Fluorescent PAS-reaction study of the epithelium of normal rabbit ileum and after challenge with enterotoxigenic Escherichia coli

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Fluorescent PAS-Reaction Study of the Epithelium of Normal Rabbit Ileum and After Challenge with Enterotoxigenic Escherichia coli T. N. KHAVKIN, M. V. KUDRYAVTSEVA, E. M. DRAGUNSKAYA, YU. E. POLOTSKY, and B. N. KUDRYAVTSEV Institute

for Experimental

Medicine,

Institute

Fluorescent periodic acid-Schiff reaction (FPR) was used in the study of the normal rabbit ileal epithelium and its changes after injection of living cultures or enterotoxins of enterotoxigenic Escherichia coli. This reaction, with the use of auramine 00-SO, complex as a Schiff-type reagent, demonstrates gut epithelium periodate-reactive mucosubstances more distinctly and brightly than does the common periodic acid-Schiff (PAS) reaction. It permitted the quantitative assessment of polysaccharide content in the gut sections by micro@orimetry, and examined extensively the mucosal structures, brush border, and mucous cells which participate in the interaction with enteropathogens. Fluorescent periodic acid-Schiff reaction showed that noninvasive enterotoxigenic E. coli 0248:H28 B7A organisms caused restricted damage to the intestinal epithelium brush border. Invasive enterotoxigenic E. Coli 026:K60:HlZ N3 organisms penetrated the epithelium and caused extensive brush border lesions and mucous cell hyperproduction. Importance of FPR in the complex morphologic analysis of enteric infections, pathogenesis of escherichoses under study, and some aspects of the intestinal epithelium histology are discussed. Received June 19, 1976. Accepted November 27, 1979. Address requests for reprints to: T. N. Khavkin, M.D., 62-42 Woodhaven Blvd., Apt. N-54, Rego Park, New York 11374. The authors express their deep gratitude to Dr. S. B. Formal (Walter Reed Army Institute of Research, Washington, D.C.) for the standard enterotoxigenic E. coli strain B7A (0146:H26); Prof. I. KBtyi (PBcs University Medical School, P&X, Hungary) for the enterotoxigenic E. Coli strain N3 (026:K6O:Hll); and Prof. T. A. Avdeeva and Dr. M. G. Chakhutinskaya (Department of Enteric Infections, Leningrad Pasteur Institute) for microbial cultures, enterotoxin preparations, and technical help. I. Ya. Barsky is acknowledged for his help in fluorimetry. 0 1960 by the American Gastroenterological Association 0016-5065/60/040762-09$02.25

of Cytology,

Leningrad,

U.S.S.R.

Polysaccharide-rich formations, such as the cell coat and mucous secretion, are known to play an important role in the function of the intestinal epithelium and its interaction with microorganisms.‘,’ The onset of intestinal infections is determined by the function of the cell coat (glycocalyx) to a considerable degree. The fact that enteropathogens can adhere to this coat and interact in different ways with its constituents is an important factor of their pathogenicity.” It is also known that the mucous cells of the intestinal epithelium take part in the development of inflammatory reaction.4 The present study deals with the possibilities of application of fluorescent periodic acid-Schiff (PAS) reaction (FPR) to detect selectively periodate reactive substances of the cell coat and mucous secretion of enterocytes. It is known that all fluorescence reactions are much more sensitive than dyes are.5 The former are highly specific and may be useful in quantitative assays of polysaccharides.‘,’ Therefore, the FPR was administered in the study of the rabbit ileal epithelium under normal conditions and in experimental enteric infections. For this purpose, two different strains of Escherichia coli and their enterotoxins have been used. According to the earlier results of electron microscopic study,’ the first strain, B7A (0148:H28), a well-known enterotoxigenic agent of choleralike disease in adults,g is noninvasive, capable only of attaching to and multiplying on the brush border. It evokes pronounced hypersecretion of the intestinal epithelium. The second strain, N3 (026:K60:Hll), recently isolated from an infantile diarrhea case,B has been shown to produce enterotoxins which, however, differed markedly from the above strain. The organisms multiply between the microvilli of the brush border, resulting in its thinning out and even disappearing in some

