1. The document discusses methods for testing the anti-biofilm potential of compounds. It focuses on the microtiter plate assay which allows rapid, quantitative testing of a compound's ability to inhibit or eradicate bacterial biofilms. 2. The microtiter plate assay can test a compound's minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC). It involves growing biofilms in microtiter plates and exposing them to serial dilutions of the test compound. 3. The document also describes how the microtiter plate assay can test a compound's effects during different biofilm development stages, such as adhesion, maturation, and preformed biofilm eradication. St

1. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id Creating environmental health and developing local wisdom for a better future
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Universitas Jenderal Soedirman
Fakultas Kedokteran
Dr. dr. Setiawati, M.Sc
Departemen Farmakologi FK UNSOED
Metode Uji Potensi
Antibiofilm: inhibisi dan
eradikasi

2. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id
BACKGROUND
Biofilm
Serious problem
Difficulty of
treatment
Drug resistance
kesehatan
FDA :
antibiofilm (-)
ekonomi
Antibiofilm ?

3. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id
Metode identifikasi biofilm
• Light microscopy
• SEM
• TEM
• CLSM
Direct
observation
• Roll plate method
• Congo red agar (CRA) method
• Tube method (TM)
• Microtiter plate (MtP) assay
• PCR
Indirect
observation

4. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id
SEM TEM CLSM

5. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id
Metode Uji anti-biofilm
• simple and reproducible
• Inexpensive
• accessible to any laboratory
• requires minimal technical expertise or
training
• takes 4 or 5 d to complete
• quantitative
Microtiter
plate
(MtP)
assay

6. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id
Metode Uji anti-biofilm
Minimum biofilm inhibitory
concentration (MBIC)
MBIC50
and MBIC90
🡪indicate the
minimum concentrations of agent
inhibiting 50 and 90% of
biofilm-embedded bacteria
Minimum biofilm eradication
concentration (MBEC).
MBEC50
and MBEC90
indicate the
minimum concentrations of agents
eradicate/reduce 50 and 90% of mature
biofilm formed.
Microtiter plate (MtP)
assay

7. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id
Metode anti-biofilm
Microtiter plate
(MtP) assay
Crystal violet
(CV)
MTT XTT Safranin
dimethyl
methylene blue
Biofilm biomass Metabolic activity

8. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id 07/02/20 8
► Biofilm inhibition activity test by microbrothdilution method (MtP)
Penyiapan
bakteri uji
Suspensi
bakteri Senyawa uji
Incubation 24-48 h
Removed
planctonic cells
+ washing
CV 0.1 % : 30 min
MTT 5%: : 2 h-4h
Elisa reader :
CV : 595 nm
MTT : 570 nm
Ethanol 70% for
30 min

9. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id

10. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id 07/02/20 10
► Biofilm reduction activity test by microbrothdilution method
Mikroba
Suspensi
bakteri+media
Senyawa uji
Incubation 24 h
Incubation 24 h
Removed
planctonic cells
+ washing
CV 0.1 % : 30 min
MTT 5%: : 2 h-4h
Elisa reader :
CV : 595 nm
MTT : 570 nm
Ethanol 70% for
30 min

11. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id

12. Universitas Jenderal Soedirman
Fakultas Kedokteran
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1. Anti-adhesion test
Mikroba + senyawa uji
1-4 jam
Elisa reader :
CV : 595 nm
MTT : 570 nm
07/02/20 12
Pencucian
CV 0.1 % : 30 min atau
MTT 5%: : 2 h-4h
Ethanol 70% for 30
min
► Uji Antibiofilm berdasar tahap perkembangan

13. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id 07/02/20 13
2. Anti-maturation test
Mikroba
2 h
Pencucian
Penambahan
senyawa uji
24 h
Elisa reader :
CV : 595 nm
MTT : 570 nm
Pencucian
CV 0.1 % : 30 min
MTT 5%: : 2 h-4h
Ethanol 70% for 30
min

14. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id 07/02/20 14
Anti-preformed biofilm test
24 h 24 h
Elisa reader :
CV : 595 nm
MTT : 570 nm
3. Anti-preform biofilm
Mikroba
Suspensi
bakteri+media
Senyawa uji
Removed
planctonic
cells
+ washing
CV 0.1 % :
30 min
MTT 5%: : 2
h-4h Ethanol
70% for 30
min

15. Universitas Jenderal Soedirman
Fakultas Kedokteran
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16. Universitas Jenderal Soedirman
Fakultas Kedokteran
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17. Universitas Jenderal Soedirman
Fakultas Kedokteran
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References
• Haney, E.F., Trimble, M.J. & Hancock, R.E.W. Microtiter plate assays to
assess antibiofilm activity against bacteria. Nat Protoc 16, 2615–2632
(2021). https://doi.org/10.1038/s41596-021-00515-3
• Brożyna, M.; Żywicka, A.; Fijałkowski, K.; Gorczyca, D.; Oleksy-Wawrzyniak,
M.; Dydak, K.; Migdał, P.; Dudek, B.; Bartoszewicz, M.; Junka, A. The Novel
Quantitative Assay for Measuring the Antibiofilm Activity of Volatile
Compounds (AntiBioVol). Appl. Sci. 2020, 10, 7343.
https://doi.org/10.3390/app10207343
• Latka, A., Drulis-Kawa, Z. Advantages and limitations of microtiter biofilm
assays in the model of antibiofilm activity of Klebsiella phage KP34 and its
depolymerase. Sci Rep 10, 20338 (2020).
https://doi.org/10.1038/s41598-020-77198-5

18. Universitas Jenderal Soedirman
Fakultas Kedokteran
fk.unsoed.ac.id