Electrospun Nanofibrous Scaffold as a Potential Carrier of Antimicrobial Therapeutics for Diabetic Wound Healing and Tissue Regeneration

Charu Dwivedi , ... Sandip Patil , in Nano- and Microscale Drug Commitment Systems, 2017

7.1 Agar Diffusion Test

Agar diffusion test is the primary method to determine the antimicrobial action of the nanofibrous scaffolds. Information technology is important to mention hither that information technology is but suitable for the diffusive test materials. The agar diffusion examination is qualitative, like shooting fish in a barrel to perform, and uncomplicated. The methodology includes the inoculation of bacterial cells on food agar Petri dishes and test samples are laid over these dishes. Subsequently, the dishes are incubated for xviii–24 h at 37°C and thereafter, the growth of bacteria is adamant below the nanofibrous scaffolds (zone of inhibition). The presence of antimicrobial activity is indicated past the absence of bacterial growth straight below the test sample (Bauer et al., 1966).

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Diagnosis of constitute virus diseases

Anupam Varma , Manoj Kumar Singh , in Applied Plant Virology, 2020

half dozen.2.1.2 Agar-gel double diffusion tests

Agar-gel double diffusion tests, likewise known as the "Ouchterlony examination," were widely used (Ouchterlony, 1968), particularly for the diagnosis of the diseases caused past the isometric viruses, owing to the convenience of performing the tests in semisolid stage using 0.vii%–1.0% agar gel. In this test, generally the antibody is put in a fundamental well cutting in the gel set in a Petri dish and antigen (purified virus or constitute extract containing the virus) to be tested in the four to eight outer wells equally required (Gibbs and Harrison, 1976). The beauty of this exam is not merely that the positive reaction appears as a distinct precipitin band midway between the antibody and antigen wells, but that it tin also be used for titrating the virus preparations and determining the serological affinity of the viruses (Fig. vi.1C). A major limitation of this exam is the nontestability of the filamentous viruses, which do not lengthened in the agar gel. However, this limitation tin can be easily overcome past breaking the virus particles to diffusible sizes by treatments similar sonication (Varma et al., 1970).

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Preliminary Studies into Wash-Fast Antimicrobial Treatments of Polyester

O. Hauck , ... J. Verran , in Medical and Healthcare Textiles, 2010

Microbiological testing methods

SN 195 920 'agar diffusion test '

The agar diffusion test is used to qualitatively assess the efficacy of textiles treated with diffusible biocides. Samples are placed in the centre of food agar plates which have been inoculated with the test leaner. The samples are incubated at 37°C for 18-24  hours. The evaluation of this test is based on the level of growth both nether and effectually the sample. (Fig. 2) The 'zone of inhibition' around the test material is measured and any growth present underneath the sample is scored.

Fig 2. Case of untreated textile (a) showing no effect on bacterial growth, and treated textile (b) showing a 'zone of inhibition' around the sample.

JIS Fifty 1902:2002 'quantitative exam for non-leaching actives '

This method is used to determine the antibacterial effectiveness of textiles treated with non diffusing active substances. The exam material is sterilised and inoculated with a standard number of bacteria The samples are incubated at 37°C for 18   hours. The difference in number of colony forming units (cfu) between 0 and 18   hrs contact with the sample indicates the efficacy of the test cloth.

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The Clinician and the Microbiology Laboratory

Patrick R. Murray , in Mandell, Douglas, and Bennett's Principles and Do of Infectious Diseases (Eighth Edition), 2015

