Extraction of bacteria from soil: Efficiency of shaking or ultrasonication as indicated by direct counts and autoradiography

Soil Bid. Eiochem. Vol. 16, No. 5, pp. 475-481, 1984

0038-0717/84$3.00+ 0.00 Copyright 0 1984Pergamon Press Ltd

Printed in Great Britain. All rights reserved

EXTRACTION OF BACTERIA FROM SOIL: EFFICIENCY OF SHAKING OR ULTRASONICATION AS INDICATED BY DIRECT COUNTS AND AUTORADIOGRAPHY ANGELA J. RAMSAY Soil Bureau, Department of Scientific and Industrial Rese,rch, Lower Hutt, New Zealand (Accepted 24 November 1983)

Summary-Blending, shaking with a mechanical wrist-action shaker or ultrasonication were compared for removing bacteria from a sandy soil and a silt loam soil. Bacteria were counted microscopically after staining with a&dine orange. There was no significant difference in the numbers of bacteria released from the sandy soil by the different treatments but ultrasonication extracted more bacteria from the silt loam. Highest recoveries of bacteria were obtained with a combination of Tris buffer as extractant, and 25% Ringer’s solution as diluent. Ultrasonication of an irradiated sandy soil containing added BaciNus cereus removed 97% of the bacteria from the sand grains, but lysed some cells. Members of the indigenous soil population were more firmly held by the soil than added bacteria. Optimum conditions for recovering soil bacteria by ultrasonication were either 10.5 pm for 30s or 5pm for 2min. With the natural population, the numbers of bacteria and the proportion of metabolizing bacteria increased a little after ultrasonication as shown by labelling cells with 3H-glucose and detecting uptake by autoradiography. However, three isolates of bacteria tested showed varying amounts of injury after ultrasonication, with 5 pm for 2 min causing more damage than 10.5 pm for 30 s. Ultrasonication was a more efficient means of extracting the natural bacterial population of soil than shaking but the time and amplitude must be adjusted to avoid injury to cultured bacteria1 cells.

INTRODUCI’ION Many bacteria in soil are surrounded by soil debris and are not readily distinguished microscopically, or extracted from soil (Balkwill et al., 1977). Early work

on the extraction of bacteria from soil (Stevenson, 1958) estimated those organisms which could be cultured on agar media. However, cultured soil bacteria comprise only about 0.01-o. 1% of direct microscopic counts (Faegri et al., 1977; Zvyagintsev and Zeitseva, 1979). Estimates of the whole bacterial population are needed to relate to soil microbial activity and to measure soil bacterial biomass (Jenkinson and Ladd, 1981). The validity of the direct microscopic method for enumerating soil bacteria depends on the suitability of the stain (Ramsay and Bawden, 1983) and on the efficiency of the extraction method. A number of studies have compared shaking methods to extract bacteria from soil or aquatic sediments. Homogenization (Dale, 1974), ultrasonication (Stevenson, 1958; Zvyagintsev and Galkina, 1967; Anderson and Slinger, 1975; Weise and Rheinheimer, 1979) or cycles of blending and centrifugation combined with ultrasonication (Balkwill et al., 1977) have all been recommended for recovering bacterial from soil. While ultrasonication has often been the method of choice, direct comparisons between results in the literature are often difficult because different, sometimes unspecified, amounts of energy are used. The dislodged bacteria have in addition been estimated using different techniques such as plate counts (Stevenson, 1958; Zvyagintsev and Galkina, 1967) or direct microscopic observation (Anderson and Slinger, 1975; Weise and 475

Rheinheimer, 1979). While some bacteria firmly attached to soil particles may be released by ultrasonic treatment, excessive treatment may damage cells (Zvyagintsev and Galkina, 1967). The advantage of increased extraction efficiencies offered by ultrasonic treatment is, nevertheless, thought to outweigh the disadvantage of any loss of cells (Jenkinson et al., 1976; Weise and Rheinheimer, 1979). My purpose was to find a suitable method to extract bacteria from soil efficiently and reproducibly so that there was little change in their metabolic acitivity. Uptake by soil bacteria of 3H-glucose was used to assess their metabolic activity, and they were enumerated by direct counts of acridine orangestained bacteria collected on polycarbonate filters (Ramsay and Bawden, 1983).

