Antimicrobial Assays

3.3.1. Microbial Culture Conditions

The different strains must be cultured twice for activation in Erlenmeyer flasks (125 mL) containing 25 mL of Mueller-Hinton broth at 37°C for 12 h in a shaker, except for Lactobacillus rhamnosus, L. plantarum, Zymomonas mobilis, and Sac-charomyces cerevisiae (see Note 10). Stop the cultures at 0.8-1 OD600 (equivalent to 108 CFU ), dilute until 0.1 OD600, and use for antimicrobial testing (see Note 11).

3.3.2. Detection of Antimicrobial Activity in Solid Medium

The agar well diffusion method, previously described (41,42), with some modifications (16), is used to detect antimicrobial activity of plant extracts.

1. Inoculate 3 mL of 0.6% M-H agar media with 30 ^L of an overnight culture of each microorganism prepared as described in Subheading 3.3.1.

2. Overlay onto plates containing 2% Mueller-Hinton agar media, except for Proteus vul-garis, for which 4% agar must be used.

3. Make wells of 3 mm in the agarized medium after inoculation with the microorganism to be tested. More than five wells per plate are not recommended.

4. Fill the wells with 20 ^L of total extract of flavonoids, fractions, or pure compound at a range of concentrations from 5 to 1.000 ^g/mL. The solvent for resuspending the extract should be MeOH 80% instead of pure MeOH because the latter produces irregular inhibition zones.

5. Carry out a negative control with each solvent.

6. Maintain the culture Petri dishes at 37°C, and do not invert them until the solvent is evaporated. After that, invert the plates to avoid water vapors condensation.

7. Measure the inhibition zones from one side to the other of the circle three times and discount the well diameter (Figs. 2 and 3).

8. Use chloramphenicol, as a standard antibiotic, at a range of 0-250 ^g/mL in order to make a calibration curve. The results are the averages of three individual experiments.

Fig. 3. Inhibition zones of fractions from T. minuta against E. coli. Solvent as negative control (1); chloroform fraction (2); ethyl acetate fraction (3); and aqueous fraction (4).

Table 2

Microorganisms Suitable for Antimicrobial Screening



Gram-negative rods

Gram-positive rods

Gram-positive cocci


Escherichia coli Salmonella enteriditis Shigella sp.

Pseudomonas aeruginosa Proteus vulgaris Zymomonas mobilis Bacillus subtilis Lactobacillus rhamnosus Lactobacillus plantarum Staphylococcus aureus Staphylococcus epidermidis Micrococcus luteus Saccharomyces cerevisiae

3.3.3. Minimum Inhibitory Concentration (MIC) in Liquid Medium

MIC values against bacterial strains are performed using the Ericcson and Sherris broth dilution method (43). Inocula suspensions are prepared from 6-h broth cultures and adjusted to 0.5 McFarland turbidity equivalents. Substances and extracts must be sterilized by Millipore filtration (0.45 ^m) and add to the MH broth medium. MIC determination are performed using serial dilutions of each extract and pure substance at a range of 0-1.000 ^g/mL.

4. Notes

1. Caution: chloroform is irritating to the skin, eyes, mucous membranes, and respiratory tract. Chloroform is a carcinogen and may damage the liver and kidneys. Methanol is poisonous and can cause blindness if ingested in sufficient quantities. Adequate ventilation is necessary to limit exposure to vapors. Glacial acetic acid is volatile. Concentrated acids should be handled with great care. Wear gloves and safety glasses and work in a chemical fume hood.

2. Microorganisms suitable for antimicrobial screening are shown in Table 2.

3. The possible enzyme action occurring during this early period of isolation, leading in particular to hydrolysis of glycosides, may be avoided by plunging the plant material into boiling solvent or by rapid drying prior to extraction. The success of the extraction is directly related to the extent that chlorophyll is removed from the solvent and when the tissue debris, on repeated extraction, is completely free of green colors.

4. Depending on the room temperature, it will take the solvent approx 24 h at 30°C to approach 2 cm from the end of sheet.

5. The run is developed in about 3-4 h. It is remarkable that with 15% or 30% AcOH the run typically forms a rounded front effect.

6. Spots detected should be circled with a pencil, and Rf values are determined as follows:

Rf = Distance between origin and the centre of concentration of the flavonoid spot Distance between origin and solvent front

7. Is important to minimize contamination of a flavonoid to be used for UV spectral studies. For purification of the flavonoid used in the spectroscopic investigation, it should be extracted from the chromatographic paper with spectroscopic methanol for a few minutes only.

8. The band should be located at approx 8 cm from the top of the chromatographic paper.

9. Flavonoids isolated by PC may still contain some soluble polysaccharide impurities; it is best to purify them further by Sephadex LH-20 chromatography.

10. Lactobacillus strains are cultured aerobically in MRS broth at 30°C for 12 h at 250 rpm. Zymomonas mobilis should be grown in Zym broth at 30°C for 12 h without agitation. Saccharomyces cerevisiae should be cultured at 30°C for 12 h at 200 rpm.

11. Prior to carrying out the antimicrobial screening, let the microbial dilution sit for 10 min at 37°C without agitation. This culture could be maintained at 4°C for a week for further antimicrobial analyses.


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