A fluorescence-staining method for microscopically counting viable microorganisms in soil

C . I . MAYFIELD Depcrrtmetlt of Biology, Ut~iversi ty of Writerloo, Wrrterloo, Otl t . , Cco~crclo

Accepted September 28, 1976

MAYFIELD, C. I. 1977. A fluorescence-staining method for microscopically counting viable microorganisms in soil. Can. J . Microbiol. 23: 75-83.

A fluorescence-staining method using the magnesium salt of I-anilino-8-naphthalene sulfonic acid is described. It allows the numbers and types of viable microorganisms in soil sections to be determined by direct microscopy. About 8% ofthe bacteriain the soils studied were able to divide when a nutrient solution was applied. A higher percentage of the fungal propagules in these soils were viable. The effects of environmentalfactors, chemical compounds, and soil amendmentson the ability of soil microorganisms to undergo division or mycelial extension it1 sitrr can also be determined.

Introduction tions consisting of glucose, both phosphates, and ammo- nium nitrate with the basic solution were also prepared.

Direct microscopic determination of the via- The following series of solutions were applied to thesoil bility of microorg~nisms in soils with minimum disturbance of those organisms in the soil matrix would enable the variation usually observed between counts obtained from dilution-plating experiments and direct counts obtained by microscopy to be more readily explained. ~ h k assumption has often been made that direct microscopic counts include a large proportion of non-viable cells. The fluorescence-staining method using the magnesium salt of l-anilino-8- naphthalene sulfonic acid (9) stains proteins on contact; otherwise it does not fluoresce. This stain has now been adapted to provide estimates of the numbers of viable microorganisms in soil samples by direct microscopy. The staining solu- tion is applied directly to soil samples with no pretreatment or washing required, and so pro- vides an estimate of the viability of microorgan- isms in situ in the soil.

Materials and Methods Three soil types were used in the study. The first was a

sandy-clay loam, originally with a grass cover, containing 2.8% C and 0.21% total N with an average pH of 7.3. The other soils were a loam and a sand loam with 4.2% and 2.2% C, 0.6% and 0.21% total N, and pH values of 7.2 and 6.9 respectively.

The magnesium salt of 1-anilino-8-naphthalene sul- fonic acid (Mg-ANS, Nutritional Biochemicals, Cleve- land, OH) was dissolved in glass-distilled sterile water (3 mg/ml) and used throughout the study as the basic staining solution. This solution can be stored in dark bottles at 4 "C for 7 days.

The staining solution was modified by the addition of glucose (5 gllitre), KH,P04 (1.25 g/litre), K2HP04 (1.25 gllitre), and NH4N03 (2.5 gllitre). Separate solu-

samples; M ~ ~ A N S alone, Mg-ANS plus all nutrients; Mg-ANS plus glucose; Mg-ANS plus both phosphates; Mg-ANS plus ammonium nitrate. These solutions were used immediately after preparation.

Soil aggregates were selected from the soil samples and sectioned with a sterile scalpel so that they presented two parallel surfaces. These aggregate 'slices' were about 0.25 cm in thickness and were placed onto microscope slides and 3 drops of each of the staining solutions were applied to different aggregate 'slices.' In the case of the Mg-ANS plus all nutrients, and the Mg-ANS alone, the aggregate 'slices' were arranged so that the stained faces of the slices were those originally in contact in the aggre- gate. Any size of aggregate 'slice' can be used up to the maximum thickness which can be accommodated on the microscope. Cover slips were placed on the aggregates which were then examined with incident illumination. A Zeiss Photomicroscope I1 with a mercury-arc light source was used for observation and photography with Zeiss UG1, UG5, BG3, and BG12 epiillumination excitation filters (a BG 38 filter was always in the light path) and Zeiss 47, 50, or 53 eyepiece barrier filters. All photo- graphs were taken with a high-speed film (Kodak Tri-X) to minimize exposure times. Randomly chosen fields on the aggregate surface were examined and the coordinates of each field on the vernier scale of the microscope stage were noted. T o ensure positive identification of the parti- cular field and the microorganisms, diagrams of the arrangement of microorganisms and recognizable soil particles in that field were also prepared.