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areas. In addition, the bacteria penetrate the epithelial cells, being enclosed in phagosomelike vacuoles, and cause desquamation of enterocytes. The enterotoxigenicity of strain N3 has been proved by rabbit gut loop, skin permeability, and suckling mouse tests.’ Recently, it has been repeatedly confirmed.‘O

Materials and Methods Microorganisms A 24-hr broth culture of E. co8 0148:H28, B7A strain9 and 028:K6O:Hll, N3 strain,’ were used for infection. The enterotoxins of these serotypes were obtained by means of Millipore filtration of the culture supernatant and ultrasonic lysates. For further details of enterotoxin isolation, see Refs. 8 and 10.

Fluorescent Study

and Sampling of Material

The rabbits, weighing 1.7-2 kg, were challenged in ligated ileal loops. This technique permits following the interaction of organisms with the epithelium at the earliest postchallenge stages as well as during development of infection. The method of infection is described elsewhere.’ The animals were fasted for 4 days before operation. The lo-cm-long ligated loops were given injections of 5 ml of bacterial culture containing 1.1-1.3 X 10” organisms or enterotoxin preparations. For control, sterile meat-peptone broth was injected into one of the loops in each rabbit; a total of ten rabbits were operated on. Additionally, we used paraffin sections of the same ileal samples, which were studied earlier by electron microscopy.’ The ileum was also examined in three intact animals. The animals were killed 3, 6, 9, and 18 hr postchallenge. Particular attention was given to the ileal loops which exhibited evident signs of pathology such as congestion and fluid accumulation in the lumen. Real samples were fixed in 10% formalin and embedded in paraffin; also, cryostat sections were prepared and fixed in ethanol or 10% formalin.

Untreated paraffin section of the rabbit ileal mucosa showing autofluorescence of the epithelium (E) and lamina propria (LP). Fluorescence intensity of some areas in the conventional units: a. (terminal web zone of epithelial cells), 3.78 f 1.02; b. (brush border and apical cytoplasm), 8.51 f 2.31. In the right corner, image of the step wedge is seen, x 1000.

PAS-Reaction

and Fluorescence

On the whole, FPR is similar to the reaction suggested by McManus” and Hotchkiss.” Contrary to the common PAS reaction, in FPR, the Schiff-type reagent with auramine 0 (00) instead of basic fuchsin, is used. The advantage of auramine application is that it, unlike other fluorochromes used for similar purposes, ensures a low absorption rate in exciting light and bright fluorescence at the same time. An auramine-based fluorescent Schiff-type reagent (Au-SO,) produces bright fluorescence in its interaction with polysaccharides and DNA, without forming metachromatic products. Moreover, it is highly specific.‘3,‘4 The procedure of FPR was developed by M. V. Kudryavtseva, B. N. Kudryavtsev, and others, and is described elsewhere.” It is as follows: 1. Oxidize

2.

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3. 4. 5. 6. 7. 8.

sections with 0.8% solution of potassium periodate for 90 min. Rinse in running water and in one portion of distilled water. Treat with freshly prepared Au-SO, (see below) for 98 min. Wash in distilled water. Treat with three portions of freshly prepared sulfur water. Wash in running water. Dehydrate in alcohol for 36 min. Immerse in nonfluorescent Vaseline oil.

For control purposes, some of the sections were not oxidized before treatment. The fluorescent Schiff-type reagent (Au-SO,) was a 8.3% solution of auramine 00 (Reanal, Hungary), containing 6.2 ml of thionyl chloride per 166 ml of dye solution. Microscopy and photography were performed by the ML-3 type fluorescence microscope (LOMO, U.S.S.R.), supplied with an opaque illuminator with a dichroic plate for excitation of fluorescence by incident light. Fluorescence was induced by ultraviolet light, using FS-1, SS-2, and SZS-14 exciter filters, and a GS-18 + GZS-19 barrier filter. Objectives: 100x/1.25and 40X/0.70.

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Figure

2.

