) The Dopa reacting granules in salivary corpuscles were generally less distinct than the peroxidase reacting granules, and never

indicated the so-called double phase" appearing in the peroxidase staining.

b) Protoplasma of salivary corpuscles occasionally stained homo-

genously brown with the Dopa solution, indicating no reacting granules.

c) The Dopa reacting granules were frequently localized in the extreme margin of the protoplasma, in the perinuclear site,

or in the endoplasmatic zone. d) Salivary corpuscles possessing Dopa reaction positive nuclei

were distinctly found although in a small number ; these nuclei stained blackish brown homogenously. In this case, it is worthy

of special comment that the Dopa reaction of the nuclei was

generally more intense than that of the protoplasma. These reaction positive nuclei were never found in the peroxidase

staining commonly used. Both protoplasma and nucleus of the oral epithelial cell were al-

ways peroxidase negative and Dopa negative.

4) Plasma membrane. (Fig. 1). The surface membrane of a cell is called the plasma membrane,

and is believed to consist fundamentally of a lipoid film to which is adsorbed a layer of protein. It is important in the consideration of the metabolic activity of a cell in relation to its immediate environment in living tissues. All interchange between the cell and the surrounding

medium must take place through the plasma membrane, whether this involves the transfer of nutrient substances into the cell or the dis-

256 S. Maruyama

charge of metabolites and other products of cell activity. In most cases, this membrane is difficult to delimit for morphological purposes, and

yet its existence cannot be doubted. However the position of the plasma membrane is made clear by the difference in properties of the cyto-

plasma which it limits and of the fluid without, and also in the dark-field by the light reflected from its surface "). The plasma membrane, indeed, is the anatomical and physiological boundary between the cell and its environment. In this paper, the tannin fuchsin method was utilized for staining of the plasma as well as nuclear membranes . By this method, these membranes appear in red fine lines.

The findings can be summarized as follows : a) The plasma as well as nuclear membranes of a salivary cor-

puscle were generally unstained whether it is degenerated by saliva or not. Very rarely, however, the plasma membrane

was found in the fresh salivary corpuscle, in which case the extreme margin of the protoplasma stained less distinctly than

in the oral epithelial cell. The leucocytes in the degenerating stages never showed plasma membrane.

b) The plasma membrane of the oral epithelial cell was stained very distinctly with the tannin fuchsin. The distinct lines

stained deep red, were recognized not only at the extreme margin of the cell as a common plasma membrane but also

on the surface of the cells in the form of folds, or edges of a cube. Although the oral epithelium is stratified squamous,

it is of interest that an oral epithelial cell is not a mere flat cell, but an almost cuboidal thin cell which possesses numerous

sharp edges straight or curved. These edges are also demonstrable by means of a phase microscope without any stain-

ing") 12). Regarding the plasma membranes of the epithelial cells, three sites of the oral mucosa, namely, palatine tonsils ,

gingival sulci and hard palate were compared. Of these, the hard palate was covered with extremely characteristic epithelial

cells ; their plasma membranes were most distinctly stained, and the edges or crests appearing on their surface structure

were most numerous and almost rectilineal, furthermore non- nucleated epithelial cells were most frequently isolated from the

hard palate. The nuclear membrane of the epithelial cell was

generally indistinct. It can be. said that the stainability of the plasma membrane is one

of the important cytological differences between the salivary corpuscles


and the oral epithelial cells.

5) Iron reaction. Neither salivary corpuscles nor oral epithelial cells were stained

with the Turnbull blue method.

Fig. 1. Various stainings of the cells in the oral cavity. A (Iodine reaction of salivary corpuscles and oral epithelial cells), B (Iodine reaction of oral epithelial cells without previous hematoxylin staining of nucleus), C (Peroxidase reaction of salivary corpuscles, NAS-Benzidine method), D (Dopa reaction of salivary corpuscles, Bloch's modification), E (Plasma membranes of oral epithelial cells, Tannin-fuchsin method).

