Isolation and identification of microflora
Samples of dental plaque were collected with the help of a dental clinician in Pune. The observable plaque present at the gingival was collected using a sterile probe, and dispensed in sterile Eppendorf tubes containing 1mL sterile Phosphate Buffered Saline (PBS). These collected samples were then transported to the laboratory at 6 °C to 10 °C and were processed immediately.

In the laboratory, samples were first homogenized using vortex mixer. 100 µL of sample was inoculated in the liquid enrichment broth and incubated at 37 °C for 24h. The enriched broth was again mixed by vortexing and a loopful (10 µL) of sample was streaked on sterile Mannitol Salt Agar, sterile de Man, Rogosa and Sharpe agar (MRS), sterile Mitis Salivarius (MS) agar. The plates were incubated for 24 to 48 hours at 37 °C in aerobic conditions. However, for MRS medium, incubation was carried out in microaerophilic conditions. Colony characteristics were noted down after incubation and saline suspension of overnight culture was prepared. Gram staining was carried out and the hanging drop technique was followed for checking motility.

Molecular characterization to identify selected isolates was carried out using 16S rRNA gene amplification procedure as illustrated by Pidiyar et al.⁷ Bacterial isolates were recognized based on 16S rRNA gene sequence homology by means of the reference sequences available in GenBank. A strain is regarded as a member of species if the sequence homology is more than 98.2%.⁸ Accordingly the three isolates got identified as Streptococcus agalactiae, Staphylococcus epidermidis and Lactobacillus fermentum all with homology ≥ 99%. The gene sequence data of these isolates was submitted to the GenBank with accession number as mentioned in Table 1. Using the 16S rRNA, a phylogenetic tree of the three identified microorganisms is depicted in Fig. 1.

Finalization of the microorganisms for the study
Various bacterial species take part in the early stages of biofilm formation. Three species of bacteria that are chiefly responsible for the biofilm formation were isolated. To scrutinize the complete range of organisms that are responsible in the early biofilm formation leading to dental plaque, the other likely organisms were procured. Therefore, the Microbial Type Culture Collection (MTCC) cultures were included which were acquired from the Institute of Microbial Technology (IMTECH), Chandigarh, India. The MTCC cultures comprised of 5 microorganisms, of which all were bacteria except Candida albicans (4748) which was a fungus. Accordingly, a total of 8 organisms denoted as ‘test organisms’ were finalized for the study (Table 1). All cultures were cultivated in the various culture media as recommended by IMTECH. Streptococcus mutans (890), Streptococcus oralis (2696), Lactobacillus acidophilus (10307), Lactobacillus rhamnosus (1408), and Candida albicans (4748) were recovered in Brain Heart Infusion Medium, Trypticase Soy Broth, de Man, Rogosa and Sharpe Medium and Yeast Extract Peptone Dextrose respectively. All cultures were held in reserve on their particular solid media.

Table (1):
Finalized test organisms for the study.

Development of mouthwash formulation
Lemongrass essential oil (LGEO) is insoluble in water. By definition, mouthwash must be a hydroalcoholic solution. Thus, direct formulation of mouthwash is not possible and hence, lemongrass must be solubilized by various co-solvents and surfactants to make the mouthwash formulation.⁹

The formula used for mouthwash was as follows: Table 2.

Table (2):
Composition of mouthwash.

For evaluating the stability, the sample was kept for 3 months at room temperature and the concentration of LGEO was monitored along with the following parameters as

1. Physical parameters,
2. Stability studies
3. Antimicrobial activity
4. Biofilm formation inhibition (Anti-biofilm activity)

