A novel, quantitative clot retraction assay to evaluate platelet function.

Blood platelets are crucial to prevent excessive bleeding following injury to blood vessels. Platelets are crucial for the formation of clots and for clot strength. Platelet activation involves aggregation, attachment to fibrin and clot retraction. Most assays that address platelet function measure platelet aggregation, not clot retraction. Here, we describe a 96-well-based clot retraction assay that requires a relatively short runtime and small sample volume. The assay involves continuous optical density monitoring of platelet-rich plasma that is activated with thrombin. The data can be analyzed using time-series analytical tools to generate quantitative information about different phases of clot formation and clot retraction. The assay demonstrated good repeatability and reproducibility and was robust to different calcium concentrations. Impairment of platelet bioenergetics, actin polymerization, fibrin interaction, and signaling significantly affected clot retraction and was detected and showed good agreement with light transmission aggregometry, suggesting that clot retraction is predictive of platelet function. Using this microplate clot retraction assay, we showed a significant difference in platelets stored in autologous plasma compared with platelet additive solution after 7 days of room temperature storage.

Platelets are cell fragments in the blood that are necessary for clot formation. They are crucial to preventing excessive bleeding following trauma. To form clots, platelets clump (aggregate) and attach to fibrin protein and cells inside the blood vessels to form strong web-like structures. Platelets also contract to pull the edges of the wound close. Most measurements of platelet function involve aggregation. This paper focuses on platelet contraction. Here, we describe a new assay to measure platelets contraction that is repeatable and reproducible. The assay uses standard and common laboratory equipment and can be performed by most laboratory personnel and has the potential to detect clinical pathologies of clot formation. The assay could be developed for bedside patient care where platelet function could be assessed rapidly and assist in the diagnosis of coagulation and platelet disorders.

Non-destructive investigation of extracellular enzyme activities and kinetics in intact freshwater biofilms with mineral beads as carriers.

Current procedures for fluorometric detection of extracellular hydrolytic enzyme activities in intact aquatic biofilms are very laborious and insufficiently standardized. To facilitate the direct determination of a multitude of enzymatic parameters without biofilm disintegration, a new approach was followed. Beads made of different mineral materials were subjected to biofilm growth in various aquatic environments. After biofilm coating, the beads were singly placed in microplate wells, containing the required liquid analytical medium and a fluorogenic substrate. Based on fluorometric detection of the enzymatically generated reaction products, enzyme activities and kinetics were determined. Mean enzymatic activities of ceramic bead-attached biofilms grown in a natural stream followed the decreasing sequence L-alanine aminopeptidase > L-leucine aminopeptidase > phosphomonoesterase > ß-glucosidase > phosphodiesterase > α-glucosidase > sulfatase. After one week of exposure, the relative standard deviations of enzyme activities ranged from 21 to 67%. Sintered glass bead-associated biofilms displayed the lowest standard deviations ranging from 19 to 34% in all experiments. This material proved to be suitable for short-time experiments in stagnant media. Ceramic beads were stable during more than three weeks of exposure in a natural stream. Biofilm formation was inhomogeneous or poorly visible on glass and lava beads accompanied by high variations of enzyme activities. The applicability of the method to study enzyme inhibition reactions was successfully proven by the determination of inhibition effects of caffeine on biofilm-associated phosphodiesterase.Key points• Optimized method to determine enzymatic parameters in aquatic biofilms• Direct investigation of bead-bound biofilms without biofilm disintegration• Fluorometric detection offers high sensitivity and sample throughput.

Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with Staphylococcus aureus.

With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC50 values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD600 measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against Staphylococcus aureus, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution S. aureus growth inhibition profiling was evaluated on a crude extract of an inactive Alternaria sp., spiked with ciprofloxacin.

Nonconventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP.

G protein-coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted from pancreatic α-cells may accumulate at high concentrations to exert promiscuous effects at the ß-cell GLP-1R, as may occur in the volume-restricted microenvironment of the islets of Langerhans. Furthermore, systemic administration of GluR or GLP-1R agonists and antagonists at high doses may lead to off-target effects at other receptors. Here, we used molecular modeling to evaluate data derived from FRET assays that detect cAMP as a read-out for GluR and GLP-1R activation. This analysis established that glucagon is a nonconventional GLP-1R agonist, an effect inhibited by the GLP-1R orthosteric antagonist exendin(9-39) (Ex(9-39)). The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His1-[Glu9]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R. When testing Ex(9-39) in combination with des-His1-[Glu9]glucagon in INS-1 832/13 cells, we validated a dual agonist action of glucagon at the GluR and GLP-1R. Hybrid peptide GGP817 containing glucagon fused to a fragment of peptide YY (PYY) acted as a triagonist at the GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). Collectively, these findings provide a new triagonist strategy with which to target the GluR, GLP-1R, and NPY2R. They also provide an impetus to reevaluate prior studies in which GluR and GLP-1R agonists and antagonists were assumed not to exert promiscuous actions at other GPCRs.

