THE USE OF PERIODATE IN MICROBIOLOGICAL STAINING

DERROL PENNINGTON Department of Microbiology, University of Washington School of Medicine, Seattle 6, Washington

Received for publication November 1, 1948

The publications of Hotchkiss (1948) and McManus (1948) relating to the use of periodic acid in detecting polysaccharide structures in fixed tissue preparations prompt us to record the results of a similar technique applied to microorganisms. The staining procedure developed independently in this laboratory is essentially the same as that of Hotchkiss with the exception that aqueous solutions are used and a reducing rinse has been found unnecessary. The staining of polysaccharide structures depends upon the specific oxidizing action of sodium periodate toward such chemical configurations as a,#-glycols, a-hydroxy ketones, and related structures (Jackson, 1946). In these configurations at least one of the oxidation products will be an aldehyde that can be detected by treating the oxidized material with sulfite-decolorized basic fuchsin (Feulgen's reagent). Those portions of the cell that contain material oxidizable by periodate to yield an aldehyde will thus appear stained red while the remainder of the cell will remain colorless. It can be presumed from our knowledge of the specificity of the reactions involved that only polysaccharide material such as cellulose, dextrans, levans, starch, glycogen, pectins, and hyaluronic acid will be stained in this manner. An excellent discussion of the specificity of the reaction is given by Hotchkiss (1948). Bacteria to be examined are fixed either by heat, in Bouin's solution, or other fixative. They are then immersed in a 1 per cent aqueous solution of sodium metaperiodate for 5 to 15 minutes at room temperature. After thorough washing with water the preparations are stained for 15 minutes in sulfite-decolorized basic fuchsin solution (Coleman, 1938). The slides are then washed for 10 minutes in sulfur dioxide water ( 5 ml of 10 per cent K2520r and 5 ml 0.1 N HCI in 100 ml water), finally in water, and dried.
RESULTS AND DISCUSSION

In applying this technique to a variety of organisms a number of apparently significant structures have been observed. None of the structures described below are stainable with iodine, nor are they stained with sulfite-decolorized basic fuchsin prior to periodate oxidation. The following observations illustrate the results obtained with this procedure. Further work will be necessary to identify and determine the significance of many of the structures observed. Unless otherwise stated, preparations were made on 24-hour cultures of the organisms grown on nutrient agar at the optimum temperature for each organism. Saccharomyces cereiswe. (Yeast extract glucose agar.) The surface of the
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cell stains very intensely red. A number of dark red granules are apparent within the cell. Baci11us cereus. (Heart infusion agar.) In very young cells (2 hours) the cytoplasm is stained lightly and uniformly red; the cell surface is somewhat darker. At 6 hours the cytoplasm stains more intensely and numerous small granules are apparent. By 10 hours definite polysaccharide structures have developed (figure 1). BaciUus mycoides. (Yeast extract glucose agar; 4-hour culture.) The cytoplasm is stained lightly, the cell surface is somewhat darker. Septa across cells undergoing division are very prominent. Bacilus sp. (Heart infusion agar; 3-hour culture.) Similar in appearance to B. mycoides (figure 2). BaciUus megatherium. Many intensely stained red granules are present in an unstained cytoplasm. The cell surface is stained. Clostridium 8porogene. The cells do not stain. Myxococus sp. The cells take a very faint uniform stain. No structure is discernible. Vibrio sp. Young cells (24 hours) are stained uniformly red with a very intensely stained cell surface. In older cells (3 to 4 days) no continuous surface is visible, the cytoplasm is not stained, but many large intensely stained bodies (or surface areas) are visible. Stir older cells (2 weeks) do not stain at all. Spirillum sp. (Ten-day culture.) The cells take only the faintest stain when oxidized by periodate and treated with Feulgen's reagent. No wall or granules are visible. However, periodate oxidation exerts a profound effect on the metachromatic (volutin) granules. After oxidation with periodate and staining with methylene blue, no metachromatic granules are visible. Instead the entire cell is stained the violet metachromatic color, and the positions once occupied by granules appear as empty spaces. Corynebacterium diphtlweriae. (Ten-day culture on Loeffler's medium.) The cells show only the faintest stain. The prominent metachromatic granules show the same reaction with periodate that was observed in the spirillum. The granules have apparently disintegrated and the metachromatic material has diffused throughout the cell. Mycobacteium phlei. (Twenty-hour culture on glycerol agar.) The cells are stained moderately red, with an intensely stained body at each end of the cell. (Thirty-day culture on glycerol agar.) The cells are stained intensely red with the exception of a centrally located vacuole. Diplococcus sp. (Yeast extract, glucose, calcium carbonate broth.) The cell surface and particularly the septa across cells undergoing division are stained red (figure 2). Pneumococcus type III. (Yeast extract, glucose, calcium carbonate broth.) After hydrolysis in 0.2 N HCI at 60 C for 5 minutes the cells stain uniformly red. After hydrolysis in 0.2 N NaOH at 60 C for 5 minutes only the cell outline and the septa across the cells are apparent and are stained red. Unhydrolyzed cells show no reaction whatever.

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age of the cells has been demonstrated. In the case of the diplococcus mentioned above a variation with the pH of the medium has been noted. Cells grown in yeast extract glucose broth without the buffering action of calcium carbonate do not deposit polysaccharide stainable by this technique. The failure of the type III pneumococcus to stain without previous hydrolytic treatment is interesting in view of the fact that Hotchkiss (1948) found the isolated type III
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polysaccharide to react readily with periodate and Feulgen's reagent. This indicates that the polysaccharide as it occurs on the surface of the cell is substituted on the second or third carbon atom of the glucose residues, which make up the polysaccharide chain, with some substituent that is lost during the process of isolation. That this is the case is indicated by the fact that hydroiysis renders stainable the polysaccharide still attached to the cell. In this respect it should be emphasized that the failure of any cell to show stained structures by this

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technique does not necessarily mean the absence of polysaccharide of any type, but only the absence of polysaccharide having two free adjacent hydroxyl or carbonyl groups. The discovery of more specific hydrolytic procedures which would render substituted polysaccharides reactive toward periodate would be most desirable. It is of some interest to consider whether the pronounced surface stain observed in a variety of organisms is due to polysaccharide components of the cell wall or of the cytoplasmic membrane. In the case of yeast it is relatively easy to rupture the cells during fixation. The empty sacs so obtained stain intensely red, indicating that in this instance the cell wall itself contains a large proportion of polysaccharide material. Plasmolysis experiments with Bacillus cereus indicate that at least a portion of the observed reaction with this organism is due to the cell wall, although additional polysaccharide seems to be concentrated in the cytoplasmic membrane.
ACKNOWLEDGMENT

The author wishes to express his thanks to Dr. C. F. Robinow for cultures and for his advice in making preparations.
SUMMARY

Polysaccharide structures in many bacteria can be detected by oxidizing fixed preparations of the cells with sodium metaperiodate followed by staining with sulfite-decolorized basic fuchsin. Volutin granules in spirilla and in the diphtheria bacillus appear to disintegrate when the cells are treated with sodium metaperiodate.
REFERENCES COLEMAN, L. C. 1938 Preparation of leuco basic fuchsin for use in the Feulgen reaction. Stain Tech., 13, 123-124.

HoTcxss, R. D. 1948 A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem., 16, 131-141. JACKSON, E. L. 1946 In Adams, Roger: Organic reactions, Vol. II. John Wiley and Sons, Inc., New York, N. Y. Refer to p. 341. McMANus, J. F. A. 1948 Histological and histochemical uses of periodic acid. Stain Tech., 23, 99-108.