JOURNAL OF BACTERIOLOGY, Mar. 1973, p. 1462-1473Copyright O 1973 American Society for Microbiology

Vol. 113, No. 3Printed in U.S.A.

Responses of Indigenous Microorganisms to SoilIncubation as Viewed by Transmission Electron

Microscopy of Cell Thin Sections1H. C. BAE AND L. E. CASIDA, JR.

Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication 13 October 1972

Air-dried soils were adjusted to 50% moisture-holding capacity and incubatedfor 2 weeks at 30 C. Samples were removed at intervals, and their total microbialpopulations were physically separated and concentrated from the soil debris forsectioning and ultrastructure examination. Although the total numbers of cellsections in these preparations remained relatively constant during the soilincubations, the percentages of dwarf cells (<0.3 gm in diameter), minute cells(<0.2 ,um in diameter), and cells with a cystlike structure decreased with timefollowed by a slow increase. During this period, a corresponding increase anddecrease occurred in the percentages of cells in the 0.3- to 0.5-,um diameterrange, but dividing cells were rarely observed. The percentages of spores and ofcells with electron-transparent areas also increased and then decreased duringincubation. When nutrients were added to these soils, the initial increases in cellsize occurred at what appeared to be a faster rate. But this probably was relatedto a corresponding increase in total cell numbers which also occurred. Theresponses of the spores, cystlike cells, and cells with electron-transparent areas

to nutrient additions were not consistent although all conditions of incubation,regardless of nutrient addition, increased the occurrence of an enlarged diffuseintine-like layer for the cystlike cells. In addition to the above, incubated soilscontained cells, which were mainly in the 0.3- to 0.5-,um cell diameter range, thathad an internal membrane surrounding the general area of the nuclear material.Changes in additional fine structure features of the microbial populations are

described.

Bae, Cota-Robles, and Casida (1) utilizedtransmission electron microscopy to study thefine structure of the indigenous microorga-nisms of soil. The cells were first physicallyseparated and concentrated from this habitatwithout the occurrence of growth, and thenfixed, embedded, and sectioned. This studyrevealed that soil harbors large numbers ofdwarf cells (<0.3 Am in diameter) and cystlikecells, as well as cells with unique cytologicalfeatures. It was not known whether any of thesecells had ever been cultivated in the laboratory,or whether they might undergo changes duringadaptation to laboratory cultivation so thatthey no longer could be recognized as belongingto the groups observed in the thin section fromsoil.

'This research was authorized for publication as paper no.4309 in the journal series of the Pennsylvania AgriculturalExperiment Station on 3 October 1972.

In the present study, these indigenous micro-organisms were cultivated within their soilhabitat. Changes in the cells which occurredduring incubation were followed by transmis-sion electron microscopy thin-section evalu-ations. Soils which had been stored were usedso that dormant cells would comprise a majorportion of the microflora. Of particular interestwas whether the small cell sizes noted previ-ously (1) were of a stable characteristic, andwhether the cystlike cells were able to germi-nate and then reencyst within the soil habitat.The responses to soil incubation of other groupsof soil microorganisms were also noted.

MATERIALS AND METHODSSoil samples and incubation. Soil A (1) was a

Hagerstown silty clay loam: pH 5.4, organic content3.6%, water content 16.0%, and plate count 8.5 x 106.Soil B was a Webster silt loam obtained from L. R.

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Frederick (Iowa State University, Ames). It wascollected in July 1969 and was stored in a poly-ethylene bag at room temperature. Its pH was 6.6,organic content 10.6%, water content 3.1%, and platecount 3.7 x 106. Studies of soil A were completedbefore those of soil B were initiated.

