Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria

Abstract

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.

The metabolism, phylogeny, and geomicrobiology of acidophilic, iron- and sulfur-oxidizing microorganisms have been studied for 50 years (1, 9, 12, 13, 14, 15, 16, 29). Many of these studies have focused on gram-negative, mesophilic, iron- and sulfur-oxidizing bacteria such as Thiobacillus ferrooxidans, Thiobacillus thiooxidans, and Leptospirillum spp. and their role in mineral leaching environments. Since then, the significance of other mineral-oxidizing microorganisms has been recognized (6, 7, 14, 18). Among these are the gram-positive, moderately thermophilic and mesophilic bacteria, which are now being studied to better understand their metabolic role in biological leaching processes (4, 8, 10, 18, 24).

Moderately thermophilic, gram-positive bacteria have been isolated from a number of acidic environments such as coal dumps, ore deposits, mining operations, and hot springs (Table 1) (2, 4, 10, 21, 24, 27, 32). These microorganisms vary in their ability to oxidize iron, sulfur, and pyrite and to grow heterotrophically and autotrophically (4, 22, 24, 27, 28). Mesophilic, gram-positive bacteria have recently been isolated and characterized for their metabolic attributes and phylogenetic placement (33).

TABLE 1.

Bacterial cultures

Organism	Strain	Original source	Reference
Acidiphilium sp. strain SJH	NCIB 12826	Disused pyrite mine, North Wales	17
Acidiphilium angustum (KLB)	ATCC 35903	Coal mine drainage, Pennsylvania	30, 31
Acidiphilum cryptum (Lhet2)	ATCC 33463	Culture of T. ferrooxidans	13
Acidiphilium facilis (PW1)		Abandoned deep mine, Pennsylvania	30, 31
Acidimicrobium ferrooxidans (ICP)	DSM 10331	Pyrite enrichment culture	4
Ferromicrobium acidophilus (T23)	DSM 11138	Cae Coch pyrite mine, North Wales	18
Leptospirillum ferrooxidans (MK)	DSM 2705	The original Markosyan isolate, mine, Armenia	1
Sulfobacillus acidophilus	DSM 10332	Coal spoil, United Kingdom	24
Sulfobacillus thermosulfidooxidans	DSM 9293	Spontaneously heated ore deposit, East Kazakhstan	10
Sulfolobus acidocaldarius	ATCC 33909	Hot spring, Yellowstone National Park, Wyoming	3
Sulfolobus shibatae	DSM 5389	Geothermal mud hole, Japan	11
Thiobacillus ferrooxidans	ATCC 23270	Coal mine effluent	12
Thiobacillus thiooxidans	ATCC 8085	Soil	29
L-15		Galways Soufrière, geothermal area, Montserrat, West Indies	33
RIV-14		White River Mouth, geothermal area, Montserrat	33
Newmont enrichment culture		Heap leaching operation, Nevada	26

Based on 16S rRNA sequencing, many moderately thermophilic, iron-oxidizing bacteria appear to be phylogenetically clustered among or near gram-positive microorganisms (20, 24). However, the traditional Gram-staining technique frequently gives variable or unrecognizable results for gram-positive mining bacteria (data not shown). In an earlier study (26), the lectin-based Gram-staining technique of Sizemore et al. (25) was modified to selectively stain a moderately thermophilic, acidophilic mining bacterium in mixed cultures that also contained T. ferrooxidans. The mechanism for staining is the binding of the wheat germ agglutinin (WGA) to the exposed n-acetylglucosamine residues of the peptidoglycan layer of the gram-positive bacteria (25). Thus, the WGA would not be able to bind to the peptidoglycan layer of cells with an outer membrane, i.e., gram-negative bacteria.

The purpose of this study was to evaluate the lectin-based stain for its suitability for gram-positive, acidophilic mining bacteria as well as bacteria associated with mineral-bearing samples. The technique was evaluated using acidophilic microbial cultures (Table 1), environmental samples, enrichment cultures, and samples collected from laboratory ore leaching studies. Cultures L-15, Riv-14, and T-23 were obtained from Barrie Johnson, University of Wales, Cardiff, United Kingdom. The Newmont culture was a subculture from an enrichment culture used in an earlier study (26).