METHODS Soils

Waitarere sand (Typic Udipsamment) was collected 500 m inland from Hokio Beach (Manawatu) from a south facing dune. Kaitoke silt loam (Typic Dystrochrept) from near Wellington, and a silty clay loam (Typic Haplaquoll) from Castlepoint, Southern Wairarapa were sampled beneath pasture. Chemical data are given in Table 1. After removing earthworms, the soils were broken up by hand and stored, field moist, at 5°C for 2-10 weeks (Kaitoke samples) and up to 8 days (Castlepoint samples). The Waitarere sand was analysed within 24 h.

ANGELA J. RAMSAY

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Table 1. Chemical properties of soils depih Organic C Total N Soil (cm) PH (%) (%) Waitarere sand’ 1.25 &5 6.9 0.09 2-l Kaitoke silt loam2 5.1 6.2 0.43 Silty clay loam’ &2 6.1 6.4 0.55 (Castlepoint) ‘Approximate values based on analyses of A and B horizons (Cowie, 1968). *Ramsay and Bawden (1983). ‘Values given are for samples from &5cm taken over one year (Ross et al., 1984). Extraction of bacteria from soil

Soil (1 g) was suspended in 9 ml of Tris buffer, pH 7.5, 50 mM (Niepold et al., 1979). The suspension was either shaken for 20 min on a wrist-action shaker (Griffin Flask Shaker, Griffin and George Ltd, England) at half the maximum speed, comminuted in a blender (Measuring and Scientific Equipment Ltd, London) at half the maximum speed for 1 or 2 min or dispersed by ultrasonic sound (MSE 150 W Ultrasonic Disintegrator; probe dia, 19 mm). A range of amplitudes and lengths of time of ultrasonic treatment was compared. For ultrasonication, samples were placed in 25 ml beakers (32 mm dia) in an ice bath. In one experiment, three extractants were compared: Tris buffer, 25% (v/v) strength Ringer’s solution (g l-‘, NaCl, 2.25; KCl, 0.105; CaCl,, 0.12; NaHCO,, 0.05) (Dickinson et al., 1975) and basal salts (gl-‘, KzHP04, 5.0; MgSO,, 2.5; NaCl, 2.5; Fe2(S04)3, 0.05; MnSO,, 0.05) (Pochon and Tardieux, 1962). Recovery of added bacteria from soil

A 48 h culture of Bacillus cereus grown at 28°C in nutrient broth (Difco) was washed twice by centrifugation. The cells were resuspended in 25% strength Ringer’s solution and 0.4ml (0.61 x 10’ bacteria ml-‘) was added to 1 g of the sandy soil in duplicate universal bottles. Quarter-strength Ringer’s solution only was added to a further bottle as a control. The soil had been sterilized by gamma-irradiation of 2.5 Mrad from a @‘Co source 2 months before use. The inoculated bottles were left for 2 h at 18°C after which the bacteria were harvested. The treatments were (a) wrist-action shaker for 20 min at half the maximum speed; (b) ultrasonication of the inoculated bottles and the control for 30 s and a further 30 s at 1Opm amplitude. A sample was withdrawn after each shaking step and the bacteria were counted by direct microscopy. Enumeration of bacteria

Ten-fold dilutions of the soil suspension were prepared in 25% strength Ringer’s solution. In one experiment, the diluents Basal Salts, deionized water and phosphate buffer pH 8.0, 0.1 M were compared with 25% strength Ringer’s solution. The numbers of bacteria were estimated by the multiple tube dilutionmost probable number technique (MPN) or by direct microscopy. MPN counts

For the MPN method, the medium was peptone, yeast extract broth (Goodfellow et al., 1968). The

sandy soil samples were held at 15°C for 13 days and the silt loam samples at 28°C for 7 days. Direct counts

Bacteria were counted directly by fluorescence microscopy using acridine orange (Ramsay and Bawden, 1983). Bacteria were not fixed with formalin as such preparations of fixed bacteria fluoresced weakly. Instead soil suspensions were filtered and stored dry for up to 7 days until stained and counted. At least 350 bacteria or the bacteria within the squares of the eyepiece graticule of 40 fields were counted on duplicate filters for each treatment. All results were related to oven (105’C) dry weight of soil. Assessment of the proportion of metabolizing bacteria

