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Europium chelate and fluorescent brightener staining of soil propagules and their photomicrographic counting—II. Efficiency
EUROPIUM CHELATE AND FLUORESCENT BRIGHTENER STAINING OF SOIL PROPAGULES AND THEIR PHOTOMICROGRAPHIC COUNTING-II. EFFICIENCY Plant Protection
Limited.
JOHN R. ANDEKSON* and JANLT M. SLINGER Jealott’s Hill Research Station. Bracknell, Berkshire (Acwptetl
RGl2
6EY. England
1 October 1974)
Summary~~The efficiency of staining Lipor,trce.\ sfarkc,!? and Pseudon~nu,s ueruyinosa cells added to agar and soil agar films and soil smears was examined using 2 rnM europium(l1 I) thenoyltrifluoroacetonate together with 25 P(Mdisodium salt of 4,4’-bis (4-aniline-6.bis(2-hydroxyethyl) amino-S-triazin-2-ylamino) 22.stilbcne disulphonic acid. abbreviated to Eu(TTA),, and FB. respectively. The combined stains, or DFS (ditferential fluorescent stain). gave recoveries of added cells close to the expected values, but prestained cells lost their Eu(TTA), fluorescence when added to agar at 5556O.C. Aqueous extracts of agar powder quenched the Eu(TTA), Ruoresccncc of cells. an effect which was attributed to water-soluble organic or inorganic ions in agar. It is suggested that the method of preparing soil-agar films and the long staining period required, effectively overcomes the quenching effect. Pre-stained cells were not affected by IO- dilutions in soil. Photomicrographically recorded counts were not significantly different from visual counts made on the same fields. Comparative counts with phenol&aniline blue. fluorescein iso-thiocyanate and DFS showed the latter stain to be more efficient in differentiating soil propagules on and in soil organic matter.
/LM, respectively. in 50”;; (v/v) ethanol in sterile distilled water. Phenollaniline blue (PAB) and fluorescein iso-thiocyanate (FITC) stains were prepared and used as recommended by Jones and Mollison (1948) and Babiuk and Paul (1971) respectively.
INTRODUCTlOh
A method for the differential fluorescent staining of soil propagules, their photomicrographic recording and subsequent enumeration by projection was described by Anderson and Slinger (I 975). The differential fluorescent stain (DFS), originally suggested by Anderson and Westmoreland (I 971) is composed of a europium chelate and a fluorescent brightener, abbreviated to Eu(TTA), and FB. respectively. This paper presents the results of experiments concerned with assessing the efficiency of the method and also compares DFS with the phenol-aniline blue stain of Jones and Mollison (1948) and the fluorescein isothiocyanate method described by Babiuk and Paul ( 1970).
A sandy clay loam (pH 6.0) was used for preparing soil-agar films and soil smears. Dilutions of 5 x. IO- ’ of the soil in sterile 10 mM piperazine-N,N’-bis-2-ethane sulphonic acid (PIPES) buffer. adjusted to pH 6.8 with 0.1 N NaOH was sonicdted at a low amplitude (4-6 jlrn) for 2 min with cooling, on an MSE I50 W “Ultrasonicator” to liberate and disperse soil propagules from clay mineral, and other soil particles. Soil suspensions were prepared immediately before forming soil-agar films or smears and were kept shaken by hand during the intervening period of ca. 20 s before adding I ml to the 9 ml sterile, filtered agar held at 55. 60°C. For soil smears, the initial (sonicated) suspension was further diluted to 5 x 10-I in sterile PIPES buffer. Soillagar films were formed in the 20 ILrn sample chamber of haemocytometers as previously described (Anderson and Slinger, 1975). The 5 x 10e2 dilutions for soil smears were thoroughly mixed for 60 s on a wrist-action shaker, allowed to settle for 30 s and IO ~1 samples used for preparing smears on 1.0 or I ,25 cm’ areas of glass slides as previously described (Anderson and Slinger. 1975).