Portions (20 g) of soil in 50-ml polycarbonatecentrifuge tubes were adjusted to 50% moisture-hold-ing capacity by addition of distilled water or nutrientsolutions (1% glucose, 1% NH4Cl, or a mixture of 1%of each). The tubes were incubated at 30 C with theircaps adjusted to impede moisture loss but still allowsome air exchange.Fractionation, fixation, and electron micros-

copy. The above 20-g portions of soil were prefixedin the centrifuge tubes by shaking for 1.5 h at roomtemperature with 20 ml of buffered 1% OSO4 (8).Residual fixative was washed from the fixed soilswith distilled water by using four 10-min centrifuga-tions at 12,000 x g. The soils were then suspended in0.1% sodium pyrophosphate and subjected to exhaus-tive centrifugal washing (1) with this solution. Thefinal cell pellet was suspended in 15 ml of distilledwater, washed three times by centrifugation in water,and then centrifuged on a Ludox density gradient (1).Embedding was in Spurr's low-viscosity resin (10).Procedures for electron microscopy and size meas-urement of cells were as previously described (1).However, measurements of cell diameters for thecystlike cells did not include the intine or exinematerials because of fluctuations encountered in thesize and consistency of these structures.

RESULTSThe exhaustive centrifugal washing of soil

(1) as modified in the present study yielded aslight increase in numbers of cell sectionsobserved per field at 25,500-fold scope magnifi-cation. The amount of contaminant soil debrisin the final cell preparations was considerablyreduced, however.During incubation of unamended soils A and

B at 50% moisture-holding capacity, the totalnumbers of cells recovered from the soil sam-ples and viewed as thin sections remainedrelatively constant. The size distributions ofthese cells, however, changed as incubationprogressed. The percentage of dwarf cells (0.30,um or less in diameter) and of the yet smaller"minute" cells (0.2 ,um or less in diameter) inboth soils decreased, followed by a slow in-crease (Fig. 1 and 2). Many of these cells in soilA (Fig. 1) would appear to have first enlargedin size into the 0.31 to 0.50 ,um range and thendecreased to their original size range as incuba-tion progressed. For soil B (Fig. 2), some ofthese cells apparently increased in size into thegreater than 0.50-,um range before decreasingin size.The initial apparent increase in cell size for

the small cells was more rapid when the soilswere amended with nutrients (12-h values,Table 1). However, during these trials the totalnumbers of cell sections for the amended sam-ples increased by approximately 15%. Also,partially lysed large cells having a diameter of1.20 jsm or greater were observed occasionallyin these preparations.The percentage of cells exhibiting a cystlike

structure for unamended soil B decreased dur-ing the first 24 h of incubation and then, pos-sibly through reencystment, rose over a periodof time approximately to the original level (Fig.3). Soil A, which contained fewer cystlikecells, behaved in a similar manner. In contrast,incubated amended soils did not present aconsistent picture of disappearance or reap-pearance of the cystlike cells. The cystlike cellsobserved in all soil samples before incubationhad an intine layer whose texture was rathercompact; examples are presented in Fig. 4a, b.In contrast, many of the cystlike cells in theincubated soils, either with or without nutrientamendments, were characterized by an en-larged, diffuse, intine-like layer (Fig. 5a-c), andby parital ruptures of the intine- and exine-likelayers (Fig. 6a-d). The appearance of the lattercells resembled that of germinatingAzotobacter cysts (13). In some of the cystlikecells (Fig. 7), structures were observed whichwere similar to the intine vesicles (12) or blebs(5) of an Azotobacter cyst, or which bore someresemblance to a bacterial forespore (Fig. 8; see

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FIG. 1. Effect on cell size of 30 C incubation of un-amended soil A at 50% moisture-holding capacity. Atotal of 450 cell sections was examined. Cell diame-ters: 0, <0.20 Atm; 0, <0.30 um; *, 0.31 to 0.50Am; 0, >0.51 .tm. The <0.30 Am curve includesthose cells <0.20 gm in diameter.

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FIG. 2. Effect on cell size of 30 C incubation ofunamended soil B at 50%o moisture-holding capacity.A total of 225 cell sections was examined. Celldiameters: 0, <0.20 Am; 0, <0.30 Am; *, 0.31 to0.50 Mm; 0, >0.51 Am. The <0.30-Am curve in-cludes those cells .0.20 Am in diameter.

TABLE 1. Effect of nutrient amendments to soils Aand B on percent occurrence of small bacterial cells.

