Sabouraud dextrose broth (sdb)

The reagents used for the study include; Sabouraud dextrose agar- SDA (Oxoid, England), Sabouraud dextrose broth- SDB (Oxoid, England), Amphotericin B powder (Sigma- Aldrich), Fluconazole powder (Sigma- Aldrich), Terbinafine powder (Sigma- Aldrich), Dimethylsulphoxide (Sigma- Aldrich). All other reagents were of analytical grade.

The reference fungal strains used for the experiments were: Candida albicans ATCC (American Type Culture Collection) 90028, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida auris CBS (Fungal Biodiversity Centre) 10913, Candida guilliermondii 184 (the collection of the Cantacuzino National Research and Development Institute for Microbiology and Immunology, Bucharest). The strains are part of the collection from the Microbiology Department from UMPhST (George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Târgu Mureş) and the Ion Ionescu de la Brad University of Life Sciences, Iași, stored at −70 °C. For each experiment, the strains were revitalized in Sabouraud Dextrose Broth (SDB, Oxoid, Basingstoke, United Kingdom) and incubated for 24–48 h, at 37 °C, in an orbital shaker.

The essential oil’s antimicrobial effectiveness was assessed against various strains of bacteria and yeast. Gram-positive bacteria included Micrococcus luteus CCM 732, Listeria monocytogenes CCM 4699, and Staphylococcus aureus subsp. aureus CCM 3953, while Gram-negative bacteria included Escherichia coli CCM 3953, Salmonella enterica subsp. enterica CCM 3807, and Yersinia enterocolitica CCM 7204T. Yeast strains included Candida albicans CCM 8186, C. glabrata CCM 8270, C. krusei CCM 8271, and C. tropicalis CCM 8223. All microbial strains were obtained from the Czech Collection of Microorganisms in Brno, Czech Republic. Prior to analysis, bacterial and yeast cultures were incubated for 24 h at 37 °C and 25 °C in Mueller–Hinton Broth (MHB, Oxoid, Basingstoke, UK) and Sabouraud Dextrose Broth (SDB, Oxoid, Basingstoke, UK), respectively. The bacterial and yeast inoculum’s optical density was standardized to 0.5 McFarland standard on the day of the experiment [22 (link)].

Twelve commercial essential oils (Essential Oils Direct Ltd., Oldham, UK) (Table 5), two terpenes (E-cinnamaldehyde and linalool (Sigma-Aldrich, Gillingham, UK)), chlorhexidine digluconate (CHX) (Sigma-Aldrich, Gillingham, UK) and triclosan (Irgasan from Sigma-Aldrich, Gillingham, UK) were evaluated.
The commercial essential oils were tested at a range of concentrations against planktonic growth (2% (v/v) to 0.007% (v/v) and biofilms (8% (v/v) to 0.125% (v/v)). All agents were prepared in Sabouraud Dextrose Broth (SDB; Oxoid Ltd, Basingstoke, UK). To enhance dispersion of essential oils in the medium, 1% (v/v) Tween 80 (Sigma-Aldrich, Gillingham, UK) was added. In the case of biofilm studies, 0.015% (w/v) Agar Bacteriological (LP0011 Oxoid) was added to SDB [53 (link)]. CHX was used in SDB at concentrations between 0.04% (v/v) to 3.1 × 10⁻⁴% (v/v) and from 0.08% (v/v) to 6.2 × 10⁻⁴% (v/v) for planktonic and biofilm growth experiments, respectively. A 20% (w/v) stock solution of triclosan was prepared in Dimethyl Sulfoxide (DMSO) (Fisher Chemical, Loughborough, UK). Serial doubling dilutions of the stock solution were prepared in SDB yielding final concentrations from 5.2 × 10⁻⁶% (v/v) to 6.7 × 10⁻⁴% (v/v) and from 1.7 × 10⁻⁴% (v/v) to 5 × 10⁻³ (v/v) for planktonic and biofilm experiments, respectively.

The microtiter plate method was used to screen biofilm formation [34 (link),35 (link)]. Sabouraud dextrose broth (SDB) (Oxoid, Basingstoke, UK) was used with a final concentration of 8% w/v glucose (Sigma-Aldrich, St. Louis, MO, USA). The candida inoculum was adjusted to approximately 3 × 10⁷ CFU/mL. Biofilm production was performed in flat-bottom microtiter plates (Hyundai Micro Co., Seoul, LTD., Korea). Sabouraud dextrose broth (180 μL) was inoculated with 20 µL cell suspension and incubated at 37 °C for 24 h. The plates were rinsed once with distilled water after incubation, and the microtiter plate was stained for 45 min with crystal violet solution. Each well was thoroughly rinsed with sterile distilled water and then destained with ethanol (95%) (Sigma-Aldrich, St. Louis, MO, USA). The destaining solution was quantified using an ELISA plate reader (Euroclone BIOTEK, Pero, Italy) at 595 nm [34 (link),35 (link)]. Positive control for biofilm development was C. albicans ATCC 10231. The isolates were classified as non-biofilm producers, weak, moderate, and strong biofilm producers according to their OD590 nm values. IF OD590 nm ≤ ODc the isolate is considered (None-biofilm producers), weak biofilm producers, when OD ˂ OD590 nm ≤ 2 × ODc, moderate biofilm producers, when 2 × ODc ˂ OD590 nm ≤ 4 × ODc, and strong biofilm producers when OD590 nm ˃ 4 × ODc [34 (link),35 (link)].