A simple colorimetric method for the determination of tryptophan

A simple, sensitive, and reproducible colorimetric method for the determination of tryptophan in amounts as low as 2 μg is described. It is based on the oxidation of tryptophan by sodium nitrite and the coupling of the oxidized product to the leucodye N-1-(naphthyl)ethylenediamine dihydrochloride. The purple-pink product has an absorption maximum at 550 nm. There is no interference by carbohydrates, other amino acids, neutral salts, or a number of other compounds likely to be found in tissue hydrolysates. A number of indole derivatives including indole-3-acetic acid also react to give a colored product. Dipeptides containing tryptophan are much less reactive than free tryptophan; hence proteins must be hydrolyzed completely for the method to be useful. The assay is carried out at room temperature and can be modified easily to increase or decrease its sensitivity. It has been employed to determine the tryptophan content of a number of proteins following alkaline hydrolysis. Generally, values obtained were in close agreement with values reported in the literature.     

A simple colorimetric method for determination of hydrogen peroxide in plant tissues

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 μmol g⁻¹ FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.     

A simple colorimetric method for testing antimicrobial susceptibility of biofilmed bacteria

This study introduces a simple colorimetric method which can measure the antimicrobial susceptibility of bacteria in biofilms using trimethyl tetrazolium chloride (TTC) as an indicator of viable bacteria. The new method was utilized for the evaluation of antibiotic susceptibility of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus biofilms.     

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.     

A simple colorimetric method for detecting cell viability in cultures of eukaryotic microorganisms

The dye methylene blue can be taken up by dead or severely damaged cells, but not by living cells. Based on this fact, a method was devised which permits quantitative determinations of injured cells in populations of microorganisms such asSaccharomyces cerevisiae, Rhodotorula glutinis, andEuglena gracilis. The percentage of damaged cells was determined by measuring, at 664 nm, the optical density of cell suspensions pretreated with 0.15 mM methylene blue for 6 min, a condition that does not affect cell integrity as determined by oxygen consumption and release of potassium ions. This technique is faster and simpler than the classical dye-exclusion and plate-counting methods.     

Detecting mycotoxins in agricultural commodities

It is difficult to obtain precise and accurate estimates of the true mycotoxin concentration of a bulk lot when using a mycotoxin-sampling plan that measures the concentration in a small portion of the bulk lot. A mycotoxin-sampling plan is defined by a mycotoxin test procedure and a defined accept/reject limit. A mycotoxin test procedure is a complicated process and generally consists of several steps: (a) a sample is taken from the lot, (b) the sample is ground (comminuted) in a mill to reduce particle size, (c) a subsample is removed from the comminuted sample, and (d) the mycotoxin is extracted from the comminuted subsample and quantified. Even when using accepted test procedures, there is variability associated with each step of the mycotoxin test procedure. Because of this variability, the true mycotoxin concentration in the lot cannot be determined with 100% certainty by measuring the mycotoxin concentration in a sample taken from the lot. The variability for each step of the mycotoxin test procedure, as measured by the variance statistic, is shown to increase with mycotoxin concentration. Sampling is usually the largest source of variability associated with the mycotoxin test procedure. Sampling variability is large because a small percentage of kernels are contaminated and the level of contamination on a single seed can be very large. Methods to reduce sampling, sample preparation, and analytical variability are discussed.     

A simple and rapid method for screening amylolytic bacteria

A laboratory class was designed for the study of the ecology of amylolytic bacteria in soil, although other sources may be equally suitable for this purpose. Groups of three students carried out the following: (a) preparation and sterilization of medium and plates, (b) collection and preparation of soil samples, spreading the samples on the plates, (c) incubation of the plates at 37 degrees C overnight, a further 1 h incubation at 60 degrees C to observe amylolytic activity due to thermophilic bacteria, and (d) interpretation and discussion of the results. These tasks are accomplished in two periods of 4h on consecutive days. No sophisticated instruments are required for these experiments, which can be carried out in three classes of 4h each. On the first day the students prepare culture media, buffers and reagents, as well as collect and grow soil samples. The second day is spent for both taxonomic identification of colonies and the HAI determination.     

Hydroxamate-based colorimetric method for direct screening of transglutaminase-producing bacteria

Microbial transglutaminase (MTGase) is a commercial enzyme that has been applied to many protein containing foods to improve their textural property. The screening of MTGase-producing microorganisms from various sources might lead to the discovery of a new MTGase with different characteristics. This report demonstrates the use of a direct detection method for MTGase-producing bacteria grown on an agar plate by filter paper disc (FPD) assay. The principle of the assay is the formation of a red burgundy color by the hydroxamate-ferric complex. The color developed intensity was linearly correlated by the concentration of hydroxamic acid in the range of 0.1-0.8 μM and was visually scored at 4 levels: 0, 1, 2 and 3. Streptoverticillium mobaraense DSM 40847, a positive MTGase-producer, was chosen for the verification and improving of the proposed method. The colonies grown on the nutrient agar plate at 37°C for 24 h were covered with FPDs and 30 μl of substrates (CBZ-Gln-Gly and hydroxylamine). After incubation, 10 μl of the ferric-TCA-HCl solution was placed on the FPD. The optimal time taken to catalyze the formation of CBZ-Gln-Gly-hydroxamic acid by the MTGase and the time taken for the hydroxamate-ferric complex to form color were 180 and 60 min, respectively. Using this assay, 30 of 189 colonies isolated from wastewater and floating-floc samples showed MTGase-positive colonies which were well correlated to the quantitative screening of MTGase activity (R(2) = 0.9758). The results revealed that the FPD assay could be used for the qualitative screening of MTGase-producing bacteria.     

A colorimetric assay for screening transketolase activity

A tetrazolium red-based colorimetric assay has been devised to screen for transketolase activity with a range of aldehyde acceptors. The colorimetric TK assay is able to detect >8% bioconversion using non-alpha-hydroxylated aldehydes as acceptor substrates and is significantly faster and more convenient to use than chromatographic procedures.     

A simple method for the screening of lactic acid bacteria for the production of exopolysaccharides in liquid media

A rapid and convenient screening metnod for the production of exopolysaccharides (EPS) from lactic acid bacteria based on the measurement of ef_ux time in Microhaematocrit capillaries was developed. The traditional pick test was more sensitive in detecting evidence of polysaccharide production on solid media but did not re_ect the potential for EPS production in liquid media. Measurement of ef_ux time in Microhaematocrit capillaries provided quantitative results which were correlated with viscosity measurements and EPS concentration and were amenable to statistical analysis.     

A high-throughput screening method for amino acid dehydrogenase

A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)–calcein complex and amino acid (e.g., l-2-aminobutyric acid, l-ABA). When ABA was introduced to a Cu(II)–calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l-ABA detection and screening for leucine dehydrogenase mutations.