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Selective fluorescence of the periodate-reactive substances in the brush border (BB) and goblet cells (GC) in the normal ileal epithelium. Fluorescence intensity of brush border (bracket), 92.54 f 2.69; CC, 101 f 14.16. Cytoplasm of the columnar epithelial cells (E) shows a slight autofluorescence. Paraffin section treated with FPR, x 1600.

Fluorescence intensity (FI) was measured by the photographic method of fluorimetry, which permitted separate evaluation of fluorescence intensity near closely located structures and the pattern of fluorescence intensity distribution in the object. To this end, the ML-2 microscope was provided with a barrier filter partly substituted by a step wedge.18 When the preparations were photographed through this device, care was taken to locate the object so as to ensure that the image of the step wedge was in the part of the picture area where the object is absent (Figure 1). Fluorescence intensity was measured in the MF-4 type microdensitometer (diameter of the photometric diaphragm was 50 pm) on the basis of density of emulsion of the negatives in the images of the object and of the step wedge, and was expressed in conventional units (CU). Assays, using conventional PAS reacfion,” were conducted along with FPR, and sections were stained with Mayer’s mucicarmine and according to RomanovskyGiemsa.

Results Paraffin

Sections of Normal Rabbit Small

Intestine Sufficiently distinct and reproducible results were obtained in paraffin sections. The sections showed some autofluorescence. Its intensity (FI) varied from 2 to 9 CU (Figure 1) and appeared weaker in the nuclei than in the cytoplasm. Autofluorescence did not interfere with the investigation and even contributed additional information on the site of PAS-positive formations. Specific fluorescence

was characteristic of the brush border and mucous secretion of enterocytes, reticular fibers, and scarce clusters of glycogen in cells. The brush border appeared as a homogeneous, somewhat striated bright fluorescent band (Figure 2). A more intense fluorescent basal part of the brush border could be discerned in some areas. The FI of the brush border was fairly uniform throughout the entire length of intestinal villi. The scanning fluorimetry along the brush border, using a standard photometric diaphragm, showed mean FI about 7090 CU. The FI of the mucous secretion in the goblet cells was 90-120 CU.

Cryostat

Sections

The autofluorescence of these sections was lower than that of paraffin sections, the brush border being wider by 10%. Although the fixing agent involved (ethanol, formalin) did not affect the structure of the brush border, it had some effect on mucous cells. When fixed in ethanol, the mucous secretion contained by these cells preserved its globulelike appearance, which agreed with the data obtained by electron microscopy.1e After formalin fixation, mucous accumulations looked like a vacuolized mass. Ethanol-fixed cryostat sections revealed glycogen in the cells of the lamina propria and muscles better than in formalin-fixed frozen or paraffin sections. The fluorescence pattern of cryostat sections was

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Ggure 3. Decrease in the fluorescence intensity of the brush border around attached bacteria E. coli B7A (0148zH28) (arrow). The other parts of the brush border (BB) and the goblet cells (CC) retained their fluorescence intensity (81 f 13.4), FPR, X 1100.

much influenced by changes in heat conditions of preparations, such as thawing and placing sample sections on warm glass slides. This resulted in a sharp decrease in the fluorescence intensity of the brush border and was probably responsible for the diffusion of the PAS-positive material. It was only the most superficial part of the brush border that retained a bright fluorescence in some areas. The fluorescence intensity of the mucous secretion was not affected by the changes of temperature. Since the fluorescence spectra maximum invariably remained within 524-527 nm, despite cerintensity, Au-SO, tain fluctuations of fluorescence of the incomplex containing the polysaccharides

Figure4. The ringlike fluorescence of PAS-positive substances in the organism E. coli B7A (048:H28) (arrow), and retention of PAS-positive substances in the bases of the microvilli (BMV). FPR, x 2600.

testinal mucous matic properties.

