Scanning electron freeze-etching · Brit.J. vener. Dis. (1975) 51, 83 Scanning electron microscopy...

Brit. J. vener. Dis. (1975) 51, 83

Scanning electron microscopy and. freeze-etching ofgonorrhoeal urethral exudateHOMAYOON FARZADEGAN* AND IVAN L. ROTHDepartment of Microbiology, University of Georgia, Athens, Georgia 30602, U.S.A.

The pathology of gonorrhoea and the mechanismsby which Neisseria gonorrhoeae establishes diseasehave not been thoroughly elucidated. It has beenreported that the entrance of the N. gonorrhoeae cellsinto the epithelial tissue of the urethral tract in themale takes place through the intercellular spaces andreaches the subepithelial connective tissue 3 or 4 daysafter exposure to an infected female (Harkness, 1948).The urethral discharge in the male is the result of themigration of polymorphonuclear leucocytes, lympho-cytes, and plasma cells to the site of infection beneaththe columnar epithelium. The presence of gonococciin leucocytes in the urethral exudate is the mostimportant diagnostic evidence of gonorrhoeal infec-tion in the male (Ov6innikov and Delektorskij, 1971;Spink, 1937). Thomas, Hill, and Tyeryar (1973)reported that cells from type 1 colonies of N.gonorrhoeae strain F62 (Kellogg, Peacock, Deacon,Brown, and Pirkle, 1963; Kellogg, Cohen, Norins,Schroeter, and Reising, 1968) survived phagocytosisby human leucocytes better than cells from type 4colonies. The ultrastructure of phagocytes andgonococci in urethral exudate has been studied byobservation of thin sections (Ovcnikov andDelektorskij, 1971; Ward and Watt, 1972; Ward,Glynn, and Watt, 1972). Although these reportscontain valuable information, thin sections provideonly a two-dimensional image of dehydrated,embedded specimens. It was the purpose of thisinvestigation to develop methods for the study ofphagocytes and the relationship between N. gonorr-hoeae and phagocytes in the urethral discharge ofmales suffering from gonorrhoea, at the three-dimensional level by scanning electron microscopyand by the use of the freeze-etch technique and theobservation of replicas in the transmission electronmicroscope.

Material and methodsGONOREHOEAL URETHRAL EXUDATEUrethral exudate was obtained from male patients withcharacteristic symptoms of gonorrhoea who reported tothe Clarke County Public Health Department, Athens,

Received for publication July 24, 1974*Present address: Department of Microbiology and Immunology,School of Medicine, University of Tehran, Tehran, Iran

Georgia, U.S.A., for treatment. Diagnosis of gonorrhoeawas made by microscopical examination of a Gram-stainedsmear of the exudate and by culture on modified Lester-Martin medium (Martin and Lester, 1971). The micro-organisms on the plate were identified as Gram-negativediplococci which showed a positive oxidase reaction withN, N'-dimethyl-P-phenylenediamine monohydrochloride(Eastman Kodak Co., Rochester, New York). Whencultures were obtained on a transparent medium (GC base,Difco, Detroit, Mich.) supplemented with supplements Iand II (Difco), approximately 85 per cent. of the colonieswere Type 1, 12 per cent. were Type 2, and the remaining3 per cent. were Types 3 and 4. These organisms werefurther identified as N. gonorrhoeae, in that they fermentedglucose, but failed to ferment lactose, sucrose, maltose,fructose, or mannitol.

