APPUED MicRoBImoLY, July 1967, p. 935-639 Vol. 15, No. 4 Copyright 1967 American Society for Microbiology Printed bi U.S.A. Electrical Sensing Zone Particle Analyzer for Measuring Germination of Fungal Spores in the Presence of Other Particles' T. SAN7TORO, G. STOTZKY, AND L. T. REM Kitchawan Research Laboratory of the Brooklyn Botanic Garden, Ossining, New York 10562 Received for publication 8 March 1967 Microscopic, respirometric, and electronic sizing methods for measuring germination of fungal spores were compared. With the electronic sizing method, early stages of germination (i.e., spore swelling) were detected long before germ tube emergence or significant changes in respiratory rates were observed. This method, which is rapid, easy, sensitive, and reproducible, also permits measuring the germination of spores when similar-size particles are present in concentrations considerably in excess of the number of spores. The germination of fungal spores is commonly measured microscopically or respirometrically, although other methods have been used (1, 2). In the microscopic method, the emergence of the germ tube is the usual criterion for germination. Prior to germ tube emergence, however, spores undergo many physiological. changes, and the germination processes are initiated long before the germ tube becomes evident (2). In many species (2), one of these changes is the increase in size that results from imbibition of water (1), which is apparently an active process occurring only in viable spores (3). The microscopic measurement of germ tube emergence and, especially, of spore swelling becomes more difficult when other particles of approximately equal size and shape (e.g., bacteria, clay minerals) are present. The use of differential stains is usually ineffective, as both the spores and the other particles retain the same dyes (unpublished data). The use of respirometric techniques eliminates these difficulties, but, because only gross metabolic changes are measured, it is not possible to distinguish changes in spore size nor to determine accurately the extent of germination of a specific spore batch. Furthermore, although the respiration of spores can be measured easily in the presence of inanimate particles (e.g., clay minerals) it is difficult to distinguish the respiration of spores from that of other microbial populations that may be present. During studies on interactions between clay minerals and microorganisms and their metabolites (Santoro and Stotzky, in press), changes in 1 Contribution no. 183 from the Brooklyn Botanic Garden. the particle size distribution of the individual component populations comprising mixtures of clays and cells were easily measured with an electrical sensing zone particle analyzer (Coulter counter). Consequently, studies were conducted to determine whether this technique could be used to measure the germination of fungal spores in the presence of other particles and to compare its efficacy with the microscopic and respirometric methods. Germination in this study is defined as an increase in spore size (when using the Coulter counter) or in oxygen uptake (when measured respirometrically), or emergence of the germ tube (when observed microscopically). MATERIALS AND METHODS Spores. Spores were harvested with sterile water and gentle agitation from cultures grown at 30 C on glucose-peptone-agar (1% glucose, 0.5% peptone, 0.1% yeast extract, 0.1% KH2P04, 0.05% MgS04.7H20, 2% agar). The spores were filtered through glass wool to remove any mycelia, and then were washed at least twice by centrifugation with sterile water. Clay minerals. The fractions (2 to 5,u) of kaolinite and montmorillonite (Fisher Scientific Co., Pittsburgh, Pa.) were made homoionic to sodium by washing three times, by centrifugation, with 0.5 N NaCl, followed by three washings with glass-distilled water. The homoionic clays were resuspended in a small volume of glass-distilled water and stored at 5 C until used. Coulter counter. A Coulter counter (Industrial model A) was used with an aperture of 100,u. The instrument was calibrated with monosized ragweed pollen and polystyrene latex particles. Details on the Coulter counter and its use in these studies have been described elsewhere (in press). A 1% sodium metaphosphate (Calgon) solution, filtered (Millipore HA, 0.45-jL pore size), and stored in capped bottles (Pyrex), was used as the electrolyte. 935
