APPLIED MICROBIOLOGY, Dec. 1969, p. 1065-1071 Vol. 18, No. 6Copyright © 1969 American Society for Microbiology Printed in U.S.A.

Observation of Microorganisms in Soiland Other Natural Habitats1

L. E. CASIDA, JR.Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication 24 September 1969

A procedure is described for visually observing and following the activities andinteractions of bacteria, actinomyctes, fungi, protozoa, nematodes, and plant roots inmasses of soil. Specific microscope components and objectives are used, and thenumerical apertures are adjusted such that light diffraction colors are produced toallow differentiation of the various biological entities and their habitat materials.Strains or other alterations in the organisms and their habitat are not employed,and time-lapse photography can be used to follow the activities of soil microor-ganisms and plant roots. As a result of the use of this technique, it is apparent thatin situ indigenous soil microorganisms differ from similar organisms grown inthe laboratory, but that, under the proper conditions, the state of the organism ineither habitat can be altered to match that which occurs in the contrasting habitat.

Casida (2) described the use of color infraredphotography for the microscopic visualizationof nonstained microorganisms in soil and otherhabitats. Although this procedure allows photo-graphic visualization of the microorganisms, itdoes not allow either short- or long-term con-tinuous observations of a single cell or group ofcells, because differentiation of microorganismsand habitat materials can be made only afterdevelopment of the film. Another feature of thistechnique is that use of the thin films of soilrequired for slide preparations for transmittedlight microscopy markedly alters the soil andsoil atmosphere conditions both for the initialobservation and for further observations duringincubation of the soil.The present study describes a visual micro-

scopic technique, not requiring photography orstains, for the continuous observation of micro-organisms residing in a mass of soil or otherhabitat materials. This method of observationalso allows an evaluation of the metabolic stateof the organism in its habitat.

MATERIALS AND METHODSSoil preparation. For observation, soil is placed as a

mound on the surface of a no. 1 cover slip, or it isplaced in a soil incubation chamber. The latter isfabricated by using epoxy cement to attach approxi-mately a 1-inch cylinder (2.54 cm) cut from an 18-mm

' This research was authorized for publication as paper no.3583 in the journal series of the Pennsylvania Agricultural Ex-periment Station on 9 April 1969.

(outside diameter) test tube to a no. 1 cover slip (Fig.1). For both procedures, the soil is moistened, since amoisture bridge is required between the soil andcover slip. For the incubation chamber, either a cottonplug is inserted in the upper portion of the container,or a polyethylene cover is secured with a rubber band.The polyethylene allows continuous incubation withatmospheric interchange but without an appreciablemoisture loss. The soil incubation chamber can beautoclaved if aseptic conditions are desired. Also,seeds can be planted in this chamber to allow observa-tions of microbial activity in the rhizoplane or rhizo-sphere.

Microscopy. Basically, the procedure employs aZeiss metallurgical reflected-light microscope withan unencumbered floating stage (Fig. 2). The objec-tives provided with this microscope are removed andreplaced by a Zeiss lOOX Apochromat, Planapo-chromat, or Planachromat objective containing adiaphragm which allows numerical apertures (NA)from 0.8 to 1.25 or 1.32, depending on the objective.The apochromatic objectives are preferred; anachromatic objective cannot be used because of itslesser color correction. Lower magnifications arepossible by employing a Zeiss 40X apochromatic oilobjective with iris diaphragm. All of the above objec-tives normally are employed for transmitted lightand not for reflected light microscopy.

This microscope is used by placing a soil sampleon a cover slip or in an incubation chamber on thesurface of the microscope stage with an immersionoil bridge between the objective and the undersideof the cover slip. The sample can be moved in anydirection merely by applying slight pressure to theedges of the floating stage. The aperture diaphragm(in this case, located in the base of the microscope) isclosed to its limit, and the objective diaphragm is

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FIG. 1. Soil incubation chamber on microscope stage. The objective is beneath the stage, and a slight pressureapplied at any edge of the floating stage brings a new area of the sample into view for observation.

set at 0.8 NA. A 12-v light source operating at 5 ampis required so as to obtain sufficient illumination forreflected light microscopy.

For observation of laboratory cultures, an aqueouscell suspension is placed on the surface of a cover slipeither with or without a second cover slip or slideplaced above. Growth on the surface of agar isobserved by cutting out a block of agar and invertingit on a cover slip.

Photography was accomplished by replacing thebinocular head with a monocular tube, into whichwas inserted a Leitz MIKAS with attached Leica MIcamera body. The film was Kodak Panatomic X,Ektachrome X, or High Speed Ektachrome type B.The growth and activities of indigenous soil micro-organisms were followed by sequentially photograph-ing fields of interest at time intervals ranging from15 min to 2 days.