PREPARATION FOR SCANNING ELECTRON MICROSCOPYSpecimens of urethral exudate were prepared forscanning electron microscopy by using the critical pointdrying technique ofAnderson (1951). The specimens werefixed in 2 per cent. glutaraldehyde (Polysciences, Inc.,Warrington, Pennsylvania) in 01M cacodylate buffer atpH 7-0 for 2 hrs at room temperature. Dehydration wasachieved by passing the specimens through 70 per cent.ethyl alcohol (U.S. Industrial Chemical Company, NewYork) for 15 min., 95 per cent. ethyl alcohol for 15 min.,and finally 100 per cent. ethyl alcohol for 30 min., withtwo changes. Then the specimens were suspended in a1:1 solution of ethanol and amyl acetate (J. T. BakerChemical Company, Phillipsburg, New Jersey) for30 min. with two changes. The specimens were nextplaced in undiluted amyl acetate for 30 min. with twochanges. Then the specimens were placed on a scanningelectron microscope stub and the excess amyl acetate wasdrained. The stubs were then placed on a specimen holderand inserted into the critical point bomb. The amylacetate was replaced by liquid CO,. After all of the amylacetate was exhausted from the bomb, it was sealed andheated to 40-42°C. The pressure in the bomb wasmonitored and maintained in excess of 1200 lb./sq. in.When the temperature was stabilized at 40-420C., the gaswas released slowly via the exhaust valve. The temperaturewas maintained above 40°C. until all the gas was removed.The dried specimens were then metal coated with gold:palladium (60: 40) (Ladd Research Industries, Burlington,Vt.) at two different angles on a rotary stage in a vacuumevaporator (Varian Model Number VE10). Specimenswere observed at an angle of 450 in the CambridgeStereoscan Mark 2A scanning electron microscope at anaccelerating voltage of 8 kV. Photographs were made with

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84 British Journal of Venereal Diseases

a Polaroid 4 x 5 Land Film Holder 500 using PolaroidP/N 55 film.

PREPARATION OF SPECIMENS FOR FREEZE-ETCHINGSpecimens of urethral exudate were fixed in 2 per cent.glutaraldehyde in 01 M cacodylate buffer at pH 7-0 for2 hrs at room temperature. A 30 per cent. (v/v) aqueoussolution of glycerol was used as a cryoprotective agent.Then specimens were frozen in Freon 22 for 40 sec.,and then kept in liquid nitrogen. A Balzer freeze-etchingunit was used in this investigation. Etching times were8 to 10 min. Shadowing time was 8 sec. Carbon coatingwas continued for 10 sec. The replica was washed in100 per cent. Clorox (5-2 per cent. sodium hypochlorite,Clorox Company, New York) ovemight at 0°C. and in48 per cent. hydrofluoric acid (Mallinckrodt ChemicalWorks, St. Louis, Missouri) for 4 hrs at room temperature.Grids (400 mesh) were used to support the replica. APhilips 200 transmission electron microscope was used toobserve the replicas at 80-100 kV.

ResultsFigs 1 and 2 show scanning electron micrographs ofurethral exudate from a male with gonorrhoea. Theexudate was prepared for observation by the criticalpoint drying technique. Fig. la shows at low magnifi-cation such a preparation. Most of the phagocytes(P) are stuck to each other and are seen as a clump ofcells. Fig. lb shows at higher magnification the

surface of the phagocytes. The surface appears tLube extensively ruffled. Fig. 2 shows scanning electronmicrographs of epithelial cells. The cell in Fig. 2aappears to be flattened against the supporting surfaceof the stub. The large protrusion at the right of theepithelial cell appears to be somewhat different fromthe rest of the cell. The epithelial cell in Fig. 2b doesnot show such a protrusion but does have a flattenedappearance. The surface of both cells (Figs 2a, b)is extensively convoluted.

Fig. 3 shows a freeze-etched preparation ofurethral exudate from a male patient. A phagocyte,which appears to be a polymorphonuclear phagocyte,is seen in this micrograph. This phagocyte does notcontain identifiable cells of N. gonorrhoeae in theplane of fracture. Three lobes of the nucleus (N) arevisible. The nuclear membrane (nm) and nuclearpores are seen in this figure. Pseudopodia (ps) andthe smooth outer surface (os) structure of thisphagocyte are readily seen. The inner track of thenuclear membrane has a rough appearance ascompared to the outer layer which is smooth. Thecytoplasm is filled with granules (G) of differentsizes.

