# Chemical Composition and Antimicrobial Properties of Honey Bee Venom
## 3 Materials and Methods
### Determination of Antimicrobial Activity of Bee Venom and Extracts  
Dry BV, as well as diethyl ether and methanol extracts obtained from it, was tested against microorganisms originating from the _American Type Culture Collection_ (LGC Standards Sp. z o.o., Lomianki, Poland): Gram-positive bacteria _Staphylococcus aureus_ ATCC 6538, _Paenibacillus larvae_ ATCC 9545, _Bacillus cereus_ ATCC 10987, and _Bacillus subtilis_ ATCC6633 and Gram-negative bacteria _Escherichia coli_ ATCC 11229 and _Pseudomonas aeruginosa_ ATCC 19582, as well as the fungus _Candida albicans_ ATCC 90029. All microorganisms stored in a 1:1 mixture of Luria-Bertani (LB) broth and glycerol were inoculated into either nutrient agar (bacteria) or Sabouraud agar (C. _albicans_) and incubated overnight at 37 \({}^{\circ}\)C.  
The antibacterial activity of the tested preparations was assessed by determining the minimal inhibitory concentration (MIC) in accordance with the Clinical and Laboratory Standard Institute (CLSI) protocols [56]. Bee venom extracts were dissolved in DMSO at a concentration of 8 mg mL\({}^{-1}\), filtered with a 0.22 \(\upmu\)m pore-size Rotilabo(r) syringe filter and serially twofold diluted in Mueller-Hinton broth, ranging from 4000 to 0.0002 \(\upmu\)m mL\({}^{-1}\), in a U-shaped 96-well microtiter plate with a final volume 100 \(\upmu\)L. The bacteria were cultured overnight in Mueller-Hinton broth at 37 \({}^{\circ}\)C with shaking at 200 rpm and then suspended to a final optical density of 0.2-0.3 at 600 nm wavelength measured with a V-670 spectrophotometer (Jasco, Tokyo, Japan). For the assay, 100 \(\upmu\)L of the bacterial suspensions was added to each well in the microtiter plate containing diluted bee venom extracts and incubated overnight at 37 \({}^{\circ}\)C. In order to obtain comparable data, all the bacteria were treated under the same conditions. The MIC values were determined as the lowest concentration of the extracts in the wells with no bacterial growth observed visually. All the tests were carried out in quadruplicate, and the results were averaged.  
In addition, the minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) of the extracts were assessed. For this purpose, 5 \(\upmu\)L of the overnight culture from each well in the microtiter plate with extracts of concentration equal to and higher than the MIC value was inoculated onto BHI (Brain Heart Infusion) agar with the use of a sterile plastic spreader and incubated overnight at 37 \({}^{\circ}\)C. The MBC/MFC values were determined as the lowest concentration of the extracts in the wells with no bacterial growth on plates observed visually. All the tests were carried out in quadruplicate, and the results were averaged.  
As a positive control, microorganisms cultured in the Mueller-Hinton broth and on the BHI agar without the bee venom extracts were applied. Mueller-Hinton broth supplemented with 10% dimethyl sulfoxide (DMSO) was used as solvent control, while Mueller-Hinton broth with 10% DMSO and extracts was used as the bee venom extract control. The MIC and MFC values for C. _albicans_ were assessed as above, but with the application of Sabouraud broth and Sabouraud agar instead of Mueller-Hinton broth and BHI agar, respectively.