Yeast Yeast 2012; 29: 111–117. Published online 23 March 2012 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.2894
Research Article
Improvement of FISH–FCM enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion Cancan Cui, Yanyan Zhang, Hui Han and Shaokui Zheng* MOE Key Laboratory of Water and Sediment Sciences/State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, People’s Republic of China
*Correspondence to: S. Zheng, MOE Key Laboratory of Water and Sediment Sciences/ State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, People’s Republic of China. E-mail:
[email protected]
Received: 24 July 2011 Accepted: 1 March 2012
Abstract This paper developed a novel strategy to improve the fluorescence in situ hybridization– flow cytometry (FISH–FCM) enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion to fully disaggregate filamentous yeast chains into single cells. A 2 h 2% snailase partial digestion liberated more rod-shaped yeast single cells from intertwined filamentous yeast samples than did sonication disaggregation, based on an optical microscopic observation and the forward-light-scatter frequency histogram of FCM analysis. However, adding snailase resulted in a fluorescencequenching phenomenon of the hybridized filamentous yeast cells, which was minimized by lowering the snailase concentration. An approximately 3 h 0.5% snailase partial digestion conducted between sonication and hybridization significantly improved the FISH–FCM enumeration performance for filamentous yeast species by 37%. The results presented here will facilitate the rapid detection, identification and exact enumeration of specific filamentous fungal species in environmental samples. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: enumeration; filamentous yeast; fluorescence in situ hybridization (FISH); flow cytometry (FCM); snailase
Introduction As a rapid and highly reliable noncultural molecular fingerprinting method, the fluorescence in situ hybridization-flow cytometry (FISH–FCM) technique has recently gained widespread acceptance to rapidly detect, identify, and enumerate specific microorganisms such as bacteria (Vaahtovuo et al., 2005; Foladori et al., 2010), yeast (Zheng et al., 2010), and even algae (Simon et al., 1995; Cellamare et al., 2010) in a number of environments, including blood samples(Kempf et al., 2005), human fecal samples (Zoetendal et al., 2002; Vaahtovuo et al., 2005), air (Lange et al., 1997), water (Tang et al., 2005; Foladori et al., 2010), soil (Thomas et al., 1997) and activated sludge (Foladori et al., 2010; Zheng et al., 2010). FISH–FCM Copyright © 2012 John Wiley & Sons, Ltd.
overcomes the limitation of polymerase chain reaction (PCR)-based community analytical approaches that cannot accurately detect and enumerate specific microorganisms (Zoetendal et al., 2002) and provides new insights into the microbial community structure of environmental samples. Filamentous yeast occur widely in environmental samples including drinking water (De Vos and Nelis, 2006; Pereira et al., 2009) and activated sludge (Zheng et al., 2010). The traditional enumeration method for yeast cells, i.e. microscopic techniques, is often slow, tedious and rather imprecise (Prigione et al., 2004), since environmental samples often contain plentiful bacteria and fungal species. To gain a better understanding of the content of specific yeast species in microbial community and its evolution in response to environmental conditions,
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it is often necessary and effective to use FISH–FCM to distinguish between the specific yeast species and bacteria or other fungal species and to further enumerate it (Zheng et al., 2010). However, FCM is an established cell-analysis technique that permits rapid analysis and quantitative evaluation at singlecell level, based on light-scattering and fluorescent signals (Deere et al., 1998; Ruiz et al., 1999; Foladori et al., 2010). At that time, filamentous fungus or algae that form chains are often counted as single entities during the sorting step if they are not separated (Cellamare et al., 2010). Therefore, disaggregation of fungal chains into single cells, i.e. the