APPuED MICROBIOLOGY, Sept., 1966Copyright © 1966 American Society for Microbiology

Vol. 14, No. 5Printed in U.S.A.

Histochemical Observations on Mycoplasma AfterStaining with Acridine Orange

D. E. JASPER AND N. C. JAIN

Department of Clinical Pathology, School of Veterinary Medicine, University of California, Davis, California

Received for publication 10 March 1966

ABSTRACTJASPER, D. E. (University of California, Davis), AND N. C. JAIN. Histochemical

observations on Mycoplasma after staining with acridine orange. Appl. Microbiol.14:720-723. 1966.-Mycoplasma colonies and Mycoplasma cells in preparationsfrom infected milk and lymph nodes were observed for their fluorescent qualitiesafter treatment with acridine orange. Mycoplasma colonies fluoresced brilliant red or

red-orange. When treated after exposure to ribonuclease, the colonies fluorescedlime-green. There was no fluorescence when both ribonucleic acid and deoxyribo-nucleic acid were destroyed by perchloric acid. Detection of Mycoplasma in smears ofmastitic milk or smears of infected lymph nodes was not definitive because of thelarge amount of nonspecific ribonucleic acid-rich material present during inflamma-tory reactions.

Acridine orange is unique in that it will fluo-resce bright red under ultraviolet light when com-bined with ribonucleic acid (RNA) at an appro-priate pH, and will fluoresce bright lime-greenunder the same circumstances when combinedwith deoxyribonucleic acid (DNA). As a conse-quence, acridine orange has been used extensivelyto demonstrate the presence of these nucleic acidsin organisms, cells, and tissues. No reference toits application to Mycoplasma colonies has beenfound, although mycoplasmas have been reported(Hers et al., unpublished data) to fluoresce lime-green in frozen sections of infected chick embryowhen stained by the acridine orange method atpH3.8 or 5.4 (4).The purpose of the present study was to deter-

mine the staining characteristics of Mycoplasma,to demonstrate histochemically the DNA andRNA components of Mycoplasma, and to explorethe possibility that staining characteristics mightbe specific enough to be of value for identificationor location of Mycoplasma in biological fluids,cells, or tissues.

MATERIALS AND METHODS

Twelve isolates associated with one outbreak ofbovine mastitis (3) and one strain of Mycoplasma gal-isepticum (obtained from H. E. Adler, Department ofAvian Medicine, School of Veterinary Medicine, Uni-versity of California, Davis) were used. Mycoplasmacolonies growing on PPLO Agar (Difco) containing15% horse serum were transferred to a glass cover slipby pressing the cover slip gently over an appropriate

area of the plate, and then carefully lifting the coverslip.

Smears of milk from normal cows, from cows withnon-Mycoplasma mastitis, and from cows with Myco-plasma mastitis were prepared either by spreading witha loop, or by sliding a small drop between two coverslips, as for making a blood smear. Tissue imprintswere made from the cut surfaces of normal mammarytissue and normal supramammary lymph nodes, fromthe same tissues of cows affected with acute non-Mycoplasma mastitis, and from cows affected withsevere Mycoplasma mastitis.

Air-dried slides were fixed by passing them quicklythrough a low flame followed by immersion for 1 minin ether-alcohol. Staining with acridine orange thenproceeded according to the methods of Armstrong (1)at pH 3.8 orof von Bertalanffy (7, 8) atpH 3.8 and 6.0.Use of Camoy's fixative did not improve the results.RNA was destroyed in preparations from five dif-

ferent bovine isolates by incubation in 1% ribonu-clease (Nutritional Biochemicals Corp., ClevelandOhio) at 37 C for 2 hr (2, 7). Both DNA and RNAwere destroyed in other preparations from the samefive bovine isolates by treatment with 10% perchloricacid at 60 C for 30 min (2). Suitable control slides wereincubated in distilled water. Treated preparations werethen stained with acridine orange at pH 6.0 (7, 8).

After observation of fluorescence, colony prepara-tions were decolorized in 50% ethyl alcohol and re-stained with Wright-Leishman stain to verify thepresence or absence of Mycoplasma on the slide.

REsuLTs

The results of staining Mycoplasma colonieswith acridine orange are shown in Table 1. My-

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ACRIDINE ORANGE STAINING OF MYCOPLASMA

coplasma colonies normally fluoresced a brilliantfiery-red or orange-red, indicative of high RNAcontent (Fig. 1). Intensity of staining was greaterat pH 6.0 than atpH 3.8. Occasionally, thin edges

TABLE 1. Fluorescent qualities of Mycoplasmacolonies after staining with acridine orange by

various procedures

Fluorescence

Method of staining colonies Yel-Red Green low- Nega-

Green tive

A. Armstrong, pH 3.8.. 2 0B. Bertalanffy, pH 6.0.. 20a 0C. Bertalanffy, pH 3.8.. 0D. Bertalanffy, pH 6.0 5 0

after ribonuclease. 5E. Bertalanffy, pH 6.0

after perchloric acid 5F. Bertalanffy, pH 6.0

after incubation inwater ............ 3b 0

'Includes 3 preparations from one strain of M.gallisepticum and 17 preparations from among 12bovine isolates, 5 of which were repeated in con-junction with methods C, D, E, and F.bTwo control slides could not be read as the

colonies had wasted away.