Fig. 4 shows a phagocyte revealing the appearanceof nuclear pores (unlabelled arrows) along withinvaginations of the membrane toward the centre ofthe nucleus.

5 _ Wl -.-.-...-......;

FIG. 1 Scanning electron micrographs of urethral (b) Higher magnification of phagocyte in urethralexudate from male patient with gonorrhoea prepared exudate. The ruffled membrane surface of thefor observation by the critical point drying technique phagocyte membrane is evident. Marker= 1 Fm.(a) Cluster of phagocytes (P). Marker= l,0um

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Electron microscopy of urethral exudate 85

_ W _ = ., I_. --=FIG. 2 Scanning electron micrographs of urethral (a) Convoluted surface of epithelial cell along withexudate from male patient with gonorrhoea prepared phagocytes (P). Marker= 10 [±mfor observation by the critical point drying technique (b) Another epithelial cell, showing rough and

convoluted surface structure, Marker= 10 Fm.



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86 British J7ournal of Venereal Diseases

FIG. 3 Freeze-etched preparation of urethralexudate from male gonorrhoea patient, showing apolymorphonuclear cell without engulfed N. gonorrhoeae,a multilobed nucleus (N), and a nuclear membrane (nm)fractured at several places. Smooth outer surface (os)

and pseudopodia (ps) clearly visible. Cytoplasm filledwith granules (G) of different sizes. Marker= 1 pLm.Arrow on upper right corner offreeze-etchedpreparations (Figs 3 to 8) indicates direction in whichplatinum was evaporated onto the specimen.

F I G. 4 Freeze-etchedpreparation ofphagocyte in urethralexudate. Noteinvagination ofnuclearmembrane aroundnuclear pores (arrows).N= nucleus, nm=nuclear membrane,os= outer surface.Marker= 1 Fm.



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F I G. 5 Freeze-etched preparation of urethral visible. Seven gonococci were phagocytosed individuallyexudate from male patient with gonorrhoea. while four were phagocytosed together. N= nucleus.Phagocyte with several N. gonorrhoeae (GC) inside Marker= 1 Fm.the phagosomes. Phagosome membranes (Phm) clearly

In Fig. 5 a phagocyte with several gonococci (GC)inside the phagocyte is seen. As is shown in themicrograph, seven N. gonorrhoeae cells (GC) werephagocytosed individually, and each one appears tobe surrounded by an independent phagosomemembrane, while four gonococci were phagocytosedtogether and were surrounded by one phagosomemembrane (Ph m) in another part of the cytoplasm.In this micrograph, the phagocyte still possessessome granules in the cytoplasm.

Fig. 6 shows at higher magnification six separatephagosomes, each with a bacterial cell containedwithin it. Several of the phagosomes have granuleswithin the phagosome membrane. On some of thephagosomes, granules (possibly lysosomes) appearto be attached to the outside of the phagosomemembrane (Ph m).

Fig. 7 shows at higher magnification the phago-some from Fig. 5 containing four gonococci (GC).Some granules (g) occur within the phagosome.



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88 British J'ournal of Venereal Diseases

FIG . 6 Higher magnification of area in Fig. 5. inside phagosome. Large granules (G) attached toN. gonorrhoeae (GC) cells, phagocytosed individually outside of phagosome membrane (Phm). The cell isin separate phagosomes. Small granules (g) also seen dividing (unmarked arrow). Marker= 1 [.m.

|{4 ;S 5~s' X > g g } .-W i \ /} 2S ;. W ' 9

FIG. 7 Higher magnification of area in Fig 5,showing four N. gonorrhoeae (GC) phagocytosedin one phagosome. Small granules (g) visible inside

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phagosome and large (G) granules in phagocytecytoplasm. Note lack of septation cell wall (unmarkedarrows). Phm=phagosome membrane. Marker= 1 j'm



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In Fig. 8 the presence of granules (g) inside aphagosome is depicted even more clearly. In thismicrograph, two phagosomes are seen, one con-taining three such cells. Apparently, the phagosomecontaining only one gonococcus contains moregranules within it than the phagosome with threegonococci.

