Den 1 mcfarland densitometer

The antifungal activity of the studied chitosan-gelled MEs loaded with FZ was evaluated against Candida albicans ATCC-90028 strains using the agar plate diffusion method with the modification that the gel samples added to wells made in the agar medium in Petri dishes. Sabouraud agar medium containing chloramphenicol and gentamicin was used as culture medium. The colonies were diluted with 0.85% sodium chloride sterile solution, and the concentration of the obtained suspensions was adjusted to approximately 3 × 10⁸ colony-forming units/mL using a turbidimeter (DEN-1 McFarland Densitometer, Biosan, Riga, Latvia). The plates with Sabouraud agar medium were inoculated with the C. albicans suspension, then allowed for 5 min to adsorb the inoculum. In the next step, a well (one for the control gel and the other for the test gel) was made in the middle of each half of the plate by cutting under sterile conditions an 8 mm diameter portion of medium, then each well was filled with the sample. The plates were then incubated at 37 ± 0.1 °C for 48 h. After the incubation period, the inhibition zones diameters (mm) were measured and the mean values of three measurements (±SD) were used to express the antifungal activity of the chitosan-gelled MEs.

Haemophilus parasuis serotype 5, strain HP 132 (CAPM 6475) originating from a pig with meningitis was obtained from CAPM. Bacteria were grown on Mueller–Hinton agar broth with yeast extract and 5% sheep blood (LabMediaServis) overnight at 37 °C, and non-confluent growth was harvested and resuspended in calcium–magnesium free Dulbecco’s phosphate-buffered saline (D-PBS, Lonza). Bacteria were washed twice with D-PBS and resuspended in D-PBS, with the final concentration adjusted to optical density of 2.5 equivalent to 10⁹ CFU/mL, using a turbidimeter (DEN-1 McFarland densitometer, Biosan).

Antimicrobial activity was evaluated using a broth microdilution method [55 ]. Bacterial suspensions were made from overnight cultures in Mueller–Hinton broth (mold suspension was made in SDB), and their turbidity was standardized to 0.5 McFarland using densitometer (DEN-1 McFarland Densitometer, Biosan). The final density of bacterial inoculums was 5 × 10⁵ CFU (colony forming units), while mold’s final inoculum size corresponded to 1 × 10⁴. Stock solutions of the DME were prepared in pure dimethyl sulfoxide (DMSO) and serially diluted (the diluting factor 2) with sterile saline in the concentration range 0.001–20.00 mg/mL. The highest final concentration of DMSO in each well was 10%. After making dilutions of the test substances, the inoculum was added to all wells and the plates were incubated at 37 °C during 24 h. Streptomycin and nystatin served as positive controls as antibacterial and antifungal agents, respectively, while one non-inoculated well, free of antimicrobial agent, was included to ensure the medium sterility. The bacterial growth was determined by adding 20 μL of 0.5% triphenyltetrazolium chloride (TTC) aqueous solution. MIC was defined as the lowest concentration of the test compound that inhibited visible growth (red colored pellet on the bottom of the wells after the addition of TTC). All experiments were done in triplicate.

The samples were anaerobically incubated on an agar–blood culture medium (Columbia Agar + 5% ram blood, Mediclim, Romania) for 24 h at 37 °C in a thermostat (Jouan IG150 Infrared-Controlled CO₂ Incubator, Germany). An ATCC 25175 suspension of S. mutans obtained from the Microbiology Department of General Medicine was prepared at 0.5 McFarland [12 (link)] with a densitometer device (DEN-1 McFarland Densitometer, Biosan, BS-050102-AAF, LATVIA). Eighteen teeth were contaminated with 0.2 mL of S. mutans suspension (10⁷ CFU/mL) [13 (link)], and all the samples were placed in a sterile container individually for 48 h at 37 °C in a thermostat (Figure 1 and Figure 2).