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4.2. DPPH 2,2-diphenyl-1-picrylhydrazyl Assay Molyneux, 2004
The trolox stock solution 10,000 µM was prepared by dissolving trolox 0.0250 g with methanol in 10 mL volumetric flasks. Then, the working standards 200-1000 µM were prepared
by diluted trolox stock solution with methanol in 10 mL volumetric flasks. The DPPH solutions were prepared by dissolving DPPH 0.0024 g with methanol in 100 mL volumetric flask.
Standard or diluted extracts 50 µL were transffered into the tube and followed by DPPH solutions 1950 µL. The mixture was kept in the room temperature for 30 min before analysis.
The absorbance of solution was measured at 517 nm. Every step in DPPH assay must be avoided from the light. The antioxidant capacity of coffee ground residue extracts were expressed in mg
trolox in 100 g dry basis.
5. Antimicrobial Activity
Three CGR extracts with highest bioactive compounds were measured the antimicrobial activity against several bacteria culture such as Bacillus subtilis, Listeria monocytogenes, and
Staphylococcus aureus as Gram-positive bacteria; Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa as Gram-negative bacteria.
There were several preparations before starting antimicrobial activity as follows: a.
Antibiotic Preparation Ampicillin and polymycin B sulphate were used in this work as positive control in
agar well diffusion method. Ampicillin was used for all bacteria except P. aeruginosa, while Polymycin B sulphate was used for P. aeruginosa. The ampicillin stock solution 100mgmL
was prepared by dissolving 1000 mg ampicillin powder with 10 mL sterile distilled water. Furthermore, the polymycin B sulphate solution 10mgmL was prepared by dissolving 100
mg polymycin B sulphate powder with 10mL sterile distilled water. The antibiotic solution was filtered into filter syringe before put in sterile sentrifuge tubes. The ampicillin stock
solution 40 µL was diluted with distilled water in 10mL sterile volumetric flask to get 20µg50µL concentration of amphicilin. Then, the polymycin B sulphate stock solution
300µL was diluted with distilled water in 10mL sterile volumetric flask to get 300 unit50µL concentration of polymycin B sulphate.
b. Mc Farland Preparation
0.5 Mc Farland solution was made by mixing 0.5 mL 1.175 wv BaCl
2
and 99.5 mL 1 vv H
2
SO
4
. The solution was checked the absorbance at 625 nm. The absorbance of 0.5 Mc Farland solutions must be between 0.08-0.13. Mc Farland solutions were kept in the
room temperature at dark condition until used. c.
Cultures Preparation The culture was activated in Nutrient Broth NB and incubated at 37
o
C for 18-24 h. Then, the activated culture was subcultured before using in the antimicrobial assay. Muller
Hinton Agar MHA was poured into petri dish and the culture was scratched on the agar. The petri dish was incubated at 37
o
C for 18-24 h. After preparation, antimicrobial activity of CGR extracts was determined with two
different methods as follows:
5.1. Agar-Well Diffusion Assay Lalitha, 2004
Agar well diffusion assay measured the effect of an antimicrobial agent against bacteria. The diameter of inhibition zone was measured by seeing the ability of the samples to inhibit the
growth of the bacteria. The microorganism was cultured in Muller Hinton Broth MHB. Then, the turbidity of the culture was compared with 0.5 Mc Farland in black-white paper. If the cultures and
Mc Farland had the same turbidity, the culture could be used in the analysis. The sample was prepared with different concentration 0.75 gmL, 1 gmL and 1.5 gmL. Before put into sterile
vials, the sample was filtered into filter syringes.
In agar well diffusion method, MHA was poured into petri disc. After 5-10 min, the fresh culture was swabed in the agar with cotton stick and kept until dry for 5 minutes. The well was
made by using cork borer. After that, 50 µL of extract with different concentration, positive control, and negative control sterile water were added into the wells. The dish was placed in the
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laminar for 2 h until the solution was absorbed into the agar. Then, the dish was incubated at 37oC for 18-24 h. Antimicrobial activity of the extract was determined by measuring clear zone
diameter around the well.
5.2.Micro-Broth Dilution Assay Lalitha, 2004
Micro-broth dilution assay 96-wells plate method in the CGR is determined antimicrobial activity by measuring minimum inhibition concentration MIC and minimum
bactericidal concentration MBC of the extracts. Extract was prepared in several concentrations. The initial concentration of each extract
was 1500 mgmL. Further 1:2 serial dilution was performed by addition of culture broth to reach concentrations ranging from 500 to 0.98 mgmL. Extract 100 µL was transffered in 96-wells
plates and 100 µL of bacterial culture in MHB was added in each well 2nd-11st well. Sterile control 200µL MHB and growth control 100 µL MHB and 100 µL bacterial cultures were also
added in the well 1st well and 12th well, respectively. Positive control 0.01 BHT was also added in a well by added 100 µL BHT and 100 µL MHB. Then, 96-wells plates were incubated at
37oC for 18-24 h.
The MIC value was evaluated after incubation, as the lowest concentration that completely inhibited the formation of visible growth have a clear turbidity compared with sterile
controls and growth controls. The MBC determination was used to assess if the inhibitory effect observed in MIC determinations was through a lethal bactericidal action. Extract from well
where the MIC results showed no bacterial growth was removed with a loop, inoculated into Muller-Hinton Agar MHA plate, and incubated at 37
o
C for 18-24 h. MBC was considered to be the concentration at which microorganisms were totally unable to grow.
6. Statistical Analysis
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