Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1-3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

Light-Addressable Actuator-Sensor Platform for Monitoring and Manipulation of pH Gradients in Microfluidics: A Case Study with the Enzyme Penicillinase.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte's pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.

Alkaline-promoted regulation of the peroxidase-like activity of Ni/Co LDHs and development bioassays.

Nanozymes' activities could be regulated by a simple and effective pH change in an in situ manner. In this work, for the first time, the peroxidase-like activity of Ni/Co layered double hydroxides (LDHs) was regulated via the alkaline-promoted reaction of fluorogenic substrate homovanillic acid and H2O2, and a promising tool for pH sensing was developed over the pH range of 8.3-9.6. As peroxidase nanozyme model, Ni/Co LDHs showed ease of preparation, low-cost, and water-solubility, which played an important role in this luminescence system. Based on the pH-dependent regulation of the Ni/Co LDHs activity, we constructed the bioassay platform for the determination urea, urease, penicillin G, and penicillinase with a wide linear range of 17-1000 µM, 3.3-270 mU mL-1, 3.3-1300 µM and 3.3-100 mU mL-1, respectively. This study not only demonstrated the alkaline-promoted modulation the nanozymes' activities, but also established a facile approach to develop novel bioassays.

Comparison of phenotypic methods for the detection of penicillinase in Staphylococcus aureus and proposal of a practical diagnostic approach.

Objectives: Disc diffusion is a cost-efficient, low-complexity, reliable method for detection of blaZ -mediated benzylpenicillin resistance in Staphylococcus aureus if the zone edge is inspected. EUCAST breakpoints cannot fully separate ß-lactamase-positive from ß-lactamase-negative strains, and EUCAST recommends the zone edge test. Literature on nitrocefin-based testing and the zone edge test is scarce with wide variations in reported assay performance. Methods: This study compared two different nitrocefin-based commercial and in-house tests and the EUCAST-based zone edge test for penicillinase detection in S. aureus applying a PCR-based gold standard. Results: In total, 215 non-duplicate clinical S. aureus isolates were included in the study, of which 127 (59.1%) did not harbour a blaZ gene, whereas 88 (40.9%) were blaZ positive. This study showed that for blaZ detection the zone edge test is more sensitive (96.6%) than nitrocefin tests independent of using nitrocefin discs (87.5% sensitivity) or solution (89.8% sensitivity), and that the significant inter-person variations of the zone edge test are probably related to the training level of the individual investigators (individual sensitivity ranging from 68.2% to 96.6%, specificity ranging from 89.8% to 100%). Conclusions: In addition to continued and strict training of investigators, we propose mandatory checking of benzylpenicillin zone edges, particularly in an investigation zone from 26 to 30 mm, which can result in improved specificity/positive predictive value of the zone edge test (from 98.4% to 100%) but retains the high sensitivity/negative predictive value of the method.

Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody.

A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1-128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93-97.5%, and the coefficient of variation [CV (%)] were from 5.55-8.38%. For interassay reproducibility, recoveries were from 89.5-95.1%, the coefficient of variation [CV (%)] were from 5.26-9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

Genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae from England and Wales.

OBJECTIVES: The objective of this study was to investigate the genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in 2012 from England and Wales. METHODS: PPNG isolates were from the 2012 Gonococcal Resistance to Antimicrobial Surveillance Programme (GRASP). Their susceptibility to seven antimicrobials was determined using agar dilution methodology. ß-Lactamase production was detected using a nitrocefin test. ß-Lactamase plasmid types were determined and blaTEM genes were sequenced. Isolates were also typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST). RESULTS: Seventy-three PPNG isolates were identified in the 2012 GRASP collection (4.6%, 73/1603). Three different blaTEM alleles were identified, encoding three TEM amino acid sequences: TEM-1 (53%), TEM-1 with a P14S substitution (19%) and TEM-135 (27%). The blaTEM-135 allele was present in nine different NG-MAST types and was found mostly on Asian (60%) and Toronto/Rio (35%) plasmids. By contrast, most TEM-1-encoding plasmids were African (98%). All the TEM-135 isolates displayed high-level ciprofloxacin and tetracycline resistance. CONCLUSIONS: The high proportion of blaTEM-135 alleles (27%) demonstrates that this variant is circulating within several gonococcal lineages. Only a single specific mutation near the ß-lactamase active site could result in TEM-135 evolving into an ESBL. This is concerning particularly because the TEM-135 isolates were associated with high-level ciprofloxacin and tetracycline resistance. It is encouraging that no further TEM alleles were detected in this gonococcal population; however, vigilance is vital as an ESBL in N. gonorrhoeae would render the last remaining option for monotherapy, ceftriaxone, useless.