MATERIALS AlVD METHODS Preparatiorl
of’uyur
artrl staim
The 1.5% (w/v) “Oxoid No I” sterile filtered agar used for agar films was prepared as previously described (Anderson and Slinger, 1975). The Eu(TTA), and FB components of DFS were europium(l11) thenoyltrifluoroacetonate and the disodium salt of 4,4’-bis (4-anilino-h-bis (2-hydroxyethyl) amino-s-triazin-2-ylamino) 2,2’-stilbene disulphonic acid, respectively, and were used at concentrations of 2 mrvr and 25 * Present address: Ministry of Agriculture. Plant Protection Research Institute, Box 8100 Causeway. Salisbury, Rhodesia 211
JOHN
212
R. ANIXXSON and
SoilLagar films and soil smears were stained in DFS for 18 h and 2 h, respectively, at room temperature and then gently rinsed with 12.5 ml and 5.0 ml of SW/, ethanol, respectively (Anderson and Slinger, 1975). For soilLagar films stained in PAB. the method of Jones and Mollison (1948) was followed and soil smears were stained with FTTC, freshly prepared daily, according to the method of Babiuk and Paul (1970). After rinsing, soil-agar fihns and soil smears were thoroughly air-dried. before mounting with Gurr “Fluormount”. a U.V. inert mountant, and grade “0” cover slips. Microscopy,
photornicroscopy
and prqjection
The incident U.V. light photomicroscope and the optical and filter system used for DFS preparations were as previously described (Anderson and Slinger, 1975). For FITC stained preparations, incident U.V. light with a BG12 (4mm thick) exciter filter and an 0G4 barrier filter were used. Transmitted visible light was used with a “Wratten” gelatin filter (Kodak No. SO), inserted in the sub-stage condenser, for PAB stained preparations. Daylight “Ektachrome High Speed” (used at 160 ASA) photographic film was used in all cases of photomicrography and projection of the chemically processed film was as previously described (Anderson and Slinger, 1975).
JANET
M.
SLINGEH
merated by repeated haemocytometer counts using phase-contrast lighting. A I ml aliquot of the suspcnsion was added to 9 ml of a lo-’ dilution of soil in PIPES buffer and briefly sonicated to disperse the cells. The soil suspension was then used to form soilL agar films and soil smears which, after drying and mounting, were enumerated under incident U.V. lighting. The accuracy of photomicrographs to record fluorescent soil propagules was examined by visually counting, and immediately photographing, each of 20 random fields on each of triplicate soil-agar films. The visual counts were carried out in a fixed focal plane corresponding to that which was photographed. Comparutivr
coum
A comparison of PAB and DFS stained soilLagar films was made using three soil-agar films prepared at the same time. The agar films were cut diagonally and the two half-films, in each case, positioned well apart on the glass slides. Three half-films were stained in DFS for 18 h and after rinsing, drying and mounting, 20 random microscope fields per half-film were counted by eye. Ten soil smears were prepared on I cm’ areas of glass slide. Five were stained with FITC and five with DFS. Twenty random microscope fields per soil smear were enumerated using the prescribed optical systems for each method of staining. RESULTS AND DISCUSSION
Cells of Lipornycrs starkeyi girzosa were added to individual
or Pseudomonas aeru9 ml volumes of sterile agar at 55°C. Each suspension was vortex mixed before forming agar films in 20 ;tm deep sample chamber haemocytometers. For each organism, three haemocytometers were used to form the agar films which were floated-free and mounted on glass slides, and three were used for counting cells by phase contrast lighting after the agar had dried in situ on the sample chamber “island”. Those on glass slides were stained for 18 h in DFS, rinsed with 12.5 ml 50’:; ethanol, dried and mounted prior to enumerating either IO or 20 random microscope fields per agar film using the area delineated by the microscope’s camera framing graticule as previously described (Anderson and Slinger, 1975). A known dilution of Lipomyces sturkeyi cells, enumerated by repeated haemocytometer counts, was added to 9 ml of a 5 x 10e3 dilution of soil in agar at 55 C. The suspension was thoroughly vortex mixed before preparing soilLagar films in 20 /Irn sample chambers. The agar films were stained in DFS as before and 20 random microscope fields on each were counted by eye. Washed cells of P.m~./o~~7or~r.s aeruyir7osa were first stained in DFS for 5 min and then rinsed with 5.0 ml of 50’,,, ethanol on a membrane filter under vacuum. The cells were re-suspended in PIPES buffer and enu-
The enumeration of Lipornycrs or Pseudomonas cells in agar films dried in situ on haemocytometer counting chambers showed an average of 1235.9 Lipornycrs cells and 4636.0 P.seudornonas cells in an area of 6.4 x IO5 pm2. The corresponding agar films mounted on glass slides and stained in DFS should. on this basis. have shown 8-9 Lipornyces cells or 3&40 Psrudornonas cells per camera graticule area of agar film of 4.62 x IO3 pm’. In practice. an average of 20 fields on each of three agar films per organism revealed 7.4 Lipornyc~~,~ and 39.5 P.srudomo~~u.s cells per field. In each case. red (europium) and green (fluorescent brightener) fluorescent cells were counted. Ten to twenty per cent of the total number of cells in each case fluoresced green indicating the loss of cell constituents essential for chelation with the Eu(TTA), component of DFS. Such cells were presumed to be non-viable. a factor which could not have emerged with the initial phase contrast counts of agar films in situ on haemocytometers or with PAB staining. The DFS stained soil&agar films containing a predetermined number of Lipomyces cells showed an average of 0.89 cells per camera framing graticule field from 60 random fields on three replicate soil--agar films. The expected number of 1.0 cell per field was dcliberatcly selected to test both the efficiency of recovery of low numbers and the distribution of cells in the initial soil-
Staining
and photomicrography
of soil propaguk-effkiency
213
214
JOHN R. ANDERSON
and JANIX M.