Because of the generally low cell densities observed in acidophilic mining microorganisms, the method of Sizemore et al. (25) was adapted for use with filters. In addition, a dilute acid rinse was used to remove residual iron-containing media and iron precipitates. WGA labeled with fluorescein isothiocyanate (Molecular Probes, Eugene Oreg.) was diluted to 200 μg/ml in phosphate buffer (pH 7.2), filter sterilized, and stored frozen in 1-ml aliquots. Cultures were grown in a variety of acidic salt media (17; website www.dsmz.de) and filtered onto black polycarbonate membrane filters (25-mm diameter; 0.2-μm pore size) (Poretics, Livermore, Calif.). Each filter was rinsed twice with 2 to 3 ml of water that had been adjusted to pH 1 with H₂SO₄ and then with 2 to 3 ml of phosphate buffer (pH 7.2). Cells were stained for approximately 1 min with 1 to 2 ml of WGA stain (100-μg/ml final concentration in phosphate buffer). Direct light was avoided as much as possible. The filters were placed directly onto glass slides with a drop of immersion oil between the cover glass and filter. Cells were viewed using an epifluorescence microscope (model 1100; Carl Zeiss, Inc., Thornwood, N.Y.) with a filter set for blue excitation wavelengths (450 to 490 nm) or a confocal microscope system (model PCM 2000; Nikon Instrument Group, Melville, N.Y.).

Bacteria classified as gram positive by rRNA analyses stained with the fluorescently labeled WGA (Table 2). When stained the cells have a characteristic “outline” appearance, if the microscope is focused near the central region of the cell (Fig. 1). Both Sulfobacillus acidophilus and Sulfobacillus thermosulfidooxidans, which have been determined to be gram positive by 16S rRNA sequence analysis (20, 24), were stained by the fluorescent lectin. The newly described mesophilic, gram-positive cultures, designated RIV-14 and L-15 (33), also stained. RIV-14 and L-15 are phylogenetically related to Sulfobacillus (33). Cultures T23 and Acidimicrobium ferrooxidans stained weakly. The bacterium T23, tentatively identified as Ferromicrobium acidophilus, was reported to be closely related to A. ferrooxidans and has a phylogenetic placement near the divergence of the gram-positive branch (18). The archaea Sulfolobus acidocaldarius and Sulfolobus shibatae stained with a positive result. The highly irregular coccoid cell shape of the acidophilic archaea rendered them easily distinguishable from the rod-shaped, gram-positive bacteria.

TABLE 2.

Results of staining of cells with fluorescent WGA

Culture	Gram classification (reference)	Staining result
Acidiphilium sp. strain SJH	− (17)	−
Acidiphilium angustum	− (20)	−
Acidiphilium cryptum	− (20)	−
Acidiphilium facilis	− (20, 31)	−
Acidimicrobium ferrooxidans	+ (4)	+/−
Ferromicrobium acidophilus	+ (18)	+/−
Leptospirillum ferrooxidans	− (20)	−
Sulfobacillus acidophilus	+ (18)	+
Sulfobacillus thermosulfidooxidans	+ (18)	+
Sulfolobus acidocaldarius	Archaea (34)	+
Sulfolobus shibatae	Archaea (11)	+
Thiobacillus ferrooxidans	− (20)	−
Thiobacillus thiooxidans	− (20)	−
L-15	+ (33)	+
RIV-14	+ (33)	+
Newmont culture	+ (26)	+

FIG. 1.

Image of the thermophilic Newmont enrichment culture stained with fluorescein isothiocyanate-conjugated WGA. The image was captured by conventional light microphotographic methods and digitized using Adobe Photoshop software. Size bar, 10 μm.

Bacteria classified as gram negative (20, 30, 31), such as Thiobacillus, Acidiphilium, and Leptospirillum, did not stain and were essentially not visible. Some of the gram-negative cultures, including those from environmental samples, exhibited some weak autofluorescence, but these cells lacked the characteristic yellow-green “outline” appearance of a typical positive result.

Overall, the positive staining reaction indicates that an outer membrane is not present and that the cell surface layer has an affinity for the WGA (25). Presumably, for Sulfolobus the outer surface is the glycoprotein surface layer, while for the gram-positive bacteria the outer surface is the peptidoglycan layer. The outer membrane of the gram-negative bacteria would prevent the binding of the WGA to the peptidoglycan, thus producing a negative result.

Staining patterns were consistent for all phases of growth (lag, log, and stationary) (data not shown), which is comparable to the results obtained by Sizemore et al. (25). In addition, little or no nonspecific binding of the WGA stain was observed for ore-containing environmental and laboratory samples. Thus, gram-positive bacteria were easily detected on particles of the evaluated ore-containing samples.

In conclusion, the fluorescent wheat germ staining technique was effective for staining gram-positive, acidophilic mining bacteria and archaea. The lectin-based staining technique is an improvement over the traditional Gram-staining technique, which is unreliable and generally not applicable to mining bacteria and archaea. The lectin-based staining technique would be useful for characterizing new isolates as well as estimating the number of gram-positive bacteria and archaea in laboratory experiments, acidic mining environments, and ore-leaching bioprocesses.