Comparison of phenotypic tests for detecting penicillin G resistance with presence of blaZ gene in Staphylococcus aureus isolated from bovine intramammary infections.

Few studies have described the relationship between genotypic and phenotypic methods for detecting penicillin resistance in Staphylococcus aureus isolated from bovine intramammary infection (IMI). Six phenotypic methods for penicillinase detection were compared with a genotypic method testing the presence of the ß-lactamase gene blaZ in Staph. aureus (n = 150) isolated from bovine IMI. Highest sensitivities and specificities were observed for disk diffusion (DD) (93 and 97.4%), minimum inhibitory concentration (MIC) (90.3 and 97.4%), Cefinase™ (85.9 and 97.4%) and Diatabs™ (85.7 and 98.7%). The estimated cut-off points estimated in the present study can be considered close to the ones indicated by CLSI (2013). The molecular detection of blaZ gene is the only method that may indicate the real or potential capacity of producing ß-lactamase in Staph. aureus. Considering that from a clinical standpoint a false negative result from a phenotypic test is the most unfavourable situation, a combination of standard DD with Diatabs™ or Cefinase™ should be performed by routine mastitis laboratories to minimise false negative results.

Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors.

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)µM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality.

Performance of phenotypic tests for detection of penicillinase in Staphylococcus aureus isolates from Australia.

Recent studies have shown that chromogenic cephalosporin tests are inferior to disc zone edge tests in detecting penicillinase in Staphylococcus aureus isolates, resulting in a change to CLSI and EUCAST guidelines in 2012. We sought to confirm these findings using Australian isolates and compare the performance of the CLSI and EUCAST methods, which use different disc strengths, penicillin at 10 units (P10) and 1 unit (P1), respectively. Using blaZ PCR as the reference standard, the sensitivities of the tests for detection of penicillinase production were as follows: Cefinase disc test, 24/38 isolates (63%); P10 disc zone edge test, 34/38 isolates (89%); P10 disc diameter test, 25/38 isolates (66%); P1 disc zone edge test, 38/38 isolates (100%); and P1 disc diameter test, 38/38 isolates (100%). We also found that the P10 disc zone edge test reading was interpreted differently by the clinical laboratory and the study investigators in 11% of instances. Our findings support those of previous studies showing that chromogenic cephalosporin-based ß-lactamase tests are inferior to disc methods in detecting S. aureus penicillinase. We also conclude that the EUCAST method using the P1 disc has the best performance, particularly because the P1 disc zone diameter reading closely correlated with penicillinase production and reading of the disc zone diameter is less subjective than reading of the zone edge.

Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae / Disseminação horizontal de plasmídios de resistência contendo genes de beta-lactamase dos tipos TEM e SHV entre isolados clínicos de Enterobacteriaceae

The extended-spectrum â-lactamase (ESBL)-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS) testing. Twenty ESBL producing strains (15 percent) including Escherichia coli (n = 9), Klebsiella pneumoniae (n = 7), Klebsiella oxytoca (n = 2) and Enterobacter aerogenes (n = 2) were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR) and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L) were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospital

O isolamento de bactérias produtoras de beta-lactamases de espectro expandido (ESBL) está aumentando no mundo todo. Freqüentemente, a expressão de ESBL está associada com resistência a múltiplas drogas e disseminação por plasmídios de resistência. Durante um período de dois meses em 2000, 133 isolados clínicos de cepas de enterobactérias foram obtidos aleatoriamente de pacientes internos e externos de um hospital universitário na Turquia. As cepas produtoras de ESBL foram identificadas pelo teste de sinergia em disco-duplo (DDS). Foram detectadas vinte cepas produtoras de ESBL, entre as quais Escherichia coli (n=9), Klebsiella pneumoniae (n=7), Klebsiella oxytoca (n=2) e Enterobacter aerogenes (n=2), que foram posteriormente analisadas quanto a suas características de transferência de resistência, perfil plasmidial e natureza dos genes de resistência. Os testes de transferência de plasmídios foram realizados empregando técnicas de conjugação em caldo. Os genes TEM e SHV foram analisados pela reação da polimerase em cadeia (PCR) e hibridização com sondas especificas. A detecção de plasmídios epidêmicos foi feita por análise dos plasmídios R com a enzima de restrição EcoRI. Através desta análise, foram obtidos catorze perfis plasmidiais (A, B1, B2, C1 e C2 até L).Observou-se pela PCR que a maioria dos plasmidios carregavam genes derivados de TEM e SHV, confirmados através da detecção dos genes pelos testes de hibridização. As evidencias epidemiológicas indicaram que havia uma aparente transferência horizontal dos plasmídios R conjugativos entre as enterobactérias multiresistentes neste hospital.

Comparison of phenotypic methods for penicillinase detection in Staphylococcus aureus.

Penicillinase testing is required for Staphylococcus aureus isolates with a penicillin MIC of

Amoxicillin-clavulanate therapy increases childhood nasal colonization by methicillin-susceptible Staphylococcus aureus strains producing high levels of penicillinase.

We examined factors associated with penicillinase production by nasal carriage Staphylococcus aureus strains in 648 children aged 3 to 6 years attending 20 randomly sampled playschools. The children were prospectively monitored for drug use and medical events for 6 months and were then screened for S. aureus carriage. Isolates were tested for their susceptibility to penicillin G and methicillin, and penicillinase production by methicillin-susceptible, penicillin-resistant strains was quantified. S. aureus was isolated from 166 children (25.6%). Exposure to amoxicillin-clavulanate during the previous 3 months was associated with higher penicillinase production by penicillin-resistant, methicillin-susceptible strains (odds ratio, 3.6; P = 0.03). These results suggest that use of the amoxicillin-clavulanate combination could induce a herd selection process of S. aureus strains producing higher levels of penicillinase.

[Correlation between sensitivity to fosfomycin and the presence of penicillinase PSE-1 in Pseudomonas aeruginosa]. / Corrélation entre la sensibilité à la fosfomycine et la présence d'une pénicillinase PSE-1 chez Pseudomonas aeruginosa.

A prospective survey was carried out during three three-weeks periods in May, October 1997 and October 1998 in 13 teaching hospitals. All non-repetitive isolates of P. aeruginosa collected were subject to serotypage and determination of the inhibiting minimal concentrations for ticarcillin, piperacillin, piperacillin + tazobactam, ceftazidime, imipenem, amikacin, ciprofloxacin and fosfomycin. Identification of the betalactamases and quantification of the cephalosporinase were done for the strains intermediate or resistant to ticarcillin. The most frequent serotypes were O: 6 (17%), O: 11 (13%), O: 1 (10%) and O: 12 (9%). Serotype O: 12 was the least susceptible to antibiotics except for fosfomycin. Whatever the serotype, 76% of P. aeruginosa strains with bla PSE-1 are susceptible to fosfomycin, when only 29.8% of non bla PSE-1 producing strains were susceptible to this antibiotic. Integron encoding bla PSE-1 could be implicated in susceptibility to fosfomycin of P. aeruginosa strains. The associations fosfomycin + imipenem or fosfomycin + ceftazidime could be proposed in case of infections due to P. aeruginosa O: 12.

A bioelectrode for penicillin detection based on gluten-membrane-entrapped microbial cells.