The indigoidine synthetase BpsA provides a colorimetric ATP assay that can be adapted to quantify the substrate preferences of other NRPS enzymes.

OBJECTIVES: To develop a colorimetric assay for ATP based on the blue-pigment synthesising non-ribosomal peptide synthetase (NRPS) BpsA, and to demonstrate its utility in defining the substrate specificity of other NRPS enzymes. RESULTS: BpsA is able to convert two molecules of L-glutamine into the readily-detected blue pigment indigoidine, consuming two molecules of ATP in the process. We showed that the stoichiometry of this reaction is robust and that it can be performed in a microplate format to accurately quantify ATP concentrations to low micromolar levels in a variety of media, using a spectrophotometric plate-reader. We also demonstrated that the assay can be adapted to evaluate the amino acid substrate preferences of NRPS adenylation domains, by adding pyrophosphatase enzyme to drive consumption of ATP in the presence of the preferred substrate. CONCLUSIONS: The robust nature and simplicity of the reaction protocol offers advantages over existing methods for ATP quantification and NRPS substrate analysis.

Rolling circle amplification based colorimetric determination of Staphylococcus aureus.

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.µL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum.

OBJECTIVE: To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. RESULTS: Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain. CONCLUSION: A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

Truncated aptamers for total and glycated hemoglobin, and their integration into a graphene oxide-based fluorometric method for high-throughput screening for diabetes.

The authors describe the identification of an effective binding region of aptamers against glycated (HbA1c) and total haemoglobin (tHb) by using a fluorometric assay. Truncation of the originally selected aptamers from 60 to 46 and 34 nucleotides for HbA1c and tHb, respectively, enhances the affinity for their targets. Moreover, shortening the aptamer sequences leads to a better conformational change after target binding which enabled the integration of the aptamers in a graphene oxide (GO)-based fluorometric assay. First, fluorescein-labelled truncated aptamers were physically absorbed onto the surface of GO surface via π-stacking interaction. This leads to quenching of fluorescence. Once the truncated aptamers bind the target protein, a conformational change is induced which results (a) )in the release of the aptamers from the surface of GO and (b) in the restoration of green fluorescence that is measured at 515 nm. The assay can be carried out in a microtiter plate format in homogeneous solution, this avoiding the steps of immobilization, incubation, and washing that are often necessary in immunoassays. This also reduces the time and the costs of the overall assay and allows for high throughput screening for diabetes. HbA1c can be detected in the range from 5.4 to 10.6%. The assay is selective for HbA1c over other proteins that commonly exist in blood. The results obtained by using this method compare well with those of a turbidimetric immunoassay that is typically applied in clinical laboratories. Graphical abstract Truncated aptamers for total and glycated hemoglobin were selected and integrated into a graphene oxide-based fluorescence detection assay for high-throughput screening for diabetes.

Green Fluorescent Protein-Based Viability Assay in a Multiparametric Configuration.

Green fluorescent protein (GFP) is considered to be suitable for cell viability testing. In our study, GFP transfected A549 lung carcinoma cell line was treated with sodium fluoride (NaF), cycloheximide (CHX) and ochratoxin A (OTA). GFP fluorescence, intracellular ATP, nucleic acid and protein contents were quantified by a luminescence microplate assay developed in our laboratory. Flow cytometry was used to confirm the findings and to assess the intensity of GFP during different types of cell death. A 24 h NaF and CHX exposure caused a dramatic decrease in ATP contents (p < 0.05) compared with those of the controls. GFP fluorescence of the cells was in close correlation with total protein; however, GFP/ATP increased at NaF and decreased at CHX treatments (p < 0.05). ATP/protein and ATP/propidium iodide (PI) were largely decreased at NaF exposure in a dose-dependent manner (p < 0.05), while CHX and OTA showed markedly fewer effects. Both treatments caused apoptosis/necrosis at different rates. NaF induced mainly late apoptosis while OTA, mainly apoptosis. CHX effects varied by the incubation time with 100-fold elevation in late apoptotic cells at 24 h treatment. GFP intensity did not show a significant difference between live and apoptotic populations. Our results suggest when using GFP, a multiparametric assay is necessary for more precise interpretation of cell viability.

Direct Resazurin Microplate Assay in Drug Susceptibility Testing of Smear-Positive Sputum Samples against Mycobacterium tuberculosis.