Thirty-five fields at 1000 x magnification were exam- ined, diagrams prepared, and representative fields were photographed for each soil with each stain. Since photo- graphic methods can only examine one focal plane at one time, the organisms in the microscope field were counted visually, refocussing the microscope as required. The slides were then carefully removed from the microscope and incubated at 100% relative humidity a t 20°C in sealed plastic boxes. At intervals, (usually 8,24, and 48 h), each of the previously examined microscope fields was located and the total number of microorganisms again

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76 CAN. J. MICROBIOL. VOL. 23, 1977

TABLE 1. The effect of Mg-ANS on the numbers of microorganisnis determined in soil samples incubated for 24 h by the FITC staining technique with different nutrient solutions

Soil type

Sandy clay Loam Sandy loam

Time of incubation, h

Microorganisms g- ' dry weight soil, x A. Mg-ANS alone 1.2. 1.4, 4 . I d 4 .2d 0.7, O.gh

Distilled water control 1 . 1 . 1.3, 4 . I d 4.2d 0.7, 0.8,

B. Glucose + Mg-ANS 1 . 1 . 2.2, 4 .2d 5.6, 0.7, 1.2, Glucose 1.1. 2.1, 4 . ld 5.7, 0.8, 1.2,

C. Phosphates + Mg-ANS 1.2. 1.4, 4 .2d 4.4, 0.7, 0.8, Phosphates 1 . 1 . 1.3, 4 . I d 4.3f 0.7, O.gh

D. Nitrate + Mg-ANS 1.2. 1 .4, 4 .1d 4.5f 0.7, O.gh Nitrate 1 . 1 . 1 .4, 4 . ld 4.6, 0.7, O.gh

E. All nutrients + Mg-ANS 1 . 1 . 2.2, 4.1d 5.9, 0.8, 1.5t All nutrients 1.1 . 2.3, 4 . l d 6.0, 0.7, 1.5k

NOTE: Similar subscripts within one soil type indicate that there was no significant difference in means a1 the 99% confidence level. Means were not compared between soil types.

counted. From the diagrams and the series of photo- graphs, the percentage of bacteria dividing and the num- ber of fungus and actinomycete spores germinating were also determined. This counting procedure was repeated on five sets of five aggregate 'slices' from each of the three soils. Ten of the aggregate 'slices' were incubated for longer periods and examined after 60- and 72-h incuba- tions. The development of fungal hyphae, sporulation of bacteria, and large microcolony development were ob- served and photographed.

The morphological types of bacteria in the loam soil and any subsequent changes during incubation were also noted and any unusual morphological types of micro- organisms and colonies of microorganisms developing in the aggregates were photographed.

To compare total viable counts obtained with the Mg-ANS method and with normal dilution-plating pro- cedures, 10 g of the loam soil was placed in 90 ml of sterile 0.85% NaCl and serial decimal dilutions prepared. Ten plates were prepared at three appropriate dilutions for fungi, actinomycetes, and bacteria on Czapek-Dox agar (lo), starchxasein agar with actidione (a), and a soil extract medium (6). Bacteria were also counted on a medium containing glucose, phosphates, and nitrate-the same medium used to modify the basic Mg-ANS staining solution. After incubation at 25 "C for 3, 8, and 14 days, the bacteria, fungi, and actinomycetes on the plates were counted.

To determine whether the Mg-ANS solutions had any significant effect on the growth and multiplication of soil microorganisms, they were applied as described above to soil aggregates, and incubated for the same periods. Another set of aggregates was also prepared and the same solutions applied, omitting the Mg-ANS stain. In each case, subsamples of the soil were removed and lo-' dilutions in sterile distilled water were prepared. Soil smears were then made and stained with fluorescein

isothiocyanate (FITC) and the numbers of microorgan- ism in the soil samples determined (1).

Results Regarding the effects of Mg-ANS stain on the

microorganisms in soil, no significant difference existed between the means of counts in soil with or without the presence of Mg-ANS in the solu- tions applied (Table 1). However, there were sig- nificant differences between the means of the counts of the microorganisms using the FITC method and the different nutrient solutions re- sulting from the different responses of the organ- isms to the various components of the solutions. The application of distilled water led to a small increase in numbers, due possibly to the distur- bance of the soil during preparation of the aggre- gates (1 1).