DiscussionScanning electron microscopy of urethral exudatereveals the presence of epithelial cells in addition tophagocytes (Figs. 1 and 2). The epithelial cells arepossibly detached from the epithelial tissue of theurogenital tract of male patients as a result of theinflammation associated with the disease. Sincegonorrhoea has been reported (Harkness, 1948) tostart with a superficial infection, one might expect tosee the epithelial cells in the urethral discharge frommale patients. Ultra-thin sections of exudateobtained from male patients by gently scraping theepithelial tissue of the urinary tract showed the

attachment of gonococci to the surface of epithelialor mucus-secreting cells (Ward and Watt, 1972).Different steps of fixation, dehydration, and critical-point drying in the preparation of specimens forscanning electron microscopy may have removed thegonococci from the surfaces of these cells. Therough surface of the epithelial cells (Fig. 2), whenfilled with mucus, provides an ideal site for attach-ment of gonococci. They can then establish theprimary infection and are protected from removalduring micturition (Ward and Watt, 1972).The surface of the phagocytes was extensively

ruffled (Figs. la, b). These ruffles, which are three-dimensional views of the pseudopodia, have beenobserved by scanning electron microscopy on peri-toneal macrophages of the mouse (Klainer andRectenwald, 1974) and the rabbit (Warfel, Elberg,and Hayes, 1973). The two dimensional views ofpseudopodia provided by the light microscope andby transmission electron microscopy of ultrathinsections of phagocytes give the impression of much

F I G. 8 Freeze-etched preparation of urethral contains more granules (g) than that with severalexudate from male gonorrhoea patient. Two gonococci. Note presence of septation cell wallphagosomes are seen, one containing one N. gonorrhoeae (unmarked arrow). Phm = phagosome membrane.(GC), and the other three gonococci. Marker=1 iLm.The phagosome containing one micro-organismB



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90 British Journal of Venereal Diseases

less complexity in the surface of phagocytes and thenumber of pseudopodia. Pseudopodia were alsoseen on the surface of freeze-fractured, freeze-etchedpreparations of phagocytes (Fig. 3). Most of thephagocytes in specimens prepared by the criticalpoint technique were found to be in clumps (Fig. la).The clumping of the phagocytes in urethral exudatewas possibly due to the coagulation of extracellularfluids and mucus secretions during fixation anddehydration. It was our desire to handle cells aslittle as possible before observation; thus theurethral exudate was introduced directly from thepatient into the fixative. The clumping of phago-cytes could perhaps be eliminated by washing thecells before fixation as was done by Klainer andRectenwald (1974). In freeze-etched preparations ofurethral exudate, we found some phagocytes con-taming no gonococci in the plane of fracture (Figs. 3and 4) and other phagocytes with as many as elevenN. gonorrhoeae diplococci in the plane of fracture(Fig. 5). Phagocytes with no visible bacterial cellsin the fracture plane appeared to be highly granulatedas seen in Fig. 3. This is in agreement with observa-tions of phagocytes by Hirsch and Cohn (1960), whoreported from their studies by phase microscopythat phagocytes degranulate after engulfing micro-organisms. Ovcinnikov and Delektorskij (1971)reported granules in phagocytes without engulfedgonococci.