compartment between two adjacent septa, is very important to quantify specific filamentous fungal species in environmental samples by FISH–FCM. Sonication has been widely applied to separate samples into single cells prior to FCM analysis (Foladori et al., 2007; Zheng et al., 2010). A fundamental difficulty in efficiently dissociating microorganisms from flocculated clumps lies in the balance between using sonication power high enough to achieve nearcomplete cell detachment from their matrix and the concomitant risk of cell disruption that occurs at higher power levels (Buesing and Gessner, 2002). For example, the maximum concentration of free total cells for activated sludge was reached at a sonication time of 90 s (Falcioni et al., 2006) or a specific ultrasonic energy of 120 kJ/l (Foladori et al., 2007), while a prolonged sonication caused a strong and progressive loss of total free cells (Falcioni et al., 2006; Foladori et al., 2007). These results demonstrate that more strategies should be considered to disaggregate environmental samples to improve the sonication-based FCM enumeration performance in filamentous fungal samples. Trichosporon asahii is a clinically important yeast species that can cause life-threatening infections in immunocompromised patients (Karashima et al., 2002). Additionally, different strains of T. asahii have extensive uses in mashita fermentation (Ongol and Asano, 2009) or lipase production (Kumar and Gupta, 2008). It has been reported that T. asahii can spontaneously switch its cell morphology from conidia (blastoconidia or arthroconidia) to hyphae in many environmental samples (Karashima et al., 2002). Therefore, dispersion of the yeast hyphae into single cells is important for diagnosis or analysis of the specific fungal species in environmental samples (Karashima et al., 2002). In this study, snailase was used to partially digest hyphae walls of the Copyright © 2012 John Wiley & Sons, Ltd.
filamentous yeast species and to break its chains into single cells to improve its FISH–FCM enumeration performance. It is well known that protoplast fusion has been widely used since the 1950s as an effective technique for fungal strain improvement, which requires the release of viable wall-less protoplasts by snailase (i.e. glusulase) digestion of fungal cell walls (Eddy and Williamson, 1957).
Materials and methods Filamentous yeast samples and pretreatment Filamentous yeast samples dominated by T. asahii were collected from a laboratory-scale activated sludge system for high organic strength industrial wastewater in our laboratory. Microscopic observations were conducted with an optical microscope (Olympus B41) at 400 magnification. The filamentous yeast samples were centrifuged at 10 000 g for 3 min, washed three times with phosphate-buffered saline (PBS) and then sonicated for 75 s, using an ultrasonic liquid processor (VCX105, Sonics, Newtown, CT, USA) at 100 W. Next, the cells were fixed at 4 C overnight with fresh 4% w/v paraformaldehyde, centrifuged, washed three times with PBS and then resuspended in 50% ethanol–PBS solution to give a final concentration of 109–1010/ml.
Snailase digestion Following fixation with paraformaldehyde, snailase solutions (Beijing Baitai Biological Technology Co., China) were added to the samples to achieve a final snailase concentration of 2% and incubated at 30 C with gentle shaking. Additionally, the effect of snailase concentration (0.1%, 0.2%, 0.5%, 1%, 2% and 5%) on fluorescence expression and the effect of snailase digestion time (0–4 h at a snailase concentration of 0.5%) on the sonication–snailase digestion–FISH–FCM enumeration performance were investigated. The snailase solutions were filtered through a 0.22 mm membrane filter to remove autochthonous microbiota prior to use.
Fluorescent staining by fluorochromes SYTOX Green (Molecule Probes, Invitrogen, Carlsbad, CA, USA) was added to samples at a final concentration of 5 mM to stain the paraformaldehyde-fixed samples and the stained samples Yeast 2012; 29: 111–117. DOI: 10.1002/yea
Snailase significantly improves FISH–FCM enumeration performance
were incubated in the dark at room temperature for 10 min prior to FCM analysis.