936 SANTORO, STOTZKY, AND REM APPL. MICROBIOL. Procedure. Washed spores were added to flasks containing 100 ml of glucose-peptone broth and montmorillonite (0.07 to 2%, w/v), kaolinite (0.03 to 2%, w/v), or no clay. The number of spores per milliliter was: Trichoderma viride, 8.50 X 106; Penicillium jenseni, 1.04 X 105; Fusarium oxysporwn, 1.71 X 106; F. nivale, 6.70 X 105; Aspergillus terreus, 1.02 X 106; Rhizopus stolonifer, 7.38 X 104; Zygorhynchus moelleri, 5.21 X 104. The flasks were incubated on a rotary shaker (180 rev/min) at 30 C. Samples (0.1 to 1.0 ml) of the mixtures, removed immediately after mixing and after various periods of incubation, were pipetted into a beaker containing 100 ml of Calgon and placed in the Coulter counter. At least two analyses of each mixture were made, usually sequentially, the first beginning with low aperture current and internal resistance settings and the second beginning with high aperture current and internal resistance settings. At least two readings were made at each setting, to minimize variations resulting from electrode polarities and to enhance precision. Analyses of the clays alone and of the spores alone were made in the same manner. All data were corrected for coincidence loss and for background counts (i.e., electrolyte alone). The data were plotted both as cumulative frequency (larger than stated size) and as differential (actual number of particles of stated size) plots. The cumulative frequency plots were put on log-log scales, to minimize the importance of the total number of particles present and to make the slopes of the curves the important criteria: the steeper the slope, the narrower 10.000 1.000 looo 00 the particle size distribution, and vice versa. As swelling proceeded, the number of larger particles increased, and the particle size distribution curves of the spore populations became wider. Because the total number of particles was not important in this type of plot, the size of the samples analyzed was chosen on the basis of counting convenience, and the resulting curves moved vertically on the cumulative plot to facilitate comparisons between them. In the differential curves, the numbers of particles were plotted on log scales and their sizes on arithmetic scales. The shapes of these curves, rather than the absolute numbers, were also the important criteria. Only the spore populations were plotted in the differential curves, as the clay populations were subtracted out. This subtraction was valid, as neither sorption between clay minerals and microbial cells nor flocculation of the clays by microbial metabolites was detected with sodium-saturated clays analyzed with sodium metaphosphate as the suspending electrolyte. Consequently, changes in the particle size distribution curves were the result of changes in spore size and not of surface interactions between or among populations of particles. No attempt was made to evaluate quantitatively the differences between slopes of the cumulative frequency curves nor between the shapes of the differential curves. The degree of swelling was expressed on an arbitrary scale ranging from 0 to 4. In the respiratory studies, changes in the rate of oxygen uptake were used as the measure of germination. When the rate attained a steep upward inflection T. VIRIDE T. VIRIDE a 10 KMOLINITE MTCE t 006 0.12 0.22 0.37 0." 1.3 2.5 5 10 0.03 0.06 0.12 022 0.37 0." 1.3 25 5 10 0.03 0.06 0.12 022023706 1.3 2.5 5 d 2.1 25 3.3 4D 4.6 6.0 7.4 9.2 11.5 I 2.1 2a 33 4.0 4.6 6.0 TA 9.2 11.5 15 2.1 2.6 3.3 4.0 4.6 6.0 74 9.2 11.5 FIG. 1. Cumulative particle size distribution curves ofspores of Trichoderma viride alone and in presence of clay minerals after various periods of incubation. Symbol t = threshold settings on instrument; d = effective spherical diameter in microns.