Cells <0.20-gm Cells <0.30-umSoil Nutrient diam (%)a diam (flaadditions

0 h 12 h 24 h 0 h 12 h 24 h

A Water" 55 36 18 83 73 56Glucose 55 18 10 83 48 47NH4Cl 55 18 16 83 55 36Glucose + 55 23 7 83 57 26NH4Cl

B Water 39 20 11 74 40 26Glucose 39 11 13 74 26 24NH4C1 39 10 11 74 27 25Glucose + 39 16 9 74 26 25NH4C1

a Percent occurrence among 90 cell-section photo-graphs examined for soil A and 45 for soil B.

h Soil moisture adjusted before incubation to 50%moisture-holding capacity with distilled water or 1%aqueous solutions of each nutrient.

references 2, 6, 9). Also noted, although rarely,were cells resembling a myxobacteria mi-crocyst, which were associated with vesicularstructures within a thick layer of capsule (Fig.9), and other encapsulated cells having (i) athick, compact capsule (Fig. 10), (ii) a diffusecapsule (Fig. 11), or (iii) a diffuse fibrous cap-sule (Fig. 12). The cells with the thick, compact

INCUBATION (hr)FIG. 3. Occurrence of cystlike cells during incuba-

tion at 30 C of unamended soils A and B at 50% ofmoisture-holding capacity. A total of 450 and 225 cellsections, respectively, were examined for soil A (solidline) and soil B (broken line).

capsule were noted with the greatest frequency(1.2% of cells from incubated soils with andwithout amendments) and were more prevalentin soil B. They were absent, however, in nonin-cubated soils.Upon incubation of nonamended soils (Fig.

13), the percentages of bacterial spores andsporulating cells (Fig. 14a-d), and of cells withelectron-transparent areas, increased and thendecreased. The amended soils, however,showed inconsistent fluctuations. Neverthe-less, no spores or sporulating cells were ob-served at 0 h incubation or at the end of the2-week incubation period for either amendedor nonamended soils.

For both soils, regardless of nutrient amend-ment, the frequency of occurrence of cells witha mesosome was not affected by the soil incu-bations, while cells in the process of divisionappeared to be slightly more prevalent uponincubation.Approximately 2.5% of the 1,485 cells from

both incubated soils photographed in thisstudy possessed an internal membrane sur-

rounding the general area of the nuclear mate-rial (Fig. 15a-d). These cells were not present innonincubated soils, and most of them were inthe 0.3- to 0.5-um diameter size range. Thisinternal membrane appeared to be in additionto the plasma membrane (Fig. 15a), althoughin about two-thirds of the examples a clearresolution of the plasma membrane was dif-ficult (Fig. 15b-d). Some of the latter cells were

1464 J. BACTERIOL.

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4Q9FIG. 4. Sections of cystlike cells from nonincubated soil B showing compact intine (arrow). a, Single central

body; b, multiple central bodies. Bar markers represent 0.20,um.

dividing, and their nuclear region surroundedby a membrane lay in the middle of thedivision plane (Fig. 15c, d).

DISCUSSIONThe concentration of OsO used in this studywas greater than that normally employed forprefixation of cells; this was because of thecrude nature of the soil samples being fixed.Other concentrations were not tested. Never-theless, the fixation procedure used provided abetter preservation of cellular fine structurethan those previously employed (1), includingglutaraldehyde prefixation followed by OSO4fixation. This was apparent from the generalappearance of sectioned cells and the smalleramounts of cellular debris observed in the cellpreparations.The modified exhaustive centrifugal washing

procedure used in this study to separate andconcentrate the soil cells utilized the exhaus-tive centrifugal washing of soil (1) and theLudox gradient centrifugation (1) in tandem.This is preferred because it yields greaternumbers of sectioned cells for viewing andsmaller amounts of soil debris than the meth-ods previously evaluated (1). A disadvantage,however, is that it is yet more time consumingthan the exhaustive centrifugal washing usedalone. In our previous study (1), it appearedthat Ludox gradients, which are quite alkaline,might have destroyed some of the cells, and