agar suspension. In this case. brief exposure of the suspension to low intensity sonication (4-6 /tm amplitude for 2 min) facilitated distribution of the cells which otherwise tended to clump. The pre-stained cells of P.scwdo771or7us trrm~qi7wscc were observed to fluoresce brightly, but when dispcrsed in agar. the resulting films failed to show Eu(TTA), Huorescencc. A suspension of prc-stained cells in PIPES buffer at 60’ C fluoresced normally but when an aqueous extract of agar powder was used. the red fluorescence again disappeared. Thus, it was not possible to prc-stain cells and add them to soilLagar suspensions as a means of testing recovery cthciency and distribution of cells. ScaK Dyer and Mori ( 1969) drew particular attention to the undesirability of phosphate in Eu(TTA), staining and since agar powders contain significant amounts of both organic and inorganic phosphate. it is suggcstcd that this may be responsible for the ctfect noted. In the cast of agar films. the procedure of floating the films free from haemocytomctcrs in sterile distilled H,O and the subsequent 1X h immersion of the dried films in DFS may effect removal of soluble phosphatc or, in the latter process, saturation of phosphate by the excess Eu(TTA), ions. With IO- ’ dilutions of soil in PIPES buffer to which was added a suspension of pre-stained P,sdowr~tr.s cells. there was no loss of fluorescence of the cells in the soil smears. The enumeration of the prc-stained cells was within 97 per cent of the calculated numbers of cells expected per camera graticule area. The accuracy of photography! in recording visually observable propagules was examined by counting propagules in a fixed focal plane and immediately photographing the same field. The results (Table 1) indicate that in some cases the sensitivity of photographic emulsion in recording the presence of very small fluorcscent cocci is greater than the sensitivity of the eye. This is particularly so after prolonged periods at the microscope when cyc fatigue is likely to result in thcsc smaller cells being overlooked. Statistical analysis of the results in Table I showed that the diffcrcnces in means of visual and photographic counts were not significantly different and the standard errors per field and per agar film for the two
SLINGER
methods of counting were also not significantly ent. In 7X.3 per cent of fields, colour reversal were within 10 per cent of the visual count and per cent of fields the photomicrographs gave which were more than IO per cent higher than counts.
Counts of soil propagules in halved soil-agar films stained with either PAB or DFS showed (Table 2) that, contrary to earlier findings (Anderson and Westmoreland. 1971) the DFS gave greater estimates of propagules. This discrepancy is attributed to the superiority of incident illumination and an improved intensity of staining with 2 tIIM Eu(TTA), compared to the earlier work using 1 mM Eu(TTA), and transmitted U.V. light, Statistical analysis of the results showed that DFS gave significantly greater estimates (P i: 0.05) than the PAB method. The standard error between fields in the PAB results was cu. 40 per cent while that for the DFS results was ~0. 20 per cent. Cells stained with PAB were frequently difhcult to dilfcrentiate from organic matter and soil particles with which they are often closely associated but in the case of DFS preparations the viable propagules were readily differentiated from organic and other soil particles. Soil smears stained with DFS or FITC were enumerated visually and with the DFS method the red and green fluorescent propagules. representing viable (or recently dead) and dead cells, respectively, were enumerated separately and the average of the total count compared against total numbers of FITC stained propagules. The results (Table 3) indicate that greater numbers of soil propagules can be detected with DFS. Statistical analysis showed that DFS derived values were significantly greater than those from FITC (P < 0.05). Possible explanations for the increase are that DFS gives a greater contrast between viable propagules and organic matter and also does not depend upon the presence ofa protein layer around cells which is the suggested mechanism of FITC staining given by Babiuk and Paul (1970). This mechanism of staining could lead to some uncertainty especially if soil particlcs possess a coating of protcm or if minute particles
Table 2. Numbers of soil propagules in diagonally divided* soil&agar films stained in PABt or DFS: Half-film no.