VOL. 15, 1967 PARTICLE ANALYZER FOR MEASURING GERMINATION 937 o10i01 A FIG. 2. 0 2 4 6 10 12 14 2 4 6 8 10 12 14 16 2 4 6 a l0 12 14 16 EFFECTIVE SPHERICAL DIAMETER {XI Diffierential particle size distribution curves ofspores of Trichoderma viride alone and in presence of clay minerals after various periods of incubation. Curves show distribution of spores only, as clay particles have been subtracted out. it was assumed that spore germination was completed and that oxygen uptake was essentially due to hyphal metabolism. The validity of this assumption was confirmed by periodic microscopic observations. Changes in respiratory rate were also expressed on an arbitrary scale ranging from 0 to 4. Microscopic measurements of germ tube emergence were made with the aid of a hemocytometer counting chamber. Although percentage germination values were obtained, these were also expressed on the arbitrary scale ranging from 0 to 4, to facilitate comparisons between methods. RESULTS AND DIscussioN Typical cumulative frequency and differential plots showing the swelling of conidia of T. viride in the presence and absence of clay minerals are presented in Fig. 1 and 2. Initially, the particle distribution of the spores was narrow, ranging in effective spherical diameter from approximately c 2.1 to 4.8 IA. After 6 hr of incubation, the spores had swelled to an effective spherical diameter ranging from approximately 3.5 to 9.2,u in the presence of the clays and to 11.5,u in their absence. After 8 hr, they had attained a diameter ranging from approximately 4.8 to 11.5,u in the presence of the clays and to greater than 15,s in their absence. Most of the particles smaller than 3.5 j in the cumulative curves were either kaolinite or montmorillonite. In addition to the increase in size of the spore populations with time, the shape of the differential curves also changed (Fig. 2). At zero-time, the curves were narrow and skewed towards sizes smaller than that of the peak populations, whereas, after 6 and 8 hr of incubation, the curves became wider and skewed toward sizes greater than the peak populations. The relative heights of the differential curves were not important in these studies, as no great care was taken in standardizing the size of samples analyzed. The major criteria were the shapes of the curves and the particle sizes at which they peaked. Although the clays reduced spore swelling, this 8 6 F.4 2 0 X TRICHODERMA VIRIDE * TRICHODERMA VIRIDE a KAOLINITE * TRICHODERMA VIRIDE S MONTMORILLONITE 0 2 4 H OURS 6 8 FIG. 3. Effective spherical diameter (d in microns) of the peak spore populations of Trichoderma viride alone and in presence of clay minerals after various periods of incubation.
938 SANTORO, STOTZKY, AND REM APPL. MICROBIOL. reduction was minimal, as the shapes of the differential curves were essentially the same after various periods of incubation. Furthermore, the majority of the spore populations swelled to the same size, regardless of the presence or absence of clay minerals (i.e., 4.0, 6.0, and 7.4,u after 0, 6, and 8 hr of incubation, respectively), and the number of spores that swelled beyond 9.2 and 11.5, after 6 and 8 hr, respectively, in absence of clays was small (approximately 2%). To facilitate presentation of these data, the peak sizes only were plotted against incubation time. This resulted in a simple plot (Fig. 3), which, however, was not sufficiently definitive, as it did not describe the spread in particle sizes nor the direction of skewness of the curves. The swelling of spores of other fungal species, both in the presence and absence of clay minerals, was analyzed in the same manner. Germination was also determined microscopically and respirometrically. A comparison of the results obtained with these three methods showed that swelling of the spores was detected several hours earlier than was germ tube emergence or changes in oxygen uptake (Table 1). As important as this early detection was the ability to measure easily the swelling in the presence of large numbers of clay particles. Observing germ tube elongation in the presence