RESULTSWhen soil or marine mud is observed by re-

flected light with this microscope, living and deadin situ soil microorganisms on soil minerals andorganic materials, or suspended in soil water,diffract light in a manner such that they appearto the eye as green, orange, yellow, red, or some-times blue (Fig. 3). The particular diffractioncolor seems to be specific for each individualmicrobial cell but, nevertheless, contrasts withsoil minerals which usually appear gray andtranslucent, and with the brown to black of soilorganic materials. Protozoa appear translucent

and colorless, but colored ingested bacteriasometimes can be observed within them. Soilnematodes also appear colorless and translucent,and the same is true for small roots and root hairsof growing plants (Fig. 4). For fungi, the lightdiffraction coloration is associated with a centralarea of the mycelium occupying approximatelyone-fifth of the diameter of the mycelium (Fig.5) but ceasing just before the growing tip. Thelight diffraction colors for both bacteria andfungi are observed by focusing at the midplaneof the cells and do not appear to be associatedwith either the upper or lower surfaces.The portion of the light spectrum responsible

for the light diffraction colors of in situ soilmicroorganisms corresponds approximately withthe green light wavelengths of 490 to 500 nm.This was determined by inserting photographicfilters of various colors into the light train abovethe aperture diaphragm while observing forlosses of specific coloration of individual micro-organisms.

Small fragments of mineral or crystallinematerials which diffract light in a manner some-what resembling that for microorganisms attimes are observed in soil and certain othernatural habitats. These can be detected by focus-ing on the artifact, followed by adjusting theobjective diaphragm to a NA of 1.25 or 1.32,depending upon the objective being used. As

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FIG. 2. Modified metallurgical microscope for observation of microorganisms in their natural habitat. Forphotography, the binocular head is replaced by a monocular tube with photographic device.

the numerical aperture is changed, the lightdiffraction picture for the artifact also changesto yield a bright multiple-diffraction patternwith a marked deterioration of image quality.Under similar conditions, the light diffractionpicture for microorganisms does not change, orit changes only slightly if the organism is em-bedded in or coated with habitat materials.The occurrence of light diffraction colors for

microorganisms in a natural habitat apparentlyis associated with a state of low metabolic activityin the habitat. This becomes more apparent whenone observes laboratory-grown cultures of bac-teria or fungi with this microscope. Thus, culturesgrown in the laboratory and observed either aswet mounts or on the surfaces of agar blocks inmost instances do not show the brilliant lightdiffraction colors associated with in situ soilmicroorganisms. Instead, their appearance re-

sembles that encountered when the cells areobserved by phase microscopy. Various treat-ments of laboratory-grown cultures, such aslyophilization, starvation, and contact with heat,solvents, and salt solutions, have not yieldedthe brilliant light diffraction colors associatedwith in situ soil organisms. However, bacteriasubjected to antibiotic stress in the laboratorydo present light diffraction colors. This is truefor bacteria growing within inhibition zonessurrounding antibiotic discs containing chlor-amphenicol, dihydrostreptomycin, erythromycin,novobiocin, and tetracycline, but not for peni-cillin, lincomycin, or neomycin. Light diffractioncolors also are observed for bacteria taken fromwithin inhibition zones on crowded plates pro-duced by plating low soil dilutions.

Several unusual cell groupings observed forcells in soil have now been observed for labora-

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FIG. 3. Soil bacteria residing on soil minerals. The cells photograph white with black-and-white film, but tothe eye appear colored in contrast to the gray minerals. X1,700.

tory cultures growing under antibiotic stress.However, stable L-form cultures of Proteusrettgeri and Staphylococcus aureus (kindlysupplied by C. W. Godzeski, Lilly Laboratoryfor Clinical Research, Indianapolis, Ind.) havenot yielded the colors associated with antibioticstress on an organism.The phase microscopy-like appearance of a

laboratory culture of Arthrobacter globiformiswas changed to that of in situ soil bacteria byadding a washed cell suspension of this organismto soil. The cells did not immediately diffractlight as did the indigenous soil bacteria. However,during a period of 1 to 2 weeks of incubation ofthe soil, those cells which did not lyse eventuallytook on an appearance resembling that for thein situ soil microorganisms.From an opposing viewpoint, at least a portion

of the in situ soil microorganisms can be madeto lose their light diffraction coloration by (i)the presence of active root hairs of a growingplant; (ii) addition of an agar block containinga carbon source such as glucose, sucrose, starch,sodium acetate, or gelatin, but not of a nitrogensource such as NH4NO3 or DL-alanine; and (iii)layering of autoclaved soil over a lower natural

soil layer. The cells (Fig. 6) then resemble thosein laboratory cultures of bacteria as viewed withthis microscope. As the available soil nutrients,added as above, become exhausted, the appear-ance of the bacterial cells reverts in a step-wisemanner to that of organisms in unaltered soil.Thus, first the central portion of the cell takes onlight diffraction color, then the outer boundary ofthe cell becomes difficult to detect.