Staining method
Total propagules in 20 fields
Av. no. per held
la
PAB DFS
1561
647
32.35
2a 2b
PAB DFS
132 1243
7X.05 36.60 62.15
3a 3b
PAB DFS
701 I739
35.05 86.95
lb
differcounts in 21.3 counts visual
* Triplicate soil ~agar films divided diagonally. t Phenol-aniline blue (Jones and Mollison, 194X). $ Europium chelate (2 mM) plus fluorescent brightener (25 I~I)
Proportional increase 2.413 I .698 2.48 I
Staining and photomicrography Table 3. Numbers Colour Slide
Stain
tl0.
used
I 2 3 la
FITC FITC FITC DFS
22
DFS
3a
DFS
of soil propagules
of soil propagulesm-efficiency
215
in soil smears stained with either FITC* or DFSt
of
fluorescent cells Green Green Green Red Green Red Green Red Green
* Method of Babuik and Paul (I 970). i Europium chelate (2 mu) plus fluorescent
Propagules
per field
32.25.31.25.26,30.30,30.32.30 30.33.29,36.31,36.41.3l.30.33 31,1X.22,28.33,29,28.30.22,29 27.21.26.18.19.22.20.22.25.20 22,16.15. I 7. I 2.16. I 3,17.72. I4 20.20.15,26.16.27.24,28.35,18 14,13.17,16.13,12,14.14~30.22 25.20,21.20,1X.16,21.20.19.25 12.10,13. 9.11. X,16.15,12,14
brightener
Total propagules ‘91 330 270 320 164 229 165 205 120
Av. no. per field 29. I 33.0 27.0 3X.4 394 32.5
(25 L(M).
of organic matter become detached from larger fragments. Soluble proteins are easily adsorbed by clay and other soil minerals, and ligno-protein complexes in organic matter are well known (Esterman. et d., 1959). CONCLUSIONS
The recovery of Liporn~~s and Psadornonus cells in prepared soil-agar fiIms and suspensions was close to the expected values indicating an acceptable degree of accuracy of the staining method. The loss of fluorescence of pre-stained cells added to agar films and the demonstration that aqueous extracts of agar powder quenched the Eu(TTA), fluorescence suggests that some organic or inorganic water soluble fraction of agar, possibly phosphate, is responsible for this effect. The presence of such a fraction in agar may account for the longer staining period required for soil-agar films. Excess Eu(TTA), would be required to overcome the effect before the soil propagules could be stained. The procedures of floating-free soil-agar films from the haemocytometer in sterile distilled water and the long immersion of the films in staining troughs probably aid in reducing the concentration of the quenching fraction in the agar film. Fluorescent staining is preferable to non-fluorescent staining since the lack of differentiation of propagules and soil minerals with the latter make enumeration more difficult. A stain that differentiates living (or recently dead) from non-living propagules is to be preferred if only for the greater degree of contrast offered by such stains.
Photomicrographic recording of numbers of soil propagules is possible. The accuracy of photography is acceptable and in some cases may be more efficient than visual counting. Errors incurred by eye fatigue during visual observations and those from photomicrography are probably self cancelling. In cases where propagules are wholly or partly obscured by organic matter the error incurred by single-plane photomicrography can be minimized by visual enumeration with in-depth focusing of the area in question followed by photomicrography of the rest of the field.
REFERENCES
AUDLKSON J. R. and WFSTMORILAYI) D. (I 971) Direct counts of soil organisms using a fluorescent brightener and a curopium chelate. So/l Bid. Bio~hrm. 3, X5X+7. A~IXRSON J. R. and SI.INC;L.KJ. M. (1975) Europium chclate and fluorescent brightener staining of soil propagules and their photomicrographic counting--l. Methods. Soil Bid. Biochem. 7, 205-209. B~~nl~ti L. A. and PAL~LE. A. (1970) The use of tluorescein isothiocyanate in the determination of the bacterial biomass of grassland soil. Curl. J. Microhid. 16, 57 62. EST~KMA~ E. F., P~T~,Rs~N G. H. and MCLARL~ A. D. (1959) Digestion of clay-prot$n. lignin-protein and silica--protern complexes by enzymes and bacteria. Pmt. Soil Sci. SW. Am. 23, 3 I 36. JONI.S P. C. T. and MOI.I.IS~S J. E. (1948) A technique for the quantitative estimation of soil microorganisms. J. (/<‘II. M~c~)hiol. 2, 54-6X. SCAFF W. L.. DYER D. L. and MORI K. (1969) Fluorescent europium chelate stain. J. BNCI. 98, 246248.