of the clay minerals was difficult or, in several instances, not possible even after repeated attempts. Although there were no difficulties in measuring respiration in the presence of the clays, the determination of time when respiration switched from that of spores to that of mycelium was, in some instances, a subjective judgment. TABLE 1. Relative germination offungal spores in presence and absence of clay minerals as determined by microscopic observation, oxygen uptake, and changes in particle size distribution Incubation time (hr) Fungus Clay3 3 6 8 12 Mb R C M R C M R C M R C Rhizopus stolonifer 0 0 1 2 4 3 4 4 4 4 4 4 4 K 0 1 2 4 3 4 4 4 4 4 4 4 M 0 1 1 4 3 3 4 4 4 4 4 4 Zygorhynchus moelleri 0 0 0 1 2 2 2 2 4 4 3 4 4 K 0 0 1 2 2 2 2 4 4 3 4 4 M 0 0 1 2 2 2 2 4 3 3 4 4 Penicillium jenseni 0 0 0 1 1 1 2 2 2 3 3 3 4 K 0 0 1 1 1 2 2 2 3 3 3 4 M ND 0 1 ND 1 2 2 2 3 3 3 4 Trichoderma viride 0 0 0 1 1 0 3 3 1 4 4 3 4 K 0 0 1 0 0 3 3 1 4 4 3 4 M 0 0 1 0 0 3 3 1 4 4 3 4 Aspergillus terreus 0 0 0-0 2 2 0 3 3 4 4 4 K 0 0 0 2 2 1 3 3 4 4 4 M 0 0 0 2 1 1 3 3 4 4 4 Fusarium nivale 0 0 0 1 2 2 3 3 3 4 4 4 4 K ND 0 0 ND 2 3 ND 3 4 ND 4 4 M ND 0 0 2 2 3 3 3 3 4 4 4 F. oxysporum 0 0 1-1 2 2 2 3 3 3 4 4 K 0 1 0 2 2 2 3 3 3 4 4 M 0 1 1 2 2 2 3 3 3 4 4 Abbreviations: 0 = no clay; K = kaolinite; M = montmorillonite. b M = microscope; R = respirometer; C = Coulter counter; - = not determined; ND = spores not distinguishable from clay particles. Results are expressed as relative germination, on an arbitrary scale ranging from 1 to 4; 0 = no germination.
VOL. 15, 1967 PARTICLE ANALYZER FOR MEASURING GERMINATION 939 TABLE 2. Incubation Swelling of spores of Rhizopus stolonifer in presence of various concentrations of montmorillonite" Montmorillonite concn (%) 0 1 2 4 6 hr 0 0 0 0 0 0 2 1 0 0 0 0 4 2 1 1 0 0 6 4 2 2 1 1 8 4 3 3 2 2 a Results are expressed as relative swelling, on an arbitrary scale from 1 to 4; 0 = no swelling. With spores of some species, montmorillonite, but not kaolinite, retarded swelling during the early periods of incubation. After 12 hr, however, no differences in the extent of swelling were appitrent in any of the treatments. To demonstrate further the efficacy of the electronic sizing technique for measuring spore swelling in the presence of other particles, sporangiospores of R. stolonifer were incubated with various concentrations of montmorillonite. In the absence of clay, swelling of the spores was detected after 2 hr of incubation and was complete after 6 hr (Table 2). When montmorillonite was present, swelling was both detected later and reduced in proportion to the amount of clay present. This inhibition of spore germination by increasing concentrations of montmorillonite has also been observed with numerous fungal species by the respirometric method (unpublished data). A comparison of the microscopic, respirometric, and electronic sizing methods for measuring germination of fungal spores has shown that, by the electronic sizing method, early stages of germination (i.e., spore swelling) can be detected long before germ tube emergence or significant changes in respiratory rates are observed. This technique, which is also rapid, easy, sensitive, and reproducible, permits measuring the germination of spores in the presence of similar-size particles in concentrations considerably in excess of the number of spores present. ACKNOWLEDGMENTS This application of the Coulter counter was suggested several years ago to one of us (G.S.) by R. E. Esposito. This investigation was supported by Public Health Service research grants AI-05810 from the National Institute of Allergy and Infectious Diseases and AP- 00440 from the Division of Air Pollution, Bureau of State Services. LITERATURE CITED 1. MANDELS, G. R., AND R. T. DARBY. 1953. A rapid cell volume assay for fungitoxicity using fungus spores. J. Bacteriol. 65:16-26. 2. SUSSMAN, A. S., AND H. 0. HALVORSON. 1966. Spores, their dormancy and germination. Harper and Row, New York. 3. TRIPATHI, R. K., AND D. GorrLEB. 1965. Physiology of swelling during spore germination. Phytopathology 55:1080.