A bioelectrode for penicillin detection was established using gluten membrane in which cells of Escherichia coli harbouring the plasmid pUC18 coding for penicillinase synthesis were entrapped. For the entrapment preparation, lyophilized cells were mixed with gluten solution during the formation of gel, and the resultant gel was hardened by the addition of oxidized starch. The immobilization procedure employed an inexpensive base material and avoided the use of purified enzyme. When the membrane with a thickness of 30 microm and a cell content of 33% (w/w) was used, the steady response of the bioelectrode to increasing concentrations of penicillin G buffered with 0.025 M phosphate was linear over the range of 1-16 mM. The response time was less than 3 min. Decreasing the cell content decreased the steady response due to a decline in enzymic activity. The treatment of lyophilized cells with ultrasonication or chemically with either N-cetyl-N,N,N-trimethylammonium bromide or guanidine hydrochloride rendered cells permeabilized and exposed the periplasmic enzyme to penicillin. The response time became shorter, but a serious decline in steady responses was observed because of the loss of enzyme activity during the cell permeabilization. The microbial electrode also showed different responses to penicillin G solution, depending on the pH and buffer concentration.

Detection of circulating antigens by ELISA using penicillinase in sera of mice infected with Toxoplasma gondii.

An enzyme linked immunosorbent assay (ELISA) has been developed using penicillinase as an enzyme marker for the detection of Toxoplasma gondii antigens during the acute stage of infection in the sera of experimentally infected mice. Circulating antigens were detected as early as 2 days after the infection and all mice had antigenemia by fourth day. The developed ELISA test is sensitive, specific and reproducible.

Study of beta-lactam resistance in ceftazidime-resistant clinical isolates of Enterobacteriaceae.

Mechanisms and transferability of beta-lactam resistance in 50 ceftazidime resistant strains of Enterobacteriaceae was studied. These strains were selected from 1991 E. coli, 1035 Enterobacter spp., 168 Citrobacter spp. and 1371 Klebsiella spp., isolated from patients hospitalized in ICUs and in the pediatric and urology departments of six hospitals in Bratislava during the years 1994-1996. The selected strains expressed the resistance not only to ceftazidime (50/50) but also to ampicillin (50/50), ceftriaxone (50/50), cefotaxime (49/50) and cefoxitin (45/50). The mechanism of resistance in all 50 strains was the production of beta-lactamases by conjugation, using either ceftazidime or cefotaxime for the selection of transconjugants and by isolation of R-plasmids ranging from to 55-87 kb from donor strains and from transconjugants. A total of 21 isolates possessed chromosomally encoded resistance and 25 clinical isolates and their transconjugants expressed ESBL sensitive to clavulanate. Selected E. coli and Klebsiella pneumoniae isolates expressed the presence of TEM and SHV enzymes determined by isoelectric focusing. The possible trends in the development of antimicrobial resistance in Slovakia in the future are indicated.

Comparison of single-dose cefuroxime axetil with ciprofloxacin in treatment of uncomplicated gonorrhea caused by penicillinase-producing and non-penicillinase-producing Neisseria gonorrhoeae strains.