Limited.
JOHN R. ANDEKSON* and JANLT M. SLINGER Jealott’s Hill Research Station. Bracknell, Berkshire (Acwptetl
RGl2
6EY. England
1 October 1974)
Summary~~The efficiency of staining Lipor,trce.\ sfarkc,!? and Pseudon~nu,s ueruyinosa cells added to agar and soil agar films and soil smears was examined using 2 rnM europium(l1 I) thenoyltrifluoroacetonate together with 25 P(Mdisodium salt of 4,4’-bis (4-aniline-6.bis(2-hydroxyethyl) amino-S-triazin-2-ylamino) 22.stilbcne disulphonic acid. abbreviated to Eu(TTA),, and FB. respectively. The combined stains, or DFS (ditferential fluorescent stain). gave recoveries of added cells close to the expected values, but prestained cells lost their Eu(TTA), fluorescence when added to agar at 5556O.C. Aqueous extracts of agar powder quenched the Eu(TTA), Ruoresccncc of cells. an effect which was attributed to water-soluble organic or inorganic ions in agar. It is suggested that the method of preparing soil-agar films and the long staining period required, effectively overcomes the quenching effect. Pre-stained cells were not affected by IO- dilutions in soil. Photomicrographically recorded counts were not significantly different from visual counts made on the same fields. Comparative counts with phenol&aniline blue. fluorescein iso-thiocyanate and DFS showed the latter stain to be more efficient in differentiating soil propagules on and in soil organic matter.
/LM, respectively. in 50”;; (v/v) ethanol in sterile distilled water. Phenollaniline blue (PAB) and fluorescein iso-thiocyanate (FITC) stains were prepared and used as recommended by Jones and Mollison (1948) and Babiuk and Paul (1971) respectively.
INTRODUCTlOh
A method for the differential fluorescent staining of soil propagules, their photomicrographic recording and subsequent enumeration by projection was described by Anderson and Slinger (I 975). The differential fluorescent stain (DFS), originally suggested by Anderson and Westmoreland (I 971) is composed of a europium chelate and a fluorescent brightener, abbreviated to Eu(TTA), and FB. respectively. This paper presents the results of experiments concerned with assessing the efficiency of the method and also compares DFS with the phenol-aniline blue stain of Jones and Mollison (1948) and the fluorescein isothiocyanate method described by Babiuk and Paul ( 1970).
A sandy clay loam (pH 6.0) was used for preparing soil-agar films and soil smears. Dilutions of 5 x. IO- ’ of the soil in sterile 10 mM piperazine-N,N’-bis-2-ethane sulphonic acid (PIPES) buffer. adjusted to pH 6.8 with 0.1 N NaOH was sonicdted at a low amplitude (4-6 jlrn) for 2 min with cooling, on an MSE I50 W “Ultrasonicator” to liberate and disperse soil propagules from clay mineral, and other soil particles. Soil suspensions were prepared immediately before forming soil-agar films or smears and were kept shaken by hand during the intervening period of ca. 20 s before adding I ml to the 9 ml sterile, filtered agar held at 55. 60°C. For soil smears, the initial (sonicated) suspension was further diluted to 5 x 10-I in sterile PIPES buffer. Soillagar films were formed in the 20 ILrn sample chamber of haemocytometers as previously described (Anderson and Slinger, 1975). The 5 x 10e2 dilutions for soil smears were thoroughly mixed for 60 s on a wrist-action shaker, allowed to settle for 30 s and IO ~1 samples used for preparing smears on 1.0 or I ,25 cm’ areas of glass slides as previously described (Anderson and Slinger. 1975).