Agar Diffusion Methods

The agar diffusion method was developed as a practical alternative to the agar and broth tube dilution procedures. The most pop agar diffusion method is the Kirby-Bauer disk improvidence method. 39 In this method, the test agar plate is swabbed with a standardized concentration of the test organism, and so newspaper disks containing a divers antibiotic concentration are placed on the lawn of bacteria. Subsequently overnight incubation, the diameter of the zone of inhibited growth around the disk is measured. This zone is influenced by a number of variables, including the susceptibility test medium (Mueller-Hinton agar is the standard for bacterial tests), the concentration of the exam organism, the charge per unit of growth of the test organism, the concentration of antibody in the disk, the improvidence of the antibiotic in the agar, and the susceptibility of the organism to the antibiotic. The first five variables are standardized by CLSI; therefore, if the exam is properly performed, the size of the zone of inhibited growth is directly related to the susceptibility of the organism—the larger the zone, the more than susceptible the organism is to the antibody. Every bit would be expected, the results of the dilution tests and diffusion tests are related. There is an inverse linear relationship between the size of the zone and the MIC value—the larger the zone of inhibited growth (more than susceptible the organism to the antibiotic), the smaller the MIC value. Thus, information technology is possible to extrapolate from the measured size of the inhibitory zone to the corresponding MIC value. In add-on, the interpretive criteria that are applied to MIC tests apply to the improvidence tests. Thus, for nigh organism-antibiotic tests, the diffusion tests and dilution tests are equally accurate in predicting antimicrobial susceptibility.

The dilution tests are commonly referred to every bit quantitative tests, whereas the improvidence tests are referred to as qualitative tests. That is, the dilution tests are unremarkably reported as a number or MIC value, whereas the diffusion exam results are reported as susceptible, intermediate, or resistant. This is an oversimplification. Both tests are quantitative; in fact, the diffusion tests appraise a greater range of quantitative values (zone sizes from 6 mm [diameter of the newspaper disk] to nearly twoscore mm), compared with dilution tests, which typically measure 2 to five concentrations of a drug in most microdilution panels. And then, the definition of a quantitative or qualitative test is adamant past the reporting method rather than the testing method.

One variation of the Kirby-Bauer deejay diffusion exam is the E exam (bioMérieux, Lyon, France), which is a gradient improvidence examination. In this exam, the antibiotic is in a commercially prepared strip, with a concentration gradient extending from the top to the bottom of the strip. When this is placed on a backyard of bacterial growth, an elliptic pattern of inhibited growth develops (greater inhibition at the end with a higher concentration of antibody). The strips are calibrated so that the area where the bacterial growth meets the strip corresponds to the organism'southward MIC value for the antibiotic. The reward of the E test is that MIC results can exist obtained easily for one or two antibiotics. However, when a large number of organism-antibiotic combinations need to be tested, the goop microdilution method is preferred.

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Sensitivity of ESBL-Producing Gram-Negative Bacteria to Essential Oils, Institute Extracts, and Their Isolated Compounds

Gy. Horváth , ... B. Kocsis , in Antibody Resistance, 2016

Agar Diffusion Method

In the other blazon of ADM technique, plant extracts or solutions of individual compounds are passed through a hole in the agar plate. During the incubation menstruum, the tested cloth diffuses from the hole into the agar medium seeded with the examination microorganism. The active antimicrobial extracts or compounds result in zones of inhibition around the pigsty, which give information near the value of the minimum inhibitory concentration (MIC). Factors influencing the size of inhibition zones in DDM and AMD include the size of the filter paper disk or pigsty, the amount of compound placed onto the disk or into the hole, the type and concentration of the agar, the thickness and pH of the medium, the microbial strain tested, and the incubation temperature. Generally, DDM and ADM tin be regarded as prescreening tests, which are appropriate for evaluating a high number of plant extracts or individual components during parallel examinations, only their results should not exist overemphsized. 13

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Laboratory Diagnosis of Bacterial Infections

