The first step carried out was the collection and preparation of Tithonia diversifolia leaves, where five kilograms of Tithonia diversifolia leaves were collected and air-dried for five to seven days, following recommendations from a lecture.
Secondly, 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 is the third step, where 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 paper tissue and set aside. Measurements were taken using a balance to precisely weigh 20 grams of the ground grey powder.
Fourthly, dissolution involved dissolving 20 grams of powder 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.
Then, extraction of active compounds took place. 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 compounds contained in the crude aqueous extracts. This concentrated extract was further processed using a rotary evaporator.
After that, preparation for the antimicrobial test began with 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 followed.
Then, agar plates using Petri dishes containing nutrient agar were prepared. 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.
Next, incubation and observation took place. 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.
Then, serial dilutions and the pour plate method were performed, where the bacterial culture was prepared using the pour plate method, and serial dilutions of the mixture sample were carried out. 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.
The final step was recording results. The zones of inhibition around the discs were recorded, and the antimicrobial efficacy was analyzed based on these observations.
The first step carried out were Collection and Preparation of Tithonia diversifolia Leaves where Collection and Drying of Five kilograms of Tithonia diversifolia leaves were collected and air-dried for five to seven days, following recommendations from a lecture.
Secondly, 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 is the third steps were, 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 and Measurement taken using a balance that is 20 grams of the ground grey powder were precisely weighed.
Fourthly, dissolution of 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.
Then extraction of Active Compounds 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.
After that, Preparation for Antimicrobial Test where 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
then, preparation of Agar Plates using the 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.
Next, Incubation and Observation where Incubation of
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.
Then, Serial Dilutions and Pour Plate Method done where 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.
The Final Step was Recording Results that was the zones of inhibition around the discs were recorded, and the antimicrobial efficacy was analyzed based on these observations.
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