Optimisation of the fluorescein diacetate antibacterial assay

Wanandy, Stephanus T.; Brouwer, Nynke; Liu, Qian; Mahon, Andrew B.; Cork, Stephen; Karuso, Peter; Vemulpad, Subramanyam; Jamie, Joanne F.

doi:10.1016/j.mimet.2004.08.010

Cited by 27 publications

(17 citation statements)

References 13 publications

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“…Thus, the nucleophilic properties of these amino acid groups are well known and are involved in FDA hydrolysis. Our results are consistent with Wanandy et al (2005) who, using a bacteriological growth peptone-yeast medium, suggested that individual amino acids or amino acid residues act as nucleophiles. As previously indicated in this study, the degree of humification increased with litter depth (Table 1).…”

Section: Interference Studiessupporting

confidence: 93%

“…FDA hydrolysis may occur in the same way as general acid/base reactions; both some lysine derivatives (Kristol et al , 1975) and some non‐enzymatic animal proteins (Clarke et al , 2001) have been described as capable of hydrolysing FDA. Wanandy et al (2005) proposed that cysteine is responsible for abiotic cleavage of FDA and our results support this. When analysing different buffer solutions, we found that hydrolysis was better promoted by imidazole buffer than by others.…”

supporting

confidence: 85%

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Non‐enzymatic hydrolysis of fluorescein diacetate (FDA) in a Mediterranean oak (Quercus ilex L.) litter

Alarcón-Gutiérrez¹,

Floch²,

Ruaudel³

et al. 2008

European J Soil Science

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We show the presence of interfering substances when the total microbial activity in litter samples is measured with fluorescein diacetate (FDA), and we propose some methodological modifications to avoid such interference. Three distinct litter layers (the OhLn, the OhLv and the OhLf) of evergreen oak (Quercus ilex L.) were characterized by 13 C CPMAS NMR and the spectra show that the recalcitrant aromatic and phenolic compounds increase with the degree of degradation of litter. A wide range of sources of interference in the hydrolysis of FDA was found. To understand the origin of this interference, sterilized litter materials (i.e. g-irradiated or autoclaved) and a wide range of organic substances (i.e. amino acids, glucose, sorbitol and organic humic acids) were investigated. Insignificant differences on the FDA hydrolysis activity (FDA activity) were found in the g-irradiated and non-irradiated OhLn litter, indicating that g-irradiation does not destroy enzymes. Conversely, after heat-sterilization of litter, samples showed FDA activity corresponding to 60, 34.8 and 30.8% (in the OhLn, the OhLv and the OhLf layers, respectively) of that of control litters. This indicates the presence of non-enzymatic interfering substances in the FDA assays. As the humification and litter depth increased, hydrolysis of FDA due to interferences decreased, indicating degradation and/or chelation of interfering substances. We hypothesize that lysine, arginine, histidine and cysteine are mainly responsible for the hydrolysis of FDA. We suggest that the use of phosphate buffer (50 mM, pH 7.0) with incubation < 30 minutes, in combination with a temperature between 30 and 40°C, produces insignificant interference in the determination of the final FDA activity in litter samples.

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Section: Interference Studiessupporting

confidence: 93%

supporting

confidence: 85%

Non‐enzymatic hydrolysis of fluorescein diacetate (FDA) in a Mediterranean oak (Quercus ilex L.) litter

Alarcón-Gutiérrez¹,

Floch²,

Ruaudel³

et al. 2008

European J Soil Science

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“…Second, substances that include nucleophilic and basic amino acids, including histidine and cysteine, have been reported to induce abiotic cleavage of FDA (Clarke et al . 2001; Wanandy et al . 2005).…”

Section: Discussionmentioning

confidence: 99%

Applicability of the fluorescein diacetate assay for metabolic activity measurement of Microcystis aeruginosa (Chroococcales, Cyanobacteria)

Zheng

et al. 2011

Phycological Research

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SUMMARYThe fluorescein diacetate (FDA) assay has been widely used to measure metabolic activity in phytoplankton. It was found that FDA fluorescence values did not decrease in some stressed cells, demonstrating that the applicability of the method needs to be assessed further in the context of growth-influencing conditions. In the present study, changes of FDA fluorescence values were studied in bloom-forming cyanobacterial Microcystis aeruginosa Kütz cells under stress conditions such as nitrogen (N) or phosphorus (P) deficiency, or darkness and low temperature (10°C), respectively. The results demonstrated that esterase activity decreased immediately in dark-stressed cells, which correlated with the decline of biomass and photosynthetic activity. Under the other three stress conditions, however, especially at low temperature, the cells lost photosynthetic activity but had the highest esterase activity, which was five times higher than the control group. These findings contrast with the assay criteria that the expression of a stain should reflect the change of photosynthetic activity and that stressed cells should have a lower staining intensity than the control cells. According to these results, the esterase activity response was dependent on environmental factors. Furthermore, higher fluorescence intensity did not mean higher metabolic activity, but a discrepant value indicated a severe stress.

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“…The standard disk diffusion and broth dilution methods are the wider extended methods used to study the bioactivity of chemical compounds (Wanandy et al, 2005). Both of them need the use of standardised microbial techniques and are time consuming.…”

Section: Introductionmentioning

confidence: 99%

A new application of a commercial microtiter plate-based assay for assessing the antimicrobial activity of Maillard reaction products

Rufián-Henares

Morales

2006

Food Research International

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Optimisation of the fluorescein diacetate antibacterial assay

Cited by 27 publications

References 13 publications

Non‐enzymatic hydrolysis of fluorescein diacetate (FDA) in a Mediterranean oak (Quercus ilex L.) litter

Non‐enzymatic hydrolysis of fluorescein diacetate (FDA) in a Mediterranean oak (Quercus ilex L.) litter

Applicability of the fluorescein diacetate assay for metabolic activity measurement of Microcystis aeruginosa (Chroococcales, Cyanobacteria)

A new application of a commercial microtiter plate-based assay for assessing the antimicrobial activity of Maillard reaction products

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