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New microorganism isolation techniques with emphasis on laser printing
2. New Isolation Methods surrounded by a ring of bacteria were formed. The ability
to manipulate hundreds of Pseudomonas aeruginosa
Nowadays, a wide variety of methods is used to expose new, bacteria simultaneously in two-dimensional (2D) and 3D
previously uncultivated microorganisms . In particular, arrays was also demonstrated. The method of holographic
[8]
new nutrient media containing specific substances are optical trapping is very accurate but technically difficult
used; cultivation is carried out under different physical to perform.
and chemical conditions of the environment, such as In the study of Rowan et al. , the method of heterogeneous
[36]
atmospheric composition, temperature, and pH. [8,25]
Furthermore, applications of diluted nutrient media in functionalization of surfaces is proposed, which is a four-
combination with long incubation periods [25,26] , as well step lithographic process based on microcontact printing
as, joint cultivation of microorganisms of different of organic monolayers, implantation of hyperbranched
species, have been studied [1,27] . In recent years, some polymer, and its further functionalization. As a result,
structures, on which the directed inoculation of bacterial
fundamentally new methods of cultivation based on the cells is carried out, are obtained. The investigations of
placement of microorganisms in the natural environment
without the usage of nutrient media have been developed. cell survival have shown that cells remain viable on the
Such methods utilize diffusion chambers [28,29] and polymer obtained structured surfaces. Large isolates of bacteria
coatings in which microbial cells are placed. In this containing 18 ± 5 bacteria and small isolates containing
[30]
case, microorganisms receive all necessary nutrients 2 ± 1 bacteria were obtained. According to this paper, the
from the natural environment, remaining isolated from it. demonstrated method can be used for high-throughput
The allocation of a new antibiotic producer (teixobactin) screening and biosensing. However, it is difficult to
using such methods is considered as an important combine the heterogeneous functionalization of surfaces
[36]
achievement . Another approach includes simultaneous using this method with routine biological research and
[14]
cultivation and screening of tens and hundreds of thousands conditions of microorganism cultivation (temperature,
of bacterial microcolonies on porous polymer or ceramic pH, nutrients, etc.). It is necessary to note that the task of
isolation chips [31,32] . For the cultivation of “unculturable” finding simple ways to provide high resolution of living
microorganisms, methods allowing isolation of single bacteria arrays with the opportunity of various biological
cells from natural environments can also be used. Among studies was solved in some publications. The approaches
these methods, the most popular is based on the dilution proposed in these works are, in fact, the harbingers of 3D
of microorganism suspension, flow cytometry and cell printing.
[37]
sorting, laser microdissection, compartmentalization, In a study of Weibel et al. , the technique of living
and application of micromanipulators [33,34] . In addition bacteria stamping on agarose plates was proposed.
to the cultivation of previously uncultivated species of Bacteria arrays were printed (the size of a single spot
2
microorganisms, the isolation of single microbial cells with bacteria >200 µm) in the area of up to 50 cm .
is necessary for the study of cell physiology, interactions Polydimethylsiloxane (PDMS) stamps were produced
between cells, as well as for the search of new metabolites, with the help of photolithographic technique. The
such as antibiotics and enzymes . achieved minimum size of the print protrusion was
[34]
Modern scientific and technological progress provides 190 µm at the height of 140 µm, which, however, is far
many opportunities in terms of the development of novel from the size required to separate bacteria. This method
methods for cultivation, isolation, manipulation, and is fast, reproducible, and convenient and can be used
study of individual bacterial cells and their consortia. New to control the pattern, spacing, and orientation between
[38]
approaches may speed up the process of working with colonies of different bacteria. In Xu et al. study ,
microorganisms significantly and allow carrying out their living bacteria arrays with cellular resolution were
more complete and comprehensive studies. It is necessary printed on agarose substrate using elastomeric (PDMS)
to note that so far very few methods have been proposed stamps with a high aspect ratio, obtained by the reverse
for positioning of bacteria arrays with micrometer in situ lithography (RISL) method. Figure 1 shows
accuracy. In Akselrod et al. , three-dimensional the advantages of the RISL method over the standard
[35]
(3D) networks of living cells in hydrogel were formed ultraviolet photolithography. The only limitation of the
without loss of their viability using arrays of multiplexed RISL technology is the protrusion diameter, which can
holographic optical traps (tweezers) with unprecedented hardly be <1 µm due to the optical diffraction limit.
accuracy (<400 nm). To form optical traps, two lasers The method of microcontact printing of bacteria arrays
were used: Ar laser (20 W, 514 nm wavelength) and works as follows: The drop of Escherichia coli in the
+
continuous wave Ti: Sapphire laser, tunable in the range culture medium LB is deposited on agarose gel (3 weight
of λ = 850–900 nm, as well as a combination of two [wt]% in LB) and, on the agarose substrate, a monolayer
diffraction elements, combined with different lenses in an of bacteria is formed (the liquid is absorbed by agarose
inverted optical microscope. Networks of 3T3 fibroblasts gel). Then, the PDMS stamp contacts with a monolayer of

2 International Journal of Bioprinting (2019)–Volume 5, Issue 1