Many of the bacteria or 'yeast-like' cells in the soil samples occurred in microcolonies when the soil was examined immediately after staining (Fig. 1). The occurrence of these microcolonies in the soil matrix was assessed in the loam soil and, in 105 microscope fields, 28% of the bac- terial cells were present as microcolonies (di- plococci and pairs of cells were not counted as microcolonies). Yeast-like cells were numerous in the soils and many of these cells were in microcolonies (Fig. 2). The larger fungal spores tended to fluoresce less brightly than the yeast- like cells and could also be distinguished during

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MAYFIELD

FIGS. 1 4 . The bar represents 5 pm and the photographs were made within 1 h of staining with Mg-ANS solution plus glucose, phosphates, and nitrate. Flg. 1. Typical bacterial microcolony in the loam soil. Fig. 2. A 'yeast-like' microcolony in the loam soil. Fig. 3. Actinoniycete mycelium in the sandy loam soil. Fig. 4. Fungus mycelium in the loam sod.

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78 CAN. 1. MICROBIOL. VOL. 23, 1977

the incubation studies because of the germina- tion of many of the spores (Table 2).

The dominant morphological types of bac- terial cells were rods and coccoid rods with many of the rods occurring in pairs. Most of the bacterial cells appeared larger than those ob- served with the FITC staining technique al- though this was not confirmed with detailed size measurements. Bacterial spores could not be distinguished from coccoid cells or actinomycete spores when examined before incubation. Ac- tinomycete mycelia (assessed simply by the dia- meter of the hyphae) were present in small quantities. A large fraction of this actinomycete mycelium could be associated with spores, and the average length of mycelium observed was about 1 1 pm. Branched actinomycete mycelium was not observed in this soil to any significant extent and Fig. 3 shows a piece of actinomycete mycelium about 20 pm in length.

Fungal hyphae were not numerous in any of the soil samples, but as the fungal spores germin- ated during incubation, the emerging mycelium became heavily stained as it ramified throughout the soil (Fig. 4).

The staining solution stained effectively during the entire 48-h incubation period, detecting dividing bacterial cells and germinating spores of actinomycetes and fungi. The most effective solu- tion was that containing all nutrients, although the solution with glucose also provided good results. The other three staining solutions did not lead to the same high percentage viability results, although even Mg-ANS alone did lead to a sig- nificant increase in the number of cells in two soils (Table 1). The results of the studies with the staining solution containing all nutrients are summarized in Table 2. Further analysis of the counts obtained and the viabilitv of different morphological types of bacteria in the loam soil is presented in Table 3. In all cases, bacterial cells were counted as viable if they underwent at least one division, and fungal spores were viable if a germ tube emerged from the spore during the incubation period. Viable bacteria were assigned to a morphological group based on morphology of the cells produced, not on the original mor- phology. This was necessary because, in some cases, coccoid rods produced rod-shaped bac- teria when they divided. The dilution plate counts are compared to the estimated viable count obtained with the Mg-ANS method in Table 4.

TABLE 2. Percentage of viable cells in the three soils based upon the response of microorganisms to incu- bation with the Mg-ANS solution containing glucose,

phosphates, and nitrate

Percentage of viable cells in soil aggregates

Fungal 'Yeast-like' Soil spores Bacteria cells

Sandy clay loam 28, 8b 70, Loam 29. 8b 84, Sandy loam 34d gb 81,

NOTE: Similar subscripts indicate no significant difference between means a t the 99% confidence level.

A representative series of photographs of the same field is presented in Fig. 5 demonstrating the development of a microcolony in the soil during incubation with the stain.

In Tables 2 and 3 the number of organisms in the soil was calculated using a conversion factor based on the water content of the soil aggregates before the staining solution was applied, the area of the microscope field, and a measurement of the average depth or thickness of that field. From these values, the volume sampled in each field was calculated and combined with the density of the soil and the water content to calculate the number of organisms in the soil. The total counts obtained, although subject to error caused by the methods and calculations used, were essentially similar to those obtained with the FITC staining method (Table 1). There was, however, greater variation between the counts of different micro- scope fields in the Mg-ANS method, because of the obvious heterogeneous distribution of cells within the soil matrix revealed with that method. This variation between fields became larger dur- ing the incubation period used to determine the proportion of viable cells, and in some cases, certain microcolonies, because of their extensive development during this period, obscured other organisms in the same field.

After the 60- and 72-h incubations, the soil was densely populated with bacteria and many of them had formed recognizable morphological structures. Chains or filaments of bacteria (Fig. 6), large colonies (Fig. 7), bacteria with some sporing cells (Fig. 8), and very extensive mycelial development of fungi (Fig. 9) were observed. In isolated cases the fungi produced structures re- sembling sporangia and spores.