that the larger cells tended to be selected bythis technique. It is now apparent, however,that a Ludox gradient, when used after theexhaustive centrifugal washing, does not des-troy cellular fine structure, and that concentra-tion of the smaller cells is not adversely affect-ed. It is believed, therefore, that the yields andtypes of cells that were obtained by Ludox gra-dient centrifugation used as the initial stageent centrifugation used as the initial stage(without preliminary cell release or centrifugalfractionations) in fractionating microbial cellsfrom soil depended on the nature of the soilsample used in terms of the degree of release ofcells from the soil particles rather than on theLudox gradient itself. Some of the cells possi-bly did not separate from the particles and,therefore, tended to settle to the bottom of thecentrifuge tube with the soil materials.The total numbers of cell sections recovered

from the final cell preparations from una-mended soils remained relatively constant dur-ing the soil incubations, and dividing cells wererarely observed. Based on this, the initialdecrease with time of the percentages of dwarfcells (<0.3 ,um in diameter) and of the "min-nute" cells (<0.2 ,um in diameter), followed bya slow increase, suggests a reversible growth insize of these cells without multiplication in situin soil. This interpretation is strengthened bythe coincident occurrence of a correspondingincrease and then decrease in the percentagesof larger sized cells. The fluctuation patterns

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FIG. 5. Sections of cyst-like cells from soil B incubated 12 h showing enlarged diffuse intine (IN). a and b,Soil amended with NH4CI; c, nonamended soil. a, Single central body; b and c, multiple central bodies. Barmarkers represent 0.30 um.

for the occurrence of cystlike cells in both soilsalso showed an initial decrease followed by aslow increase. This could reflect cyst germina-tion up to about 24 h of incubation followed by aslow reencystment. It is assumed that availablesoil nutrients were used up during this initial

period. A correlation was not evident betweenthe activities of' the small cells and cystlikecells, but some of the cystlike cells were dwarfcells.

Katznelson and Stevenson (7) and Stevenson(11) reported that remoistening of dried soils

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Nonamended soil incubated 12 h; b, soil amended with glucose plus NH4Cl, incubated 12 h; d, nonamendedsoil incubated 24 h. In d note the absence of intine although exine residues are still present. Bar markersrepresent 0.20,m.

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FIG. 7. Section of cystlike cells from soil B incubated 48 h showing intine vesicles or blebs (arrows). Barmarkers represent 0.30 ,im.

FIG. 8. Section of cystlike cells from soil B incubated 48 h showing entities bearing some resemblance to abacterial forespore (FS). Bar marker represents 0.20 ,im.

FIG. 9. Section of a myxobacteria microcyst-like cell showing membranous vesicles (arrow) within thecapsule. From soil A incubated 24 h with glucose plus NH4CI. Bar marker represents 0.30 tim.

resulted in an initial burst of respiratory activ-ity due to the soil microflora using readily avail-able nutrients which were released from thesoil upon air-drying. They concluded that thisrespiratory increase resulted from adaptiveenzymatic mechanisms rather than from cellmultiplication. Griebel and Owens (4) alsonoted no changes in numbers of soil microor-

ganisms during a transient activation of micro-bial respiration caused by ethanol or acetalde-hyde additions to soil. Stevenson postulatedthat the rapid increase in rate of oxygen uptakemight be associated with a physiological ac-tivation of various microbial elements in thesoil, such as germination of spores of fungi,actinomycetes, and bacteria, followed by rapid

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FIG. 10. Section of a cell from soil B showing a thick, compact capsule (arrow). Soil incubated 24 h withNH4CI. Bar marker represents 0.20,1m.

FIG. 11. Section of a cell from soil B showing a diffuse capsule (arrow). Soil incubated 24 h with glucose.Bar marker represents 0.20 Am.

