**3. Determination of antimicrobial activity of honey**

The in vitro antimicrobial activity of most agents is usually estimated with two methods: an agar diffusion assay and a serial dilution method in microtiter plates. Both these methods have been also used for determination of antimicrobial potential of honey. The agar diffusion assay is based on the measurement of size of growth inhibition zone around the place of loading a sample of honey (usually well, cut with a cork borer in the agar). The assay is easy and quick in performance. Unfortunately, it has several important limitations [11]:


**Figure 3.** Results of agar diffusion assay of activity of four selected honeys: (A) rapeseed honey (*Brassica napus* L.); (B) multifloral honey; (C) buckwheat honey (*Fagopyrum esculentum* Moench); (D) Manuka honey (*Leptospermum scoparium*). Definitely the highest activity was observed in the case of Manuka honey—picture D. Some characteristic halo zones are present in all pictures. 100 µl of 50% (v/v) was loaded to the wells in the agar.


growing bacteria characterize with different (lower) diameter, and some changes of color of agar can be noticed, which is difficult in interpretation (**Figure 3**). In our opinion, the presence of these halo zones is a consequence of influence of low molecular components of honey on the growth of microbial cells.

assay is based on the measurement of size of growth inhibition zone around the place of loading a sample of honey (usually well, cut with a cork borer in the agar). The assay is easy and

• high viscosity of honey and problems with loading of defined volume of the product sample to the wells in the agar. It is especially problematic when the honey is crystallized;

• problems with diffusion of active components (defensin-1 and especially glucose oxidase characterize with high molecular weight) through the agar matrix. As a result, the diameters of observed growth inhibition zones are relatively low. The honeys with evidentially different activities established with other methods give similar results in agar diffusion assay (not large differences in the diameters of growth inhibition zones are observed—based

• low reproducibility—it is difficult to get similar results (diameter of growth inhibition

**Figure 3.** Results of agar diffusion assay of activity of four selected honeys: (A) rapeseed honey (*Brassica napus* L.); (B) multifloral honey; (C) buckwheat honey (*Fagopyrum esculentum* Moench); (D) Manuka honey (*Leptospermum scoparium*). Definitely the highest activity was observed in the case of Manuka honey—picture D. Some characteristic halo zones are

• low discriminatory power—consequence of relatively low sizes of observed growth inhibition zones. It is also difficult to compare the obtained results with the results of other authors;

• problems with interpretation of obtained results. Usually except of clear, growth inhibition zones at least one halo zone can be observed (**Figure 3**). In this halo zone, the colonies of

• lack of possibilities to distinguish bacteriostatic and bactericidal activity;

quick in performance. Unfortunately, it has several important limitations [11]:

on results of own studies, **Figure 3**);

220 Honey Analysis

zone) in several independent experiments;

present in all pictures. 100 µl of 50% (v/v) was loaded to the wells in the agar.

The problems with high viscosity can be, at least partly, omitted by using honey dissolved in sterile water (e.g., 50%, w/w), as it has been proposed by several authors [15]. However, usually it does not solve other discussed above problems.

Based on our experience, we would rather recommend a serial dilution method for investigation of antimicrobial potential of honey [13]. This method allows quantitative determination of both bacteriostatic and bactericidal activity of tested honey samples, **Figures 4** and **5**. The bacteriostatic activity is characterized with MIC (*Minimal Inhibitory Concentration* the lowest concentration of honey that inhibits the growth of tested strain of microorganisms) parameter, while bactericidal activity is characterized with MBC (*Minimal Bactericidal Concentration*—the lowest concentration of an antibacterial agent required to kill a particular bacterium) parameter.

**Figure 4.** The results of determination of antistaphylococcal activity of four tested honeys: (A) rapeseed honey (*Brassica napus* L.)—rows 1–3; (B) multifloral honey—rows 4–6; (C) buckwheat honey (*Fagopyrum esculentum* Moench)—rows 7–9; (D) Manuka honey (*Leptospermum scoparium*)—rows 10–12. The concentrations of honeys in the wells of following columns were as follows: 12.5, 6.25, 3.12, 1.56, 0.78 and 0.39% (v/v). The wells of column number 7 contained only growing medium (Mueller Hinton BrothII cation adjusted) neither honey nor cells of bacteria were present in these wells—negative control. The wells of column 8 did not contain honey, and they were used as a positive control of growth of bacteria in the medium not containing any antimicrobial agent. No activity was observed in the case of rapeseed honey. The reference strain of bacteria *S. aureus* PCM1051 was able to grow in all wells of rows 1–3. The MIC value for multifloral honey (the lowest concentration of honey, which caused visible inhibition of growth of *S. aureus* strain), was 3.12% in row 4 and 1.56% in rows 5 and 6. The MIC value for buckwheat honey in all three tested rows was 1.56%, and the constant value of MIC for Manuka honey, 12.5%, was observed in the rows 10–12.

**Figure 5.** The results of determination of minimal bactericidal concentrations of tested honeys against *S. aureus*. The assay (carried out according to the procedure presented in **Figure 6**) confirmed the lack of activity of rapeseed honey. The MBC values of multifloral honey were exactly the same as MIC values for this product. In the case of buckwheat honey, MBC and MIC values were the same in the rows 7 and 9, and in the case of row 8, the MBC was twice of the MIC value, 3.12 and 1.56% (v/v), respectively. Interestingly, no bactericidal activity was observed in the case of Manuka honey. However, the intensity of growth of bacteria transferred from the wells with the highest concentration of this honey (12.5%) was evidentially inhibited in comparison with samples presenting wells containing lower concentrations of this product. Despite the fact that the same product was tested in triplicate, in the case of determination of activity of multifloral and buckwheat honeys, some differences in the obtained MIC and MBC values were observed for different rows (e.g., MBC value for buckwheat honey was 1.56% in the case of rows 7 and 9, and in the case of row 8, the bactericidal effect was achieved at the concentration of 3.12%). However, the observed differences of determined values of the parameters of interest for particular honey were not larger than two times, which is acceptable in these assays.

