Biofilm Formation Assay KitB601 Product Manual

Biofilms are biological aggregates consisting of microorganisms and extracellular polysaccharides that can exist in many environments. Bacterial contamination of medical devices, and infectious diseases such as caries and periodontal disease, are attributed to biofilms. The microorganisms in biofilms are highly resistant to antibiotics; therefore, research into medicines and food ingredients that have anti-biofilm activities is a growing area.
The Biofilm Formation Assay Kit measures the amount of biofilm formation and the anti-biofilm activity of samples. As the biofilm is formed on a peg layer on the lid of a microplate, multiple samples can easily be washed and measured at once without having to peel the biofilm off for each process.

	96 tests
Crystal Violet Solution	22 ml x 1
96-peg Lid	x 1
96-well Plate	x 10

Store in a cool and dark place

• Microplate reader (590 nm filter)
• Incubator (37 °C)
• 20-200 μl multichannel pipette
• Ethanol
• Sterile physiological saline solution

• This kit includes 10 sterile 96-well Plates. After opening, please keep the plates under sterile conditions until the experiments and use a new 96-well Plate in each step.
• The conditions for the formation of the biofilm vary by the species of microorganism. Please refer to table 1. Please optimize the conditions for the formation of the biofilm prior to measuring the anti-biofilm activity.

Measuring the amount of biofilm formation and anti-biofilm activity.

Anti-biofilm activity measurement for ε-poly-L-lysine : Staphylococcus aureus

1. The microbial cell suspension was prepared at approximately 10⁷ CFU/ml with Mueller-Hinton broth (MHB) and 180 μl of the cell suspension was added to each well of 96-well Plate ①.
2. ε-Poly-L-lysine (10,000 μg/ml) was prepared with MHB medium and sterilized by filtration. The 10,000 μg/ml ε-Poly-L-lysine solution was diluted 5-fold with the 10⁷ CFU/ml microbial cell suspension to give a 2,000 μg/ml ε-Poly-L-lysine solution. The 2,000 μg/ml ε-Poly-L-lysine solution (180 μl) was added to the leftmost well of 96-well plate ① from step 1and serially diluted to prepare a double dilution series of ε-Poly-L-lysine (180 μl in each well). The 96-peg Lid was then placed onto 96-well Plate ①, and the plate was incubated at 37°C for 24 hours.
   (e.g.1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.95, 0.98, and 0 μg/ml)
3. A double dilution series of ε-Poly-L-lysine, the same concentrations as step 2, was prepared with MHB medium on 96-well Plate ②.The 96-peg Lid from step (2) was then placed onto 96-well Plate ②, and the plate was incubated at 37°C for 72 hours.
4. A double dilution series of ε-Poly-L-lysine, the same concentrations as step 3, was prepared with MHB medium in  96-well Plate ③. The 96-peg Lid from step (3) was then placed onto 96-well Plate ③, and the plate was incubated  at 37°C for 24 hours.
5. Sterile physiological saline solution (200 μl) was added to each well of 96-well Plate ④ and 96-well Plate ⑤.Crystal Violet Solution (200 μl) was added to each well of 96-well Plate ⑥.
6. The 96-peg Lid from step 4 was washed by soaking in 96-well Plates ④ and ⑤. The 96-peg Lid was then placed onto 96-well Plate ⑥ and the plate was incubated at room temperature for 30 minutes.
7. Sterile physiological saline solution (200 μl) was added to each well of 96-well Plate ⑦ and 96-well Plate ⑧. Ethanol (200 μl) was added to each well of 96-well Plate ⑨.
8. The 96-peg Lid from step 6 was washed by soaking in 96-well Plates ⑦ and ⑧. The 96-peg Lid was then placed onto 96-well Plate ⑨ and the plate was incubated at room temperature for 15 minutes.
9. The 96-peg Lid was removed and the absorbance at 590 nm was measured.

 

Table.1 Experimental example 2

Microorganism	Microorganism dilution medium	Microorganism concentration [CFU/ml]	Incubation time [h]	Test substance dilution medium	Culture conditions
Pseudomonas aeruginosa	Brain heart infusion broth	106	24	MHB	Aerobic conditions
Escherichia coil	Brain heart infusion broth	107	24+72+24	MHB	Aerobic conditions
Streptococcus mutans	TSB+2％Sucrose	107	24+72	TSB+2％Sucrose	Anaerobic conditions
Porphyromonas gingivalis	Enriched BHI	107	24+72	Enriched BHI	Anaerobic conditions

Enriched BHI : Enriched BHI broth (3.7% brain heart infusion, 0.5% yeast extract, and 0.05% cysteine) containing 5 μg/ml of hemin and 0.5 μg/ml of menadione.

 

mutansT. Tsukatani, et al., J. Microbiol. Biotech. Food Sci., 2016, 6, 677-680

This product was developed through joint research within the Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center.