BACKGROUND: A new direct microplate-based colorimetric drug susceptibility test that omits the initial isolation of Mycobacterium tuberculosis from sputum specimens was evaluated. METHODS: A total of 51 M. tuberculosis acid fast bacilli (AFB) smear-positive sputum specimens were inoculated directly into drug-free and serial dilutions of drug-containing Middlebrook 7H9 broth media. With this direct resazurin micro plate assay (REMA) method, resazurin dye was used as a growth indicator in microplate wells. The minimum inhibitory concentrations (MIC) of isoniazid (INH) and rifampicin (RIF) were compared with those of the 'gold standard' absolute concentration method (ACM). The turnaround time (TAT) of the direct REMA and the ACM were also determined. RESULTS: At the selected cut-off points (INH: 0.0625 µg/mL; RIF: 0.125 µg/mL), good drug susceptibility test results were obtained for INH and RIF with an average sensitivity, specificity and accuracy of 90%, 100% and 97%, respectively, with a TAT of 15 days. The REMA method also correctly classified the resistant isolates with positive predictive values of 95% and negative predictive values of 98% for the two drugs. CONCLUSIONS: The direct REMA was reliable in routine diagnostic laboratories for the drug susceptibility testing of M. tuberculosis and the rapid detection of multi-drug-resistant tuberculosis.

A miniaturized assay for kinetic characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

We demonstrate the miniaturization of an enzymatic assay for the determination of NADH oxidation and quinone reduction by the Na+ -translocating NADH quinone oxidoreductase (NQR) in the 96-well plate format. The assay is based on the spectrophotometric detection of NADH consumption and quinol formation. We validated the new method with known inhibitors of the NQR and optimized conditions for high-throughput screening as demonstrated by excellent Z-factors well above the accepted threshold (≥0.5). Overall, the method allows the screening and identification of potential inhibitors of the NQR, and rapid characterization of NQR variants obtained by site-specific mutagenesis.

Fluorimetric microplate assay for the determination of extracellular alkaline phosphatase kinetics and inhibition kinetics in activated sludge.

The microbial enzyme alkaline phosphatase contributes to the removal of organic phosphorus compounds from wastewaters. To cope with regulatory threshold values for permitted maximum phosphor concentrations in treated wastewaters, a high activity of this enzyme in the biological treatment stage, e.g., the activated sludge process, is required. To investigate the reaction dynamics of this enzyme, to analyze substrate selectivities, and to identify potential inhibitors, the determination of enzyme kinetics is necessary. A method based on the application of the synthetic fluorogenic substrate 4-methylumbelliferyl phosphate is proven for soils, but not for activated sludges. Here, we adapt this procedure to the latter. The adapted method offers the additional benefit to determine inhibition kinetics. In contrast to conventional photometric assays, no particle removal, e.g., of sludge pellets, is required enabling the analysis of the whole sludge suspension as well as of specific sludge fractions. The high sensitivity of fluorescence detection allows the selection of a wide substrate concentration range for sound modeling of kinetic functions.•Fluorescence array technique for fast and sensitive analysis of high sample numbers•No need for particle separation - analysis of the whole (diluted) sludge suspension•Simultaneous determination of standard and inhibition kinetics.

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation.

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

In vitro biofilm forming potential of Streptococcus suis isolated from human and swine in China.

Streptococcus suis is a swine pathogen and also a zoonotic agent. The formation of biofilms allows S. suis to become persistent colonizers and resist clearance by the host immune system and antibiotics. In this study, biofilm forming potentials of various S. suis strains were characterized by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and tissue culture plates stained with crystal violet. In addition, the effects of five antimicrobial agents on biofilm formation were assayed in this study. S. suis produced biofilms on smooth and rough surface. The nutritional contents including glucose and NaCl in the growth medium modulated biofilm formation. There was a significant difference in their biofilm-forming ability among all 46 S. suis strains. The biofilm-forming potential of S. suis serotype 9 was stronger than type 2 and all other types. However, biofilm formation was inhibited by five commonly used antimicrobial agents, penicillin, erythromycin, azithromycin, ciprofloxacin, and ofloxacin at subinhibitory concentrations, among which inhibition of ciprofloxacin and ofloxacin was stronger than that of other three antimicrobial agents.Our study provides a detailed analysis of biofilm formation potential in S. suis, which is a step towards understanding its role in pathogenesis, and eventually lead to a better understanding of how to eradicate S. suis growing as biofilms with antibiotic therapy.

Analysis of Myxococcus xanthus Vegetative Biofilms With Microtiter Plates.

