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Improving Bacterial Detection Processes using Nano-Fiber Nonwoven Fabrics: A Preliminary Study.

 

Majdy Fahd Madanyyah^1.*,  Dr. Eng. Hani Amasha^2,1

 Dr. Eng. Basel Younes³,  Dr. Eng. Baraa Noman^1,2.

 

1.     Biomedical Engineering Department, Faculty of Mechanical and Electrical Engineering, University of Tishreen.

2.     Biomedical Engineering Department, Faculty of Mechanical and Electrical Engineering, Damascus University.

3.     Faculty of Engineering, Private University of Kalamoon; Faculty of Mechanical and Electrical Engineering University of Damascus.

* Corresponding author's email: Majdy77madanyyah@gmail.com.

 

 

Abstract

Keywords

Introduction

1.  Methods and Materials:

2.     Analyses the results:

3.     Conclusion:

Acknowledgement

References

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Abstract

 

The Polycaprolactone nano-fiber nonwoven fabric (nanonet) was prepared and provided by a local supplier.  Transplant media have been prepared as test materials. The nanonet had been placed in the mold and the test material put on top of it, thus, the test environment has become ready. Urinary samples with bacterial inflammation were taken from human urine (and sometimes from the bronchi with bacterial inflammation), then, placed in the appropriate medium (positive or negative gram), after which the mold has been inverted upside down in the incubator for 24 hours and the microbial growth was monitored until it was complete. When taking these samples from patients, the germs are usually very small and their type unspecified, therefore, the doctor cannot correctly treat the patient until the type is specified. As a result, these samples were taken and incubated in testing media until they grow enough so that their type becomes known to the doctor, in which case, he can prescribe the cure medicine to those germs. The utilization of nanotechnology has yielded good results for microbial growth and less time than those with no nano nets. Soon, the production of better nano-scale nonwoven fabric under more precise and well-controlled conditions will shorten even more the period required for full microbial growth and, hence, no need to wait until the next day to reach results.

 

 

 

 

 

Keywords:

 

Nanotechnology, electrospinning process - germ testing media - poly caprolactone – nano nets.

 

 

 

 

 

1.     Introduction:

Nanoscience is defined as the study of the basic principles of molecules and compounds not measuring more than 100 nanometers.

Nanotechnology relies on the principle of capturing, manipulating, and moving micro-atoms of any material from their original positions to other locations, and then combining them with other material atoms to form a crystalline network to obtain high-performance nanomaterials [1].

Nanomaterials are a distinct category of advanced materials that can be produced so that the scales of their dimensions or the dimensions of their inner pellets range from 1 nm to 100 nm, and the small size of these materials has resulted in their qualities being different from larger ones (greater than 100 nm). These materials are the construction base for the 21st century and an important pillar of this century's techniques. Nanomaterials vary by source (organic or inorganic) and vary by percentages (from natural to manufactured) [2].

 

1.1: Electro-spinning process

 

The electro-spinning process is a direct technique to produce microfibers and non-woven (micro- or Nanoscale) continuous nonwoven samples of a polymer solution using static electrical forces.

The spun fibers are synthetic or natural polymers, and the polymers can be mixed with nanoparticles (metals or ceramics). Individual fibers can be produced, or random or regular non-woven fabric can be produced that place fibers.

The electrospinning process enables the acquisition of nonwoven fabric with a specific structure (determining the size of the network's pores by adjusting the diameter of the fiber and the thickness of the nonwoven samples). The electro-spinning process can be generally divided into four stages [3]:

 

·         Charging the liquid drop and forming a Taylor cone.

·         The charged jet extends along a straight line.

·         Jet diameter decreases with the presence of the electric field and increases electrical bending disorder.

·         Sclerosis and a fiber-shaped jet collection on the grounded complex.

 

A wide spectrum of polymers is used in electrospinning to produce micro-fibers or nano-fibers so that they fit a lot of applications. The spectrum of these polymers extends from those of natural origin to artificial or a combination [4].

So far, nano-fibers and microfibers have been produced from more than 200 polymers, and whenever new applications for these fibers emerge, suitable polymers or mixtures of polymers have been searched for to achieve this application, and then adjust the appropriate conditions to produce fibers from them. In general, live weaving engineering, separation membranes, and medical applications are among the most important targets for which fiber is produced by the electrospinning method [5].

