Isolation of microorganisms
Microbial strains were isolated from different contaminated skin and body care products based on a method described in a previous study of Alshehrei.² Thirty-two cosmetic products used for body and skin collected from different stores and outlets in Mecca region, SA. Samples are transported to the lab in sterilized condition. Serial dilutions (10^-1, 10^-2, 10^-3, 10^-4, and 10^-5) are used to isolate microorganisms from cosmetic products, each dilution transferred to two different plates, one includes Tryptone Soy Agar (TSA) to grow bacteria, plates then are incubated for 48 hours at 37 °C, and the other one includes Sabouraud Dextrose Agar (SDA) to grow fungi, plates are incubated at 25 °C for 5-7 days. After isolation, microbial stains undergo molecular analysis; 16S rRNA in case of bacteria and 18S rRNA in case of fungi.

Identification of bacteria using 16S rRNA sequencing
Extraction of DNA
Bacterial isolates were grown in nutrient agar for 48 h at 37 °C, and then the bacterial culture was harvested using centrifugation at 5000 rpm for 10 min. One milliliter of a solution containing 5 × 10⁸ bacterial cells was prepared, and DNA was extracted using the Qiagen Bacterial DNA extraction kit, as per the manufacturer’s protocol.

PCR amplification
The universal forward primer (5′-GAG TTT GAT CCT GGC TCAG-3′), and reverse primer (5′-GTT ACC TTG TTA CGA CTT-3′) were used to amplify DNA.¹⁹

Polymerase chain reaction (PCR) was performed in a thermal cycler with the following cycle conditions: The total volume of the reaction mixture was 25 µL; PCR was conducted for 35 cycles comprising the following steps: denaturation at 96 °C for 50 s, annealing at 50 °C for 45 s, and extension at 72 °C for 2 min. In addition to the 35 cycles, the PCR also included an initial denaturation step at 96 °C for 10 min and a final extension step at 72 °C for 10 min.

Identification of fungi using 18S rRNA sequencing
Extraction of DNA
Fungal species were detected using molecular internally transcribed spacer regions. Fungal isolates were grown in Sabouraud dextrose agar for 72 h at 30 °C. After harvesting, a 1 ml saline suspension containing 1-5 × 10⁶ conidia were prepared. DNA was extracted from each sample using a DNeasy kit (Qiagen) to isolate genomic DNA from fungi (cultures and blood samples) according to the manufacturer’s instructions.

Internally transcribed spacer-PCR
A forward primer (5′-TCC GTA GGT GAA CCT GCG G-3’) and reverse primer (5′-TCC TCC GCT TAT TGA TAT GC-3’) were used to amplify ITS4 from the PCR reactions, consisting of 20 ng genomic DNA in a total volume of 25 L. PCR was performed in a thermal cycler. The reaction mixture contained 0.5 µM of both the forward and reverse primer, 50-100 ng of template DNA, 0.5 U of Taq DNA polymerase, 200 µM of dNTPs, 10 mM Tris, 50 mM KCl, and 1.5 mM MgCl₂. The total volume of the reaction mixture was 25 µL. PCR was conducted for 35 cycles, with each cycle consisting of the following steps: denaturation at 96 °C for 50 s, annealing at 50 °C for 45 s, and extension at 72 °C for 2 min. In addition to the 35 cycles, the PCR process also included an initial denaturation step at 96 °C for 10 min and a final extension step at 72 °C for 10 min.²⁰

Minimum inhibitory concentrations (MICs)
The effects of four preservatives that are used broadly in cosmetics preserving, PE, BA, PP, and MP, on seven strains isolated from contaminated cosmetics were examined.

Preparation of the bacterial inoculum
Bacterial strains were isolated from various contaminated skin and body care cosmetics, based on a previous study.² The identified strains were B. cereus, B. subtilis, S. aureus, and E. coli. For each bacterium, nutrient broth medium was inoculated with a single colony and incubated at 37 °C with shaking. Next, suspensions were adjusted to the same OD60 for quantification purposes. MIC tests were performed using bacterial strains, maintained in nutrient broth medium, and inoculated with 10⁵ CFU/mL of each bacterium. Serial dilutions were carried out in test tubes, which were incubated for 24 h at 37 °C in a rotary shaker at 180 rpm.

Determination of minimum inhibitory concentrations (MICs)
A serial dilution method was used to determine the MICs of the four preservatives (PE, BA, PP, and MP) at different concentrations (3.2, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 mg/mL). Medium without preservatives was used as the control. The MICs of the preservatives were calculated in triplicate.

MICs experiments were performed in round-bottomed 96-well plates (Figure 1). First, 100 µL of each preservative agent was aliquoted into the 96-well microplates (1 to 10 wells). Growth and control groups were included. Next, 100 µL of RPMI1640 broth medium and 100 µL of fungal/bacterial inoculum were added to the preservative agents. Additionally, a negative control was prepared using 25 µL of preservative agent and 75 µL of RPMI1640 broth medium. Culture plates were heated at 37 °C for 24 hrs.

The MICs were determined at 24 and 48 hrs as the concentrations of the preservatives that achieved 100% growth inhibition.

Preparation of fungal inoculum
Fungal strains were isolated from different contaminated skin and body care products based on a method described in a previous study.³ These strains were identified on a molecular level at the Laboratory of Microbiology, Mr. Alqura University to be A. flavus, A. fumigatus, and A. welwitschiae.

Determination of minimum fungicidal concentration (MFC)
After 48 hrs of incubation, 10 L of the contents were removed from the wells that showed no growth and spread on Sabouraud dextrose agar. MFCs were determined after incubation for 72 hrs.

Statistics
Data were analyzed by Excel program. Standard deviation for each treatment was calculated. Error bars were visualized as the value of STDEV on the charts.