FISH The paraformaldehyde-fixed (and subsequently snailase-digested in some cases) sludge samples were individually subjected to hybridization, using an 18S rRNA-targeted oligonucleotide probe with FITC or Cy3 fluorochrome at the 5′ end, PF2 (5′-CTC TGG CTT CAC CCT ATT C-3′) (Kempf et al., 2005). The fluorochrome type was determined based on which flow cytometry channel was available, i.e. FITC for the FL1 channel and Cy3 for the FL2 channel. For each hybridization reaction, 100 ml pretreated cells were centrifuged (5 min at 5000 g) and resuspended in 92.5 ml prewarmed hybridization buffer (0.9 M NaCl, 20 mM Tris–HCl, pH 7.2, 0.01% w/v sodium dodecyl sulphate, 20% v/v formamide) containing 2.5 ml PF2 probes (100 ng/ml in Tris–EDTA buffer (100 mM Tris–HCl, pH 8.0, 50 mM EDTA), after which the samples were incubated for 3 h at 46 C with gentle agitation in the dark. Additionally, negative controls were prepared, consisting of hybridization buffer with no probes. After hybridization, the probes were washed by incubating the samples for 20 min with 1 ml washing buffer (20 mM Tris HCl, pH 7.2, 0.01% SDS, 5 mM EDTA and 225 mM NaCl) at 46 C. Subsequently, the cells were centrifuged, and resuspended in 1 ml ice-cold PBS, pH 7.2, in the dark.
FCM analysis The microbial concentration of the labelled or stained cells was adjusted to keep the concentration at < 1000 events/s, after which known concentrations of 0.7 mm yellow-green fluorescent beads (TruCount Tube, Becton-Dickinson, Franklin Lakes, NJ, USA) were added, according to the manufacturer’s instructions, to determine cell concentrations. All samples were thoroughly vortexed to minimize the possibility of clumping. The labelled cells were enumerated using a FACSCalibur flow cytometer (Becton-Dickinson) equipped with an air-cooled argon ion laser (488 nm, 15 mW) and a red-emitting diode (635 nm) used for excitation. The green fluorescence of the FITC-labelled PF2 probe, SYTOX Green, and fluorescent beads were detected by excitation on the FL1 detector, whereas Copyright © 2012 John Wiley & Sons, Ltd.
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Cy3 fluorescence was detected by excitation on the FL2 channel. Commercial FACSFlow (BectonDickinson) was used as the sheath fluid. The impulses from the labelled microbial cells were discriminated from other events, including negative non-stained controls by comparing the dot plots of FL1 (or FL2) and SSC in the experimental samples. All data were collected as pulse-height signals in list mode and were further analysed using CellQuest software (Becton-Dickinson). The concentration of groupspecific cells in the sample was calculated relative to the concentration of reference microspheres and proportional to the number of events identified by the flow cytometer, as follows: Cell concentration ¼ no: of cell events=no: (1) of bead eventsbead concentration
Results and discussion The preliminary investigation showed no significant cell proliferation following the fixation of filamentous yeast samples and the filtration of snailase solution (data not shown). Furthermore, the autofluorescence of snailase was obviously observed on the FL3 and FL4 channels but not on the FL1 and FL2 channels when snailase was added to the sonicated yeast samples (see Supporting information, Figure S1). To avoid disturbance, the FITC and Cy3 fluorescence was therefore chosen and detected on the FL1 or FL2 channel in this study. The snailase digestion performance of filamentous yeast samples was investigated using optical microscopic examination (Figure 1). Generally, the 2% snailase solutions were used to release wall-less fungal protoplasts (Ferenczy et al., 1975), therefore, 2% snailase digestion was also conducted in this study to break filamentous yeast chains into single cells. After 2 h of snailase digestion, intertwined filamentous yeast samples (Figure 1A) were fully disaggregated into abundant rod-shaped yeast single cells (Figure 1B), which were significantly different from the conventional fungal protoplasts that are generally spherical in shape (Bachmann and Bonner, 1959). The enzyme used here did not completely dissolve the wall; it attacked only a minor component of the yeast wall to break the yeast chains into single cells. In many cases, during protoplast preparation the original cell wall was also only partially digested, and even a long-lasting 10% snailase Yeast 2012; 29: 111–117. DOI: 10.1002/yea
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A
B
Figure 1. Microscopic photographs of filamentous yeast samples before (A) and after (B) the 2% snailase digestion (400)
digestion did not solubilize the entire wall (Bachmann and Bonner, 1959). A preliminary investigation showed that a 75 s sonication at 100 W yielded the highest yeast cell counts from filamentous yeast samples, and cell destruction occurred after 75 s (see Supporting information, Figure S2). Therefore, the snailase digestion of the filamentous yeast samples in this study was conducted following the 75 s sonication at 100 W. The sonication–snailase digestion performance of filamentous yeast samples was further validated, based on the forward light-scatter (FSC) frequency histogram from the FCM analysis, with sonicated filamentous yeast samples (75 s, 100 W) as a control (Figure 2). Generally, coupled cells or short yeast chains are larger in size than yeast single cells and produce higher FSC signal intensity. Compared with the sonicated control, the sonication–snailase-digested
Figure 2. Forward-scatter flow-cytometrical histograms of filamentous yeast samples prepared by sonication–snailase digestion (A) and solely sonication treatment (B) Copyright © 2012 John Wiley & Sons, Ltd.