membrane

revealed

no metachro-

Rabbit Small Intestine After Challenge with E. coli and Their Enterotoxins; Infection with E. coli B7A (0148:H28) The morphologic pattern was chiefly characterized by such manifestations of enhanced secretion as the large-scale emptying of goblet cells, congestion, and slight edema of the gut mucosa. All these signs became more pronounced as infection progressed. Multiplication of bacteria was observed on the epithelial surface. In the case of FPR application, the gut structure and PAS-positive material distribution were much the same as in controls. The brush border remained unchanged, and its fluorescence intensity did not differ much from that of controls. It was easy to see the organisms on the brush-border surface, owing to the bright fluorescence of the polysaccharides they contained. At the same time, a close examination of the preparations sampled from a considerable length of the intestine revealed that in some areas, where organisms were in contact with the epithelium, the brush border contained less specifically fluorescent material (Figures 3 and 4). This phenomenon was observed 9 and 18 hr postchallenge. The enhancement of secretory activity was identified as a large-scale release of mucus from goblet cells into the gut lumen, as well as a lower content of PAS-positive material in the mucous secretion. The fluorescence intensity of some goblet cells by 18 hr postchallenge was 20-30X lower than that in control. This suggested a reduction in the PAS-positive material content of the mucous secretion, probably due to a decrease in its concentration. After challenge with enterotoxins, the ligated gut loops revealed signs of enhanced secretory activity similar to those observed after infection with a living culture.

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Figure 5. Uneven fluorescence intensity of the brush border (BB) 6 hr after challenge with E. coli N3 (026:K6O:Hll). Arrows point to the attached bacteria. FPR, x mm.

Infection

with E. coli IV3(026:K60:Hll)

The observed changes were characteristic both of the reaction to enterotoxigenic bacilli (e.g., a sharp rise in the secretory activity of the intestinal epithelium), and the pathogenic effect of invasive E. coli causing infantile diarrhea (bacterial multiplication on the epithelial surface, thinning of the brush border, and penetration of the organisms in some enterocytes). Due to FPR application, these changes were distinctly seen even in the 40x/0.75 objective. The areas of the brush border showing a lower fluorescence, as well as the first signs of its thinning, could be detected by 6 and 9 hr postchallenge (Fig-

ure 5). By 16 hr, the brush border became very thin, and looked somewhat like a thin breaking line (Figure 6). In some areas, PAS-reactive material of the brush border could not be distinguished at all. These lesions could be seen not only in the bacterial foci, but also far from them. Hypersecretion was identified in fluorescence studies when an increase in mucous cell number, their large-scale emptying, and appearance of granules of mucous secretion in many columnar cells were registered. An unusual morphologic feature was the arrangement of several goblet cells in one unbroken row. By 9 hr postchallenge, groups of goblet and oligomucous cells were seen, mainly in the

Figure6. Broken linelike appearance of the brush border 18 hr after challenge with E. coli N3 (028:K80:Hll). Fluorescence intensity of some areas: a. (thinned brush border), 30.63 f 1.48; b. (mucous secretion), 70.0f 20.28; c.(thinned brush border), 50.99 f 5.01; d. (attached organism), 69.6 f 42.82; e.(mucous secretion), 102.2f 14.33; f.(apical cytoplasm of epithelium), 6.1 2 1.87. FPR, x 1400. Inset: Electron micrograph of the same ileal loop sample. Some microvilli are thinned out and shortened. Original magnification, x 15000.

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Figure 7. Thinned brush border and mucous cells of ileal villus top 18 hr after challenge with E. coli N3 (026:K6O:Hll). Mucous secretion in the apical areas of the columnar enterocytes are seen. Fluorescence intensity of some areas: o., 69.1 f 15.7; b., 74.4 f 14.5; c., 86.9 f 11.8; d., 78.0 + 1.22; e., 71.6 f 1.33; f., 95.24 f 8.23; g., 52.5 f 11.3; h., 76.1 f 5.78; i., 47.62 jz 6.46; j., 15.4 f 6.11; k., 29.77 f 4.02; 1.. 85.8 f 3.86; M., 48.32 f 3.39; m., 113.5 f 8.58; n., 62.0 f 4.0; o., 76.5 f 22.6; p., 1.61 & 4.77; q., 31.17 % 1.97; r., 98.08 f 1?.44. a., gut lumen microbe; b.-i.; i-o., mucous secretion in epithelial cells. k., M; thinned brush border. p., basement membrane of the blood vessel. q., basement membrane of the epithelial layer. r., polymorphous leukocyte. j., autofluorescence of epithelium cytoplasm. Paraffin section, FPR X 1400.