The effect of ultrasonication on the metabolic activity of bacteria was determined by the autoradiographic method of Ramsay (1974) using D-[6-‘H]glucose (specific activity, 333 GBq mmol-‘; 1 GBq = 27.027 mCi). Experiments with [3H]glucose (specific activity, 473.6 GBq mmol-‘) and three bacterial cultures (Bacillus cereus, Nocardia asteroides and a fluorescent pseudomonad) indicated that 740 kBq ml-’ of ‘H-glucose incubated with the bacteria for 2 h gave maximum labelling (> 95% for N. asteroides and B. cereus and >80% for the pseudomonad) after exposing the cells to the photographic emulsion for 14 days. ‘H-acetate was also tested but with its lower specific activity (11.1 GBq mmol-‘), much higher concentrations (2.963.70 MBq ml-‘) were needed to give maximum labelling compared with [3H]glucose. As such additions of [3H]acetate would increase the natural pool of this substrate substantially, it was not used in further work to determine the proportion of metabolically-active bacteria. Uptake of the radioactive substrate by the bacteria was ended by the addition of 2% (v/v) glutaraldehyde in phosphate buffer giving a final concentration of fixative of 0.22%. This fixative was preferable to formalin or Lugol’s iodine. With formalin (final concentration, 2.6% v/v) there was some leakage of material from the Gram-negative radioactive fluorescent pseudomonad and, to a lesser extent, N. usteroides. This was evident from the silver grains scattered around the cells. With dilute Lugol’s iodine, when the fluorescent pseudomonad was left overnight after fixation, only 60% of the cells produced autoradiograms compared with 94% in slides prepared 3 h after fixation. Labelling soil microorganisms TO

test for damage to cells, suboptimal

conditions

Extraction

of soil bacteria

Table 2. Comparison of methods for extracting bacteria from a coastal sandy soil Direct counts of bacteria

Method of extraction Untreated Shaker’ Blender’ Ultrasonication2 30 r Ultrasonication 60 s

In suspension Meanj(lOW C.V. (%) ND 2.97 5.82 11.41 10.54

0’ 4.0 27.3 9.0

On sand grains (106cm-s) Mean4 C.V. (%) 3.43 1.49 0.97 0.79 0.88

38.5 6.6 0.7 40.0 14.5

Removal from sand grains (%) 0 57 72 77 74

‘One sample only. *Ultrasonication at 12 pm amplitude. ‘Differences not significant. 4All counts significantly (P = 0.05) less than untreated but no difference between treatments. ‘MPN (x lO’g_‘) shaker: 10.0; blender: 7.4. ND = Not determined.

for labelling were used so that any difference between treatments would be accentuated. A soil suspension was prepared by brief hand shaking. The supernatant was decanted after large particles had settled and analyzed either untreated or after ultrasonication. Soil suspension (0.1 ml) was added to 0.1 ml solution (final of [3HIglucose concentration, 370 kBq ml-‘). Dead control bottles, to which fixative was added before the radioactive substrate, were included in each experiment. Incubation was for 1 h. For the silty clay loam soil from Castlepoint the whole soil was analyzed. “Untreated” soil was labelled by adding 10 mg soil to 0.2 ml [3H]glucose solution. The optimal 740 kBq mll’ and 2 h incubation were used.

fore counted using transmitted light while unlabelled bacteria were totalled using epifluorescence microscopy. The proportion of bacteria which had taken up the radioactive substrate was then calculated. Suhicient bacteria were scored as labelled or unlabelled to give a 5% error with 95% confidence limits (Snedecor and Cochran, 1967).

Preparation of autoradiograms

Extraction of soil bacteria

The bacteria were washed free of excess radioactive substrate by centrifugation, and autoradiograms were prepared of bacteria1 smears (Ramsay, 1974). Ilford L4 emulsion was exposed to the radiation for the suboptimum 7 days or for one soil 14 days before the slides were processed (Caro and van Tubergen, 1962). Duplicate slides were prepared from each bottle.