The detailed description of procedure of performance of this assay as well as determination of both parameters MIC and MBC has been presented in **Figure 6**. The most problematic step of this assay is preparing the output solution of honey; in our laboratory, it is usually 25% (v/v). Because of high viscosity of honey, the determination of volume of the product used for preparing the solution has to be done extremely carefully. Other way, it can be a source of significant measurement errors. There are also several other advantages of serial dilution method in comparison with agar diffusion assay. The dilution assay gives more reproducible results, which are easy in interpretation. It also characterizes with much better discriminatory power, for example, results presented in **Figures 4** and **5**.

Slightly modified serial dilution method can be also used for determination of antibiofilm activity of honey. Minimum biofilm eradication concentration (MBEC—the lowest concentration of an antibacterial agent required to eradication of biofilm formed by a particular bacterium) of honey is determined in this assay. In general, the assay is performed identically as in the case of determination of MIC or MBC parameters. However, in the first step, the bacterial biofilm is grown in the wells of titration plates.

Preparing this chapter, we performed some assays of activity of four selected honeys: A, rapeseed; B, multifloral; C, buckwheat; D, Manuka honey against *S. aureus* PCM2054 reference strain. As it is presented in **Figures 3** and **4**, some important differences in the results of these two assays have been obtained. In the case of agar diffusion assay, definitely the highest activity was observed for Manuka honey, while in the case of dilution method, buckwheat

**Figure 6.** The procedure of performance of serial dilution method. The MHBII medium used for honey dilution should be prepared with using of only 75% of water volume recommended for this medium. The required volume of medium will be obtained in the consequence of adding of honey. MHBII medium used for serial dilution of honey and preparing of suspension of bacterial cells should be prepared according to manufacturer's procedure (with using recommended volume of water).

The detailed description of procedure of performance of this assay as well as determination of both parameters MIC and MBC has been presented in **Figure 6**. The most problematic step of this assay is preparing the output solution of honey; in our laboratory, it is usually 25% (v/v). Because of high viscosity of honey, the determination of volume of the product used for preparing the solution has to be done extremely carefully. Other way, it can be a source of significant measurement errors. There are also several other advantages of serial dilution method in comparison with agar diffusion assay. The dilution assay gives more reproducible results, which are easy in interpretation. It also characterizes with much better discriminatory

interest for particular honey were not larger than two times, which is acceptable in these assays.

**Figure 5.** The results of determination of minimal bactericidal concentrations of tested honeys against *S. aureus*. The assay (carried out according to the procedure presented in **Figure 6**) confirmed the lack of activity of rapeseed honey. The MBC values of multifloral honey were exactly the same as MIC values for this product. In the case of buckwheat honey, MBC and MIC values were the same in the rows 7 and 9, and in the case of row 8, the MBC was twice of the MIC value, 3.12 and 1.56% (v/v), respectively. Interestingly, no bactericidal activity was observed in the case of Manuka honey. However, the intensity of growth of bacteria transferred from the wells with the highest concentration of this honey (12.5%) was evidentially inhibited in comparison with samples presenting wells containing lower concentrations of this product. Despite the fact that the same product was tested in triplicate, in the case of determination of activity of multifloral and buckwheat honeys, some differences in the obtained MIC and MBC values were observed for different rows (e.g., MBC value for buckwheat honey was 1.56% in the case of rows 7 and 9, and in the case of row 8, the bactericidal effect was achieved at the concentration of 3.12%). However, the observed differences of determined values of the parameters of

Slightly modified serial dilution method can be also used for determination of antibiofilm activity of honey. Minimum biofilm eradication concentration (MBEC—the lowest concentration of an antibacterial agent required to eradication of biofilm formed by a particular bacterium) of honey is determined in this assay. In general, the assay is performed identically as in the case of determination of MIC or MBC parameters. However, in the first step, the bacterial

Preparing this chapter, we performed some assays of activity of four selected honeys: A, rapeseed; B, multifloral; C, buckwheat; D, Manuka honey against *S. aureus* PCM2054 reference strain. As it is presented in **Figures 3** and **4**, some important differences in the results of these two assays have been obtained. In the case of agar diffusion assay, definitely the highest activity was observed for Manuka honey, while in the case of dilution method, buckwheat

power, for example, results presented in **Figures 4** and **5**.

biofilm is grown in the wells of titration plates.

222 Honey Analysis

and multifloral exhibited much better activity in comparison with the honey produced in New Zealand. Rapeseed honey in both assays was classified as non-active. These results are in agreement with our previous observations [13]. Our previous research revealed also that activity of polish honeys is hydrogen peroxidase dependent [13]. Thus, the relatively low activity of buckwheat and multifloral honey in the case of agar diffusion method was probably a consequence of difficulties of migration glucose oxidase through the agar. Activity of Manuka honey comes mainly from high content of MGO, which is a low molecular weight component that can easily migrate through the agar generating a large growth inhibition zone. It is also worth to notice that MBC and MIC values for buckwheat and multifloral honeys are the same (1.56%, v/v). The MBC value for Manuka honey could not be determined in the tested range of concentrations; however, evident inhibition of growth of *S. aureus* in the wells containing 12.5% (v/v) of the honey is visible in both: titration plate as well as on the Petri dishes with Baird-Parker agar.