FIG. 12. Section of cells from soil B incubated 12 h showing a diffuse fibrous capsule (arrow). DPS,Electron-dense periplasmic space. Bar marker represents 0.20 tim.

assimilation and oxidation of the substrate.The results of the present study indicate ad-ditional possible explanations. It is temptingto conclude that the small cells are dormant

before they begin to enlarge. Regardless ofwhether they are or are not dormant, however,their increase in size probably coincides withan increase in respiratory activity. Increased

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INCUBATION (hr)FIG. 13. Occurrence of bacterial spores and cells

with electron-transparent areas during 30C incuba-tion of unamended soils A and B at 50% of moisture-holding capacity. A total of 450 and 225 cell sections,respectively, was examined for soil A (solid line) andsoil B (broken line). Cells containing electron-trans-parent areas, 0; spores and sporulating cells, *.

respiration would also be evident if the cystlikecells are germinating under these conditions.As to Stevenson's thoughts on bacterial spore

germination, the present study showed an ab-sence of these spores at 0 h. Their numbers,however, may have been too low for detectionat the scope magnifications used. They be-came apparent as sporulating cells at 12 and24 h of incubation, and as spores at 48 h. Thiscould reflect a cycle of germination, multipli-cation, and sporulation with an attendant in-crease in overall respiratory activity.The disappearance of the bacterial spores

between 2 and 14 days of incubation is difficultto explain. However, Gorden et al. (3) noted a

similar disappearance of Bacillus spores after 5days in a "laboratory microecosystem" study.It would appear, therefore, that bacterialspores indigenous to a natural habitat do nottend to accumulate over a period of time in thehabitat.Some of the cystlike cells (1.5% and 6.1%,

respectively, for soils A and B) resembledpublished pictures of a germinatingAzotobacter cyst (13) with its central bodyemerging from the cyst coat (Fig. 6a-c). It ispossible that in our study a fraction of the exineor intine may have broken away due to thefractionation procedure employed. We considerthis to be unlikely, however, since the apparentcyst germination in this study occurred only inincubated soil samples, and it was not observed

in our previous study (1) in which the soils werenot incubated.Germination of Azotobacter cysts in labora-

tory cultures begins with an increase in the sizeof the central body at the expense of the intine(13). The germination of cysts in soil possiblycan occur in a different manner, although it isrealized that one may not be able to differenti-ate germination from encystment. The largediffuse intine occurring in some of the cystlikecells (Fig. 5a-c) thus may illustrate a process ofgermination in which the intine polymers be-come broken down and diffuse and then are nolonger stainable (Fig. 6d).When the soils were amended with nutrients

before incubation, the initial rates of increasein cell size (decreased occurrence of small cells)appeared to be greater than in unamendedincubated soils (Table 1). This increased rate,however, coincided with a 15% increase in thetotal number of cells. It appears, therefore, thatthe initial increase in cell size was approxi-mately the same in amended and unamendedsoils. The responses in amended soils of thecystlike cells, cells with electron-transparentareas, spores, and sporulating cells were notclearly defined. It may be that the elevatedinteractions among actively metabolizing mi-croorganisms in the amended soils obliteratedthe manifestation of any one microbial activi-ty.Some of the cells in the incubated soils,

either with or without nutrient amendments,had a thick, compact capsule (Fig. 10) whichwas different from capsules previously ob-served (1) and from those shown in Fig. 11 and12. These capsules bear some resemblance tothat of a myxobacteria microcyst. It is notknown why these capsules occurred only forcells in the incubated soils, although capsuleformation might be an initial protective res-ponse of the cell to its environment whenincreased metabolic activity and growth be-come possible due to nutrients becoming avail-able.The present study revealed a group of cells,

mainly in the 0.3- to 0.5-Am cell diameterrange, which occurred only in incubated soils(regardless of the incubation conditions), andwhich appeared to have an internal membranesurrounding the general area of the nuclearmaterial (Fig. 15). A clear resolution of theplasma membrane was difficult for approxi-mately two-thirds of these cells (Fig. 15 b-d).For the rest of the cells (Fig. 15 a), however,this membrane was resolved and was separatefrom the plasma membrane. The space be-tween this membrane and the plasma mem-

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14dFIG. 14. Sections of sporulating cells and of a mature spore from soil A. a, Nonamended soil incubated 12 h;

b, soil incubated 12 h with NH4C1; c and d, nonamended soil incubated 24 h. FM, forespore membrane; IFM,inner forespore membrane; OFM, outer forespore membrane; C, cortex; arrow in d, spore coat. Bar markerrepresents 0.50 ,m for a, b, and d, and 0.30 Am for c.