MATERIALS AlVD METHODS Preparatiorl
of’uyur
artrl staim
The 1.5% (w/v) “Oxoid No I” sterile filtered agar used for agar films was prepared as previously described (Anderson and Slinger, 1975). The Eu(TTA), and FB components of DFS were europium(l11) thenoyltrifluoroacetonate and the disodium salt of 4,4’-bis (4-anilino-h-bis (2-hydroxyethyl) amino-s-triazin-2-ylamino) 2,2’-stilbene disulphonic acid, respectively, and were used at concentrations of 2 mrvr and 25 * Present address: Ministry of Agriculture. Plant Protection Research Institute, Box 8100 Causeway. Salisbury, Rhodesia 211
JOHN
212
R. ANIXXSON and
SoilLagar films and soil smears were stained in DFS for 18 h and 2 h, respectively, at room temperature and then gently rinsed with 12.5 ml and 5.0 ml of SW/, ethanol, respectively (Anderson and Slinger, 1975). For soilLagar films stained in PAB. the method of Jones and Mollison (1948) was followed and soil smears were stained with FTTC, freshly prepared daily, according to the method of Babiuk and Paul (1970). After rinsing, soil-agar fihns and soil smears were thoroughly air-dried. before mounting with Gurr “Fluormount”. a U.V. inert mountant, and grade “0” cover slips. Microscopy,
photornicroscopy
and prqjection
The incident U.V. light photomicroscope and the optical and filter system used for DFS preparations were as previously described (Anderson and Slinger, 1975). For FITC stained preparations, incident U.V. light with a BG12 (4mm thick) exciter filter and an 0G4 barrier filter were used. Transmitted visible light was used with a “Wratten” gelatin filter (Kodak No. SO), inserted in the sub-stage condenser, for PAB stained preparations. Daylight “Ektachrome High Speed” (used at 160 ASA) photographic film was used in all cases of photomicrography and projection of the chemically processed film was as previously described (Anderson and Slinger, 1975).
JANET
M.
SLINGEH
merated by repeated haemocytometer counts using phase-contrast lighting. A I ml aliquot of the suspcnsion was added to 9 ml of a lo-’ dilution of soil in PIPES buffer and briefly sonicated to disperse the cells. The soil suspension was then used to form soilL agar films and soil smears which, after drying and mounting, were enumerated under incident U.V. lighting. The accuracy of photomicrographs to record fluorescent soil propagules was examined by visually counting, and immediately photographing, each of 20 random fields on each of triplicate soil-agar films. The visual counts were carried out in a fixed focal plane corresponding to that which was photographed. Comparutivr
coum
A comparison of PAB and DFS stained soilLagar films was made using three soil-agar films prepared at the same time. The agar films were cut diagonally and the two half-films, in each case, positioned well apart on the glass slides. Three half-films were stained in DFS for 18 h and after rinsing, drying and mounting, 20 random microscope fields per half-film were counted by eye. Ten soil smears were prepared on I cm’ areas of glass slide. Five were stained with FITC and five with DFS. Twenty random microscope fields per soil smear were enumerated using the prescribed optical systems for each method of staining. RESULTS AND DISCUSSION
Cells of Lipornycrs starkeyi girzosa were added to individual
or Pseudomonas aeru9 ml volumes of sterile agar at 55°C. Each suspension was vortex mixed before forming agar films in 20 ;tm deep sample chamber haemocytometers. For each organism, three haemocytometers were used to form the agar films which were floated-free and mounted on glass slides, and three were used for counting cells by phase contrast lighting after the agar had dried in situ on the sample chamber “island”. Those on glass slides were stained for 18 h in DFS, rinsed with 12.5 ml 50’:; ethanol, dried and mounted prior to enumerating either IO or 20 random microscope fields per agar film using the area delineated by the microscope’s camera framing graticule as previously described (Anderson and Slinger, 1975). A known dilution of Lipomyces sturkeyi cells, enumerated by repeated haemocytometer counts, was added to 9 ml of a 5 x 10e3 dilution of soil in agar at 55 C. The suspension was thoroughly vortex mixed before preparing soilLagar films in 20 /Irn sample chambers. The agar films were stained in DFS as before and 20 random microscope fields on each were counted by eye. Washed cells of P.m~./o~~7or~r.s aeruyir7osa were first stained in DFS for 5 min and then rinsed with 5.0 ml of 50’,,, ethanol on a membrane filter under vacuum. The cells were re-suspended in PIPES buffer and enu-
The enumeration of Lipornycrs or Pseudomonas cells in agar films dried in situ on haemocytometer counting chambers showed an average of 1235.9 Lipornycrs cells and 4636.0 P.seudornonas cells in an area of 6.4 x IO5 pm2. The corresponding agar films mounted on glass slides and stained in DFS should. on this basis. have shown 8-9 Lipornyces cells or 3&40 Psrudornonas cells per camera graticule area of agar film of 4.62 x IO3 pm’. In practice. an average of 20 fields on each of three agar films per organism revealed 7.4 Lipornyc~~,~ and 39.5 P.srudomo~~u.s cells per field. In each case. red (europium) and green (fluorescent brightener) fluorescent cells were counted. Ten to twenty per cent of the total number of cells in each case fluoresced green indicating the loss of cell constituents essential for chelation with the Eu(TTA), component of DFS. Such cells were presumed to be non-viable. a factor which could not have emerged with the initial phase contrast counts of agar films in situ on haemocytometers or with PAB staining. The DFS stained soil&agar films containing a predetermined number of Lipomyces cells showed an average of 0.89 cells per camera framing graticule field from 60 random fields on three replicate soil--agar films. The expected number of 1.0 cell per field was dcliberatcly selected to test both the efficiency of recovery of low numbers and the distribution of cells in the initial soil-
Staining
and photomicrography
of soil propaguk-effkiency
213
214
JOHN R. ANDERSON
and JANIX M.
