This protocol presents a multilevel method, both for EST detection and the phage figure. It also introduced large index for saves to compare EMR levels of different control technology. This cost-effective technique is intelligent, high-throughput and multifunctional.
It has enabled the establishment of VAMS a veterinary monitoring network that has collected 20, 000 epidemic genes and analytic phages. This method can also be used for monitoring and controlling pathogenic bacteria in medicine, agriculture, animal heart battery, and the fishery. This protocol and this company paper should provide to be a.
We with technical training guide. Following it would help anyone master the technique in a short span. Begin by incubating the quality control organism E.coli and 93 test salmonella strains on Mueller-Hinton agar plates.
Next, dilute the prepared inoculum of each strain 10 times. Transfer 200 microliters of sterile normal saline into the a-1 well of a 96 well plate as the negative control. Then pipette two suspensions of E.coli into the a-2 and a-3 wells as positive and quality controls respectively.
To the remaining 93 wells, add 200 microliters of the diluted test strain. For the preparation of the antimicrobial agar plates. First, set the concentration ranges for the different antibacterial agents being tested, as per the LAR index.
Perform a log-two doubling dilution scheme for the antibiotic solution following the CLSI standard agar dilution method. Now add two milliliters of the antimicrobial solution to a sterilized glass bottle containing 18 milliliters of molten Mueller-Hinton agar media. After mixing thoroughly, pour into plates inside a bio safety cabinet.
Allow the agar to solidify at room temperature. Make sure to leave a gap under the lid of the plates to allow for the drying of the agar surface. Label the types of antimicrobial agents and their respective concentrations on the underside of the incubated plates.
Arrange the multiple incubated plates for each antimicrobial agent in a stack arranged in log two doubling dilution order. Finally, prepare two drug-free agar plates to act as controls for each agent. Install a sterilized inoculation pin plate on the support of a 96 dot matrix inoculator within a bio safety cabinet.
Next, arrange the prepared seed block with tested strains and an agar incubated plate on the mobile carrier. Push the mobile carrier until the seed block is below the inoculation pin plate. Now press the operating handle and lower the inoculation pin plate, guiding the 96 pins towards the inoculum in the 96 wells of the seed block.
Release the operating handle in a controlled manner and automatically reset the inoculation pin plate to stir each inoculum well and dip, push the operating handle two to three times. Afterward, shift and position the carrier plate so the incubation plate is under the inoculation pin plate. Press down the operating handle, lowering the inoculation pin plate and leaving it for one to two seconds to ensure full contact with the surface of the incubated plate.
Release the operating handle to complete one round of inoculation. Replace the incubated plate and repeat the process until the batch of antimicrobial agar plates is completed. Switch to a new inoculation pin plate and seed block to inoculate another group of tested strains.
Incubate the inoculated antimicrobial agar plates at room temperature until moisture from the inoculum spots is completely absorbed into the agar. After incubation, invert the plates and continue incubation to allow uninhibited bacteria to form colonies. To perform image acquisition and data statistics, start by clicking on the 96 dot matrix AST image acquisition system to launch the program.
From the task bar, select test information. Click new to create a new test task and fill in the required details. Then press data collection followed by photograph and test item to select the newly created task.
Choose antibiotics to select the antibiotic name and gradient to select the initial concentration of the antibiotic. Lastly, click on connect to establish a connection with the image acquisition converter. Next, place the incubated plates on the detection plate base and insert them into the image acquisition converter.
Click on collection to capture images of the plates. Press antibiotics and select the next set of plates. Choose gradient to identify the starting gradient, then proceed to the next round of image collection.
Finalize the procedure by clicking submit to determine colony formation and convert images into minimum inhibitory concentrations, that is MIC values. Click on query to access all MIC results. Begin by utilizing the double layer agar method to produce different phages.
Dilute the phages to a suitable parallel concentration and add 200 microliters of the phage inoculum into the 96 well seed block. Next, prepare a double layer incubated plate for each strain to be examined. Leave a gap under the lid of the double layered plate to allow for drying.
Place the prepared phage seed block and the double layer plate on the mobile carrier of the 96 dot matrix inoculator. Transfer all phage inocular to the semi agar surface. Let the plates remain at room temperature until all inocular moisture has been absorbed into the semi agar.
Now incubate the plates at 37 degrees Celsius for the tested strains for four to six hours. Use the image acquisition converter to obtain and save the image of the experimental result of each double layer plate. Record the number and morphologies of the different spot shapes in a spreadsheet based on the obtained images and calculate the respective proportions of the different types of phages.
Morphological study of salmonella growth on plates with ampicillin showed that the negative control in a-1 showed no growth. The MIC of the salmonella strain in a-4 was 64 micrograms per milliliter, while that of a-5 was 16 micrograms per milliliter. The percentage resistance values of ampicillin, ciprofloxacin, and amoxicillin-clavulanic acid were higher than 50%While that of doxycycline, florfenicol, cefotaxime and enrofloxacin were 30%to 50%Gentamicin, amikacin and meropenem showed percentage resistance values of less than 30%The LAR indices of ciprofloxacin and amoxicillin-clavulanic acid deferred significantly.
Morphological analyses of salmonella growth on double layer plates showed that four main spot morphology of phages were observed. The round clear lytic spot was from a phage that could reliably kill the host on the plate, but which may or may not successfully replicate at that host's expense. The collection of plaques was formed by true plaques.
The turbid lytic spot resulted from a phage that did not reliably kill the host on the plate, and which may or may not successfully replicate at that host's expense. The no lytic spot indicated non-lytic nature. This technique easily an efficiently transfers 96 agar lens at once and the instruments automatically recognizes images and then calculates the line dice.
This technology is expected to promote the research and the industrialization of phages and the application of phages is one of the amazing ways to reduce and solve the problem of AMR.