Barbara A. Byrne , in Equine Infectious Diseases (2nd Edition), 2014

Methods

The 2 major methods for testing for antimicrobial drug susceptibility are agar improvidence, nearly frequently using the Kirby-Bauer method, and broth dilution, in which microdilution techniques are most common. All testing and interpretation should be performed in accordance with recommendations of the Clinical and Laboratory Standards Institute (CLSI) so that results are repeatable and able to be compared between laboratories ( http://www.clsi.org). In the agar diffusion method, leaner, at a single concentration, are spread over Mueller-Hinton agar to create a lawn, and discs impregnated with the drugs to exist tested are placed on the lawn. The plates are incubated 18 to 24 hours, and the bore of the zone of inhibition, or the surface area where bacteria practise not grow, is measured. Zone size correlates with susceptibility (Fig. 27-4). The advantages of the Kirby-Bauer method are ease, petty need for specialized equipment, and the dissimilar drugs used in testing can exist changed readily. Disadvantages are that some bacteria abound poorly or not at all on the media, and the minimum inhibitory concentration (MIC) cannot be adamant.

When broth microdilution methodology is used, a set up concentration of bacteria is inoculated into each well of a 96-well plate containing an antimicrobial drug. A number of drugs can be tested on each plate at the discretion of the laboratory or plate manufacturer. Each drug is tested at several different concentrations; twofold dilutions are centered effectually the plasma levels expected in the patient when the drug is administered at recommended dosages. The plate is incubated xviii to 24 hours, and each well is examined for growth. Growth in a well indicates that the bacterium is not sensitive to the drug at that concentration. The lowest drug concentration at which at that place is no growth is considered the MIC (Fig. 27-five). The MIC will be unique for that detail isolate.

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Myristica fragrans: A Review

V. Kuete , in Medicinal Spices and Vegetables from Africa, 2017

four.5 Antimicrobial activities

A qualitative written report of the antibacterial activity of Thou. fragrans using agar diffusion indicated that ethanol and acetone extracts of the seed pulps and crust were active against Gram-positive leaner, Staphylococcus aureus, and Bacillus subtilis contrary to aqueous extracts (Kadhim et al., 2013). Nevertheless, no event of the aforementioned extracts was observed in Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa (Kadhim et al., 2013). The extracts from the seeds at 160 μg/mL inhibited human rotavirus past 90% indicating that information technology can be useful in the treatment of human diarrhea if the etiologic agent is a rotavirus (Goncalves et al., 2005). The methanol extract of fruit inhibited the growth of multidrug resistant Salmonella typhi B330 and M531 strains with identical minimal inhibitory concentration (MIC) value of 64 μg/mL (Rani and Khullar, 2004). Narasimhan and Dhake have evaluated the antibacterial action of trimyristin, myristic acid, and myristicin isolated from nutmeg confronting Gram-positive and Gram-negative microorganisms (Narasimhan and Dhake, 2006). Results showed that trimyristin was highly active against Eastward. coli and Micrococcus luteus (MIC: 1.250 μg/mL), S. aureus (MIC: ane μg/mL), B. subtilis, and P. aeruginosa (MIC: 0.6 μg/mL); myristic acid was also highly active against Eastward. coli and B. subtilis (MIC: 1.250 μg/mL), S. aureus and G. luteus (MIC: 0.75 μg/mL) and P. aeruginosa (MIC: 0.650 μg/mL); myristicin was active against E. coli (MIC: 1.250 μg/mL), B. subtilis (MIC: 1.00 μg/mL), South. aureus (MIC: 0.75 μg/mL), Yard. luteus (MIC: 0.625 μg/mL), and P. aeruginosa (MIC: 0.6 μg/mL). The elective of nutmeg, ten displayed antimicrobial effects against Porphyromonas gingivalis ATCC33277 with a MIC of 0.39 μg/mL, P. gingivalis 381 (MIC: 0.098 μg/mL), Fusobacterium nucleatum ATCC25586, Streptococcus mutans IFO13955 (MIC: fifty μg/mL), Actinomyces viscosus ATCC15987 (MIC: 13 μg/mL), various strains of Helicobacter pylori (MIC: 13–25 μg/mL), and South. aureus (MIC: 13 μg/mL) (Shinohara et al., 1999). Malabaricone C irreversibly inhibited Arg-gingipain and selectively suppressed the growth of P. gingivalis growth (Shinohara et al., 1999). Malabaricone B and malabaricone C exhibited stiff antifungal and antibacterial activities confronting Southward. aureus and Streptococcus durans (MICs of 1 and 4 μg/mL, respectively), B. subtilis (MICs of 1 and 2 μg/mL, respectively), and against various strains of C. albicans (MIC of eight–32 μg/mL) (Orabi et al., 1991). Lignans isolated from seeds, 12–fourteen displayed moderate antimicrobial activeness against Alternaria alternata, Colletotrichum coccodes, Colletotrichum gloeosporioides, Magnaporthe grisea, Agrobacterium tumefaciens, Acidovorax konjaci, and Burkholderia glumae (Cho et al., 2007).