Discussion.

It has long been believed that the salivary corpuscles are the

lymphocytes arising from the palatine tonsils"")19, while it is obvious

to-day that the salivary corpuscles are predominantly the neutrophils

arising from the palatine tonsils as well as the gingival sulci7)16) 17) 18) 19)

The secretions on the various mucosae are generally unsuitable

environments for the survival of leucocytes. When leucocytes are

258 s Maruyama

mixed with saliva in the oral cavity, they. are gradually 'inactivated . The morphological alterations of salivary corpuscles are characterized by loss of their , affinity. for R o m a n o w s k y stain, loss of segmentation and of filaments in the nucleus, karyorrhexis and vacuolation of the

protoplasma, finally they undergo fragmentation terminating in autolysis. The present study considers the influence of saliva upon the

glycogen-, iodine-, peroxidase- and Dopa oxidase-reactions of leucocytes, furthermore considers the differences between the staining qualities in the salivary corpuscl6 and the oral epithelial cells. It was revealed here that glycogen-, Dopa- and peroxidase-reactions of the salivary corpuscles are generally weaker than those of the blood leucocytes , but that more intense reactions of the salivary corpuscles than in the blood leucocytes may occasionally be found. The glycogen reaction intense positive leucocytes in saliva, demonstrating deep purple, large, numerous granules cannot be found in the peripheral blood. It is of prominent interest to find such a strong glycogen reaction of salivary corpuscles , because the saliva is generally regarded as one of digestives of glycogen and the salivary digestion test is usually employed in the glycogen staining. This is one of the proofs that the periodic acid leuco-fuchsin method can also stain the substances other than glycogen -which is digested - by saliva. It was already described that the halo of the saliva

surrounding salivary corpuscles stained purple by this method . It • is well known that the periodic acid leuco-fuchsin solution can stain not only glycogen but also other polysaccharides such as starch , and mucin which resists salivary digestion" "). The neutrophils demonstrating brown coarse or large conglomerate iodophil granules within the proto-plasma as well as the neutrophils with iodophil nucleus, were occasion-ally found from the saliva but not from the peripheral blood where the neutrophils showed only homogenous yellow iodophil protoplasma .

Furthermore the neutrophils with Dopa reaction positive nuclei were occasionally 'found from the saliva but not from the peripheral blood. On the other hand, the intracellular vacuoles of salivary corpuscles did not stain with the . glycogen-, iodine-, Dopa- or peroxidase-reactions, namely, always remained reaction negative with any reagents . Sasaki

(1952)19) already pointed out that the vacuoles appear first in the endo-plasmatic zone of the salivary corpuscles and subsequently within the nucleus and the ectoplasmatic zone. All these characteristic findings of salivary corpuscles are presumably indications of degeneration

whetiier abnormally intense reactions or weak ones. Although the essential , nature of the iodophil granules in the salivary cotpuscle's is


unknown, these granules can not be identified in the normal blood cells21)22); it is thought that the iodophil granules may occur in the

leucocytes in cases of infections or toxic. diseases, and that the appear-ance of them means an activation of the cellular function21. Howevef,' this activation theory can not be accepted concerning the salivary corpuscles at least. M itsu i et al."' "n" already proved by the use of tissue sections and saliva smears that the salivary corpuscles can be derived from almost all sites of the oral mucosa, and that the upper pole of the palatine tonsils as well as the -gingival sulci are the greatest source of them. According to the results of the present study, intense degenerative leucocytes are most frequently found from the gingival sulci, while infrequently from the palatine tonsils .

