Biofilms are biological aggregates consisting of microorganisms and extracellular polysaccharides that can exist in many environments. 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.Our assay kits for the quantitative measurements of biofilm formation/inhibition of biofilm formation (Biofilm Formation Assay Kit) and anti-biofilm drug efficacy (Biofilm Viability Assay Kit) adopted a protocol which is designed to use the peg plate from biofilm formation. Our assay kits combined all the necessary components into each package.
Feature 1: Measurement time can be significantly shortened.In existing methods, biofilms are formed on the bottom of the well of a microplate, and additional work is required to change microbial culture media and wash sample several times before and after staining. However, our assay kits allow you to form biofilms on the pegs attached to the plate’s lid. Media change and staining process can be done simply by transferring the lid; therefore, our assay kits are very easy to use.
Feature 2: Our assay kits can reduce measurement variation.In existing methods, biofilm formation occurs on the bottom of the well of a microplate, which made biofilm easier to peel off because of the processes such as washing. This causes measurement variation, and this measurement variation was an issue for the quantification of biofilm. Our assay kits allow you to form biofilms on the pegs attached to the plate’s lid and make it possible to avoid peeling off biofilm which occurs during the process of the formation.
Two different types of assay kits for different purposes.We offer two types of assay kits that measure the amount of biofilm formation or the metabolic activities of live microorganisms within biofilms by using the same measurement method. You can select the one that best suits your needs.
Selection of 2 types of assay kits
*Conditions on biofilm formation vary according to microbial species and strains. When you need to examine these conditions, we first recommend you use the Biofilm Formation Assay Kit.*The Biofilm Formation Assay Kit is a product developed together with Fukuoka Industrial Technology Center.
Procedure
Example of measurementIndicators for drug-mediated inhibition of biofilm formation or antimicrobial effect of biofilms include MBIC (minimum biofilm inhibitory concentrations) and MBEC (minimum biofilm eradication concentrations). MBIC and MBEC of S. aureus were measured using each kit.
Measure the amount of biofilm formation – ①Under the following optimized conditions: types of culture media, seeding density of microorganisms, and media changes, the amount of biofilm formation can be confirmed by the experiments shown below.
Example of plate arrangement
Experimental schedule
Procedure(Step 1) Day 1: 96-well plate ①In accordance with the reference layout shown above, fill each well (rows A and B) of the 96-well plate with 180μl of microbial cell suspension with concentrations ① and ② made in culture media A through D.Leave the wells (rows C through H) blank. Place a 96-peg lid on the 96-well plate and incubate the plate at the optimum growth temperature for the microorganism for 24 hours.※ ExamplesMicrobial concentrations ① and ②: Approximately 107 CFU/ml and 106 CFU/ml, respectively.(Step 2) Day 2: 96-well ②In accordance with the reference layout shown above, fill each well (rows A and B) of the 96-well plate with 180μl of culture media without microorganism.Fill each well (rows C and D) of the same 96-well plate with 180μl of microbial cell suspension with concentrations ① and ② made in culture media A through D. Leave the wells (rows E through H) blank.Place the 96-peg lid (Step1) on the 96-well plate ② and incubate the 96-well plate ② at the optimum growth temperature for the microorganism for 24 hours.(Step 3) Day 3: 96-well plate ③In accordance with the reference layout shown above, fill each well (rows A through D) of the 96-well plate with 180μl of culture media without microorganism.Fill each well (rows E and F) of the same 96-well plate with 180 μl of microbial cell suspension with concentrations ① and ② made in culture media A through D. Leave the wells (rows G and H) blank.Place the 96-peg lid (Step1) on the 96-well plate ③ and incubate the 96-well plate at the optimum growth temperature for the microorganism for 24 hours.(Step 4) Day 4: 96-well plate ④In accordance with the reference layout shown above, fill each well (rows A through F) of the 96-well plate with 180μl of culture media without microorganism.Fill each well (rows G and H) of the same 96-well plate with 180μl of microbial cell suspension with concentrations ① and ② made in culture media A through D.Place the 96-peg lid (Step1) on the 96-well plate ④ and incubate the 96-well plate ④ at the optimum growth temperature for the microorganism for 24 hours.(Step5) Perform experiments using a new 96-well plate, in accordance with Steps 2 through 6 which describe the measurements of biofilm formation/inhibition of biofilm formation in the technical manual.Measure the amount of biofilm formation – ②Under the following optimized conditions: types of culture media, seeding density of microorganisms, and media changes, the amount of biofilm formation can be confirmed by the experiments shown below.
Example of plate arrangement
Experimental schedule
Procedure(Step 1) Day 1: 96-well plate○1In accordance with the reference layout shown above, fill each well (rows A and B) of the 96-well plate with 180μl of microbial cell suspension with concentrations ① and ② made in culture media A through D.Leave the wells (rows C through H) blank.Place a 96-peg lid on the 96-well plate and incubate the plate at the optimum growth temperature for the microorganism for 24 hours.※ ExamplesMicrobial concentrations ① and ②: Approximately 107 CFU/ml and 106 CFU/ml, respectively.(Step2) Day 2: Remove the 96-peg lid. Allow cells to grow in the rows A and B of the 96-well plate○1 without changing the culture medium.Fill each well (rows C and D) of the same 96-well plate with 180μl of microbial cell suspension with concentrations ① and ② made in culture media A through D. Leave the wells (rows E through H) blank.Place the 96-peg lid back to the 96-well plate ①. Continuously incubate the 96-well plate ① at the optimum growth temperature for the microorganism for 24 hours.(Step3) Day 3: Remove the 96-peg lid. Allow cells to grow in the rows (A through D) of the 96-well plate ①without changing the culture medium.Fill each well (rows E and F) of the same 96-well plate with 180μl of microbial cell suspension with concentrations ① and ② made in culture media A through D. Leave the wells (rows G and H) blank. Place the 96-peg lid back to the 96-well plate ①. Continuously incubate the 96-well plate ① at the optimum growth temperature for the microorganism for 24 hours.(Step 4) Day 4: Remove the 96-peg lid. Allow cells to grow in the rows (A through F) of the 96-well plate ① without changing the culture medium.Fill each well (rows G and H) of the same 96-well plate with 180 μl of microbial cell suspension with concentrations ① and ② made in culture media A through D. Leave the wells (rows G and H) blank. Place the 96-peg lid back to the 96-well plate ①. Continuously incubate the 96-well plate ① at the optimum growth temperature for the microorganism for 24 hours.(Step 5): Perform experiments using a new 96-well plate, in accordance with Steps 2 through 6 which describe the measurements of biofilm formation/inhibition of biofilm formation in the technical manual.
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