filamentous yeast samples obviously showed superior yeast single-cell liberation. Consistent with the FSC data, more ternate cells or shorter chains were observed in the microscopic images of the sonication–snailase-digested samples than those of the sonicated controls (data not shown). Figure 3 shows the effect of 2% snailase digestion on the green fluorescence emission of yeast cells hybridized with the PF2 probes or stained with SYTOX Green. It includes filamentous yeast samples following the sonication–snailase digestion– FISH–FCM procedure (Figure 3A, 2) with a negative control without FISH (Figure 3A, 1) and another control treated with sonication–snailase digestion–fluorescent staining–FCM procedure (Figure 3A, 3), and following the sonication– FISH–FCM procedure (Figure 3B, 2) with a negative control without FISH (Figure 3B, 1) and another control treated by the sonication–fluorescent staining–FCM procedure (Figure 3B, 3). It appears that the sonication had no significant influence on fluorescence intensity of the PF2 probes (Figure 3B, 2) compared to the negative control (Figure 3B, 1). Moreover, the fluorescence of the dyes was also not quenched by either snailase digestion (Figure 3A, 3) or sonication (Figure 3B, 3). However, the snailase digestion of filamentous yeast cells (Figure 3A, 2) obviously resulted in no fluorescence or a significant decrease in the fluorescence intensity, i.e. a fluorescence-quenching phenomenon, possibly due to an interaction between the snailase and a nucleobase of the PF2 probes. This decrease in fluorescence intensity will result in lower fluorescence signal: noise ratios, which makes it impossible to discriminate microbial cells from background noise (Lenaerts et al., 2007). Additionally, it is suggested that snailase digestion could be combined with Yeast 2012; 29: 111–117. DOI: 10.1002/yea
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Figure 3. Fluorescence expression of filamentous yeast samples following the sonication–snailase digestion–FISH–flow cytometry (FCM) procedure (A, 2) with a negative control without FISH (A, 1) and another control treated with sonication–snailase digestion–fluorescent staining (fluorochromes SYTOX Green)–FISH–FCM procedure (A, 3) and following the sonication–FISH–FCM procedure (B, 2) with a negative control without FISH (B, 1) and another control treated by the sonication–fluorescent staining–FISH–FCM procedure (B, 3)
Number of cells
fluorescent staining to achieve a better FCM analysis for filamentous yeast samples than that achieved solely by sonication in some cases. Subsequently, a series of snailase concentrations were prepared to investigate their effects on the fluorescence expression of hybridized filamentous yeast samples following the combined sonication– snailase digestion–FISH–FCM process (Figure 4). It seems that the fluorescence signal intensity increased significantly when the snailase concentrations were lowered stepwise from 5% to 0.1% (Figure 4). In other words, the target yeast cells could be discriminated from the negative control at a low snailase concentration. However, as shown in Figure 4, not only did the fluorescence intensity
FL1-H
Figure 4. Effect of snailase concentration on the fluorescence expression of filamentous yeast samples following the sonication–snailase digestion–FISH–flow cytometry (FCM) analysis Copyright © 2012 John Wiley & Sons, Ltd.