middle and upper areas of villi. By 18 hr, rows of such cells filled the tops of the villi. Apart from typical goblet cells, there were columnar ones which contained mucous secretion in the apical areas only (Figure 7). Bacterial fluorescence permitted us to fol-

low the distinctly mucous discharge from some enterocytes, together with the attached organisms. Fluorescence distinctly showed the bleblike protrusions of cytoplasm (Figure 8), as well as bacterial invasion into epithelial cells. All the changes pro-

of the cytoFigure 8. Bleblike protrusion plasm (arrow) of damaged columnar enterocyte 18 hr after challenge with E. coli N3 (026:KBO:Hll). GC, goblet cells; LP, laminar propria containing some leukocytes (L). FPR, X 1600. Inset: Electron micrograph image of the same ileal loop shows electron transparent cytoplasmic matrix in similar protrusion (arrow), and adherence of organisms between some microvilli. Original magnification, X 15,ooO.

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Figure 9. Abundant mucous secretion in the ileal villi (V) 18 hr after challenge with E. coli N3 (026:K60:Hll). Unbroken rows of mucous cells (arrows) in the top of villi. FPR, x 266.

duced by strain N3 (026:K6O:Hll) organisms became most pronounced at 16 hr postchallenge (Figures 9 and 10). Erosion and slight ulceration were observed on the site of the most abundant proliferation of bacteria. Leukocyte reaction was more marked than after challenge with serotype 0146:H26. The reaction was easy to evaluate by the polynuclear glycogen fluorescence. The results of the study using FPR were found to coincide completely with those of conventional PAS-reaction and staining with mucicarmine. However, the fluorescent pictures were more distinct, particularly those of separate granules of mucin in the cells. The enterotoxins of E. coli 026:K6O:Hll caused diffuse epithelial changes similar to those caused by living culture challenge, except that the former is not followed by cell desquamation and erosion. The alterations occurring after enterotoxin challenge developed at the same rate, and were seen along a considerable length of mucosal surface (Figure 11).

Figure 16. Erosion on the villus top (VT), and abundant multiplication of organisms (arrows) 16 hr after challenge with E. coli N3 (026:K60:Hll). FPR, x 866.

Discussion Application of FPR allows the demonstration of polysaccharide-rich substances in the rabbit ileal epithelium, and it produces more distinct and bright pictures, as compared with the “classical” PAS-reaction. This is particularly true for the pictures of single granules of mucous secretion in mucous cells and polysaccharides of the microvillus coat. Due to the high sensitivity of fluorescence reaction, fluorimetry may be used for assessment of the polysaccharide content of the gut sections. Both cryostat and, especially, paraffin sections are suitable for FPR studies. The examination of cryostat sections showed that the ability of PAS-positive

Figure 11. Abundance of mucous cells (arrows) in the ileal villus 18 hr after challenge with enterotoxin of E. cob N3 (026:KBO:Hll). FPR, x 800.E. autofluorescence of epithelium cytoplasm. LP, laminar propria.

