The Impact of High Fidelity Simulation on Stress Level in Medical Students.

Α-amylase activity in saliva [U/ml]

Saliva was collected using a disposable Salivette tube (Sarstedt AG \& Co, Germany) by placing a sterile tampon under the tongue or chewing for 30-45 seconds. Next the tube was centrifuged (1000 x g for 10 min.) to obtain a ready to test saliva supernatant. Samples were frozen after centrifugation at - 85°C until performing laboratory tests. Α-amylase activity assay was performed by a static method with AMYLAZA kit (Aqua-Med Łodz, Poland). The samples were diluted 100 times using 0,9% chloride solution. 2-chloro-4-nitrofenylo-maltotrioside is a substrate in this method. The reaction was performed in pH 6,0 MES buffer at 37 ° C rendering a colored reaction product. The product was then analyzed via spectrophotometry at 405 nm.

Time frame: 120 minutes

Secretory immunoglobulin class A concentration in saliva [ug/ml]

Saliva was collected using a disposable Salivette tube (Sarstedt AG \& Co, Germany) by placing a sterile tampon under the tongue or chewing for 30-45 seconds. Next the tube was centrifuged (1000 x g for 10 min.) to obtain a ready to test saliva supernatant. Samples were frozen after centrifugation at - 85°C until performing laboratory tests. Determination of secretory immunoglobulin class A (sIgA) concentration was established using an ELISA (Immunodiagnostic AG, Germany). The analytical procedure was carried out in accordance to the instructions provided by the manufacturer in the user manual. Absorbance readings were taken using a μQuant reader (BioTek, USA), the results were processed using the KCJunior program (BioTek, USA).

Time frame: 120 minutes

Cortisol concentration in saliva [ng/ml]

Saliva was collected using a disposable Salivette tube (Sarstedt AG \& Co, Germany) by placing a sterile tampon under the tongue or chewing for 30-45 seconds. Next the tube was centrifuged (1000 x g for 10 min.) to obtain a ready to test saliva supernatant. Samples were frozen after centrifugation at - 85°C until performing laboratory tests. The commercial ELISA (Diapra, Italy) were used to determine the concentration of cortisol in saliva. The analytical procedure was carried out with accordance to the manufacturer's instructions in the technical manual supplied with the kit. Absorbance readings were taken using a μQuant reader (Biotek, USA), while results were processed using KCJunior (Biotek, USA).

Time frame: 120 minutes

Testosterone concentration in saliva [pg/ml]

Saliva was collected using a disposable Salivette tube (Sarstedt AG \& Co, Germany) by placing a sterile tampon under the tongue or chewing for 30-45 seconds. Next the tube was centrifuged (1000 x g for 10 min.) to obtain a ready to test saliva supernatant. Samples were frozen after centrifugation at - 85°C until performing laboratory tests. The commercial ELISA (Diapra, Italy) was used to determine the concentration of testosterone in saliva. The analytical procedure was carried out with accordance to the manufacturer's instructions in the technical manual supplied with the kit. Absorbance readings were taken using a μQuant reader (Biotek, USA), while results were processed using KCJunior (Biotek, USA).

Time frame: 120 minutes

Total protein concentration [mg/ml]

Saliva was collected using a disposable Salivette tube (Sarstedt AG \& Co, Germany) by placing a sterile tampon under the tongue or chewing for 30-45 seconds. Next the tube was centrifuged (1000 x g for 10 min.) to obtain a ready to test saliva supernatant. Samples were frozen after centrifugation at - 85°C until performing laboratory tests. To determine the total protein concentration the Lowry method was used. This method base on the reactions between peptide bonds, tyrosine and Folin-Ciocalteu reagent. The absorbance of the resulting color was read at a wavelength of 650-750 nm, 30 minutes after the addition of the reagent. Bovine serum albumin water solution (BSA - Sigma Aldrich, Germany) at slightly basic pH was used as standard.

Time frame: 120 minutes