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MAYFIELD

TABLE 3. Percentage viability of various morphological types of bacteria in the loam soil as determined with the Mg-ANS staining

solution containing glucose, phosphates, and nitrate

Total number z of total Morphology g-' dry wt. bacterial z of viable

of cells by direct count nos. cells

R O ~ S 1.48 x 109 39 Sporing rods 1.90 x lo7 0.5 Long rods (> 5 wm) 1.90 x lo7 0 .5 Coccoid rods 1.44 x lo9 3 8 Diplococci 7.60 x lo7 2 Cocci 1.52 x lo8 4 Actinomycete sporesn (7.60 x lo6) (0.2) Unidentified 6.08 x lo8 16 Total bacteria 3.80 x lo9 100

nActinomycete spores could only be distinguished from cocci after germination. They were thus originally counted as part of the cocci group and so are excluded from calculations o f percentage o f viable cells.

TABLE 4. A comparison of the viable counts of microorganisms from the loam soil with dilut~on-plating methods and the Mg-ANS method

Ratio of counts, No. of viable % viabilitya Mg-ANS method:

microorganisms in Mg-ANS dilution-plate Microorganisms Method g-I dry wt. soil method method

Bacteria 1. Soil extract medium 1.18 x I0 2 .6 2. Glucose, phosphates, 1.04 x 10 29

and nitrate medium 3. Mg-ANSb 3.03 x 10 8

Fungi 1. Czapek-Dx medium (a) Yeasts 1.10 x 10 39 (b) Mycelial fungi 1.20 x 10 10

2. Mg-ANSb (a) Yeasts 4.37 x 10 84 (b) Mycelial fungi 1.19 x 10 29

Actinomycetes 1. Starch-casein medium 2.20 x 10 3.5 2. ME-ANSb 7 .6 x 10 100' -

nFrom Table 2. bMg-ANS statning solution plus all nutrients. Viable counts der~ved from the % viability o f each group and the total direct

Count. 'Act~nomycete spores were ident~fied solely by germtnat~on dur~ng incubatton

Discussion Most counting methods for microorganisms in

soil rely on either direct observation or culture methods. The counts by direct microscopic methods are often in the range of 108 or lo9 per gram of soil while dilution-plate methods usually yield results of 1 to 10% of these values (12). Most stains d o not differentiate between living and dead cells, although this property was claimed for acridine orange used as a fluorescent stain (13). Most fluorescent stains, such as FITC (1) and fluorescent brighteners (3) are themselves fluorescent when exposed to ultraviolet or blue

light. The Mg-ANS stain is not fluorescent until it comes into contact with hydrophobic groups, typically those of proteins (5, 14). This property means that normal fixation and washing pro- cedures, used with other fluorescent stains, are not required with Mg-ANS. Soil can be stained intact, and no disruptive procedures are necessary which would disturb the growth pattern or form of the soil microorganisms. The stain has n o dis- cernable toxic effects in short-term incubations with soil microorganisms, although there may be some selective mechanisms occurring for cer- tain types of microorganisms, but which were

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FIGS. 6-8. The bar represents 5 Kin. Fig. 6. Chains of bacteria in the loam soil after 72 h of incuba- tion. Fig. 7. A large niicrocolony of bacteria which developed in the loani soil during 72 h of incuba- tion. Fig. 8. Sporulating bacterial cells in the loalii soil after 96 h of incubation.

FIG. 9. The bar represents 50pni. Extensive developnient of fungus niyceli~~m at the periphery of a loani soil aggregate after 96 h of incubation.

not apparent with the techniques used in the present study. This use of Mg-ANS as a protein stain, coupled with ultraviolet epiillumination, was able to detect the viability of soil microor- ganisms it? situ. The values obtained for viable cells must be interpreted with some caution. Only one nutrient-rich medium was used, and many of the soil microorganisms may have dif-

ferent nutritional requirements to those provided in the glucose-phosphates-nitrate solution.

I11 a comparative study on methods to esti- mate numbers and activity of bacteria in soil (7), an agar-slide culture method designed to detect viable cells in the soil indicated that many of the cells in the soil were in the spore state and that, after incubation for 8 to 12 h, rod-shaped bac-

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82 C A N . J . MICRO

teria appeared in the soil. Coccoid cells were not observed in the agar-slide cultures although they were abundant' in FITC-stained preparations of the same soil (7). Coccoid rods and cocci were observed in the present study since a protein stain was used, but they exhibited a lower per- centage viability than the rods.

