To begin, pour 100 milliliters of distilled water into a beaker containing 4 grams of sugar and 0.8 grams of agar. Heat the mixture to 100 degrees Celsius while stirring continuously. Remove the mixture from the heat to cool it down.
Lower the heat to maintain a temperature of 80 degrees Celsius. Then, add 7.4 grams of flour, followed by 2.8 grams of yeast to the mixture while stirring. Next, add the Moldex propionic acid solution into the mixture, then cool the temperature to 50 degrees Celsius.
Add the psidium guajava plant extract solution followed by 0.5 grams of bromophenol blue powder. Pour the prepared food mixture into Petri dishes until each dish is full. After the dishes have cooled to room temperature, seal them with their lids and store them in a refrigerator at 4 degrees Celsius.
To prepare the vials for testing, first prepare the carbon dioxide tanks, carbon dioxide blowgun with needles, fly pad, paintbrush, and microscope for handling the flies. Select vials containing at least 10 Drosophila pupae to standardize the age of the flies. To remove the adult flies, invert the vials and insert a needle between the cotton plug and the vial's sidewall.
Anesthetize the adult flies using the carbon dioxide blowgun until they are unconscious on the cotton plug. Open the vial above a glass bottle containing 70%ethanol and drop the flies into it. Seal the vial with the cotton plug.
Then, incubate it at 25 degrees Celsius with 60%humidity and a light cycle of 12 hours light and 12 hours dark. Next, sort the flies into virgin females and males with the help of a microscope within eight hours of incubation to prevent mating. The female genitalia can be identified by their pale color while the male genitalia have a reddish hue.
Males can also be recognized by the sex combs on their front legs. Divide the sorted flies into two fresh tubes, one for each sex, and incubate them for 6-8 days at 25 degrees Celsius. Start by stacking labeled Petri dishes on top of each other.
Invert the Petri dishes with the dyed food on blotting paper to soak up any excess liquid. Then, use a spatula to divide the food in each dish into 12 equal segments. Transfer one slice of food into an empty Petri dish.
Next, anesthetize the flies using carbon dioxide until they are asleep on the cotton plug. Carefully transfer six healthy flies into each Petri dish. Place the sealed dishes in an incubator.
To prevent the flies from escaping during the experiment, secure the top and bottom covers of each Petri dish with tape. After a rearing period of 24 hours, anesthetize the flies again using carbon dioxide. Then, transfer the flies into a container filled with 70%ethanol for disposal.
Also, discard any remaining food from the dishes. To facilitate the quantification of the Petri dishes, launch the Epson Scan 2 software, assign a file name, and perform a preview scan. Individually scan both the top and bottom covers of each Petri dish.
Carefully crop the scan to images with an open source image editor to eliminate any artifacts and food residue, then save the cropped image as a TIFF file. Next, launch the T.U.R.D.software. Click on File to create a new experiment document, assign a name to it, and save it in the Analysis sub folder.
Click on the Plates option and then select Add Plate to choose the Petri dish for analysis. A new window will appear displaying the names of selected plates along with new parameter settings. Set the Block Size to 21, Offset to 5, Minimum Size to 20, and Maximum Size to 600.
To confirm the accuracy of fecal deposit detection, navigate to Plates then click on Inspect Selected Plates followed by Graphics and View Annotated Images. Zoom into the images to review the deposit counts. If necessary, deselect any deposits that should not be included in the analysis.
Following the analysis with T.U.R.D.software, adjust the number of flies by clicking on the number flies. Change the group name by clicking on Plates, followed by Edit Groups, then press Add. Select the appropriate group name in the Group column.
Next, click on Analyze, followed by Descriptive Statistics and select Group to export the data for each replicate separately. Save all resulting spreadsheet files in the same designated folder. To compile all data into a single spreadsheet, open the application Excel Merge v4.
When prompted with select the path of the folder with CSV files, input the folder address and press Enter twice to create a new spreadsheet in the same folder. Add a new sheet in the spreadsheet with the merged data to calculate the mean of each parameter across all replicates. Utilize the VLOOKUP function to gather the mean of each parameter of all replicates.
Launch the GraphPad Prism software and input the dataset names. Transfer the data from the Excel spreadsheet to the Prism data chart. Click on Analyze to choose the datasets for analysis and press OK.Choose the appropriate test parameters, analyze the P-value.
The number of fecal deposits, total area of the deposits, and the IOD was significantly higher in the normal food group as compared to the P.guajava extract in both virgin males and females. Tracking of the consumption of solid food showed that there were no significant differences in the food consumption between the drug supplied and non-drug supplied groups. However, the male flies that consumed loperamide appeared to take in less food than normal.