Natural polymers have shown to be more suitable for uses related to weaving engineering and pharmaceutical applications because they are biocompatible and do not cause immunopathy in vivo compared to synthetic polymers.

One of the most used natural polymers in electric spinning is collagen, keto zyme, polymer poly-lactic acid, and polycaprolactone [6].

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.2: Factors affecting the electro-spinning process

Factors affecting the electrospinning process are divided into three types: polymeric solution factors, technological factors (technical factors), and engulfing medium factors.

Polymeric solution coefficients include concentration or viscosity, electrical conveyance, molecular weight, and surface tension, while technological process coefficients include: the applied high voltage value responsible for the generation of the electric field and the distance from the injector's metal head to the collector, the collector shape and the polymeric solution flow rate. Each of the previous coefficients significantly affects the form and structure of the resulting fibers, so it is necessary to find a proper relationship between all those coefficients to get the fibers with the right diameter and structure. The engulfing coefficients affecting the electrospinning process are the relative temperature and humidity in which the process takes place [7].

 

1.3: Solvents for the electro-spinning process

 

The solvent type used in the preparation of polymeric solution significantly affects the conditions of electrospinning and the quality and structure of the resulting fiber. The most important characteristics of a solvent used in electro-spinning fiber are volatility speed, high saturation pressure, low boiling temperature, and the ability to achieve homogeneity and compatibility among all components of the polymeric solution when there is more than polymer or when adding some salts to improve the electrospinning process. In many recent studies, the most important characteristics of the solvent used for electrospinning have been found to be surface tension, electric tolerance constant, saturation pressure, and boiling temperature [8].

 

1.4: Germ Testing media

 

There are several types of media, three of which will be addressed in this research [9]:

A.    McConkey Agar's medium allows the growth of gram-negative bacteria.

B.     The EMB medium allows the growth of gram-negative bacteria.

C.     The blood glue medium allows the growth of gram-positive bacteria.

 

A.   MacConkey Agar's medium-growing gram-negative bacteria:

 

It is the medium of bacterial and selective cultivation that only develops gram-negative bacterial species, whereas gram-negative organisms are more distinct based on lactose metabolism. This MacConkey Agar media results from lactose fermentation and gives organic acids, especially lactic acid, reducing the pH of agar. MacConkey has a pH that turns pink under acidic conditions, so the gram-negative lactose (lactose fermentation) will form pink colonies, while non-lactose ferments will form non-transparent white colonies. The main ingredients of McConkey's media include violet crystal dye, bile salts, lactose salts, and neutral red which all together stop the growth of gram-positive bacteria, so it only allows gram-negative bacterial species to form colonies on it [10].

 

B.   The EMB medium for growing gram-negative bacteria

 

EMB stands for Eosin Methylene Blue and is a differential bacterial medium, slightly inhibiting the growth of gram-positive bacteria and providing a color indicator that distinguishes between organisms that brew lactose (for example E. coli), this medium was originally created by Holt Harris and Teague and was modified by Levin. EMB is recommended as a means of isolating and distinguishing gram-negative intestinal bacteria from clinical and non-clinical samples of lactose in the medium. Also, it helps to isolate and differentiate fermented intestinal Bacilli of lactose and non-lactose and serves to distinguish gram-positive bacteria from gram-negative bacteria, and helps isolate and differentiate intestinal and gram-negative bacilli [11].

 

C.   The blood glue medium for growing of gram-positive bacteria:

 

Blood agar (BA) is a fertilizing medium used to grow bacteria or microbes that do not grow easily, these bacteria are called "sensitive" because they require a special and fertilized dietary environment compared to routine bacteria, blood agars are used in cultivation of a wide range of pathogens especially those that are difficult to grow such as Hemorrhagic, Streptococcus pneumoniae, and it is a differential means to detect  Hemolysis (Destruction of red blood cells) by toxins of cells secreted by certain bacteria, such as certain strains of bacillus, streptococci, intestinal macrophages, staphylococci and pneumococci [12].

 

 

1.5: Preparing nanonets:

  Polycaprolactone (PCL) was dissolved in (chloro + acetone) Solvent at 8% concentration. Electro spinning instrument contains a pair of electrodes, a high-voltage source (8kV), a control unit for producing nonwoven fabric (Figure 1).