Antimicrobial Assay
Antimicrobial activity of undiluted formulated mouthwash against test organisms was established by means of the standard disc diffusion technique as per Clinical and Laboratory Standards Institute (CLSI) guidelines.¹⁰ Saline suspension of 24 h old culture of all the 8 test organisms was prepared according to 0.5 McFarland standards. This 750 μL of saline suspension was mixed with 20 mL of sterilized, cooled Mueller-Hinton agar butt and dispensed in a sterile Petri plate. The plates were set aside to solidify at room temperature. Sterile Whatman filter paper discs pre-soaked (10 μL) in mouthwash were positioned on agar surface. Chlorhexidine gluconate, a commercially available mouthwash was used as a positive control. For pre-diffusion, plates were refrigerated at 4 °C for 30 min. Plates were incubated at 37 °C for 24 h. The diameter of zone of inhibition was determined in millimeter (mm) and noted. The diameter of zone of inhibition was measured with the help of HI MEDIA antibiotic zone measuring scale. All exposures were carried out in triplicates and average value was considered for further evaluation.

Determination of growth of biofilm and anti-biofilm activity
Assay for biofilm growth and anti-biofilm activity
The growth of biofilm was quantitatively determined according to the “Protocols to study the physiology of oral biofilms” by Lemos et al.¹¹ The principle of assay is that the biofilm produced by the organism binds to the crystal violet and the bound crystal violet is later eluted The absorbance of eluted crystal violet is proportion to the amount of biofilm. Assay was carried out in 96 well microtiter plates. Saline suspension of 24 h old culture of all 8 test organisms was prepared; according to 0.5 McFarland standards. Biofilm medium comprising 1 M glucose (source of carbohydrate) was formulated. 20 μL of each 0.5 McFarland standard of microbial culture was dispensed into each well with 180 μL of biofilm medium. Wells with a 200 μL uninoculated biofilm medium served as negative controls while positive control consisted of 20 μL of chlorhexidine with 180 μL of medium. Each experiment was carried out in triplicate. Plates were sealed with an adhesive microtiter plate sealer and without any agitation incubated for 24 h at 37 °C. After incubation, the plates were first blotted on a paper towel to remove culture media, and then were carefully immersed in a large dish with deionised water to remove the loosely bound cells and again blotted on a paper towel. This step was repeated twice. 50 μL of 0.1% crystal violet was added to the test wells. Plates were then incubated for 15 min at room temperature. The washing was repeated and the plates were later air-dried. To elute the crystal violet bound to the biofilm formed in the wells, 200 μL of 33% acetic acid solution was added to all the wells. Plates were again incubated for 10 min at room temperature. Using a multi-channel micropipette, the entire content of each well was transferred in the respective wells in a new blank microtiter plate. The absorbance was measured at 570 nm using ELISA reader (Thermo Lab systems Model No. 352). In anti-biofilm studies, 160 μL of medium was subjected to 20 μL of culture with 20 μL of formulated mouthwash keeping all other steps in the protocol the same. The ability of the mouthwash to inhibit the formation of biofilm was determined as “anti-biofilm activity of mouthwash”. Chlorhexidine gluconate was taken as a positive control.

MTT Assay ( 3-(4,5-dimethylthiazole-2-yl)- 2,5-diphenyltetrazolium bromide cell proliferation assay)
The primary mouse embryonic fibroblast cells NIH 3T3 were procured from NCCS, Pune. Cell lines were preserved in a humidified incubator in a 5% CO₂ atmosphere at 37 °C in Dulbecco’s Modified Eagle’s medium (DMEM). 96 well plates seeded with NIH 3T3 cell lines were incubated at 37 °C in humidified 5% CO₂ atmosphere for 24 h. The cell lines exposure was carried out for cell control, DMSO control, formulated mouthwash and LGEO 1.5%. The exposure was made in triplicate and incubated further for 48 h. Then 10 μl of 5 mg/ml of MTT dye was added to all the wells and incubated for 4 h in dark. Formazan crystals were solubilised using 100 μl of DMSO and after keeping plates in dark for 15 min, the absorbance was measured at 570 nm. To confirm the results the assays were performed at least twice. The survival of cells was measured as absorbance of the mean of triplicate wells in comparison to that of control.