Although this outer boundary of the bacterialcell residing in soil is difficult to detect, it cansometimes be seen by adjusting the NA of theobjective to 1.32. In contrast, the outer boundaryfor cells of laboratory cultures under antibioticstress can easily be observed at 1.32 NA, and,in this instance, it is obvious that the light diffrac-tion colors are associated with that portion ofthe cell which does not include the cell wall.

Laboratory cultures of fungi do not demon-strate a central core of light diffraction color.Also, segments of both septate and nonseptatemycelium in soil sometimes lack this charac-teristic, and, as mentioned previously, this alsoapplies to the growing tip. The outer boundaryfor mycelia in soil is clearly visible, however,regardless of its internal light diffraction charac-

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FIG. 4. Bacteria on the root surface of clover growing in a soil incubation chamber. The bacteria are coloredby diffracted light, whereas the root surface is colorless. X 2,000.

teristics. The effects of antibiotics on fungi inrelation to light diffraction colors have not beenstudied.

DISCUSSIONThe microscopic technique described allows

the examination and continuous observation ofthe growth, activities, and interactions in soilof bacteria, actinomycetes, fungi, nematodes,protozoa, and plant roots without the use ofstains or other alterations in the microorganismsof their habitat and environment. Also, by useof the soil incubation chamber described, or ofvarious possible modifications of it, observationsof microorganisms and roots should be possiblewhile changing any combination of the soilatmosphere, temperature, and available nutrientlevel, and during addition of microbial or plantgrowth inhibitors or stimulators. Specific ex-amples of such studies might be (i) determiningthe effects of added pesticides on the activities

of the soil microflora, and (ii) following undervarious conditions the rhizosphere activities andinfection processes for pathogens of growingplant roots.

This microscope allows a three-dimensionalobservation of the microorganisms and theirhabitat, with the depth of the soil which can bevisually penetrated being approximately equiva-lent to the working distance of the objectiveused. Obviously, the 40 X objective, with itsgreater working distance, allows a greater visualpenetration into the soil than does the 100Xobjective. Regardless of its lower magnification,this objective still allows observation of thevarious living forms in soil, including bacteria.The three-dimensional viewing aspect at times

causes difficulty in time-lapse photography. Thus,growing plant roots may push soil particlesaround, or soil particles may move or rotateslightly due to other stresses in soil, with theresult that sequential photography of particular

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FIG. 5. Fungal hyphae in soil in an incubation chamber containing decomposing pea seeds. Note the coloredlight diffraction area running through the central portion of the mycelium, and the colorless outer area of themycelium. X 1,700.

FIG. 6. Bacterial microcolonies in natural soil with overlay of autoclaved soil. The bacteria are colorless andresemble laboratory-type growth, but a few of the cells have started to take on light diffraction colors. X 1,700.

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cells at a given plane within the soil may haveto be terminated before it is desired to do so.As a result of these studies, it is apparent that

indigenous soil microorganisms undergo con-siderable change when the nutritional status oftheir environment is altered, or when they areisolated and grown in the laboratory. It is notknown whether this reflects merely a responseof the organism to altered nutrient availability,or whether it indicates a release from a soil stasiscaused by antibiotics or other possible factorsin the soil environment. These observations,nevertheless, tend to corroborate the conclusionsof Gledhill and Casida (3) that soil microorgan-isms can occur in soil in a state different fromthat which they take on during adaptation to andgrowth on laboratory media.The relationship of the optical phenomena

observed for microorganisms in soil and thechemical and physical states of their cell consti-tuents is not clear at present. It is known, how-ever, that various of the antibiotics which inducelight diffraction colors for laboratory culturescause changes in cell nucleic acids which arevisible be light and electron microscopy [seeBrock (1)].

It would appear that use of the particularcombination of microscope components andobjectives, as described in this study, is a require-

ment for obtaining light diffraction colors ofmicroorganisms residing in masses of soil, andfor viewing the relationships of these organismsto the roots of growing plants. Other combina-tions of equipment and objectives do not providethe specific light path through the microscopeand sample, nor do they allow the requiredadjustments of NA.

It also should be noted that the light diffractioncolors observed for in situ soil microorganismsare not to be confused with the false red colorobserved when photographing soil bacteria withKodak Ektachrome Infrared film and trans-mitted light (2). The false red color for theinfrared photography occurs only at the lowersurface of the cell, the surface closest to the lightsource, and it is caused by a portion of the lightspectrum different from that for the visuallyobserved light diffraction colors. Thus, use ofcolor infrared film has not been successful withthe reflected light microscopy described in thisstudy.

LITERATURE CITED

1. Brock, T. D. 1961. Chloramphenicol. Bacteriol. Rev. 25:32-48.2. Casida, L. E., Jr. 1968. Infrared color photography: selective

demonstration of bacteria. Science 159:199-200.3. Gledhill, W. E., and L. E. Casida, Jr. 1969. Predominant

catalase-negative soil bacteria. I. Streptococcal populationindigenous to soil. Appl. Microbiol. 17:208-213.

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