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15 dFIG. 15. Sections of cells from soil A showing an internal membrane surrounding the general area of the

nuclear material. a, Nonamended soil incubated 48 h; b, soil incubated 12 h with NH4CI; c, soil incubated 12 hwith glucose plus NH4Cl; d, nonamended soil incubated 12 h. M, Internal membrane; PM, plasmamembrane; arrow, membranous vesicles. Bar markers represent 0.20 ,.m.

brane (or cell wall when the plasma membranewas not clearly resolved) was uniformly filledwith what appeared to be cytoplasmic mate-rial. This area thus did not seem to be anextensive periplasmic space containing elec-tron-dense material as in Fig. 12, nor due toplasmolysis. In the latter instance, it would beunlikely that the cytoplasmic material wouldbe released through the contracting plasmamembrane in such a manner that it woulduniformly fill the periplasmic space. The possi-bility that these cells might represent bacterialforespore formation was ruled out since fore-

spore formation should not occur in the middleof a division plane. Also, at the onset offorespore formation, that portion of cytoplasmsurrounded by the forespore membrane (Fig. 14a) is similar in appearance to the rest of thecytoplasm. It is possible that these cells repre-sent a hitherto unknown developmental stagewhich occurs mainly, or only, in soil habitatswhich are biologically active.

ACKNOWLEDGMENTS

This work was supported by National Science Foundationgrant GB-14487 and contract NGR 39-009-180 with the

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National Aeronautics and Space Administration.We thank E. H. Cota-Robles for stimulating discussions

conceming this study.

LITERATURE CITED

1. Bae, H. C., E. H. Cota-Rohles, and L. E. Casida, Jr.1972. Microflora of soil as viewed by transmissionelectron microscopy. Appl. Microbiol. 23:637-648.

2. Freese, E. B., R. M. Cole, W. Klofat, and E. Freese.1970. Growth, sporulation, and enzyme defects ofglucosamine mutants of Bacillus subtilis. J. Bacteriol.101:1046-1062.

3. Gorden, R. W., R. J. Beyers, E. P. Odum, and R. G.Eagon. 1969. Studies of a simple laboratory micro-ecosystem: bacterial activities in a heterotrophicsuccession. Ecology 50:86-100.

4. Griebel, G. E., and L. D. Owens. 1972. Nature of thetransient activation of soil microorganisms by ethanolor acetaldehyde. Soil. Biol. Biochem. 4:1-8.

5. Hitchins, V. M., and H. L. Sadoff. 1970. Morphogenesisof cysts in Azotobacter vinelandii. J. Bacteriol.104:492-498.

6. Hoeniger, J. F. M., P. F. Stuart, and S. C. Holt. 1968.Cytology of spore formation in Clostridium

perfringens. J. Bacteriol. 96:1818-1834.7. Katznelson, H., and I. L. Stevenson. 1956. Observations

on the metabolic activity of the soil microflora. Can. J.Microbiol. 2:611-622.

8. Kellenberger, E., A. Ryter, and J. Sechaud. 1958.Electron microscope study of DNA-containing plasms.II. Vegetative and mature phage DNA as comparedwith normal bacterial nucleoids in different physiolog-ical states. J. Biophys. Biochem. Cytol. 4:671-687.

9. Koroch, C. T., and R. H. Doi. 1971. Electron microscopyof the altered spore morphology of a ribonucleic acidpolymerase mutant of Bacillus subtilis. J. Bacteriol.105:1110-1118.

10. Spurr, A. R. 1969. A low-viscosity epoxy resin embed-ding medium for electron microscopy. J. Ultrastruct.Res. 26:31-43.

11. Stevenson, I. L. 1956. Some observations on the mi-crobial activity in remoistened air-dried soils. Plantand Soil 8:170-182.

12. Vela, G. R., G. D. Cagle, and P. R. Holmgren. 1970.Ultrastructure of Azotobacter vinelandii. J. Bacteriol.104:933-939.

13. Wyss, O., M. G. Neuman, and M. D. Socolofsky. 1961.Development and germination of the Azotobactercyst. J. Biophys. Biochem. Cytol. 10:555-565.

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