One of the first systematic in vitro examinations of the antimicrobial activity of essential oils dates back to the late nineteenth century when Buchholtz studied the growth inhibitory properties of caraway oil, thyme oil, phenol, and thymol on bacteria having been cultivated in a tabac decoction. In this examination, thymol turned out to be 10-fold stronger than phenol (Buchholtz, 1875), which was in use as surgical antiseptic at that time (Ashhurst, 1927). The German pharmacopoeia "Deutsches Arzneibuch 6" (DAB 6) issued in 1926 and later supplements (1947, 1959) listed together 26 essential oils, and by this it has become obvious that essential oils have a long history in pharmaceutical practice due to their pharmacological activities. The European Pharmacopoeia 6th edition issued in 2007 lists 28 essential oils. Among them are 20 oils already present in DAB 6 (anise, bitter fennel, caraway, cassia, cinnamon bark, citronella, clove, coriander, eucalyptus, juniper, lavender, lemon, matricaria, neroli, peppermint, pine needle, pumilio pine, rosemary, thyme, and turpentine), three oils have been previously listed in the British Pharmacopoeia in the year 1993 (dementholized mint, nutmeg, and sweet orange), one in the French Pharmacopoeia X (star anise), and five oils were added later (cinnamon leaf, clary sage, mandarin, star anise, and tea tree).
Since essential oils are subject for pharmacological studies, tests on their antimicrobial activities have been done frequently. In consequence, a comprehensive data material exists, which, however, has never been compiled together for important essential oils, like such listed actually in the European Pharmacopoeia. Therefore, an attempt was done to collect and examine such information from scientific literature to obtain an insight into the variability of test parameters, data variation, and the significance of such factors in the interpretation of results.
Among the testing methods used to characterize in vitro antimicrobial activity of essential oils, the three main methods turned out to be agar diffusion test, serial broth or agar dilution test, and the vapor phase test. Further tests comprise various kill-time studies, for example, the activity of a compound relative to phenol after 15 min (phenol coefficient) (Rideal et al., 1903), killing time determination after contact to a test compounds using contaminated silk threads (Koch, 1881), recording of growth curves and determination of the amount of a compound being effective to inhibit growth of 50% of test organisms (Friedman et al., 2004), poisoned food techniques in which the delay of microbial growth is determined in presence of growth inhibitors (Kurita et al., 1983; Reiss, 1982), spore germination, and short contact time studies in fungi (Smyth et al., 1932; Mikhlin et al., 1983). Other studies monitor presence or absence of growth by measuring metabolic CO2 in yeast (Belletti et al., 2004) or visualize growth by indicators, such as sulfur salts from sulfur-supplemented cow milk as growth medium (Geinitz, 1912), 2,3,5-triphenyltetrazolium chloride (Canillac et al., 1996), or p-iodonitrophenyltetrazolium violet (Al-Bayati, 2008; Weseler et al., 2005). The bioautographic assay on thin-layer chromatography plates has been developed for identification of active compounds in plant extracts (Rahalison et al., 1994), but was later also taken for the examination of essential oils (Iscan et al., 2002).
12.1.1 Agar Diffusion Test (ADT)
In the agar diffusion test, the essential oil to be tested is placed on top of an agar surface. Two techniques exist: In the first one, the essential oil is placed onto a paper disk; in the second, a hole is made into the agar surface and the essential oil is put into the hole. In the following, the essential oil diffuses from its reservoir through the agar medium, which is seeded with microorganism. Antimicrobially active oils cause an inhibition zone around the reservoir after incubation, respectively, and normally the size of inhibition zone is regarded as measure for the antimicrobial potency of an essential oil. However, lipophilic compounds such as farnesol cause only small inhibition zones against Bacillus subtilis in the agar diffusion test (Weis, 1986), although the compound resulted in a strong inhibition in the serial dilution test (MIC = 12.5 ^g/ml) (Kubo et al., 1983). Thus, strong inhibitors having low water solubility gave a poor or even negative result in the agar diffusion test. It is therefore wrong to conclude that an essential oil without resulting in an inhibition zone in the agar diffusion test is without any antimicrobially active constituents; or in other words, antimicrobially active compounds are easily overlooked by this method. Another problem is the interpretation of the size of inhibition zones, which depend on both, the diffusion coefficient plus antimicrobial activity of every compound present in an essential oil. Beside generally unknown diffusion coefficients of essential oil constituents, the size of inhibition zones is influenced by several other factors: volatilization of essential oil, disk or hole sizes, amount of compound given to disk or into hole, adsorption by the disk, agar type, agar-agar content, pH, volume of agar, microbial strains tested (Janssen et al., 1987). Taken together, this test method can be used as a pretest, but the results should not be over-rated. In the following data schedule the experimental conditions are briefly given (culture medium, incubation temperature, incubation time, disk or hole size, amount of essential oil on disk or hole). The amount of compound in microgram added to a reservoir (disk or hole) is recalculated from microliter with a density of 1 for all essential oils.
