Review
Bioautography detection in thin-layer chromatography

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Abstract

Bioautography is a microbial detection method hyphenated with planar chromatography techniques. It is based mainly on antimicrobial or antifungal properties of analyzed substances. The review discusses three versions of bioautography, i.e. contact, immersion and direct bioautography. The more concern is given to the last one. Many applications are quoted, not only for testing various groups of compounds, but also for investigating biochemical processes and factors influencing bacterial growth. Additionally, related methods, which can be included into direct bioautography, are discussed. The most promising among them seems to be TLC-bioluminescence screening.

Introduction

Bioautography belongs to microbiological screening methods commonly used for the detection of antimicrobial activity (Fig. 1). The screening can be defined as the first procedure, which is applied to an analyzed sample, in order to establish the presence or absence of given analytes [1]. Basically speaking, it is a simple measurement providing a “yes/no” response [2]. Quite often, screening methods give higher sensitivity than any other methods. Moreover, they are simple, cheap, time-saving and do not require sophisticated equipment. Bioautography screening methods are based on the biological activities, e.g. antibacterial, antifungal, antitumour, and antiprotozoae of the tested substances [3]. This detection method can be successfully combined with layer liquid chromatography techniques, such as thin-layer chromatography (TLC), high-performance thin-layer chromatography (HPTLC), overpressured-layer chromatography (OPLC) and planar electrochromatography (PEC). In this review, the name TLC-bioautography is used mostly in its wide-ranging meaning concerning any planar technique linked to bioautography. In so-called direct bioautography, i.e. bioautography hyphenated directly with thin-layer chromatography (TLC-DB), both separation and microbial detection are performed on the same TLC plate. Generally, the method measures antibacterial properties of analyzed substances, i.e. changes in bacterial growth. However, other mechanisms of action can be considered, e.g. disturbing vital cell processes as it takes place when bioautography is performed using luminescent bacteria, in so-called TLC-bioluminescence method [4], [5]. Both TLC-DB and TLC-bioluminescence enable searching for biological active substances in complicated mixtures and matrices, and can be included into effect-directed analysis (EDA), a new approach in environmental and hazard management based on biological response [6], [7].

Section snippets

Diffusion methods

Diffusion methods are frequently used in testing antimicrobial susceptibility of pure substances, preferably polar than non-polar ones [8], [9], [10]. The disc method is the official one for quantitative detection of inhibitory substances in milk in the USA [11], [12]. In this procedure, filter paper discs (about 6 mm diameter), containing the test compound, are placed on the agar surface previously inoculated with the test microorganisms (dipping a filter paper into a test compound solution

Thin-layer chromatography-direct bioautography

The beginnings of coupling microbiological assay with planar chromatography date back to 1946, when Goodall and Levi [27] combined paper chromatography method (PC) with contact bioautography detection for the determination of the different penicillins. Fifteen years later, Fisher and Lautner [28], and Nicolaus et al. [29] introduced thin-layer chromatography (TLC) in the same field. The methods were described as simple, reproducible and highly sensitive. The first review of the application of

Conclusions

Thin-layer chromatography-bioautography enjoys its great come-back now. The success of TLC with microbial detection is connected mainly with growing interest in an effect-directed analysis. The presence of increasing number of the known and unknown pollutants in the environment imposes the necessity for searching potentially harmful substances in various matrices. TLC-bioautography, including TLC-bioluminescence, accomplishes this task successfully. The bioassay, together with spectroscopic

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