The efficacy of compound xv (macelignan), isolated from nutmeg, was evaluated; results showed that at 24 h of biofilm growth, Due south. mutans, A. viscosus, and Streptococcus sanguis biofilms were reduced past upward to thirty, xxx, and 38%, respectively, after treatment with 10 μg/mL of 15 for 5 min; increasing the treatment fourth dimension to 30 min resulted in a reduction of more than 50% of each of the single primary biofilms (Rukayadi et al., 2008). These results point that fifteen is a potent natural antibiofilm agent against oral main colonizers.

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Gatifloxacin

Ebtehal South. Al-Abdullah , in Profiles of Drug Substances, Excipients and Related Methodology, 2012

v.2.8 Microbiological assay method

Salgado et al . used a simple, sensitive, and specific agar improvidence bioassay for gatifloxacin using a strain of Bacillus subtilis ATCC 9372 as the test organism. Gatifloxacin could be measured in tablets and raw material at concentrations over the range of 4–16   μg/mL. The calibration graph for gatifloxacin was linear over 4.0–xvi.0   μg/mL, and the method validation demonstrated that the method was linear (r 2  =   0.9993), precise (RSD   =   1.14%), and authentic. The results confirmed precision and did non differ significantly from those obtained using other methods described in the literature. The validated method yielded proficient results in terms of the range, linearity, precision, accuracy, specificity, and recovery. Information technology was ended that the microbiological assay is satisfactory for in vitro quantification of the antibacterial activity of gatifloxacin [twoscore].

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Anti-Carbohydrate Antibodies with Specificity for Monosaccharide and Oligosaccharide Units of Antigens

John H. Pazur , in Advances in Carbohydrate Chemistry and Biochemistry, 1998

ii Agar Diffusion

A method used extensively for the identification and characterization of antibodies is agar diffusion. The method was originally devised by Ouchterlony. 27 The process is readily performed on a microscope slide. Agarose (0.5 one thousand) is suspended in 50 mL of 0.1 Chiliad phosphate buffer of pH 7.0 and 5 mg of merthiolate is added. The mixture is heated to boiling until the agarose is in solution. To ready a slide, a hot solution of agar is introduced in a sparse layer on the slide. The agar is allowed to solidify and wells are punched at appropriate distances autonomously in a circular fashion on the slide with a well puncher. The plug of agar is withdrawn by suction and immune serum or purified antibodies are placed in the center wells. A solution of the antigen at concentrations of 0.ii to one% is placed in the outer wells. The slide is placed on moist filter paper in a petri dish, and the dish is covered. Diffusion is allowed to proceed for time varying from 6 to 24 h. Visible precipitin bands develop on the plate (Fig. 2), and the plate is photographed for a permanent tape and for comparison with results obtained in other tests.

Fig. 2. Agar-diffusion blueprint of allowed serum (Ab) and antigenic polysaccharides from Streptococcus faecalis. Wells ane and 2 incorporate a tetraheteropolysaccharide, wells three and 4 comprise a diheteropolysaccharide, and wells 5 and six incorporate a mixture of the two antigenic polysaccharides.

