Table of Contents
- Introduction
- Principle of TLC–Bioautography
- Materials Required (with Functions)
- Step-by-Step Procedure of TLC–Bioautography
- Interpretation of TLC–Bioautography Results
- Advantages of TLC–Bioautography
- Applications of TLC–Bioautography
- References
Introduction
- Microorganisms such as bacteria, fungi, and actinomycetes are well known for producing bioactive secondary metabolites.
- These metabolites include antibiotics, antifungals, antivirals, and other antimicrobial compounds that help the organism survive in competitive environments.
- During growth in liquid culture media, many of these compounds are released extracellularly into the culture broth, forming part of the culture supernatant after removal of cells.
- Detecting these antimicrobial substances is important for:
- Antibiotic discovery
- Natural product research
- Pharmaceutical screening
- Studying microbial interactions
- One powerful technique used for this purpose is TLC–Bioautography, which combines:
- Chemical separation of compounds using Thin Layer Chromatography (TLC), and
- Biological detection of antimicrobial activity using a test microorganism.
- This method not only shows whether antimicrobial activity is present, but also helps identify:
- The number of active compounds
- Their relative mobility (Rf values)
- Their separation pattern
Principle
- Microorganisms synthesize secondary metabolites that may possess antimicrobial properties.
- These bioactive compounds are secreted into the culture medium during growth and accumulate in the culture supernatant.
- The supernatant is extracted using an organic solvent to concentrate these compounds.
- Thin Layer Chromatography (TLC) is used to:
- Separate the mixture of compounds based on:
- Polarity
- Solubility
- Affinity for stationary and mobile phases
- Each compound moves a different distance on the TLC plate, forming separate spots.
- The developed TLC plate is then subjected to bioautography, where:
- A test microorganism (indicator strain) is applied over the plate using an agar overlay.
- During incubation:
- The antimicrobial compounds diffuse from the TLC spots into the agar.
- These compounds inhibit the growth of the indicator organism.
- After incubation, clear zones of inhibition appear at locations where active antimicrobial compounds are present.
- The position of inhibition zones corresponds to:
- Specific separated compounds
- Their Rf values, helping in compound characterization.
Materials Required (with Functions)
1. Culture & Extraction Materials
These are used to grow the producer microorganism and obtain antimicrobial metabolites from the culture supernatant.
Microbial culture producing antimicrobial compound
- The test organism (bacteria, fungi, or actinomycetes) suspected of producing bioactive secondary metabolites.
Nutrient broth / suitable production medium
- Provides essential nutrients for microbial growth.
- Specialized production media may enhance secondary metabolite (antibiotic) synthesis.
Centrifuge tubes
- Used to hold culture broth during separation of cells from liquid medium.
Centrifuge
- Separates microbial cells (pellet) from the culture supernatant, which contains extracellular metabolites.
Whatman filter paper or membrane filter
- Removes remaining cell debris to obtain a clear, cell-free supernatant.
Organic solvent (Ethyl acetate / Methanol / Chloroform)
- Extracts antimicrobial compounds based on their polarity and solubility.
- Ethyl acetate is commonly used for moderately polar metabolites.
Separatory funnel
- Allows liquid–liquid extraction, separating aqueous supernatant from organic solvent layer.
Rotary evaporator or water bath
- Removes solvent to concentrate the extract.
- Leaves behind crude antimicrobial residue.
2. TLC Setup Materials
These materials are required to separate compounds present in the crude extract.TLC plates (Silica gel 60 F254)
- Silica gel acts as the stationary phase.
- F254 indicator allows visualization under UV light.
Capillary tubes or micropipette
- Used to apply small, precise spots of extract onto the TLC plate.
Developing chamber
- A sealed container that holds the mobile phase solvent and ensures uniform chromatographic development.
Mobile phase solvent system
- Mixture of solvents that moves up the plate by capillary action.
- Separates compounds based on polarity differences.
3. Bioautography Materials
These are used for biological detection of antimicrobial activity after chemical separation.Test pathogen (e.g., E. coli, S. aureus)
- Indicator organism to test antimicrobial effect of separated compounds.
Soft agar (0.7% agar)
- Semi-solid medium that allows:
- Diffusion of antimicrobial compounds
- Growth of indicator bacteria
Nutrient agar plates
- Support the growth of the test microorganism during incubation.
Incubator
- Maintains optimal temperature for growth of the indicator organism.
Step-by-Step Procedure of TLC–Bioautography of Culture Supernatant
Step 1: Preparation of Culture Supernatant
- Inoculate the producer microorganism into a suitable production broth.
- Incubate under optimal conditions:
- Temperature
- pH
- Aeration/shaking (if required)
- These conditions promote secondary metabolite production.
- After 24–72 hours of incubation:
- Transfer culture into centrifuge tubes.
- Centrifuge at 8,000–10,000 rpm for 10–15 minutes.
Purpose:
- Centrifugation separates:
- Cell pellet (microbial biomass)
- Supernatant (contains extracellular antimicrobial metabolites)
- Carefully collect the clear supernatant.
- Filter through Whatman filter paper or membrane filter.
- This produces a cell-free filtrate containing antimicrobial compounds.
Step 2: Extraction of Antimicrobial Compounds
- Transfer supernatant into a separatory funnel.
