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To evaluate the acidity degree, 50 mL of 67% neutralised ethyl alcohol was added to ten g of the sample, this solution was shaken in a closed container for five min, and then it was filtered.
Spore suspension was prepared from spores harvested from five days cultured slants by adding 10 ml of 0.1%% sterile Tween 80, vortexed for five min, and filtering the resulting spore suspension through a sterilized 8 μm membrane filter disk.
The cells were fixed in 4% paraformaldyhyde for 15 min followed by three washes with PBS for five min each.
Saliva was collected immediately following injection for five min and chilled on ice as previously described [14].
Blood samples were collected using intravenous canula from individuals and immediately centrifuged for five min at 14,000 rpm to separate serum from other cellular materials.
The obtained cell lysate was sonicated two times for five min (50% duty cycle, 1 s on, 1 s off) on ice.
The supernatants were retained, spun for five min and the protein concentration of the samples was measured using the BCA method (Pierce, Cheshire, UK).
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When the first number-symbols were written down some 5,000 years ago in Sumer, a region of what is now Iraq, the word for one (ges) also meant man, or erect phallus, while the word for two (min), also meant woman.
The reduction in absorbance was measured spectrophotometrically after one minute of mixing the solutions and continued for six min. Ascorbic acid was used as a positive control.
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