Figs 3 and 4 show interesting anatomical charac-teristics for human polymorphonuclear leucocytes.Several pseudopodia of the phagocytes were seen atany angle or level of fracture. The cell in Fig. 3clearly reveals a trilobed nucleus. The two layers ofthe nuclear membrane are evident in Figs. 3 and 4.The outer layer appears smooth (Fig. 4). When theouter layer was removed as a result of freeze-fracturing the specimen, the inner layer was revealedand showed a rough appearance. The presence ofnuclear pores was always accompanied by indentationof the pore toward the centre of the nucleus.The majority ofphagocytes with engulfed gonococci

showed individual N. gonorrhoeae cells in onephagosome (Fig. 5). However, phagosomes con-taining three or more gonococci were also seen(Figs 5 and 8). Ward and others (1972) reportedthat, during natural infection, polymorphonuclearleucocytes are continuously phagocytosing gonococci.They showed that phagocytes are capable of des-troying gonococci in 30 minutes. Polymorphonuclearcells containing gonococci showed evidence ofdegranulation (Figs 5 to 8), while those withoutgonococci were highly granulated (Figs 3 and 4).This supports the suggestion of Ward and Watt(1972) that gonococci cannot block the degranulationof polymorphonuclear leucocytes.

Several N. gonorrhoeae cells in the phagosomeappear to lack the ceU wail septation between the two

cells and possibly these gonococci were engulfedduring the process of cell division. There is also apossibility that, during natural infection, gonococciby some unknown mechanism can survive in thephagocyte and continue to divide. When exudatewas studied at the level of light microscopy, as manyas 25 to 50 gonococci were observed in one smallarea of the phagocyte, presumably in one phagosome.The preparative techniques employed for trans-mission electron microscopy show only a small areaof the phagocyte, since both fracturing and thinsectioning reveal only a single plane through thephagosome. This might account for the lowernumber of gonococci seen within the phagocytewhen studied by these methods.Thomas and others (1973) showed that cells from

type 1 colonies of N. gonorrhoeae strain F62 canresist the phagocytic action of leucocytes more thancells from type 4 colonies. Observation of thecolonial type of N. gonorrhoeae isolated from thepatients revealed 80 to 85 per cent. of colonies weretype 1. Therefore, it is possible that several gono-coccal cells found in one phagosome can initiallyresist the phagocytic action of the leucocyte, buteventually the leucocyte overcomes the resistanceand destroys the engulfed micro-organisms. Destruc-tion of gonococci by phagocytes is accompanied bymigration of cytoplasmic granules, mostly lysosomes(Hirsch and Cohn, 1960) into the phagosome (Figs6, 7, and 8). Lysosomes inside the phagosome werefound to be smaller in size than those in the cyto-plasm. Ward and others (1972) reported the ruptureof lysosomes in the phagosome. Possibly the small-sized granules in the phagosome are the result ofpartially used or ruptured lysosomes. The presenceof these small granules inside the phagosome mem-brane containing gonococci was reported by Wardand others (1972) and Ovcinnikov and Delektorskij(1971).The interesting finding in the observation of

freeze-etched preparations of phagosomes was thatthe degree of granularity in the phagosomes withonly one gonococcus (Fig. 8) was much greater thanin those containing several N. gonorrhoeae. It ispossible that these granules which contain differentenzymes, such as Rnase, Dnase, alkaline phosphatase,lysozyme, etc. (Hirsch and Cohn, 1960), have beendepleted during destruction of the gonococcal cells.We recognize that we observed only a small portionof the phagosome, and the number of bacterial cellsand granules in a given phagosome cannot beestablished from one fracture. In freeze-etchingthere is no equivalent to the serial-sectioning tech-nique used with plastic-embedded specimens.

This is the first demonstration of N. gonorrhoeaewithin polymorphonuclear leucocytes in urethralexudates by the freeze-etch technique. The appear-ance and surface morphology of phagocytes and



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epithelial cells m urethral exudates ofmale gonorrhoeapatients are also shown here for the first time byscanning electron microscopy. The phagocytesobserved had been exuded through a natural bodyopening as a result of the gonorrhoeal infection.This allowed us to observe phagocytes which werenaturally produced as a result of disease. In addition,the specimens were minimally manipulated aftercollection from the patients, being fixed with themildest of fixatives, 2 per cent. glutaraldehyde. Inthe case of the freeze-etch preparations, evendehydrating agents were avoided, thus eliminatinganother insult to cellular structures. The use ofthese two methods of study with minimal manipula-tion of specimens may lead to a better understandingof phagocytosis in general and more specifically to abetter understarlding of the pathogenesis of gonorr-hoea. Unfortunately we have been unable, as yet,to observe the gonococcal cell in the process of beingphagocytosed by either method. This would prob-ably require the observation of many more specimens.

