STAINING MATERIAL CONTAINING ASTRAZON BLUE FGGL, BASIC BLUE 41 , C.I.11105/11154 FOR IDENTIFICATION OF FILAMENTOUS BACTERIA IN

ACTIVATED SLUDGE OF BIOLOGICAL TREATMENT PLANTS

TECHNICAL FIELD

Invention; It is related with a staining (or dyeing) material containing Astrazon Blue FGGL, Basic Blue 41 , C.1.1 1 105 / 1 1 154, which will be used to determine the morphology of filamentous bacteria in wastewater.

PRIOR ART

Inexpensive water supply and provide high quality water in various sectors are becoming a bigger problem in our age. Human population is increasing and they need more water. Big part of the world is covered with water, but only 3% of this part is fresh water. Freshwater resources become insufficient to meet human needs. Therefore, at least in some sectors, it is considered that we can provide some of our increased water demands by wastewater treatment systems and reuse the effluent waters of these systems.

Biological wastewater treatment systems are the most prominent environmental approaches in recent and are the most commonly used systems. Bacteria are the most used organisms in biological treatment systems, and while they purify the wastewater, other living specimens accompany by them. The main role in the treatment processes is belong bacteria.

It is a very important issue to predict the problem of the system so that the system can provide a desired quality of effluent. If the problems cannot be solved during the operation of the treatment plants, desired quality of effluent cannot be obtained. Microbiological examinations are carried out to find the problem, and it is understood that the species cause of the problem is usually living organisms called "filamentous bacteria". The identification of filamentous bacteria is particularly constitutive for understand the cause of the problem and thus indicates the precautions to be taken as well as what needs to be done to resolve it after the problem has arisen. Today more than thirty types of filamentous bacteria that are found in treatment plants and some of them usually show that system is not in the optimum conditions and they show causes of the problems. In this respect, detection of the difference between the specimens is of great importance.

There are more than one staining materials and other chemical substances in the staining methods used for identification of species, and their solutions must be prepared carefully (e.g. Gram staining). In addition, the staining process must be applied with carefully in a certain order and thus, the species can be determined. Generally, bacteria are stained to obtain information about their microscopic morphology (Shapes, sizes, arrangement of the objects in the cells, the existence and structure of certain cell organelles) and behavior against various dyes. The stain can be defined as a compound carrying chromophore and oxochrome groups linked to a benzene ring (ΟβΗβ). Different colors are formed by substituting different elements and groups instead of H atoms in the benzene ring. Stains are separated into acidic, basic and neutral dyes according to the electric charge of the stain molecule. In bacterial staining, basic dyes are generally used.

Filamentous organisms are difficult to identify by light microscopy and staining methods are used for their identification. The most common staining methods used are Gram and Neisser. These traditional staining methods consist of various steps, which must be applied with care. The contents of their solutions are shown below:

Solutions of gram staining reagent

1 . Part of solution:

Crystal violet), 2g

Ethanol, 95% (vol/vol), 20 ml_

2. Part of solution:

Iodine, 1 .0 g

Potassium iodide, 2.0 g

Distilled water, 300 ml_ 3. Part of solution:

Ethanol, 95% (vol/vol)

4. Part of solution:

Safranin O (Basic Fuchsin) 2.5 g

100 ml 95% Ethanol

Solutions of Neisser staining reagent: 1 . Part of the solution:

Methylene blue 0.1 g

Glacial Acetic acid 5 imL

Ethanol 96% 5 mL

Distilled water 100 mL

2. Part of the solution:

Crystal violet, 10% in 96% ethanol 3.3 mL

Ethanol 96% 6.7 mL

Distilled water 100 mL

3. Part of the solution

Chrysodin Y, 1 % aqueous solution 33.3 mL

Distilled water 100 mL It is possible to examine the morphology of filamentous organisms when the above solutions are used at appropriate concentrations. It is necessary to prepare the solutions carefully and to apply the staining procedure properly. The staining procedures consist of 3-5 steps for both dyes and take time.

It has been reported in the scientific resources that Indian ink can be used to better see the exopolysaccharides (capsules) of bacteria (Richard et al., 2003). In the same study it is reported that Gram and Neisser stains are used especially for identification of filamentous bacteria. It is known that Nocardia is a Gram (+), which is responsible for the formation of foam, and thus is well detected by Gram stain (Richard et al., 2003). It has been reported that the same organism can be examined with Ziehl-Neelsen staining method (Muricy et al., 2014). In another research, Guan et al. (2009) reported that Haloglycomyces albus, a halophilic filamentous bacterium, responds to gram staining (+). Fungi may also be present as activated filamentous filamentous organisms and may cause problems in the sludge of the treatment plant from time to time and their identification is also important. They usually give respond weakly to traditional staining methods. However, Hoch and Galvani (2005) reported using Solophenyl Flavine 7GFE500 and Pontamine Fast Scarlet 4B fluorescent stains to examine their cell walls and septa (cell compartment). Hoch and Galvani (2005) have reported that they can use these stains on at least 18 fungal species. For the staining of Nocardia, Onuma et al. (2006) used Toluidine Blue, Papanicolaou, Silver (Grocott) staining methods. Fourest et al. (2004) reported that Gram, Neisser and Crystal Violet stains were used in the identification of Thiothrix, Type 021 N, Haliscomenobacter hydrossis and Type 0092 from filamentous bacteria in activated sludge. Fischer and Sawers (2013) reported that Streptomyces and other filamentous organisms could be examined by staining with Methylene Blue.