agar suspension. In this case. brief exposure of the suspension to low intensity sonication (4-6 /tm amplitude for 2 min) facilitated distribution of the cells which otherwise tended to clump. The pre-stained cells of P.scwdo771or7us trrm~qi7wscc were observed to fluoresce brightly, but when dispcrsed in agar. the resulting films failed to show Eu(TTA), Huorescencc. A suspension of prc-stained cells in PIPES buffer at 60’ C fluoresced normally but when an aqueous extract of agar powder was used. the red fluorescence again disappeared. Thus, it was not possible to prc-stain cells and add them to soilLagar suspensions as a means of testing recovery cthciency and distribution of cells. ScaK Dyer and Mori ( 1969) drew particular attention to the undesirability of phosphate in Eu(TTA), staining and since agar powders contain significant amounts of both organic and inorganic phosphate. it is suggcstcd that this may be responsible for the ctfect noted. In the cast of agar films. the procedure of floating the films free from haemocytomctcrs in sterile distilled H,O and the subsequent 1X h immersion of the dried films in DFS may effect removal of soluble phosphatc or, in the latter process, saturation of phosphate by the excess Eu(TTA), ions. With IO- ’ dilutions of soil in PIPES buffer to which was added a suspension of pre-stained P,sdowr~tr.s cells. there was no loss of fluorescence of the cells in the soil smears. The enumeration of the prc-stained cells was within 97 per cent of the calculated numbers of cells expected per camera graticule area. The accuracy of photography! in recording visually observable propagules was examined by counting propagules in a fixed focal plane and immediately photographing the same field. The results (Table 1) indicate that in some cases the sensitivity of photographic emulsion in recording the presence of very small fluorcscent cocci is greater than the sensitivity of the eye. This is particularly so after prolonged periods at the microscope when cyc fatigue is likely to result in thcsc smaller cells being overlooked. Statistical analysis of the results in Table I showed that the diffcrcnces in means of visual and photographic counts were not significantly different and the standard errors per field and per agar film for the two
SLINGER
methods of counting were also not significantly ent. In 7X.3 per cent of fields, colour reversal were within 10 per cent of the visual count and per cent of fields the photomicrographs gave which were more than IO per cent higher than counts.
Counts of soil propagules in halved soil-agar films stained with either PAB or DFS showed (Table 2) that, contrary to earlier findings (Anderson and Westmoreland. 1971) the DFS gave greater estimates of propagules. This discrepancy is attributed to the superiority of incident illumination and an improved intensity of staining with 2 tIIM Eu(TTA), compared to the earlier work using 1 mM Eu(TTA), and transmitted U.V. light, Statistical analysis of the results showed that DFS gave significantly greater estimates (P i: 0.05) than the PAB method. The standard error between fields in the PAB results was cu. 40 per cent while that for the DFS results was ~0. 20 per cent. Cells stained with PAB were frequently difhcult to dilfcrentiate from organic matter and soil particles with which they are often closely associated but in the case of DFS preparations the viable propagules were readily differentiated from organic and other soil particles. Soil smears stained with DFS or FITC were enumerated visually and with the DFS method the red and green fluorescent propagules. representing viable (or recently dead) and dead cells, respectively, were enumerated separately and the average of the total count compared against total numbers of FITC stained propagules. The results (Table 3) indicate that greater numbers of soil propagules can be detected with DFS. Statistical analysis showed that DFS derived values were significantly greater than those from FITC (P < 0.05). Possible explanations for the increase are that DFS gives a greater contrast between viable propagules and organic matter and also does not depend upon the presence ofa protein layer around cells which is the suggested mechanism of FITC staining given by Babiuk and Paul (1970). This mechanism of staining could lead to some uncertainty especially if soil particlcs possess a coating of protcm or if minute particles
Table 2. Numbers of soil propagules in diagonally divided* soil&agar films stained in PABt or DFS: Half-film no.