Bacteria were extracted from a sandy soil by blending, shaking or by dispersion by an ultrasonic probe. The direct counts of bacteria in suspension and attached to individual sand grains were recorded (Table 2). After blending or shaking, the MPN estimates of bacteria were only a small fraction of the direct counts. Ultrasonication apparently released more bacteria into suspension than either blending or shaking although differences were not significant. The counts of bacteria on the sand grains indicated the efficiency of the extraction method. None of the methods removed all the bacteria. However, over 70% were released by both ultrasonication and blending. After shaking, 57% of the bacteria were removed from the sand grains. Although the blending gave slightly higher numbers of bacteria than shaking, the latter was chosen for comparison with the ultrasonic probe as it is more convenient for processing large numbers of samples. When a suspension of a silt loam soil was prepared by shaking, 0.43 x 10” (SE, 0.055 x 10”) bacteria g -’ were counted directly while the MPN estimate was only 1.3 x 1O’gg’ (SE, 0.26 x 10’). After further ultrasonic treatment for 1 min at 10 pm, significantly (P = 0.01) more bacteria were counted directly (2.82 x lO’Og_‘; SE, 0.33 x 10”). No MPN count was made at this time. The numbers of bacteria released by ultrasonication were influenced by both the extractant and diluent (Table 3). Of the extractants tested, the two treatments using Tris buffer gave significantly higher (P = 0.05) numbers of bacteria. Quarter-strength Ringer’s solution, while not as efficient an extractant,

Staining the autoradiograms

Developed slides were stained with a&dine orange (Meyer-Reil, 1978) but using a low concentration of strain (0.003x, w/v) in 0.1 M citrate buffer pH 6.7. Microscopic examination

Each field was examined with incident U.V. radiation and also transmitted visible light. Many of the bacteria which had taken up [3H]glucose had so many silver grains over them that their fluorescence was not visible. The numbers of labelled bacteria were thereTable 3. Effect of different extractants and diluents on the numbers of bacteria released’ from a silt loam

Extractant Tris buffer Tris buffer 25% Ringers Basal salts

Diluent 25% Ringers Basal salts 25% Ringers Basal salts

Direct counts of Mean WOg-‘) C. 2.82b 2.14b 1.13c 0.88c

bacteria v. (%) 16.5 7.9 15.0 25.7

‘Samples were shaken for 20 min on wrist-action shaker and then sonic&d at 10nm for 1 min. Differences significant (P = 0.05) between suffixes of different letter names.

Statistics

Differences between treatments were analyzed using Scheffe’s test (Snedecor and Cochran, 1967). Where necessary, log transformation of data was used to equalize the variances. RESULTS

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478

Table 4. Efficiencv of shakine or ultrasonication

for removine bacteria from irradiated sand erains

Bacteria attached to sand grains (104ctn2) B. cerew

Treatment

Mean ~___~~ 10.6~ 3.0d 0.31d

Untreated Shaker Ultrasonication’

C.V. (%) 17.9 51.8 45.6

Removal (%)

Natural population Mean C.V. (%) 28.4 02 22.8 25.1 10.5 47.1

Natural

B.cereus population 0 72 97

0 19.5 63

’ 1 min at 10.5 pm amplitude. ‘One replicate only. Differences significant (P = 0.05) between suffixes of different letter names. Table 5. Effect of different amounts of ultrasonication

on direct counts of bacteria

Numbers of bacteria 10’Og-’

IO*ml-’ Soil suspension’ B. cereus

Soil’ 5 Time (s) 0 30 60 120

Mean

7.5 C.V. (%)

Mean

ND 1.70 1.99 2.94f

C.V. (%) ND

16.0 6.7 3.0

2.07 1.84

8.1 9.0 ND

Amplitude (pm) 10.5 Mean C.V. (%) ND 2.74f 12.4 ND ND

10.5 Mean 0.35 0.33 0.27 0.36

10.5 C.V. (%) 6.0 6.3 26.2 5.8

Mean 0.42~ 0.39c 0.20d 0.041e

C.V. (%) 8.7 13.3 10.3 14.1

‘Wholesoil was treated. ‘A soil suspension was decanted and this was extracted. Bacihs eereus: differences significant (P = 0.01) between suffixes of different letter names. Soil suspension: NSD. Soil: two counts designated “f” together significantly (P = 0.01) greater than remainder of counts. ND = not determined.

