1. firstly, fresh leaves of Tithonia diversifolia were carefully cut and thoroughly washed with distilled water. Subsequently, the leaves were boiled, and the extraction process was carried out using dichloromethane (DCM) and ethanol. The resultant extracts were then prepared and stored for future use.

Question

Secondly, to ensure aseptic conditions, all materials were disinfected using 75% alcohol, and the spirit lamp was used to sterilize the forceps and other tools.
Thirdly, for Cultivation and Inoculation, Petri dishes containing nutrient agar were inoculated with E. coli using swab sticks and small discs soaked in DCM and ethanol extracts were then placed onto the agar plates. Additionally, positive and negative controls were set up on separate plates. Finally Incubation and monitored of all plates commenced at 11:50 AM. Zones of inhibition around the discs were measured at 24 and 48 hours, with estimations made for the 72-hour mark.
2. Firstly, 5kg of Tithonia diversifolia leaves collected and air dried them for five to seven days as recommended during a lecture. After drying, the leaves gathered and used scissors to chop them finely. Then conducted a quality check to ensure only pure, high-quality leaves were selected for blending.
In the extraction phase, the blender was opened and placed the first batch of air-dried Tithonia diversifolia leaves cuttings inside. After securely fastening the lid, plugged in the blender and turned it on, blending the leaves for 3 to 5 minutes. Closely monitored the process until all the dried leaves were transformed into a grey powder. Once blending was complete, switched off the power and transferred the leaf powder onto a paper tissue, setting it aside. This repeated process for the second batch of dried leaves.

Next, measured the ground grey powder. Then placed the powder on a balance, ensuring an exact mass of 20g, and then dissolved it into 200ml of dichloromethane and 200ml of ethanol. Wrapped the solutions in foil and labeled the beakers accordingly. These beakers were then preserved for 3 to 4 weeks to allow for absorption before extracting with dichloromethane and chloroform.

After 4 weeks, the evaporation process began. The mixture containing dichloromethane and Tithonia diversifolia placed on a heater or heating plate to obtain the antibacterial activity of the crude aqueous extracts of Tithonia diversifolia from the Zocki Zoi area in Goroka, EHP. The concentration or crude extract was processed using rotary evaporating iron. While the liquid extract was evaporating, prepared discs for testing the inhibition of microbial growth and then used the test compounds on agar plates.

The final phase of the experiment was the antimicrobial test which 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. Lastly, gently streaked the surface of an agar plate with the inoculating loop or used swabs and results were recorded.

correctly compare the above two procedures and fit in the procedure one into procedure two and make it sense.

Answer 1

Certainly! Below is a combined and cohesive procedure that integrates the steps from Procedure 1 into Procedure 2, ensuring logical flow and clarity:

### Combined Procedures

#### Collection and Preparation of Tithonia diversifolia Leaves

1. **Collection and Drying**:
- Collected 5 kg of Tithonia diversifolia leaves and air-dried them for five to seven days as recommended during a lecture.

2. **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.

3. **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.

4. **Measurement**:
- Measured and precisely weighed 20g of the ground grey powder using a balance.

5. **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

6. **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

7. **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

8. **Preparation of Agar Plates**:
- Prepared Petri dishes containing nutrient agar.

9. **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

10. **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.

11. **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

12. **Recording Results**:
- Recorded the zones of inhibition around the discs and analyzed the antimicrobial efficacy.

By aligning the steps from both procedures logically, you can achieve a clear and coherent experimental workflow that ensures accurate and repeatable results.