The differences between the staining qualities in the salivary cor-puscles and the oral epithelial. cells were as follows :

a) In the epithelial cells, the glycogen reaction positive granules' are of various sizes and forms more often than in the salivary corpuscles.

b) Both nucleus and protoplasma of the epithelial cells are general,- ly more intense iodophil than those of the salivary corpuscles;

c) Neither protoplasma nor nucleus of the epithelial cells shows peroxidase- and Dopa oxidase-reactions. Namely, the epithelial

cells are enzyme-reaction negative cells, while the salivary corpuscles are rich in the peroxidase as well as oxidase reacting substances.

d) The plasma membrane of the epithelial cells is generally stained very distinctly with the tannin fuchsin stain, while that of the

salivary corpuscles is generally not. e) The iron reaction of both the salivary corpuscles and the

epithelial cells is negative. According , to Kobayakawa (1938)n) the neutral fat is found in the protoplasma as well as

in the nucleus of both the salivary corpuscles and the epithelial cells.

As to the biological significance of the salivary corpuscles, nothing can be said with certainty. In general, the leucocytes can be discharged into the urine (Yokota et al., 1950)24), into feces (Sugiyama, 1953)25), into the semen (I sh i k a w a et al., 1953)26) or into the mucous membranes of alimentary, respiratory tracts, conjunctiva and vagina z7)28).29). The mechanical as well as the bacteriological stresses on the 'mucous mem-branes are important factors for the development of the Wandering' leucocytes but these stresses may- not be the only factors involved."

260 S. Maruyama

Are the wandering leucocytes merely accidental, waste cells similar to the shed epithelial cells, or are they indispensable cells on the various mucosae ? These questions remain unanswered, although, as has already. been stated by Mori t a (1952)Th, the discharging phenomenon of the leucocytes may have a certain relationship with the human hemato-

poiesis. In any case, it was confirmed-here that the leucocytes ranging from the fresh active stage to the degenerative dying stage, can be

isolated from the saliva, and that the peroxidase-, Dopa oxidase-,

glycogen- and iodine-reactions are recommended for the demonstration of the salivary corpuscles in any stages, because the Romanowsky Staining hematological granules excepting the eosinophils are highly

water-soluble").

Summary.

In order to obtain a better understanding of the essential nature of the cellular component in the saliva, reactions of glycogen, iodine,

peroxidase, Dopa oxidase and of iron as well as staining of the plasma membrane were tested on the saliva smears derived from 114 human subjects aged from 6 to 56 years. It was made clear through the pre-sent study that the glycogen-, peroxidase-, Dopa oxidase-reactions of

the .salivary corpuscles generally became weaker with increase of the degeneration in the saliva, and that, to our surprise, a very small

fraction of the salivary corpuscles indicated a more intense glycogen reaction than normal neutrophil leucocytes in the peripheral blood,

furthermore that the nuclei of the salivary corpuscles were occasionally Dopa and iodine reactions positive, which was never seen in the leuco-cytes of the peripheral blood. Most of the salivary corpuscles are thought to be the neutrophil leucocytes which stain in considerably different degrees from those of the peripheral blood. The peroxidase and Dopa reactions of the oral epithelial cells were negative, while

glycogen, and iodine reactions of them were positive, in addition to this, the plasma membrane of them was stained very distinctly as compared with that of the salivary corpuscles.

The author wishes to express his gratitude to Prof. Mitsui for his kind advice during the course of this work.

This study was carried out with the grant in aid for fundamental scientific research of the Ministry of Education.


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25) S u g i yam a, K., Leucocytes in human feces, unpublished. 26) I s hi k a w a, K. & Asakur a, T. (1953), Alterations of human semens in atomic

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Explanation of figures.

Fig. 2. Glycogen staining of various salivary corpuscles (periodic acid leucofuchsin method). E is an eosinophil leucocyte in the saliva, othera are all neutrophil

leucocytes. Fig. 3. Glycogen staining of various oral epithelial cells. Fig. 4. Glycogen staining of salivary corpuscles. Vacuolations in the protoplasma are

distinctly shown. N is an intensely stained neutrophil leucocyte. Fig. 5. Glycogen staining of salivary corpuscles and oral epithelial cells.

Plate I

• Flit . 2. Fig. 3.

, _

Fig. 5.

S. Maruyama.

cytological study on the salivary corpuscles as well as

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