12.1.2 Dilution Test (DIL)
In the dilution test, the essential oil to be tested is incorporated in a semisolid agar medium or liquid broth in several defined amounts. Absence of growth in agar plates or test tubes is determined with the naked eye after incubation. The minimum inhibitory concentration (MIC) is the concentration of essential oil present in the ungrown agar plate or test tube with the highest amount of test material. When essential oils are tested, the main difficulty is caused by their low water solubility. The addition of solvents (e.g., dimethylsulfoxide and ethanol) or detergents (e.g., Tween 20) to the growth medium are unavoidable, which however influences the MIC (Hili et al., 1997; Hammer et al., 1999; Remmal et al., 1993). Another problem is the volatilization of essential oils during incubation. Working in a chamber with saturated moistened atmosphere (Pauli, 2006) or high water activity levels (Guynot et al., 2005) improved the situation. The MIC is additionally influenced by the selection of growth media, for example, in RPMI 1640, the MIC toward yeast is about 15 times lower than in Sabouraud medium (Jirovetz et al., 2007; McCarthy et al., 1992). Further MIC-influencing test parameters are size of inoculum, pH of growth medium, and incubation time. Nevertheless, the serial dilution test in liquid broth was recommended for natural substances (Boesel, 1991; Hadacek et al., 2000; Pauli, 2007) and is standardized for the testing of antibacterial and antifungal drugs in liquid broth and agar plates (Clinical & Laboratory Standards Institute, 2008). Its use enables a link to data of pharmaceutical drugs and an easier interpretation of test results. In the data section, all concentrations are recalculated in pg/ml. To distinguish between the agar dilution test and the serial dilution test, the growth medium is abbreviated either as agar (A) or broth (B). Test parameters— exceptionally citations—are systematically given and comprise growth medium, incubation time, incubation temperature, and MIC in pg/ml or ppm.
12.1.3 Vapor Phase Test (VP)
In the vapor phase, a standardized method does not exist among tests to study antimicrobial activity of essential oils. In most of the examinations, a reservoir (paper disk, cup, and glass) contains the sample of essential oil, and a seeded agar plate was inverted and covered the reservoir. After inoculation, an inhibition zone is formed, which is the measure of activity. Most of the data listed in the following tables have been worked out by such methods. Because these methods allow only the creation of relative values, a few examiners defined the MIC in atmosphere (MICair) by using airtight boxes (Inouye et al., 2001; Nakahara et al., 2003), which contained a seeded agar plate and the essential oil on the glass or paper. Otherwise, the results were estimated with +++ = normal growth, ++ = reduced growth, + = visible growth, and NG = no growth. The test parameters given in the following tables are growth medium, incubation time, incubation temperature, and activity evaluation or MICair in pg/ml or ppm.
To give detailed information, the following abbreviations are used in Tables 12.1 through 12.80: (h), essential oil was given into a hole; BA, Bacto agar; BHA, brain-heart infusion agar; BlA, blood agar; CA, Czapek's agar; CAB, Campylobacter agar base; CDA, Czapek Dox agar; DMSO, dimeth-ylsulfoxide; EtOH, ethanol; EYA, Emerson's Ybss agar; germ., germination; HIB, heart infusion broth; HS, horse serum; inh., inhibition; ISA, Iso-sensitest agar; KBA, King's medium B agar; LA, Laury agar; LSA, Listeria selective agar; MA, malt agar; MAA, medium A agar; MBA, mycobiotic agar; MCA, MacConkey agar; MEB, malt extract broth; MHA, Mueller-Hinton agar; MHA, Mueller-Hinton broth; MIA, minimum inhibitory amount in pg per disk; MIC, minimum inhibitory concentration in pg/ml or ppm; MICair, minimal inhibitory concentration in pg/ml or ppm in the vapor phase; MYA, malt extract-yeast extract-peptone-glucose agar; MYB, malt extract-yeast extract-glucose-peptone broth; NA, nutrient agar; NB, nutrient broth; OA, oat agar; PB, Pennassay broth; Ref., reference number; SA, Sabouraud agar; SB, Sabouraud broth; sd, saturated disk; SDA, Sabouraud dextrose agar; SGB, Sabouraud glucose broth; SMA, Sabouraud maltose agar; sol., solution; TYA, tryptone-glucose-yeast extract agar; THB, Todd-Hewitt broth; TSA, trypticase soy agar; TSB, trypticase soy broth; TYB, tryptone-glucose-yeast extract broth; VC, various conditions: not given in detail; WA, sucrose-peptone agar; WFA, wheat flour agar; YPB, and yeast extract-peptone-dextrose broth.
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