increase with decreasing snailase concentrations but also the distribution of the fluorescence histograms changed (from 5% to 0.5% a single population was observed, but < 0.5% approximately two or three subpopulations were observed). Therefore, a snailase concentration of 0.5% was used in the subsequent investigations. Finally, the improvement in sonication-based FISH–FCM enumeration performance by 0.5% snailase digestion was further investigated in terms of snailase digestion time or lytic incubation time (0, 1, 2, 3 and 4 h). The snailase digestion performance at 0 h was used as the control without snailase digestion, i.e. the conventional sonicated-based FISH–FCM performance. The effect of snailase digestion time on the enumeration performance was evaluated by a one-way analysis of variance (ANOVA) at p < 0.05, using the SPSS software program (SPSS 10.0, SPSS, Inc.). It appears that the yeast cell yield varied with the duration of lytic incubation (Figure 5). At a snailase digestion time of < 3 h, the maximum number of free total yeast cells amounted to 6.85 109 cells/ml due to the further enzymatic separation of the sonicated filamentous yeast samples, representing a 37% improvement compared with the control (5.01 109 cells/ml). The snailase digestion performance of the sonicated yeast sample makes it possible to prepare more yeast single cells from yeast hyphae by the combined sonication and snailase digestion pretreatment than by the conventional sonication pretreatment. In other words, the snailase partial digestion can significantly (p = 0.00) improve the Yeast 2012; 29: 111–117. DOI: 10.1002/yea
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Concentration of fungal cells (cells × 109/ml-1)
identification and enumeration of specific filamentous yeast species in environmental samples.
Acknowledgements This study was supported by the New Century Excellent Talents in University (Grant No. NECT-11-0044) and the Natural Science Foundation of China (Grant No. 21077011).
Supporting information on the internet Snailase digestion time (h) Figure 5. FISH–flow cytometry (FCM) enumeration performance in filamentous yeast samples prepared by ultrasonication (100 W, 75 s) and snailase digestion (0–4 h) in turn (these data are shown as averages and standard errors; n = 3)
sonication-based FISH–FCM enumeration performance for specific filamentous yeast species in environmental samples. However, a longer lytic digestion time of 4 h led to a marked decrease in yeast cell concentration. Therefore, the snailase lytic time was also a critical factor for further liberating single cells from the sonicated filamentous yeast samples. A similar phenomenon also occurred during the release of fungal protoplasts following enzyme treatment. For example, the maximum number of fungus Trichothecium roseum protoplasts was observed after a 4 h incubation with a lytic enzyme combination, but a prolonged incubation resulted in the steady loss of protoplasts due to lysis (Balasubramanian et al., 2003). Additionally, it is reported that the results of the conventional FISH–FCM were comparable to those of microscopy–FISH; however, the coefficients of variation of the conventional FISH–FCM analyses (ca. 2–5%) were approximately 10 times lower than the corresponding values obtained using microscopy-based FISH (ca. 16–29%) (Vaahtovuo et al., 2005). Likewise, the coefficients of variation of the sonication–snailase digestion–FISH–FCM analyses presented here (6–8%) were also extraordinarily low. The results presented here show that the snailase partial digestion significantly improved sonicationbased FISH–FCM enumeration performance for specific filamentous yeast species in environmental samples, which will facilitate the rapid detection, Copyright © 2012 John Wiley & Sons, Ltd.
The following supporting information may be found in the online version of this article: Figure S1. Autofluorescence detection of snailase on four channels when it was added into the sonicated fungal samples with no snailase digestion as negative control Figure S2. Total fungal cell counts after treatment with sonication (100 W, 15–135 s)
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