 Figure )1 (Rotary electro-spinning device [13]

Nozzle: nozzle is black and caliber 25. The nozzle is far from the assembler: it is 5 cm away, so as to help control the diameter and structure of the fiber produced, and it allows sufficient time for the fiber to recover well, dispose of the solvent and stiffen before reaching the assembler.

Electro spinning is usually performed at temperatures close to the temperature at which the polymeric solution (25 °C) was prepared to maintain the same value of viscosity imported during the spinning process.

Moisture: (60-70%) to impede and slow down the evaporation of the solvent from the fiber rushing towards the assembler, and reaches it non-rigid.

 

 

 

 

1.6: Electrical incubator:

 

For the incubation of agricultural media (with a temperature of 37 °C for most germs) in which the planting dishes are placed in an inverted position to prevent the condensation of water vapor on the medium and prevent the vandalism of colonies.

 

 

 

 

2.  Methods and Materials:

 

The nanonet is placed in the middle, then, the test material is placed on it, and the sample was planted on it. The medium is placed in the incubator. The samples are urinary samples as shown in figure 2

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 Figure (2) put the grid in the middle

 

Two different samples were tested in three media:

1.      McConkey Agar's medium allowing the growing of gram-negative bacteria.

2.      The EMB medium allowing the growing of gram-negative bacteria.

3.      The blood glue medium allowing the growing of gram-positive bacteria.

 

In the EMB medium, a sample of E-coli (gram-negative bacilli) (eco-intestinal bacilli) was implanted, and another sample of the same type was grown in another medium that did not contain a net, and the two media were inverted and embraced within the incubator for 24 hours at a temperature of 37 °C. After 18 hours it was observed that the microbial growth had been complete on the medium containing a net and gave a clear form to that growth (Figure3).

 

 

  Figure (3) E-Coli sample growth in

                    EMB medium

 

In the McConkey Agar negative gram medium, the same sample was cultivated. In the same way, another sample of the same type was planted in another medium that did not contain a net under the same conditions and placed inverted upside down in an incubator for 24 hours at a temperature of 37°C. It was observed that the microbial growth was also completed at 18 hours in the medium containing the net (figure 4).

 

    Figure (4) E-Coli sample growth in

             McConkey agar medium

 

 

In the medium of the blood glue, a staphylococcus sample was implanted. Another sample of the same type was grown in another medium that did not contain a net.

 

The two bodies were inverted upside down and embraced within the incubator for 24 hours at a temperature of 37 degrees Celsius. Nearly 19 hours later, it was observed in the network medium that germ growth had been completed and given a clear form to that growth (Figure 5).

 

      Figure (5) staphylococcal sample

        Growth in blood glue medium

 

 

 

 

 

3.     Analyses the results:

 

Table (1) shows the various samples. If there were too many colonies on the plate, they can work together and become indistinguishable as sole colonies in which case the plate is called stacked or too many to count. Usually, the number of colonies lies  between 30 and 300 colonies on the plate, where, it would be difficult to count when there are more than 300 colonies, or very small colonies of sample size less than 30.  To provide accurate representation of the original sample population, the number of colonies is the number of colonial formation units representing the number of microorganisms per ml.  When comparing the growth of microbial colonies in nanonets with those in which the nanonet is absent (Figure (6)) found that growth in web-based media was dense (thick) and very obvious. It means that germs are strong because growth has given the characteristics of the germ, which is represented by metal glitter very clearly, and the fermentation shown in the E-Coli germs in McConkey Agar medium indicates the visibility of the germ's growth. Table 2 shows the results obtained.

 

 

Table (1) Samples setup and medium for each

Samples		Medium Used
E-Coli	Gram-Negative Bacillus	Mcconkey Agar And EMB
Staphylococcus	Gram-Positive Corpses	Blood Glue

 

 

Table (2):  Shows the duration in hours for each case with or without a nanonet.

Duration Time (Hours)	McConkey Agar		EMB	Blood Glue
	Without a nanonet	22	22	23
	With a nanonet	18	18	19
Growth Of Germ Colonies		Growth is Clear and Accurate	Growth is Clear and Accurate	Growth is Clear and Accurate

 

 

 

 

                                          Figure (6) the length of time until bacterial growth is complete