SummaryUrethral exudates from male patients with gonorrhoeawere studied by use of the critical point dryingtechnique for scanning electron microscopy. Thesurfaces of phagocytes were extensively ruffled. Thiswas interpreted to be the three-dimensional appear-ance of pseudopodia. Epithelial cells present in theurethral exudate showed a highly convoluted surfacestructure.Specimens ofurethral exudate prepared by the freeze-

etch technique showed that most N. gonorrhoeae wereengulfed individually in phagocytes. Some phago-somes contained two or more gonococcal cells.Phagocytes containing gonococci were degranulated,but small granules were present in the phagosomes.Nuclear pores on the surface of the nuclear mem-brane were always associated with an indentation ofthe nuclear membrane. The outer layer of thenuclear membrane was smooth compared to theinner layer.

ReferencesANDERSON, T. F. (1951) Trans. N.Y. Acad. Sci., 13, 130HARKNESS, A. H. (1948) Brit. J. vener. Dis., 24, 137HIRSCH, J. G., and COHN, Z. A. (1960)J. exp. Med., 112,

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KELLOGG, D. S., COHEN, I. R., NORINS, L. C.,SCHROETER, A. L., and REISING, G. (1968) J. Bact.,96, 596

PEACOCK, W. L., DEACON, W. E., BROWN, L., andPIRKLE, C. I. (1963) Ibid., 85, 1274

KLAINER, A. S., and RECTENWALD, M. (1974) 'ScanningElectron Microscopy', ed. 0. Johari and I. Corvin, p.822. Illinois Institute of TechnologyResearch Institute,Chicago

MARTIN, J. E., Jr., and LESTER, A. (1971) HSMHA HlthRep., 86, 30

OV6INNIKov, N. M., and DELEKTORSKIJ, V. V. (1971) Brit.J. vener. Dis., 47, 419

SPINK, W. W., and KEEFER, C. S. (1937) J. clin. Invest.,16, 169

THOMAS, D. W., HILL, J. C., and TYERYAR, F. J., Jr (1973)Infect. and Immun., 8, 98

WARD, M. E., GLYNN, A. A., and WATT, P. J. (1972)Brit. J7. exp. Path., 53, 289

- and WATT, P. J. (1972) J. infect. Dis., 126, 601WARFEL, A. H., ELBERG, S. S., and HAYES, T. L. (1973)

Infect. and Immun., 8, 665

IEtude par microscope e1ectronique a balayage etcryo-decapage* des secretions uretrales gono-cocciques

SOMMAIRE

Des secretions uretrales d'hommes atteints de gonococciefurent etudiees a l'aide de la technique du pointcritique pour le balayage en microscopie electronique. Lessurfaces des phagocytes etaient extremement ridees. Cecifut interprete comme l'apparence tri-dimensionnelle depseudopodes. Les cellules epitheliales presentes dans lasecretion uretrale montraient une structure de surfacefortement convolutee. Les specimens de la s6cr6tionuretrale etudiees par cryo-decapage montraient que laplupart des N. gonorrhoeae etaient inclus individuellementdans les phagocytes. Quelques phagosomes contenaientdeux ou un plus grand nombre de cellules gonococciques.Les phagocytes contenant des gonocoques avaient perduleurs granulations mais de petits granules etaient presentsdans les phagosomes. Les pores nuckaires a la surface dela membrane nucleaire correspondaient toujours avec uneempreinte en creux de la membrane nucleaire. La coucheexterne de la membrane nucleaire etait lisse par com-paraison avec la couche interne.

*NDT - attaque par congelation



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