- Add an equal volume of organic solvent (e.g., ethyl acetate).
Why?
Most antimicrobial metabolites are more soluble in organic solvents than water.- Shake vigorously for 5–10 minutes.
- Allow the layers to separate.
- Collect the organic (upper) layer.
- Repeat extraction 2–3 times for maximum recovery.
- Pool all organic extracts.
- Evaporate solvent using:
- Rotary evaporator (preferred) or
- Water bath at low temperature
Purpose:
- Removes solvent and concentrates bioactive compounds.
- Dissolve dried residue in 1–2 mL methanol.
- This is the crude antimicrobial extract.
Step 3: TLC Separation
- Draw a pencil baseline 1.5 cm from bottom of TLC plate.
- Apply small spots of crude extract using a capillary tube.
- Air dry the spots.
- Place plate in developing chamber with mobile phase.
- Allow solvent to travel ¾ of plate length.
- Remove plate and air dry.
Purpose:
- Compounds separate based on:
- Polarity
- Interaction with silica (stationary phase)
Step 4: Bioautography (Agar Overlay Method)
Preparation of Indicator Organism
- Grow test bacterium overnight.
- Prepare suspension ~10⁶ CFU/mL.
Overlay Process
- Melt soft agar (0.7%).
- Cool to ~45°C (prevents killing bacteria).
- Add bacterial suspension to soft agar.
- Pour thin layer over developed TLC plate.
- Allow agar to solidify.
Purpose:
- Active compounds will diffuse from TLC spots into agar.
Step 5: Incubation
- Incubate at 37°C for 18–24 hours.
During this time:
- Bacteria grow to form a lawn.
- Antimicrobial compounds inhibit growth around active spots.
Step 6: Observation
- Observe clear zones against bacterial lawn.
- Each clear zone = antimicrobial compound location.
Rf Value Calculation
Rf= Distance moved by compound/Distance moved by solvent frontImportance of Rf:
- Helps characterize compounds
- Useful for comparing metabolites
Interpretation of TLC–Bioautography Results
Clear inhibition zone on TLC plate
- Confirms presence of an antimicrobial compound
- Compound diffused into agar and inhibited test organism
No inhibition observed
- No active antimicrobial metabolite present or
- Compound concentration too low or
- Compound not effective against selected test organism
Multiple clear zones
- Indicates more than one antimicrobial compound
- Compounds differ in polarity and mobility
Large inhibition zone
- Suggests strong antimicrobial activity
- May indicate higher concentration of compound
Small or faint inhibition zone
- Indicates weak antimicrobial activity
- Could be low concentration or partial inhibition
Zones appearing at different Rf values
- Shows presence of chemically different metabolites
- Useful for compound comparison and profiling
No bacterial lawn growth on whole plate
- Possible experimental error:
- Excess extract
- Toxic solvent residue
- Problem with bacterial culture
Interpretation of Rf Value
- Each antimicrobial compound has a characteristic Rf value
- Same Rf in repeated tests → likely same compound
- Different Rf values → different metabolites present
Important Concept
- TLC–bioautography confirms biological activity
- It does not identify chemical structure
- Further analysis required:
- HPLC
- Mass spectrometry
- NMR spectroscopy
Advantages of TLC–Bioautography
- Simple and cost-effective technique requiring basic laboratory equipment
- Combines chemical separation and biological activity detection in one method
- Detects biologically active compounds directly, not just chemical presence
- Allows separation and identification of multiple metabolites in a mixture
- Requires only small amounts of sample
- Rapid screening method compared to advanced analytical techniques
- Helps locate active compounds on the TLC plate using Rf values
- Useful in screening antibiotic-producing microorganisms
- Supports natural product and drug discovery research
- Suitable for preliminary analysis before HPLC or spectroscopic studies
Applications of TLC-Bioautography
- Screening of antibiotic-producing microorganisms from environmental or clinical samples
- Detection of bioactive secondary metabolites in bacteria, fungi, and actinomycetes
- Natural product research for discovery of new antimicrobial agents
- Analysis of actinomycete-derived metabolites, especially antibiotic compounds
- Evaluation of herbal and plant extracts for antimicrobial activity
- Comparative profiling of metabolites from different microbial strains
- Preliminary assessment before advanced techniques like HPLC or LC–MS
- Studying microbial competition and ecological interactions
- Quality control in research involving bioactive compound production
- Academic and pharmaceutical research in early-stage drug discovery
References
- Choma & Jesionek (2015) — TLC-Direct Bioautography as a high-throughput method for detecting antimicrobials in plant extracts (review of principles and applications).
- TLC Bioautography overview — Review on TLC bioautography combining chromatographic separation with biological detection for antimicrobials and other activities.
- Bioautography detection in TLC — Review of bioautography methods including direct and other variations used for antimicrobial screening.
- PubMed Case Example: TLC-Bioautography to isolate antibacterial compounds — Shows real application detecting antimicrobials from plant extracts.
- Direct TLC-DB investigation of antibacterial compounds — Example of using TLC-direct bioautography to locate antibacterial agents and identify them with spectroscopic tools.
- TLC separation and bioassays of plant extracts for antimicrobial identification (JoVE video/methods article).
