Chapter 1 INTRODUCTION
3.2 Glass Column Chromatography
3.3.2 Process of Bioassay
40 If the amount of the fractions was very little, it was directly dissolved with an appropriate amount of filtered methanol: water solution (2:1) to form a 10 mg/mL solution. After labelled properly, they were also kept in the fridge.
41 amphotericin B and fungus were sprayed with 70% ethanol solution for decontamination and transferred into the laminar flow cabinet. Gloves was worn when handled these works and it were also decontaminated by 70% ethanol solution.
Then, each column and wells of the 96-well plate were labeled properly according to the number of fractions, antibiotics and other aspects which was shown in Figure 3.1 below.
- 50 μL of fraction + 50 μL of medium + 50 μL of fungus
- Sterility control (100 μL medium)
- Growth control (50 μL medium + 50 μL of fungus)
42 Figure 3.1: Illustration of the utilization of 96- well plate for MIC assay
Table 3.2: Function of each control on 96-well plate
Type of control Function
Positive control - To show the expected positive result (Increased in the formation of precipitates at decreasing concentration of antibiotics)
- To determine whether the fungus is drug resistance Negative control - To show the expected negative result and to determine
whether the fraction had been contaminated by fungus Growth control - To determine whether the fungus can grow properly Sterility control - To determine whether the 96-well plate and medium is
contaminated
3.3.2.1 Orders of Addition of Reagents and Fractions
The medium, RPMI 1640 was poured into a 25 mL reservoir. Then, multichannel micropipette with 8 10-100 µL micropipette tips attached was used to transfer 50
- Negative control (75 μL medium + 25 μL of fraction) - Positive control (50 μL medium + 50 μL of antibiotic + 50 μL of fungus)
43 µL of the medium to all the wells in the fractions columns, the column for amphotericin B and the positive control. Then, the volume of the multichannel micropipette was adjusted to transfer 75 µL of the medium to the wells of negative control and 100 µL to the four wells of sterility control.
After that, a 10 – 100 µl micropipette was used to transfer 50 µL of each fraction to the first row of the fractions' well and another 25 µL for the well of negative control corresponding to each fraction. On the other hand, 50 µL of Amphotericin B was also added to its own well on first row. After all fractions and medium had been added to their corresponding wells in an appropriate amount, serial dilution was conducted for the fractions and Amphotericin B.
3.3.2.2 Serial dilution for fractions and Amphotericin B
A multichannel micropipette with appropriate number of 100 µL micropipette tips corresponding to the total number of fractions and Amphotericin B in the first row was pre-adjusted to 50 µL. Then, it was used to mix the mixture in the fractions' and Amphotericin B's well by taking in and out the mixture for roughly 15 to 20 times. After that, 50 µL of each mixture was transfered to the wells in second row.
The process was repeated again for the wells in second row and then transferered again to the subsequent row until the final row. On that time, 50 µL of the mixture in the final row was withdrew and discarded.
44 3.3.2.3 Preparation of Fungus
One sub-cultured fungus agar plate cannot be used for all sets of replicates in the bioassay. For each set of replicate, only one sub-cultured fungus plate can be used.
Thus, in order to avoid confusion, each capped test tube (used to contain the fungus solution) was labelled properly according to the number of replicate.
Approximately 3 mL of the medium was added into the capped test tube. Then, the metal loop of the inoculating loop was burned in the flame of Bunsen burner until it turned glowing-red. After it was cooled, it was used to transfer a little amount of fungus into the medium prepared in the test tube. The loop was heated with the flame again for decontamination so that it can be used for next preparation. After the fungus was transferred to the medium and mixed well, its optical density was measured with spectrophotometer at 530 nm after a blank (RPMI 1640) was used to calibrated the instrument. If its absorbance fell between 0.12-0.15, the fungal solution can be used for bioassay. If it was below the range, more fungus was added until the absorbance fall within the desired range. On the other hand, more medium was added to dilute the fungus solution if the absorbance was above the range.
Optical density measurement of microorganism cultures is common to identify the essential cultures parameters such as cell concentration. It is based on the amount of light scattered by the cells in the cultures rather than the absorbance. The
45 instrument will measure the decrease in transmittance which is caused by the scattering effect from the cell suspension. Thus, the higher the cell suspension, the greater the scattering effect and the higher the absorbance (Matlock, 2017).
For one 96-well plate, around 4995 µL of medium and 5 µL of fungus solution are required. Thus, if the replicate consists of two 96-well plates, it need to transfer 9990 µL of medium and 10 µL of fungus solution to a reservoir and mix together. The mixture need to be mix thoroughly because the fungus added was in a very small amount.
After that, multichannel micropipette was used to transfer 50 µL of the diluted fungus solution to each of the well of the plates that consists of column fractions, Amphotericin B, negative control and positive control except the wells of sterility control. Lastly, the plates were closed with the caps, sealed with parafilm and incubated in the fungus incubator oven (35 oC) for 2 days.
3.3.2.4 Addition of p-iodonitrophenyltetrazolium violet (INT) indicator During the next day of incubation, the plates were took out from the incubator oven. Firstly, INT indicator was poured on the reservoir. Then, multichannel micropipette was used to transfer 50 µL of the indicator to each of the well that consists of column fractions, Amphotericin B, negative control, positive control
46 as well as the wells of sterility control. After that, the plates were sealed with parafilm and put back into the incubator oven to incubate one more day.
3.3.3 Determination of Minimum Inhibitory Concentration (MIC) and