Simple Diffusion

In 10 g dried sediment sample added 7 ml 0.2 M NH4Cl solution. A mixture of 100 ml hexane: acetone (1:1) was used as a solvent to extract pesticides with overnight shaking for 12 h on reciprocal or wrist action shaker at 180 rpm. The extract was carefully decanted through activated florisil column (2-3 cm), giving twice wash with25 ml hexane: acetone (1:1) to the sediments. The elute was then washed with 200 ml water and then again aqueous layer was extracted with 50 ml hexane. Finally the hexane layer was washed with 100 ml water and then evaporated to dryness with a vacuum rotary evaporator.

In the round-bottom flask (100 mL), we placed p-aminobenzoic acid (1.2 g) and ethanol (12 mL). We swirled the mixture until the solid dissolved completely. We used Pasteur pipet to add concentrated sulfuric acid (1.0 mL) to the flask. We added boiling stone and assembled the reflux. Then, we did reflux for 75 minutes.

3) Centrifuged at 2500 rpm for 12 mins. Upper hexane layer (supernatant) was transferred carefully into another test tube. 4) Evaporated the hexane under a stream of grade 1 nitrogen gas and added 100 µl of methanol to the residue left and vortexed for 1 min. 5) Injected 100 µl of extract in HPLC vials and closed properly. Standard curves and calculations- Retinol was quantified from standard curves peak area for each vitamin.

The mixture was stirred until the entire dissolved and homogeneous. The materials were ready each put in a bottle 100 mL size vial, then sterilized with autoclave for 15 minutes at 121 °C. The preparations hydrogel done within aseptic at LAF cabinet. Evaluation of chloramphenicol hydrogel ophthalmic preparations Organoleptic test Organoleptic hydrogel checked by observing changes in color, odor and clarity. Clarity was checked visually by examination

The incubation mixture contained 2.5 ml of 1.2% (w/v) fibrin, 2.5 ml of 100 mM Tris–HCl buffer, 10 mM CaCl2 (pH 7.8), and 20 µg of enzyme. The incubation was carried out at 37°C for 30 min, and the reaction was stopped by adding 5 ml of 110 mM trichloroacetic acid containing 220 mM sodium acetate and 330 mM acetic acid. This reaction mixture was centrifuged at 3,000×g for 5 min, and the absorbance of the trichloroacetic acid (50 mM) soluble product was determined at 275 nm. One unit of fibrinolytic enzyme activity was defined as the amount of enzyme required to liberate 1 µg of L-tyrosine per minute at 37°C. The total protein determination was performed as described by Lowry et al.

Then, the test media is then incubated at 37 ° C, for 18-24 hours. Rinsing reusable instruments The samples were rinsed with 40 ml of pyrogen-free water using a glass beaker that is free from pyrogens. Endotoxin testing using STV

 Dissolve the salt in 60 ml of tap water. Add 30 ml 6 M Hcl and stir the mixture with a glass rod.  Add 12 g solid Nacl to the solution and stir the mixture for about 2 minutes.  Support a 250 ml separatory funnel on a ring, making sure that the stopcock is closed and that a clean beaker is placed beneath the exit tube. Transfer the aqueous solution from the beaker to the separatory funnel.

Total essential oils analysis method is conducted based on Ali [7] with modification. 0,1 gr of kaffir lime oil nanocapsule is weighed and diluted to 100 ml using aquadest, taken as much as 1 ml (100x dilution) to put in the reaction tube then 1ml of saturated NaCO3 solution is added to the test tube and incubated for 10 min at room temperature. Then 0.5 ml of the folinciocalteu (Chemix CV, Yogyakarta) reagent and 7.5 ml aquadest were added, the mixture homogenized using vortex and then incubated for 30 min at room temperature under dark environmental conditions. Absorbance of the sample was then measured using a UV-vis spectrophotometer at a wavelength of 770 nm. The total phenol content of the sample was interpreted to be equivalent to gallic acid based on the standard curve of obtained gallic

Putting the dye solution in the blank cuvette and observe the absorbance level that it dorms. Repeat the steps five times making different dilutions to maintain multiple absorbance values. The second week was making another commercial dye solution and also testing the food dye brought into class. Calibrate the spectrophotometer the same exact way so that it does not mess up the calculations gathered. This week using yellow dye #5 mix .20 milligrams of this will 250 ml of distilled water in a volumetric flask.

Top agar was dispensed into 50ml test tubes and then autoclaved for 15 minutes at 121℃ as well. Stored the top agar at -5℃ refrigerator when it cooled down. Preheated top agar for 20 minutes to liquefy before use. The optimal dispense temperature for top agar was 45℃. 3.3.4 Preparation of CaCl2 solution

The dried roots of Inula racemosa were pulverized and sieved with 100 ~ 200 mesh. The herb powder was placed into a glass bottle. Ultrasound-assisted extraction was carried out in an ultrasonic cleaner RK102H (Bandelin sonorex, Germany). The powder of Inula racemosa was extracted three times under the following conditions: the ratio of material to solvent was 10:1, undergoing ultrasonic treatment 30 minutes at 25 °C, 100 kHz /450  W.31 Before large extraction, a small-scale extraction experiments were carried out: 95% ethanol and ethyl acetate as the extractive solutions was investigated, respectively.

Using the thermometer, the temperature was measured and recorded. Then, the 25-mL graduated cylinder was filled with 25 mL of distilled water, and its mass was measured and recorded. The density of the water was found using the temperature and the Density of water index. Moreover, the calculated volume of water was calculated using the formula of density, and the difference between observed volume and calculated volume was found. This process was then repeated using the 50-mL beaker and the results were recorded.