was a good diluent and in a further experiment on diluents, 25% Ringer’s solution gave higher numbers of bacteria than phosphate buffer or water. The brightness of the fluorescence was also recorded. All the bacteria fluoresced green but those diluted with phosphate buffer fluoresced less brightly than those in 25% Ringer’s solution. Recovery of added bacteria from soil

The gamma-irradiated soil bacteria were still clearly visible but only about one-fifth as many were recorded as for an earlier sample of untreated soil (Fig. 1, Table 2). The natural population was distinguishable from B. cereus by the much smaller size of the cells. The original suspension of B. cereus contained mainly chains of cells but many of these were broken into single cells after the extraction

Fig. 1. Recovery of Bucillus anus and the natural bacterial population from an irradiated sandy soil after incubation for 2 h. WI-, wrist-shaker; US 30, ultrasonication for 30 s at lOpm, US 60, ultrasonication for 60 s at 10 pm. Differences significant between suffixes of different letter names (a,b etc., P = 0.05; A, B etc., P = 0.01).

treatment. After shaking, significantly (P = 0.01) more B. cereus were counted than in the original suspension (Fig. 1). Numbers of B. cereus declined, but not significantly after ultrasonication for 60 s. In contrast, more of the natural population was released by ultrasonication with significantly (P = 0.05) more bacteria being counted after the prolonged ultrasonication than after shaking. When individual sand grains were examined, it was found that almost all B. cereus had been removed by ultrasonication but that the natural population was more firmly attached with only 63% being removed (Table 4). Variations in ultrasonic treatment and assessment of the metabolic activity of bacteria

Significantly (P = 0.01) higher numbers of bacteria were extracted from soil after 2min of ultrasonication at 5 pm amplitude and after 30 s at 10.5 pm compared with the other treatments (Table 5). There was no significant difference between the counts of bacteria released from the prepared soil suspension treated for varying periods of ultrasonication but significantly (P = 0.1) fewer B. cereus were counted after 1 and 2min of ultrasonication. The suitability of ultrasonication at 10.5 pm or 5 pm for extracting soil bacteria was studied further with two soils and washed cells of three bacterial species. More bacteria were counted in the soil suspensions after ultrasonication (Fig. 2). Numbers of Nocardia asteroides were also higher but Bacillus cereus and the fluorescent pseudomonad were reduced in numbers after ultrasonication. To assess possible damage to cells the uptake of [3H]glucose by the bacteria was determined by autoradiography. The bacteria under the photographic emulsion were successfully stained with acridine

Extraction of soil bacteria 6-

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Fig. 2. Effect of two amplitudes of uhrasonication on direct counts and metabolic activity of bacteria. C, control, no ultrasonication; 10.5pm, uhrasonication at 10.5pm amplitude for 30s; 5pm, ultrasonication at 5 pm for 2 min. Silt loam soil suspension; decanted before treatment. Silty clay loam soil labelled with optimum amount of [)H]glucose, remainder with suboptimum (see Methods). Differences significant within each series indicated as in Fig. 1. orange, but all cells fluoresced red rather than the green obtained when bacteria were stained on membrane filters for direct counts. There was no significant change in the proportion of the cultured bacteria metabolizing [‘HIglucose after both ultrasonication treatments except for the proportion of B. cereus which declined significantly (P = 0.05) from 88 to 34% after ultrasonication at 5 pm for 2 min. Apart from the soil sample from Castlepoint, all the other bacteria were labelled for suboptimal times. This is reflected in the proportions of labelled bacteria in the controls which were considerably less than 100%. The bacteria in the soil suspension decanted before ultrasonication had a low proportion of metabolizing cells both before and after ultrasonication. In the autoradiograms of soil from Castlepoint a significantly (P = 0.01) higher proportion of bacteria (71%) was labelled after ultrasonic treatment than in the untreated soil (58%). DISCUSSION

