**2. Analysis of single bacterial isolates for PHA production using staining methods**

Two types of samples were collected for this study. The sludge samples were collected from the Petersfield Southern Water Treatment plant (Hampshire, UK). The three compost samples were collected from (1) a graveyard, (2) food-based waste and (3) garden waste (Portsmouth, UK). A mass of 0.5 g of sludge and compost was added to the 50 ml of the mineral salt medium (MSM) and Lysogeny broth (LB) medium (Fisher BioReagents, UK); each sample was inoculated in duplicate for each media and was transferred to the Laboratory of Molecular Microbiology (University of Portsmouth, UK). These bottles were incubated overnight in Innova™ 4000 incubator shaker (New Brunswick Scientific, USA) at 150 rpm at 25°C. Overnight-grown cultures were used for the isolation of single bacterial colonies. Serial dilutions were made into six concentrations using 0.9% NaCl in dH2O for each culture after incubation. Six dilution tubes were generated for each media (two types of media) and sample (two types of samples), resulting in a total of 48 tubes. Each individual sample from each of the 48 tubes was used to inoculate one LB agar Petri dish. Some of the single colonies that grew from the two most diluted cultures were collected. The collected single colonies were regrown on separate LB agar to ensure that each Petri dish contains a pure colony. The colonies were picked up from Petri dishes and inoculated in individual universal tubes with 5 ml LB, rotating at 37°C overnight at 150 rpm. As a result, 73 bacterial isolates were recovered.

Different approaches have been used for the screening of PHA-producing microbes and/or the imaging of PHA granules [17]. The methods most widely used for detecting PHAs are staining techniques using Nile Red [18], Nile Blue A [19], and Sudan Black B [20]. Due to the lipophilicity of the dyes, the tests are very useful, but they have the ability to nonspecifically bind to other lipid droplets, membranes, and cell envelopes, leading to an incorrect answer [21, 22]. Sudan Black B (SBB) is a diazo

fat-soluble dye. PHAs are observed as black granules with SBB by bright-field microscopy. Nile Blue A and Nile Red are highly fluorescent organic dyes belonging to the benzophenoxazine family. Nile Red is a neutral molecule that is poorly soluble in water, and its chromophore is highly susceptible to changes in solvent polarity, showing little or no fluorescence in most popular solvents. Nile Blue A is a cationic dye and is thus more soluble in water than Nile Red. Both fluorescent dyes are particularly useful for visualizing hydrophobic cell structures, such as membranes or lipid-like inclusions (PHA granules). PHA inclusions appear as brightly fluorescent red/orange granules with Nile Blue A and Nile Red [23].

In this study, 73 bacterial isolates were tested for PHAs production using all above mentioned staining methods. When the ability of isolates to produce PHA was screened by Sudan Black B staining, cultures showed granules filled up with dark staining, as PHA granules can be observed as bluish dark spots under a light microscope (**Figure 2**). Out of the tested 73 isolates, 48 isolates tested positive for SBB staining and were considered to produce PHA granules.

To test the possible presence of PHAs additional stains were used. The Sudan Black B-positive strains were stained by Nile Blue A and Nile Red. Both of these dyes are used to detect PHAs production in bacteria grown on solid media. Bacteria were grown on the LB agar plates in the presence of DMSO, incubated at 37°C overnight, and then exposed to UV light to check the fluorescence in the cells (**Figure 3**).

Eighteen colonies out of 73 showed bright orange fluorescence by both fluorescent dye staining. Unfortunately, the staining methods cannot give an unambiguous answer. Therefore, other testing methods were essential to confirm the presence of PHAs in bacteria on the genetic and structural levels.

#### **Figure 2.**

*Screening of bacterial strains for PHA production using Sudan Black B dye. Panel A shows the positive result with different isolates from compost samples stained with SBB. Panel B shows the positive and negative results for different isolates from the sludge sample stained with SBB.*

**Figure 3.**

*Screening of different isolates for PHA production using Nile Blue A dye (Panel A) and Nile Red dye (Panel B).*

*Bioplastics against Microplastics: Screening of Environmental Bacteria for Bioplastics… DOI: http://dx.doi.org/10.5772/intechopen.109756*