A randomized, multicenter, investigator-blind trial was conducted to compare the efficacies of cefuroxime axetil and ciprofloxacin for treatment of patients with uncomplicated gonorrhea caused by penicillinase-producing Neisseria gonorrhoeae (PPNG). A total of 832 patients (434 females and 398 males) received a single oral dose of cefuroxime axetil (1,000 mg [417 patients]) or ciprofloxacin (500 mg [415 patients]). N. gonorrhoeae was eradicated from the cervix in 114 of 118 (97%) and 118 of 119 (99%) bacteriologically evaluable females treated with cefuroxime axetil and ciprofloxacin, respectively (P = 0.213; difference, -2%; 95% confidence interval, -6 to 1%), and from the urethra in 154 of 166 (93%) and 171 of 171 (100%) bacteriologically evaluable male patients treated with cefuroxime axetil and ciprofloxacin, respectively (P < 0.001; difference, -7%; 95% confidence interval, -11 to -3%). Both treatments were effective in eradicating N. gonorrhoeae in females with rectal infections (cefuroxime axetil, 29 of 30 [97%]; ciprofloxacin, 25 of 25 [100%]; P = 1.00). In small numbers of patients, cefuroxime axetil was less effective than ciprofloxacin in treating males with pharyngeal infections (eradication in 4 of 10 and in 8 of 8 patients, respectively; P = 0.013). PPNG was eradicated from the cervix in 22 of 23 (96%) and 32 of 32 (100%) bacteriologically evaluable female patients treated with cefuroxime axetil and ciprofloxacin, respectively (P = 0.418; difference, -4%; 95% confidence interval, -13 to 4%), and from the urethra in 35 of 36 (97%) and 34 of 34 (100%) bacteriologically evaluable male patients treated with cefuroxime axetil and ciprofloxacin, respectively (P = 1.00; difference, -3%; 95% confidence interval, -8 to 3%). The incidences of drug-related adverse events were similar for the two study drugs. In summary, treatment with a single oral dose of cefuroxime axetil is as effective as treatment with a single oral dose of ciprofloxacin in eradicating PPNG from males and females with uncomplicated gonorrhea (urethral and endocervical), and both regimens are well-tolerated. However, in the present study, cefuroxime axetil was less effective than ciprofloxacin in treating urethral gonococcal infections in male patients, although both study drugs were highly effective in treating cervical gonococcal infections in female patients.

Evaluation of product analogues for purification of penicillinase by affinity chromatography.

Among different matrices prepared, ampicilloic acid-polymer matrix offered 86.7% adsorption, 95% elution and 82.4% overall recovery of penicillinase. The structure of both the side chain and penicilloic or cephalosporoic acid moieties contribute to the affinity interactions.

Fluorescent spot test method for specific detection of beta-lactamases.

A simple, sensitive fluorescent spot test method for specific detection of microbial beta-lactamases has been developed, based on the modification of a previously disclosed method (K. C. S. Chen, October 23, 1990, U.S. Patent 4,965,193). The new fluorescence developer used in the present study consisted of 0.5 mM HgCl2, in 0.5 M sodium citrate buffer, pH 4.5, prepared in 0.5% formaldehyde aqueous solution. A beta-lactam substrate solution consisting of a beta-lactam antibiotic with an acyl side chain containing an alpha-amino group and an alpha-phenyl group, or its derivatives, was incubated with a beta-lactamase-producing organism. One volume of the fluorescence developer was added to 4 vol of the incubated beta-lactam substrate solution, followed by heating the mixture at 45 degrees C for 10 min. The mixture was spotted on filter paper. Production of fluorophore indicated beta-lactamase activity. Each fluorophore was analyzed by TLC and its chemical identity was determined. Using ampicillin as the penicillinase substrate and cephalexin as the cephalosporinase substrate, the new method can be conveniently carried out by using dropping bottles for storing and dispensing the substrate solutions and the fluorescence developer. This modified method also provided more favorable conditions for the penicillinases to remain active during fluorescence development. Therefore, the sensitivity of the test was increased.

Characterization of chromosomally encoded penicillinases in clinical isolates of Klebsiella pneumoniae.

Four Klebsiella pneumoniae strains, isolated in two geographically distant French hospitals, were found to produce constitutive beta-lactamases with an unusual isoelectric point for this species (8.1). The four enzymes were chromosomally encoded and related to the Ambler's class A plasmid-mediated SHV-type enzymes. The four enzymes were predominantly penicillinases, with good activity against amino- and ureidopenicillins. They poorly hydrolysed cephalothin, cephaloridine and cefoperazone and did not hydrolyse third-generation cephalosporins and aztreonam. The four enzymes were susceptible to the inhibitory effect of clavulanic acid. Finally, the strains were all found to produce an acetyl-esterase. The acetyl-esterases catalysized hydrolysis of the acetoxy group of cephalothin and cefotaxime although this did not affect their antibacterial activities. These esterases were not susceptible to the inhibitory effect of clavulanic acid and EDTA.