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material of the brush border is higher than that in goblet cells. This reflects differences in the chemical composition of the mucoproteins of the cell coat and the mucopolysaccharides of mucous secretion.” The results of previous electron microscopy investigationsZo-22 demonstrated that the PAS-reactive material of the brush border is associated with the external leaflet of the microvillus plasmalemma. However, in fluorescence microscopy, as in any other light microscopy study, the fluorescence of microvilli assumes a diffuse aspect. The fluorescent cell coat distinguishable in paraffin sections seems to be chiefly at the sides and bases of the microvillus. The part connecting the tips of the microvilli”*z3 cannot be distinguished in paraffin sections; it can sometimes be seen in cryostat sections. Also, our observations show that the superficial layer of the cell coat can hardly be distinguished sometimes because of cell coat heterogeneity. Such a heterogeneity is also supported by the data in the literature, showing that different areas of the microvillus coat may have different affinities for reagents used for polysaccharide detection, as well as different contents of enzymes.lg The ligated rabbit ileal loop experiments with dif ferent enterotoxigenic E. coli showed that application of FPR, combined with histologic and electron microscopy procedures, may yield additional information on the infectious processes taking place in the gut and the histophysiology of the intestinal epithelium. For example, it was found that after E. coii strain B7A (0148:H28) challenge, the brush border appears to have no PAS-positive substance in the points of contact with bacteria. It is possible that this involves some damage to the microvilli, too. Such changes are rather rare. They were not detected in the investigations using conventional staining techniques, nor were they established in electron microscopy studies. Earlier, it was considered that the agents of choleralike escherichioses, which include this serotype, did not impair the brush border. Fluorescence studies vividly demonstrated the following lesions characteristic of the damage done by the penetrating enterotoxigenic E. coli N3 (028:K60:Hll) strain: hypersecretion with formation of numerous mucous cells, thinning of the brush border, and disappearance of its PAS-positive mucosubstance. It is in good agreement with the results of electron microscopy of the same ileal samples.’ The above alterations are caused by the enterotoxins of this strain, because they can be induced by challenging with microbial lysates and supernatants, too. Such changes lead specificity to the lesions involved in such an escherichiosis. As to erosion, it is not clear yet whether it is caused by infection itself or is due to the gut being extended by the fluid accumulation in the loop lumen.

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Failure to distinguish the most superficial layer of the enterocyte coat is a limitation of FPR application for evaluation of the earliest stage of enteric infection development. The results of the present investigation may elucidate certain aspects of the histophysiology of enterocytes and, chiefly, the composition of the cell coat and mucous cell production. For example, it was found that the PAS-positive substance in the microvillus bases was more resistant to enterotoxins than in the sides and tips of the microvilli. This seems to account for the coat being preserved at the bases of the microvilli and its appearance in a light microscope as a continuous thin fluorescent band. It may be supposed that the coat of the microvilli is actually more heterogeneous functionally (and probably structurally), than it was thought to be before. The detection of the hyperproduction of mucous cells, as a result of infection caused by the 026:K6O:Hll organisms pose a question as to the pathways of this reaction. According to the widespread Unitarian theory,24 the multipotent stem cells serve as a source of the four main types of enteromucous, Paneth, and entecytes, i.e., columnar, roendocrine cells. The stem cells are known to be located in the crypt bases. Leblond and Chang” believe the said Unitarian theory is substantially supported by their discovery of immature enterocytes with two different secretory granules, e.g., mucous and Paneth cells. Based on these data, as well as on mitosis counts, these authors suggested that some columnar cells may be transformed into mucous cells. However, such transformation has not yet been observed under natural conditions. Our findings related to pathology are in full agreement with this suggestion and, hence, the Unitarian theory too. However, our results show that midvillial cells are close to the so-called “committed progenitor cells,024 and are likely to retain a certain degree of multipotence. Given a certain stimulus, they may be transformed into mucous cells rather than into columnar ones only. Such stimulation was provided by the enterotoxins of the penetrating 026:K60:Hll strain in our experiments. This irritating effect of the enterotoxins may be a factor that probably plays a role in the pathogenesis of infantile diarrhea. It can cause not only hypersecretion but damage to enterocytes. We can assume that a newly described rabbit diarrhea agent, E. coli strain 015 RDEC-1,” that produces a Shigella dysenteriae-like cytotoxic enterotoxin,26 which adheres densely to the intestinal epithelium, causes degenerative changes, and penetrates its apical cytoplasm,” manifesting similar properties with the combined effect of irritating and damaging action of the toxin. Recent studies” have demonstrated frequent entero-

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toxigenicity of infantile diarrhea agents that could not be detected by conventional test systems. Their cytotoxic properties have not been investigated. However, Konowalchuk et al.*’ have shown cytotoxicity of a number of enteropathogenic E. coli isolated from infants. Peculiar invasive, enterotoxigenie, and damaging (cytotoxic) properties of enteropathogenic E. coli causing infantile diarrhea should be elucidated in further studies and compared with that of invasive enteropathogens, Shigella and Salmonella, known to produce enterotoxins.30-34 Fluorescent periodic acid-Schiff reaction can be recommended for this purpose.

