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Year : 2012  |  Volume : 1  |  Issue : 1  |  Page : 53-56

Mycobacterium under AFM tip: Advantages of polyelectrolyte modified substrate

1 Institute for Chemistry of New Materials, National Academy of Sciences, F. Skoriny 36, 220141 Minsk; The Republican Research and Practical Centre for Epidemiology and Microbiology, Filimonova 23, Minsk, Belarus
2 The Republican Research and Practical Centre for Epidemiology and Microbiology, Filimonova 23, Minsk, Belarus

Date of Web Publication28-Feb-2017

Correspondence Address:
Genady Zhavnerko
Institute for Chemistry of New Materials, Belarus National Academy of Sciences, F. Skoriny 36, 220141 Minsk
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.ijmyco.2012.01.009

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Immobilization of bacterial cells onto surfaces is critical for imaging with an atomic force microscope. In this paper, polyethylenimine (PEI) coated silicon plates are shown to be suitable for immobilizing and imaging Mycobacterium tuberculosis.

Keywords: Mycobacterium tuberculosis, Bacteria, Mutated forms, AFM

How to cite this article:
Zhavnerko G, Poleschuyk NN. Mycobacterium under AFM tip: Advantages of polyelectrolyte modified substrate. Int J Mycobacteriol 2012;1:53-6

How to cite this URL:
Zhavnerko G, Poleschuyk NN. Mycobacterium under AFM tip: Advantages of polyelectrolyte modified substrate. Int J Mycobacteriol [serial online] 2012 [cited 2021 May 13];1:53-6. Available from: https://www.ijmyco.org/text.asp?2012/1/1/53/201201

  Introduction Top

Atomic force microscopy (AFM) is one of the most effective tools for studying structure and surface properties of biological samples [1],[2],[3],[4],[5]. AFM makes possible the ability to visualize the morphology of a surface or stages of cell evolution; it may probe the separate places of a surface and can estimate cell wall elasticity and rigidity. Overall, AFM is a high-resolution imaging tool that mechanically probes a surface [6]. Basically, the successful imaging depends on the probes and the quality of the sample. The probes may have various sizes, shapes and can be made from different materials. In the contact-mode, the flexible cantilever of the ATM (normal spring constants are varied between 0.1 and 0.6N/m) works as a nanometric spring, which allows the probe to measure the interaction forces on the surface. For tapping mode, the tips are made from crystalline silicon (spring constant of the order 70N/m), which oscillate in its natural frequency. In addition to the probes and the sample preparation, the solid substrate or immobilization of the sample is very important. For bacterial cells, this is often challenging, since they have a well-defined shape and have no tendency to spread over substrates. As a result, the contact area between the cell and substrate become very small; this might lead to cell detachment during scanning. To resolve these problems, pre-treatments of samples or substrates were introduced. Later on, experiments using pre-treatment of samples (i.e., air-drying and chemical fixation) showed the denaturation of the surface molecules [7], whereas, the pre-treatment of substrates seem to yield good results. Doktycz et al. [8] described stable immobilization techniques for gram-positive and gram-negative bacteria. They showed that a gelatin coated mica surface was superior to a poly-l-lysine coated surface. In the present study, the effect of various immobilization techniques was investigated for better imaging of the Mycobacterium tuberculosis (MTB). Mycobacteriums are classified as gram-positive species, but they have special characteristic that differentiate them from other gram-positive bacterial species. Recently, a heterogeneous population for M. tuberculosis (rod and round shape TB bacilli) was proposed [9],[10],[11]. Therefore, it becomes necessary to investigate the most suitable substrate that successfully differentiates various shapes of TB bacilli. Generally, it is accepted that bacterial shape would affect the outcome of mechanical immobilization. For this purpose, M. tuberculosis were analyzed after chemical pretreatment of substrate by polyethylenimine (PEI) and polysodium-4-styrenesulfonate (PSS). The results were compared with the commonly used approach of bacteria adsorption onto a porous surface using Millipore filter membrane.

  Materials and methods Top

Bacterial cultures

A loop of Lowenstein–Jensen culture media was inoculated into Middle Brook 7H9 broth (Difco, Franklin Lakes, NJ, USA) supplemented with 0.2% glycerol and 10% Middle Brook OADC enrichment (Difco). Cells at an optical density (OD) of 0.6 at 600nm were used for further experiments. These cells were first centrifuged at 800rpm for 5min., and then the supernatant was adjusted to an OD of 580nm, corresponding to 6.3 × 107 colony-forming units of M. tuberculosis per milliliter (ml). Ten milliliters of this supernatant were subjected to acid-fast bacilli and gram staining. Nontuberculous mycobacterial contamination was checked by culturing 100ml of supernatant on 7H11 agar plates [9],[11].

Samples preparation

To image mycobacteria by AFM, the cells were immobilized by mechanical trapping onto isopore polycarbonate membrane (Millipore), with pore size 0.2 micron that is smaller than normal MTB cell size. After filtering a concentrated cell suspension, the filter was gently rinsed with deionised water, carefully cut and attached to a steel sample puck (Veeco Metrology Group) using a small piece of adhesive tape. Silicon plates from wafers<100>orientation were modified by alternative adsorption of 1mg/ml polyethylenimine (PEI) and polysodium-4-styrenesulfonate (PSS) solutions during 15min. treatment. The treated surfaces were rinsed vigorously with Milli-Q water after each step and finally the samples were dried with N2. The outermost layer becomes «negatively» or «positively» charged after the last treatment with PSS or PEI, respectively. The polyelectrolyte film assembly was always started with the first layer positively charged on silicon that was negatively charged as a result of the cleaning procedure (H2O2:NH4OH:H20 mixture) [10].