Staining method
Total propagules in 20 fields
Av. no. per held
la
PAB DFS
1561
647
32.35
2a 2b
PAB DFS
132 1243
7X.05 36.60 62.15
3a 3b
PAB DFS
701 I739
35.05 86.95
lb
differcounts in 21.3 counts visual
* Triplicate soil ~agar films divided diagonally. t Phenol-aniline blue (Jones and Mollison, 194X). $ Europium chelate (2 mM) plus fluorescent brightener (25 I~I)
Proportional increase 2.413 I .698 2.48 I
Staining and photomicrography Table 3. Numbers Colour Slide
Stain
tl0.
used
I 2 3 la
FITC FITC FITC DFS
22
DFS
3a
DFS
of soil propagules
of soil propagulesm-efficiency
215
in soil smears stained with either FITC* or DFSt
of
fluorescent cells Green Green Green Red Green Red Green Red Green
* Method of Babuik and Paul (I 970). i Europium chelate (2 mu) plus fluorescent
Propagules
per field
32.25.31.25.26,30.30,30.32.30 30.33.29,36.31,36.41.3l.30.33 31,1X.22,28.33,29,28.30.22,29 27.21.26.18.19.22.20.22.25.20 22,16.15. I 7. I 2.16. I 3,17.72. I4 20.20.15,26.16.27.24,28.35,18 14,13.17,16.13,12,14.14~30.22 25.20,21.20,1X.16,21.20.19.25 12.10,13. 9.11. X,16.15,12,14
brightener
Total propagules ‘91 330 270 320 164 229 165 205 120
Av. no. per field 29. I 33.0 27.0 3X.4 394 32.5
(25 L(M).
of organic matter become detached from larger fragments. Soluble proteins are easily adsorbed by clay and other soil minerals, and ligno-protein complexes in organic matter are well known (Esterman. et d., 1959). CONCLUSIONS
The recovery of Liporn~~s and Psadornonus cells in prepared soil-agar fiIms and suspensions was close to the expected values indicating an acceptable degree of accuracy of the staining method. The loss of fluorescence of pre-stained cells added to agar films and the demonstration that aqueous extracts of agar powder quenched the Eu(TTA), fluorescence suggests that some organic or inorganic water soluble fraction of agar, possibly phosphate, is responsible for this effect. The presence of such a fraction in agar may account for the longer staining period required for soil-agar films. Excess Eu(TTA), would be required to overcome the effect before the soil propagules could be stained. The procedures of floating-free soil-agar films from the haemocytometer in sterile distilled water and the long immersion of the films in staining troughs probably aid in reducing the concentration of the quenching fraction in the agar film. Fluorescent staining is preferable to non-fluorescent staining since the lack of differentiation of propagules and soil minerals with the latter make enumeration more difficult. A stain that differentiates living (or recently dead) from non-living propagules is to be preferred if only for the greater degree of contrast offered by such stains.
Photomicrographic recording of numbers of soil propagules is possible. The accuracy of photography is acceptable and in some cases may be more efficient than visual counting. Errors incurred by eye fatigue during visual observations and those from photomicrography are probably self cancelling. In cases where propagules are wholly or partly obscured by organic matter the error incurred by single-plane photomicrography can be minimized by visual enumeration with in-depth focusing of the area in question followed by photomicrography of the rest of the field.
REFERENCES
AUDLKSON J. R. and WFSTMORILAYI) D. (I 971) Direct counts of soil organisms using a fluorescent brightener and a curopium chelate. So/l Bid. Bio~hrm. 3, X5X+7. A~IXRSON J. R. and SI.INC;L.KJ. M. (1975) Europium chclate and fluorescent brightener staining of soil propagules and their photomicrographic counting--l. Methods. Soil Bid. Biochem. 7, 205-209. B~~nl~ti L. A. and PAL~LE. A. (1970) The use of tluorescein isothiocyanate in the determination of the bacterial biomass of grassland soil. Curl. J. Microhid. 16, 57 62. EST~KMA~ E. F., P~T~,Rs~N G. H. and MCLARL~ A. D. (1959) Digestion of clay-prot$n. lignin-protein and silica--protern complexes by enzymes and bacteria. Pmt. Soil Sci. SW. Am. 23, 3 I 36. JONI.S P. C. T. and MOI.I.IS~S J. E. (1948) A technique for the quantitative estimation of soil microorganisms. J. (/<‘II. M~c~)hiol. 2, 54-6X. SCAFF W. L.. DYER D. L. and MORI K. (1969) Fluorescent europium chelate stain. J. BNCI. 98, 246248.