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13. Kasten FU: Schiff-type reagents in cytochemistry. I. Theoretical and practical considerations. Histochemie 1:466-509, 1959 14. Kasten FU: The chemistry of Schiff’s reagent. Int Rev Cytol l&l-100, 1960 15. Kudryavtseva MV, Kudryavtsev BN, Rosanov YM, Alashbaev M: The conditions of periodic oxidation in fluorescence PAS reaction. Tsitologiya 17533-538, 1975 16. Barski IYa, Khavkin TN: A method of photographic microfluorometry for immuno-luminescent investigations. Tsitologiya 10:1501-1504,1968 17. Lillie RD: Histopathologic Technic and Practical Histochemistry. New York, McGraw-Hill Book Co., 1965 18. Neutra M, Leblond CP: Synthesis of carbohydrate of mucus in the Golgi complex as shown by electron microscope radioautography of goblet cells from rats injected with glucose-H3. J Cell Biol 30:119-138, 1966 19. Lojda Z: Cytochemistry of enterocytes and other cells in the mucous membrane of small intestine. In: Intestinal Absorption. Edited by DH Smith. New York, Plenum Press, 1974, p 43-122 20. Ito S: Form and function of the glycocalyx on free cell surfaces. Philos Trans R Sot Lond 268:55&i, 1974 21. Rambourg A: Morphological and histochemical aspects of glycoproteins at the surface animal cells. Int Rev Cytol 31:57114, 1971 A: The surface coat animal cells. Int Rev 22. Martinez-Palomo Cytol 29:29-76, 1970 23. Ito, S: The enteric surface coat on cat intestinal microvilli. J Cell Biol 27:475-491, 1985 24. Leblond CE, Chang H: Identification of stem cells in the small intestine of the mouse. In: Stem Cells: Renewing Cell Population. Edited by AB Cairnie, PK Lala, DG Osmond. New York, Academic Press, 1976, p 5-31 25. Cantey JB, Blake RK: Diarrhea due to Escherichio coli in the rabbit: A novel mechanism. J Infect Dis 135:454-462, 1978 26. O’Brien AD, Thompson MR, Cantey JR, Formal SB: Abstr Ann Mtg Am Sot Microbial B103:32, 1977 27. Takeuchi A, Inman LR, O’Hanley PD, Cantey JR, Lushbaugh WB: Scanning and transmission electron microscopic study of Escherichia cob 015 (RDEC-1) enteric infection in rabbits. Infect Immun 19:686-894, 1978 28. Klipstein FA, Rowe B, Engert RF, Short HB, Gross RJ: Enterotoxigenicity of enteropathogenic serotypes of Escherichia coli isolated from infants with epidemic diarrhea. Infect Immun 21:171-178, 1978 29. Konowalchuk J, Dickie N, Stavrie S, Speirs JI: Properties of Escherichia coli cytotoxin. Infect Immun 20:575-577, 1978 30. Keusch GT, Grady GE, Takeuchi A, Sprinz H: The pathogenesis of Shigella diarrhea. II. Enterotoxin-induced acute enteritis in the rabbit ileum. J Infect Dis 126:92-95, 1972 31. O’Brien AD, Thomspon MR, Gemski P, Doctor BP, Formal SB: Biological properties of Shigella felexneri 2a toxin and its serological relationship to Shigella dysenteriae I toxin. Infect Immun 15:796-798, 1977 32. Kbtyi I, Mdlovics I, Vertenyi A, Krontrohr T, P&csa S, Kuch B: Heat-stable enterotoxin produced by Shigella flexneri. Acta Microbial Acad Sci Hung 25:165-171.1978 33. KBtyi I, Vertenyi A, MBlovics I, Kontrohr T, Pdcsa S: Unique features of heat-stable enterotoxin of Shigeflo flexneri. Acta Microbial Acad Sci Hung 25:219-227, 1978 34. Sedlock DM, Koupal LR, Deibel RH: Production and partial purification of SoImonella enterotoxin. Infect Immun 20:375380, 1978