Disadvantages of the technique are that the soil is still disrupted to some extent to expose the organisms, thus releasing some nutrients, and that the application of the stain and the examina- tion methods may alter the responses of the microorganisms.

These objections, however, apply to all methods so far developed which can examine or extract viable microorganisms from soil, and it appears that the severity of the disruptions and interferences with growth are less with the Mg- ANS method than with the methods currently in use. Other theoretical sources of error in the technique relate to the fact that the observed percentage viability of microorganisms depends upon the provision of the required nutritional and environmental conditions, even on the micro- site scale, so that attempts to arrive at a total 'viable' count of microorganisms in soil by any method, including the present one, must be assumed to be only an approximation. It may be that combinations of methods are preferable to reliance on one particular method, whether this be a dilution-plating experiment or the method described here.

The bacteria and other microorganisms in the soil aggregates may also have requirements for different environmental conditions. It has been demonstrated that aggregates can maintain anaerobic conditions at their centres (4) and any obligate anaerobes may have been damaged by exposure to air during the preparation of the 'slices.' The percentage of viability results are therefore an average obtained under many dif- ferent environmental condition, some of them presumably affecting microorganisms on the microenvironmental level. The presence of the glass cover slip may also introduce different conditions of water content, surface structure, aeration, etc., while sectioning the aggregates may disrupt microsite habitats and release nu- trients (1 1). Autotrophic microorganisms would not have responded to the nutrient solution used in the present study, but the staining technique could possibly be applied to this group by using selective nutrient solutions with the stain. Pro-

IBIOL. VOL. 23, 1977

tein debris and dormant cells in the soil will also be stained and could be mistaken for non-viable cells and certain organisms (cysts, cells with slime layers or capsules and very small cells) may not have been detected at all.

The major sources of technical error in the procedure are the estimates of the thickness of the soil samples examined, the omission of any organisms which occur on the reverse side of opaque particles (although this could be com- pensated for by statistical treatments), any dele- terious effects of ultraviolet illumination, and problems of microscopic examination of micro- organisms in the soil 'slices' used.

In view of all of these factors, it was surprising that the estimates of viable organisms in soil were as high as they were within the 24-h period. This was especially true for the fungal spores and the 'yeast-like' cells, but was also the case for the 8% average figure for the viability of bacterial cells. A number of explanations for these high percentages are possible. It may have been due to the detection and observation of only the larger cells in soil, with many of the cells being smaller and therefore less easily observed. It has been demonstrated (2) that fluorochrome dyes such as FITC and acridine orange do not differ- entiate the dormant resident soil bacteria and that they also can fluoresce with various inani- mate proteinaceous soil materials. Differential concentration of the staining solution may have occurred in the larger cells, or in the viable cells, thus apparently increasing the percentage via- bility. Many of the cells in soil may have been capable of one or two divisions (or spore germina- tion) but did not sustain this growth on normal media because of nutritional, environmental, or competitive factors. This last reason may pro- vide: at least, part of the explanation for the marked difference in counts between the dilu- tion-plate method and the Mg-ANS method.

In summary, therefore, the main advantages of this method are that it allows estimates of the number of viable microorganisms actually in situ in soil samples, thereby removing some of the disadvantages of normal dilution plating experiments, where the organisms are exposed to severe and rapid changes in environment. It is a very simple method, and the disruption of habi- tats and growth patterns of microorganisms can be minimized. It is also extremely flexible in that many different nutrient solutions can be used to activate division or mycelial extension of soil

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microorganisms. It therefore combines many of the advantages of dilution-plating techniques with those of direct observation methods. The viability of organisms is also assessed by their ability to undergo one division or to exhibit sig- nificant mycelial growth. This is a more sensitive assay than the ability to form colonies on normal dilution plates. Because of the disadvantages of the method, it may prove most useful in studies of the larger microorganisms in soil, especially in studies on the colonization of substrates.

Acknowledgements

This work was supported by a grant (No. A-6327) from the National Research Council of Canada.

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This article has been cited by:

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2. D. R. Polonenko, C. I. Mayfield, E. B. Dumbroff. 1986. Microbial responses to salt-induced osmotic stress. Plant and Soil 92:3,417-425. [CrossRef]

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9. J. W. L. Vuurde, A. Lange. 1978. The rhizosphere microflora of wheat grown under controlled conditions II. Influence of thestage of growth of the plant, soil fertility and leaf treatment with urea on the rhizosphere soil microflora. Plant and Soil 50:1-3,461-472. [CrossRef]

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