### Collection and Preparation of Tithonia diversifolia Leaves
#### Collection and Drying
Five kilograms of Tithonia diversifolia leaves were collected and air-dried for five to seven days, following recommendations from a lecture.
#### Cutting and Quality Check
Once dried, the leaves were gathered and chopped finely using scissors. A meticulous quality check was conducted to ensure that only pure, high-quality leaves were selected for blending.
#### Blending
The air-dried, finely chopped leaves were then placed into a blender. The lid was securely fastened before the blender was plugged in and operated for 3 to 5 minutes, transforming the leaves into a grey powder. This process was repeated for the remaining dried leaves. The resulting leaf powder was transferred onto a paper tissue and set aside.
#### Measurement
Using a balance, 20 grams of the ground grey powder were precisely weighed.
#### Dissolution
The 20 grams of powder were dissolved in 200 ml of dichloromethane (DCM) and 200 ml of ethanol in separate beakers. These solutions were wrapped in foil, labeled accordingly, and preserved for 3 to 4 weeks to facilitate absorption.
### Extraction of Active Compounds
#### Extraction
After 4 weeks, the mixture containing dichloromethane and Tithonia diversifolia was placed on a heater or heating plate to evaporate the solvents and extract the antibacterial activity contained in the crude aqueous extracts. This concentrated extract was further processed using a rotary evaporator.
### Preparation for Antimicrobial Test
#### Disk Preparation and Testing
During the solvent evaporation process, discs for testing microbial growth inhibition were prepared. All materials were disinfected using 75% alcohol, and a spirit lamp was employed to sterilize forceps and other tools.
#### Cultivation and Inoculation
##### Preparation of Agar Plates
Petri dishes containing nutrient agar were prepared.
##### Inoculation
The agar plates were inoculated with E. coli using sterilized swab sticks. Small discs, soaked in the DCM and ethanol extracts, were placed onto the agar plates. Positive and negative controls were established on separate plates.
### Incubation and Observation
#### Incubation
All plates were incubated at 11:50 AM. Zones of inhibition around the discs were monitored and recorded at 24 and 48 hours, with estimations made for the 72-hour mark.
### Serial Dilutions and Pour Plate Method
The bacterial culture was prepared using the pour plate method, and serial dilutions of the mixture sample were performed. To avoid contamination, the inoculating loop was sterilized, and the caps of the tubes were carefully removed. The lip of each tube was flamed before and after dipping the sterile loop into the broth. Correspondingly, the lip of the tube was flamed again before replacing the cap. The surface of an agar plate was gently streaked with the inoculating loop, or swabs were used for accurate results recording.
### Final Step
#### Recording Results
The zones of inhibition around the discs were recorded, and the antimicrobial efficacy was analyzed based on these observations.
Collection and Preparation of Tithonia diversifolia Leaves
Collection and Drying
- Collected 5 kg of Tithonia diversifolia leaves and air-dried them for five to seven days as recommended during a lecture.
Cutting and Quality Check
- After drying, gathered the leaves and used scissors to chop them finely. Conducted a quality check to ensure only pure, high-quality leaves were selected for blending.
Blending
- Opened the blender and placed the first batch of air-dried Tithonia diversifolia leaf cuttings inside. Securely fastened the lid, plugged in the blender, and turned it on, blending the leaves for 3 to 5 minutes until all the dried leaves were transformed into a grey powder. Repeated the process for the second batch of dried leaves.
- Transferred the leaf powder onto a paper tissue and set it aside.
Measurement
- Measured and precisely weighed 20g of the ground grey powder using a balance.
Dissolution
- Dissolved the 20g of powder into 200 ml of dichloromethane (DCM) and 200 ml of ethanol in separate beakers.
- Wrapped the solutions in foil, labeled the beakers accordingly, and preserved them for 3 to 4 weeks to allow for absorption.
Extraction of Active Compounds
Extraction
- After 4 weeks, placed the mixture containing dichloromethane and Tithonia diversifolia on a heater or heating plate to evaporate the solvents and obtain the antibacterial activity of the crude aqueous extracts.
- Processed the concentration or crude extract using a rotary evaporator.
Preparation for Antimicrobial Test
Disk Preparation and Testing
- While the liquid extract was evaporating, prepared discs for testing the inhibition of microbial growth.
- All materials were disinfected using 75% alcohol, and a spirit lamp was used to sterilize the forceps and other tools.
Cultivation and Inoculation
Preparation of Agar Plates
- Prepared Petri dishes containing nutrient agar.
Inoculation
- Inoculated the agar plates with E. coli using sterilized swab sticks.
- Placed small discs soaked in the DCM and ethanol extracts onto the agar plates. Set up positive and negative controls on separate plates.
Incubation and Observation
Incubation
- Incubated all plates at 11:50 AM and monitored the zones of inhibition around the discs at 24 and 48 hours, with estimations made for the 72-hour mark.
Serial Dilutions and Pour Plate Method
- Prepared the bacterial culture using the pour plate method and performed serial dilutions of the mixture sample.
- To avoid contamination, sterilized the inoculating loop, carefully removed the caps from the tubes, flamed the lip of each tube, dipped the sterile loop into the broth, and then removed it. Flamed the lip of the tube again before replacing the cap.
- Gently streaked the surface of an agar plate with the inoculating loop or used swabs for accurate results recording.
Final Step
Recording Results
- Recorded the zones of inhibition around the discs and analyzed the antimicrobial efficacy.
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