The researches related to the subject area are to be compared with other examples; references of Gram stainers: Eikelboom (2000), Saunders et al., (2003), Wilen et al., (1999), Pandolfi and Pons, (2004), Mamais et al., (1998), Andreasen and Nielsen (1998), Knoop and Kunst (2000), Saunders et al., (1998), Bradford et al., (1998), Westlund et al., (1998), Kaiser et al., (1998), examples of those using Neisser staining; Wilen et al., (1999), Serafim et al., (2002), Hagland et al., (1998), Westlund et al., (1998), Knop and Kunst (1998), and Andreasen and Nielsen (1998), examples of Sudanese Black B users Saunders et al., (2003), Wilen et al., (1999), Pandolfi et al., (2007), examples of using periodic Acid-Schiff method; Carvalho et al., (2007), Gahrton (1964), and Carvalho et al., (2007), examples of using Methylene Blue dye Carvalho et al., (2007), Saunders et al., (2003), Smolders et al., (1994), examples of fluorescent stain DAPI users; Streichan ve et al., (1990), Gunther et al., (2009), He et al., (2008), Majed et al., (2012), Aschar-Sobbi et al., (2008), Kuruda et al., (2002), Kawaharasaki et al., (1999), Andreasen and Nielsen (1998), examples of Nile Blue stain users; Serafim et al., (2002), Betscheider and Jose (2009), Tandoi et al., (1998) and Shennawy et al., (1984) as a case study for those using aniline blue dye.

BRIEF DESCRIPTION OF THE INVENTION

The invention; provides a very good way of detecting the presence of filamentous bacteria, especially the sheath of them, which plays a major role in the identification of them. Although it is almost impossible to detect the existence of sheath by light microscopy, only the filamentous bacteria contacted with these stains are seen easily by the expert examiners and can make the identification of the bacteria in microscope by them. "Attached growth" is also a term for the "formation of single celled organisms adhering to some filamentous bacteria", and it is not visible on every filament. The staining material that we use, Astrazon Blue FGGL, Basic Blue 41 , C.1.1 1 105 / 1 1 154, also shows very clearly how it grows. MEANINGS OF THE FIGURES

Figure 1 . Open Molecular Formula of Astrazon Blue FGGL

Figure 2. Microscopic Image of Sample staining with Astrazon Blue FGGL(a) Figure 3. Microscopic Image of Sample staining with Astrazon Blue FGGL(b) Figure 4. Microscopic Image of Sample staining with Astrazon Blue FGGL(c)

DETAILED DESCRIPTION OF THE INVENTION

The staining material containing Astrazon Blue FGGL, Basic Blue 41 , C.1.1 1 105/ 1 1 154, which is the subject of our invention, is used in dyeing various textile products in the textile industry and it is very efficient to examine the morphology of filamentous bacteria even it is used as a single dye in the solution. The experiments we have done in our laboratories show that the staining method (containing Astrazon Blue FGGL) we have achieved is very simple and takes very short time. According to our opinion, researchers in this area will be able to identify the filamentous bacteria faster and easier and take advantage by this stain (Astrazon Blue FGGL) in the future. The stain we use shows very well the presence of filamentous bacteria, especially the sheath, which plays a major role in identify of filamentous bacteria. It is almost impossible to detect the presence of sheath by light microscopy. However, sheaths of filamentous organisms can easily recognized by the expert examiner when they stained with Astrazon Blue FGGL and thereby enable to identify of the bacterium. Attached growth is also a term for the formation of single- celled organisms adhering to some filamentous bacteria, and attached growth is not visible easily on every sample. Astrazon Blue FGGL also shows very clearly attached growth of bacteria.

Astrazon Blue FGGL, Basic Blue 41 , C.1.1 1 105/1 1 154; the molecular structure of which contains a single azo bond, has the chemical formula C2oH26N ₄ O6S2. It is Bright Blue, like violet powder. The solubility in water is 40 g/L and the aqueous solution is blue. Acrylic staining is possible. Color change is not observed even when staining at a high temperature of 120 ^Q C. It can be used in acrylic fiber, wool, jersey, carpet dyeing. In order to use the Astrazon Blue FGGL for examine the filamentous organisms, it is necessary to prepare the solutions first. The Astrazon Blue FGGL solution can be prepared easily as follows:

Astrazon Blue FGGL 0.8 grams

Distilled Water . 100 mL.

Astrazon Blue FGGL is dissolved at least 0.8 grams of dye in 100 mL distilled water. If the mixture is pour into in a beaker and heated in a heated magnetic stirrer at 50-60 ^Q C and dye is slowly added on it, granulation of dye is prevented and the dye is better solved. A clean slide is taken for staining and 1 drop of the activated sludge biomass to be instilled onto the slide by a pipet. Then, with the aid of a loop, the spreading of one or two drop of activated sludge is provided over the entire lamella at room temperature (smear). Fixing process is performed after the drying process is over (this is obligatory during all bacteria staining). After fixation, the slide is placed on a staining cuvvette and a certain amount of dye solution is drawn from the staining material solution containing Astrazon Blue FGGL by syringe or pipette, and the entire surface of the slide is covered with this stain and waited for 1 minute. Then the surface is washed with distilled water by holding the slide with a pliers. It is then allowed to dry on a paper towel leaving the surface up. Then identifications can be made in light microscope.