Microscopic observations

AFM images were recorded in contact mode using Nanoscope 3-D Multimode AFM (Veeco, Santa Barbara, CA, USA). The device was equipped with a <E> calibrated scanner using the manufacturer's grating. AFM images were obtained by both contact mode (CM) and tapping mode (TM). One-hundred and 200mm nanoprobe cantilevers (standard spring constants ranging from 0.12 to 0.52N/m) with oxide-sharpened Si3N4 integral tips (Veeco NanoProbe Tips NP-20) were used for CM mode. Silicon cantilevers with resonance frequency ~260kHz (Veeco NanoProbe Tips RTESP) were used for TM regime.

  Results and discussion Top

Initial studies were carried out by immobilization of bacteria by spreading 100μl of bacterial suspension onto Millipore filter membrane. Millipore filters with a pore diameter of 0.22μm is usually used as a substrate due to the sufficiently large size of mycobacterium [9]. As shown in [Figure 1], some of the TB bacilli can pass through the Millipore membrane. In addition, a considerable number of TB bacilli were lost while rinsing the filter. Therefore, the Millipore filter may not be a suitable substrate for studying M. tuberculosis. To increase immobilization in M. tuberculosis, the silicon surface modified with polyelectrolyte film was investigated. Both a positively charged layer of PEI and a negatively charged PSS surface were used. The best results of immobilization (the presence of single bacteria and their shape) were obtained on the surface of silicon with a polyethylenimine sub-layer. Overall, the proteins from culture media and waste discharges from the bacteria itself was not attached in positively charged substrate; as a result, the background of the image was very clean. In a negatively charged substrate, the background was contaminated by waste by products ([Figure 2] and [Figure 3]). Another advantage of PEI silicon-coated plate was the high resolution of images without distortion ([Figure 4]). It was further demonstrated that adhesion of bacteria to the surface of the positively charged polyethylenimine film is different in different growth condition media.
Figure 1: AFM-image (a) of the surface area of filter for sterilizing liquid after culture filtration and (b) MBT deformation due to adsorption into pore of a membrane.

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Figure 2: Shows immobilization of TB bacilli on polyethylenimine (PEI) coated silicon plates.

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Figure 3: Shows immobilization of TB bacilli on polysodium-4-styrenesulfonate (PSS) coated silicon membrane.

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Figure 4: Shows high quality (without distortion) of Mycobacterium tuberculosis image on PEI silicon coated plate.

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  Conclusion Top

The polyethylenimine substrate is suitable for M. tuberculosis imaging.

  References Top

G. Binning, C.F. Quate, C. Gerber, Atomic force microscopy, Phys. Rev. Lett. 56 (1986) 930–933.  Back to cited text no. 1
A.V. Bolshakova, O.I. Kiselyova, I.V. Yaminsky, Microbial surfaces investigated using atomic force microscopy, Biotechnol. Prog. 20 (2004) 1615–1622.  Back to cited text no. 2
G. Francius, O. Domenech, M.P. Mingeot-Leclercq, Y.F. Dufrene, Direct observation of Staphylococcus aureus cell wall digestion by lysostaphin, J. Bacteriol. 190 (2008) 7904–7909.  Back to cited text no. 3
A. Touhami, M.H. Jericho, J.M. Boyd, T.J. Beveridge, Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy, J. Bacteriol. 188 (2006) 370–377.  Back to cited text no. 4
C. Verbelen, V. Dupres, F.D. Menozzi, D. Raze, A.R. Baulard, P. Hols, et al, Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy, FEMS Microbiol. Lett. 264 (2006) 192–197.  Back to cited text no. 5
A.L. Weisenhorn, P.K. Hansma, T.R. Albrecht, C.F. Quate, Forces in atomic force microscopy in air and water, Appl. Phys. Lett. 54 (1989) 2651–2653.  Back to cited text no. 6
N.A. Burnham, R.J. Colton, Measuring the nanomechanical properties and surface forces of materials using an atomic force microscope, J. Vac. Sci. Technol. A 7 (4) (1989) 2906–2913.  Back to cited text no. 7
M.J. Doktycz, C.J. Sullivan, P.R. Hoyt, D.A. Pelletier, S. Wu, D.P. Allison, AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces, Ultramicroscopy 97 (2003) 209–216.  Back to cited text no. 8
A.A. Velayatei, M.R. Masjedi, M.A. Merza, G.K. Zhavnerko, P. Tabarsei, L.P. Titov, et al, New insight into extremely drugresistant tuberculosis: using atomic force microscopy, Eur. Respir. J. 36 (2010) 1–3.  Back to cited text no. 9
P. Farnia, M.R. Masjedi, M.A. Merza, P. Tabarsei, G.K. Zhavnerko, T.A. Ibrahim, et al, Growth and cell-division in extensive (XDR) and extremely drug resistant (XXDR) tuberculosis strains: transmission and atomic force observation, Int. J. Clin. Exp. Med. 3 (2010) 320–326.  Back to cited text no. 10
A.A. Velayati, P.F. Farnia, M.R. Masjedi, G.K. Zhavnerko, M.A. Merza, J. Ghanavi, et al, Sequential adaptation in latent tuberculosis bacilli: observation by atomic force microscopy (AFM), Int. J. Clin. Exp. Med. 4 (3) (2011) 193–199.  Back to cited text no. 11


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

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