Abstract: A simple and rapid agarose immobilization method of microbial cells was developed to study the behavior of a single living cell in the changeable environment. Using this technique, process of apotptosis of a single microbial cell and subcellular particles’ movements were recorded real-timely. The proliferation process of Pseudomonas aeruginosa was also pursued.
INTRODUCTION
Single microbial cell assays were applied extensively in various researches (Pogliano et al., 1995; Deich et al., 2004; Gato and Means, 2011; Nasseri et al., 2011; El-Ahmady et al., 2011), such as the subcellular location and cell behaviors, in which cell immobilization played an important role. Though cells had been fixed by using many methods such as with amino-silanes and flagellin antibodies (Cluzel et al., 2000) or on a poly L-lysine-treated coverslip (Pogliano et al., 1999), long-range real-time record of single microbial cell’s behaviors and the movements of subcellular structures in the adjustable environment was still challenging. Moreover, due to the small size and quick proliferation of microbial cells, many techniques performed in cases of a single animal or plant cell (Rahmat et al., 2006; Huang, 2006; Rajendran et al., 2008; Khorshid et al., 2011) were not good to the living single microbial cell (Kehr, 2003).
METHODS AND RESULTS
To realize those characteristics including long-range observations, real-time records, adjustable environment around microbial cells, we designed a square well in the center of a glass slide and immobilized the microbial cell in agarose gel at a low concentrations (Fig. 1a,b).
| Fig. 1(a-b): | Schematic view of the experimental apparatus. (a) We employed an inverted Olympus microscope to perform the research on single microbial cell. The microbial cell was immobilized in the agarose gel on a platform. An ICCD camera was used to record the movement of a bacterium immobilized in agarose gel and (b) The platform, on which the cell was immobilized with agarose gel |
The behaviors of cell were monitored and recorded by inverted Olympus microscope (FM, Olympus IX-70) with an oil immersion objective (Olympus, 100x, N.A.=1.35) and a U-MWB filter set (Olympus, 450-480/500/515 nm) and recorded by an ICCD (intensive charge-coupled device) camera simultaneously.
In initiation of an experiment, Saccharomyces cerevisiae cells were pre-cultured in YPD (1% yeast extract; 2% tryptone; 2% glucose, Sigma) at 28°C and 220 rpm overnight. 50 μL of the resulting cell suspension was inoculated into 5 mL of YPD and cultivated for an additional 1 h. One milliliter of 1% agarose gel were mixed with 1 mL of the yeast cell YPD solution/culture when the temperature of agarose gel was cooled to 40°C. The mixture was transferred into the well and covered with a microscope cover slide (Esco 24x40 No.1 thickness, ERIE SCIENTIFIC COMPANY, Portsmouth, NH, USA).
| Fig. 2: | Dynamic investigation on one yeast cell treated by H2O2. Yeast cell stained with DAPI and viewed by inverted Olympus microscope continuously. (A1-2) yeast cell immobilized in agarose gel in the first hour of investigation. (B1-2) After 1 h, the yeast cell was treated with H2O2 for about 70 min (C1-2) The same cell treated with H2O2 for about 100 min. (D1-2) The cell treated with H2O2 for a total of about 140 min. The images show the nuclear of the cell was desegregating and the body of the cell was shrinking when treated with H2O2 |
After it had been coagulated for 20 min, the cover glass was removed and 1% DAPI (4,6-diaminidino-2-phenylindole, Sigma) was dropped on the agarose gel. Then the slide was placed in a dark box for 30 min. The gel was covered again before view.
To monitor process of apoptosis, yeast cells were embedded in 0.5% agarose gel and plated on the slide we designed. One percent H2O2 was dropped on the gel to induce apoptosis (Fig. 2a). Images were collected every 1 h for a total of 4 h. The images show clearly that the nuclear of the cell was disaggregating and the cell body was shrinking after H2O2 was applied (Fig. 2b-d). In this case, it is feasible to technician to change the culture environment of the observed cell repeatedly by dropping media or any solution on the gel. The media or the solution can also be removed by being adsorbed by filter paper. It is noteworthy that the humidity and the concentration of the gel should be carefully kept during experiment.
To perform the real-time observation on movement of subcellular structures, a similar method was employed. In detail, yeast cells were immobilized on the slide with 1% agarose after cultured. Movement of subcellular structures’ were monitored and recorded with the inverted microscope. As a result, an object was discovered in the about 10% of yeast cells and its movements were recorded in a real-time manner. It is clear that the size of the object was bigger than mitochondria’s in photos (Fig. 3a) and it can be stained by DAPI (Fig. 3b). In addition, the object can move inside the vacuole as well as outside cells. Unlike the techniques such as Pendent drop and the microchannel (Xu et al., 2003; Lim et al., 2010; Luo et al., 2008). Our method was successfully used to record the dynamic process of subcellular structures. We believe that this technique would also make it easy to monitor the macromolecular movement within living cells. Moreover, life cycle of microbial cell won’t be disturbed by the procedures of our method. P. aeruginosa PAO1 cells were cultured in LB-broth medium containing 1% tryptone, 0.5% yeast extract and 1% NaCl with shaking at 150 rpm and 28°C. The cells were then inoculated and cultured for additional 1 h. The cell solution was immobilized with 0.3% agarose gel and observed at 28°C with inverted Olympus microscope for 40 min. Four of the images were selected to show the progress of proliferation of a single P. aeruginosa PAO 1 cell (Fig. 4). Different from other methods (Korobkova et al., 2004; Pogliano et al., 2001), this method has little effect on physiological behaviors of living cell, such as division, sporulation and proliferation. The entire cell cycle can thus be monitored and recorded.
Agar has long been used as a common material to immobilize the microbial cells (Perrot et al., 1998; Khattar et al., 1999).
| Fig. 3: | The movement of an object in a yeast cell. A yeast cell was stained with DAPI after immobilized with 1% agarose gel. Images were collected every 50 msec for a total of 5 sec. And 4 out of 100 photos were selected. (A) A cell was observed under bright field (B) Another cell was viewed with fluorescence. The results suggest, there was a moving object in the cell vacuole since the body of the cell was fixed |
| Fig. 4(a-d): | Dynamic investigation on the proliferation of P. aeruginosa PAO 1. The cells of P. aeruginosa PAO 1 were immobilized with 0.3% agarose gel. The photos were taken every 1 min for 40 min. (a) The photos of 30, (b) 31, (c) 32 and (d) 33 min were shown which recorded the dynamic process of the proliferation of the cell of P. aeruginosa PAO 1. The result suggests the bacterial cell can move freely in the low concentration of agarose |
CONCLUSIONS
In these experiments, higher concentration of agar restricted movements of microbial cells. Most of studies focus on population of bacteria or eukaryotic cells. Our method can be used not only to monitor real-time responses of a microbial cell toward change of environment, but also to offer a quick and simple way for investigating movement of the substance/structure within a living cell. It can also be applied to record cellular phenotypes during cell cycle detailedly.
LIMITATIONS AND RECOMMENDATIONS
The technique we mentioned here has some limitations. For example, the device that we used to monitor cell behavior or particle movement around cell is mechanically simple. It has not been combined with any robot or automatic. Hence, we have to re-localize cell of interest after we change the culture environment of the cell. But the simple device can be produced, we believe, in most of labs over the world. The technique is also feasible to almost all biological labs. Additionally, in our experiments, the performance of single cell was not consistent all along, which might be due to difference between individual cells. So repeat of experiment was strongly suggested to obtain reliable result. Although analyses and operations on single cell remain challenging, we believe experiments based on this technique can provide useful information about single-cell microbiology or medical science.