of a colony or thin layers of Mycoplasma cellswhich had washed across the slide tended to staingreen, especially when stained at pH 3.8.Mycoplasma colonies treated with ribonuclease

fluoresced a bright lime-green, indicative of sig-nificant DNA content. When both DNA andRNA were destroyed, there was no fluorescence.Presence of organisms on such slides was laterproved by use of Wright-Leishman stain. Goodcolony stains were obtained with this stain eitherbefore or after use of acridine orange. Colonies oncontrol preparations incubated in water fluo-resced red.

Smears of normal milk contained no fluorescentmaterial suggestive of Mycoplasma cells. Imprintsof normal supramammary lymph nodes containedoccasional large mononuclear cells with red-stain-ing cytoplasm.

Smears of milk and imprints of supramammarylymph nodes from severe Mycoplasma mastitis re-vealed many red-staining intracellular and extra-cellular particles (Fig. 2). Many young cells withabundant red cytoplasm were present in bothtypes of preparations. The cytoplasm of matureneutrophils was generally dark.Milk smears and supramammary lymph nodes

from acute non-Mycoplasma mastitis also con-tained red-staining intracellular and extracellularparticulate material in addition to large young

FIG. 1. Mycoplasma colony fluorescing bright red after staining with acridine orange. Note fine details offimbri-ated border. X 450

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JASPER AND JAIN

FIG. 2. Milk smear from Mycoplasma mastitis. Intracellular and extracellular particles fluoresced bright red.Cytoplasm oflarge cells fluoresced red; nuclei fluoresced green. X 280.

cells with abundant red cytoplasm. The cyto-plasm of mature neutrophils was generally dark.

DISCUSSION

The brilliant red staining of colonies reflects asignificant RNA content, which has been shownto be about double that of DNA (5, 6), althoughthe DNA present fluoresced a strong lime-greenafter RNA was destroyed. Very thin or single celllayers of organisms were sometimes observed tofluoresce green instead of red. This characteristicmay be related to the reported green staining ofMycoplasma in thin frozen sections of infectedchick embryo (4).Although the amount of intracellular and ex-

tracellular red-staining material appeared to bemore abundant in material from the Mycoplasma-infected animals than in material from acute non-

Mycoplasma mastitis, it was not possible to dif-ferentiate the Mycoplasma-infected material fromnoninfected material on this basis. Young RNA-

rich cells and RNA-containing granular debrisappeared to be common factors in inflammatoryexudate and tissues whether Mycoplasma cellswere present or not. The value of acridine orangestaining for study of Mycoplasma in cells andtissues may, therefore, be limited to situations inwhich RNA and DNA from other sources iseither lacking or well-defined in location.Some problems were encountered from the

washing away of Mycoplasma colonies duringprocessing. The fixation method outlined was bet-ter than other variations tried, but washing stilloccurred occasionally. Additional procedures,such as incubation in ribonuclease or in water, in-creased this hazard.

LITERATURE CITED

1. ARMSTRONG, J. A. 1956. Histochemical differentia-tion of nucleic acids by means of induced fluores-cence. Exptl. Cell Res. 11:640-643.

2. CULLING, C. F. A. 1963. Handbook of histopatho-

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VOL. 14, 1966 ACRIDINE ORANGE STAIl

logical techniques, 2nd ed., p. 222. Butterworthand Co., Ltd., London.

3. JASPER, D. E., N. C. JAIN, AND L. H. BRAZIL. 1966.Clinical and laboratory observations on bovinemastitis due to mycoplasma. J. Am. Vet. Med.Assoc. 148:1017-1029.

4. MARMioN, B. P., AND J. F. HERS. 1963. Observa-tions on Eaton's primary atypical pneumoniaagent and analogous problems in animals. Am.Rev. Respirat. Diseases 88(Suppl.):198-211.

5. MoRowrrz, H. J., M. E. TOURTELLOTTE, W. R.GUILD, E. CASo, ANm C. WOESE. 1962. Thechemical composition and submicroscopic mor-

KING OF MYCOPLASMA 723

phology of Mycoplasma gallisepticum, avianPPLO 5969. J. Mol. Biol. 4:93-103.

6. RAziN, S., M. ARGAMAN, AND J. AVIGAN. 1963.Chemical composition of mycoplasma cells andmembranes. J. Gen. Microbiol. 33:471-475.

7. VON BERTALANFFY, L., AND I. BICKIS. 1956. Identifi-cation of cytoplasmic basophilia (ribonucleicacid) by fluorescence microscopy. J. Histochem.Cytochem. 4:481-493.

8. VON BERTALANFFY, L., M. MASIN, AND F. MASIN.1956. Use of acridine-orange fluorescence tech-nique in exfoliative cytology. Science 124:1024-1025.

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