The bacterium Myxococcus xanthus forms both developmental and vegetative types of biofilms. While the former has been studied on both agar plates and submerged surfaces, the latter has been investigated predominantly on agar surfaces as swarming colonies. Here we describe the development of a microplate-based assay for the submerged biofilms of M. xanthus under vegetative conditions. We examined the impacts of inoculation, aeration, and temperature to optimize the conditions for the assay. Aeration was observed to be critical for the effective development of submerged biofilms by M. xanthus, an obligate aerobic bacterium. In addition, temperature plays an important role in the development of M. xanthus submerged biofilms. It is well established that the formation of submerged biofilms by many bacteria requires both exopolysaccharide (EPS) and the type IV pilus (T4P). EPS constitutes part of the biofilm matrix that maintains and organizes bacterial biofilms while the T4P facilitates surface attachment as adhesins. For validation, we used our biofilm assay to examine a multitude of M. xanthus strains with various EPS and T4P phenotypes. The results indicate that the levels of EPS, but not of piliation, positively correlate with submerged biofilm formation in M. xanthus.

Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay.

Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors.

Spectrophotometric microplate assay for titration and neutralization of avian nephritis virus based on the virus cytopathicity.

INTRODUCTION: Plaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells. METHODS: CK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated. RESULTS: The ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA. CONCLUSION: The newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.

Recent Progress in Rapid Determination of Mycotoxins Based on Emerging Biorecognition Molecules: A Review.

Mycotoxins are secondary metabolites produced by fungal species, which pose significant risk to humans and livestock. The mycotoxins which are produced from Aspergillus, Penicillium, and Fusarium are considered most important and therefore regulated in food- and feedstuffs. Analyses are predominantly performed by official laboratory methods in centralized labs by expert technicians. There is an urgent demand for new low-cost, easy-to-use, and portable analytical devices for rapid on-site determination. Most significant advances were realized in the field bioanalytical techniques based on molecular recognition. This review aims to discuss recent progress in the generation of native biomolecules and new bioinspired materials towards mycotoxins for the development of reliable bioreceptor-based analytical methods. After brief presentation of basic knowledge regarding characteristics of most important mycotoxins, the generation, benefits, and limitations of present and emerging biorecognition molecules, such as polyclonal (pAb), monoclonal (mAb), recombinant antibodies (rAb), aptamers, short peptides, and molecularly imprinted polymers (MIPs), are discussed. Hereinafter, the use of binders in different areas of application, including sample preparation, microplate- and tube-based assays, lateral flow devices, and biosensors, is highlighted. Special focus, on a global scale, is placed on commercial availability of single receptor molecules, test-kits, and biosensor platforms using multiplexed bead-based suspension assays and planar biochip arrays. Future outlook is given with special emphasis on new challenges, such as increasing use of rAb based on synthetic and naïve antibody libraries to renounce animal immunization, multiple-analyte test-kits and high-throughput multiplexing, and determination of masked mycotoxins, including stereoisomeric degradation products.

A sensitive chemiluminescence method for quantification of the oxidative burst in grapevine cells and rice roots.

Roots are prominent plant-microbe interaction sites and of great biological relevance for many studies. The root response is of interest when searching for potential systemic resistance inducers. Screening of elicitors often focuses on the oxidative burst, the rapid and transient production of Reactive Oxygen Species (ROS). However, to our knowledge, no high-throughput, sensitive methods have been developed for the quantification of ROS released by roots. Here, we report on the development of an L-012-based chemiluminescence bioassay to quantitatively determine the oxidative burst following elicitation events in roots. Rice and grapevine were used as monocot and dicot models. We demonstrate that chitosan, a recognized elicitor in rice cells, was able to elicit ROS production in rice roots. Chitosan also triggered a strong oxidative burst in grapevine cell suspension cultures, while grapevine roots were not responsive. Although this method is broadly applicable, the L-012 probe requires careful consideration of solvents and plant species. Insufficient extracellular ROS, quenching, and the interference of solvents with the probe can undermine the assay sensitivity.

Application of a robust microplate assay to determine induced ß-1,3-glucanase and chitinase activity in the cotton plant.

Resistance is induced in cotton plants as the result of either viral infection or exogenous application of elicitors. Induced resistance can be evaluated by determining the production of ß-1,3-glucanase and chitinase in plants as a biochemical parameter. The assays being used for the determination of chitinase and ß-1,3-glucanase activity are laborious and not cost-effective, as the reducing sugars produced by the substrates colloidal chitin and laminarin are very expensive. The concentration of both substrates was standardized and reduced to 0.25% from 4% in a modified microplate assay, which appeared to be more effective. The amount of ß-1,3-glucanase and chitinase produced was significant and determined by the new modified assay. The sensitivity of the microplate assay was significantly raised approximately one- to twofold.