The direct counts of bacteria showed that ultrasonication was more efficient than shaking for removing bacteria from the silt loam soil and comparable to shaking for extracting the sandy soil (Table 2). These results are in agreement with those of Zvyagintsev and Galkina (1967), Anderson and Slinger (1975) and Weise and Rheinheimer (1979), although Dale (1974) preferred high speed blending to ultrasonication for extracting bacteria from intertidal sand. A greater proportion of the population in the silt loam was released by ultrasonication than by shaking

compared with the sandy soil. It is likely that these additional bacteria had been extracted from small particles not affected by the shaking or blending treatments. There was little difference in the proportional removal of bacteria from the large sand grains by blending or ultrasonication (Table 2). There was a wide disparity between direct counts and most probable numbers of bacteria in the soils. The difference varied between 2 and 3 orders of magnitude, providing further evidence to that of Faegri et al. (1977) and Zvyagintsev and Zeitseva (1979) that cultural techniques are unsuitable to determine total bacterial numbers in soil. Attempts to extract bacteria from soil have involved manipulating factors such as pH, surface charge, use of surfactants and physical force (Niepold et al., 1979; Scheraga et al., 1979). Tris buffer, which gave the best extraction of hydrogen bacteria added to soil in the study of Niepold et al. (1979), proved to be a good extractant of the natural bacterial populations of soils. Bacillus cereus cells added to the sandy soil were readily extracted. The lower counts recorded for the original culture before shaking probably reflect the difficulty of accurately counting the individual cells of chains. Although the ultrasonic treatment removed almost all of the cells from the sand grains, fewer B. cereus were present in suspension than after shaking, suggesting some cells had been lysed by the ultrasonic treatment. The natural population remaining after irradiation was more firmly attached than the added cells, as a smaller proportion of bacteria on the sand grains were released by ultrasonic treatment. However, the higher counts after ultrasonication com-

ANGELAJ.

480

pared with shaking indicated that the extracted cells were more resistant to breakdown by ultrasonication than the cultured bacteria, as also noted by Jenkinson et al. (1976). Comparison of a range of amplitudes of ultrasonic energy showed that 10.5 pm for 30 s and 5 pm for 2 min extracted the most bacteria from soil. Longer periods of ultrasonication are not recommended as they did not extract more bacteria. Cultures of bacteria treated in the same way actually declined in numbers. These results agree with those of Anderson and Slinger (1975) who advocated 4-6 pm for 2 min LO prepare soil suspensions and found that with amplitudes of 12 pm and above some cells were damaged. The autoradiographic technique provided a useful indication of the metabolic activity of the bacteria before and after extraction. The data from the whole soil (Fig. 2) showed that a large proportion of the soil population was viable and able to metabolize after ultrasonic treatment. This proportion was higher than that from shaking alone. This may be a reflection either of more of the cells metabolizing or of more metabolizing cells being in contact with [3H]glucose after dispersion of the soil particles. Either way, the ultrasonic treatment extracted a population of bacteria which was apparently not damaged by the treatment. In contrast, the cultures showed some decline in the proportion of cells able to metabolize glucose after ultrasonication. The susceptibility of the cultures to damage varied with species. Bacillus cereus and the fluorescent pseudomonad were both far more susceptible than the actinomycete, Nocardia asteroides, which was little affected. When the direct counts of bacteria and the autoradiographic data were examined together, it was apparent that 5 pm for 2 min caused more damage than 10.5 pm for 30 s. Assessing the efficiency of an extraction method requires observation of bacteria in soil before and after treatment. This presents a dilemma with most soils because bacteria are not visible unless extracted. The bacteria attached to sand grains in the sandy soil were however able to be examined, allowing an estimation of their recovery (63-77x) by ultrasonication. Although the recovery of bacteria from smaller particles in the sandy soil, or from the other soils could not be determined, the recovery from the sand grains was high compared with the 25-50x estimated by Weise and Rheinheimer (1979) from data on removal of organic matter from marine sand grains. The assessment of metabolic activity of extracted soil bacteria showed that ultrasonication at 10.5 pm for 30 s caused little change in their metabolic activity. This indicated that these extraction conditions provided the best compromise for releasing metabolically-active bacteria from soil. However, for recovery of bacteria added to soil a less vigorous extraction treatment should be used. Acknowledgement-1 thank Dr D. A. Rhoades for help with the statistical analyses. REFERENCES Anderson J. R. and Slinger J. M. (1975) Europium chelate and fluorescent brightener staining of soil propagules and

RAMSAY

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