 [From J. H. Pazur in Sugar Analysis: A Practical Approach, (M. F. Chaplin, J. F. Kennedy, eds.) IRL Press, Oxford, England, 1986, pp. 55–96, by permission of Oxford University Press.]

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Moxifloxacin Hydrochloride

Mahmoud K.H. Al Omari , ... Adnan A. Badwan , in Profiles of Drug Substances, Excipients and Related Methodology, 2014

iv.6 Biological methods

Determination of moxifloxacin in human body fluids by a conventional cup-plate agar diffusion method with Bacillus subtilis as indicator organism was reported [108,169]. The method was used every bit a reference to a proposed HPLC method with fluorescence detection. The splendid correlation between results of both methods (gradient one.05) was constitute. The linearity range and quantitation limit are 0.06−2.0 (urine and plasma) and 0.06   μg/ml, respectively.

In vitro activeness of moxifloxacin against Legionella species and the outcome of medium on susceptibility test results were investigated using an ager dilution method with buffered charcoal yeast extract agar containing α-ketoglutarate [170]. A spore pause of Bacillus subtilis was used as the indicator organism and the medium was Iso-Sensitest agar. The study revealed that charcoal has inhibitory consequence on the test media, every bit a result the MIC was corrected to the charcoal-spring fraction of the drug.

Escherichia coli (NCTC 10418) was used as indicator organism in studying in vitro phramacodynamic action of moxifloxacin for Staphylococcus aureus and Streptococci of Lancefield Group A and G [171]. The method showed a detection limit of 0.03   μg/ml. Results of this study revealed that moxifloxacin has significant bactericidal activity against Staphylococcus aureus with MICs ≤   0.14   mg/50 but may be less bactericidal confronting β-haemolytic streptococci.

A deejay diffusion microbioassay with Bacillus subtilis (ATCC 6633) was used to evaluate the bactericidal effectiveness and the pharmacodynamic profile of moxifloxacin in cerebrospinal fluid (CSF) and to compare the bactericidal activeness with that of ceftriaxone and meropenem therapy [172]. Calibration curves were constructed using concentration ranges of 0.two−x and 0.1–6   μg/ml for plasma and CSF samples, respectively. The detection limit was 0.2 for plasma and 0.i   μg/ml for CSF specimens.

A parallel-line bioassay was used to investigate the antimicrobial activity of moxifloxacin photodegradation products [173]. A continuous menses photochemical reaction unit (Beam-Heave) was used to partially photodegrade the drug (30–83%, as confirmed by HPLC). These results were compared by parallel-line bioassays using Escherichia coli, Enterobacter cloacae, and Klebsiella oxytoca. The estimated relative potencies (ERP; the ratio of the matched command solution concentration to that of the irradiated solution concentration) for irradiated versus command solutions showed no meaning deviation after 30% or 54% photodegradation with any of the indicator organisms, nor after 83% photodegradation with Escherichia coli and Klebsiella oxytoca. The ERPs were significantly different for 83% photodegradation using Enterobacter cloacae.

Bioassay was used in studying the activities of mutant prevention concentration-targeted moxifloxacin and levofloxacin confronting Streptococcus pneumoniae in an in vitro pharmacodynamic model [174]. The concentration of moxifloxacin and levofloxacin were determined using tryptic soy agar supplemented with 5% sheep claret as the medium and Klebsiella pneumoniae (ATCC 33495) every bit the indicator organism. The detection limit was 0.31   μg/ml.

Bioassay was used for evaluation using moxifloxacin in pleural empyema, a serious complication of pneumonia, therapy [175]. Moxifloxacin concentrations in serum and pleural fluid were measured microbiologically using Bacillus subtilis (ATCC 6633) on trypticase soy agar pH   9.0. Concentration range and the detection limit were 0.15–twenty.0 and 0.15   μg/ml, respectively.

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