Automated AST Workflows for MIC & Checkerboard Assays

ANTIMICROBIAL SUSCEPTIBILITY TESTING

OVERVIEW

The reproducibility problem in antimicrobial susceptibility testing

MIC determination by broth microdilution is well-established, but the workflow is unforgiving: a single-dilution pipetting error propagates across an entire column, and when you are running multiple plates across multiple operators, inter-run variability quickly becomes the dominant source of noise in your dataset. Checkerboard assays introduce an additional challenge. Generating orthogonal two-drug dilution gradients manually (in which one compound titrates across rows while the second titrates across columns to build the full interaction matrix) requires precision at every transfer step.

Myra addresses these challenges by supporting automated antimicrobial susceptibility testing through automated serial dilution setup, inoculum dispensing, and checkerboard matrix generation within a compact portable liquid handler that fits inside many biological safety cabinets (BSCs) and laminar flow hoods. Myra reduces the technical variability that limits reproducibility without requiring the infrastructure overhead of a larger robotic system. Any deviation in the concentration series undermines the fractional inhibitory concentration index (FICI) calculation and the clinical or pharmacological conclusions that depend on it.

Containment compatibility: Myra’s compact footprint allows operation within many Class II BSCs and laminar flow hoods, an important consideration for work with ESKAPE pathogens or carbapenem-resistant clinical isolates, where containment is non-negotiable.

WORKFLOW

How the workflow runs on Myra

Myra handles the steps most susceptible to operator variability. Media preparation and post-incubation readout remain off-deck, keeping the workflow practical without over-engineering it.

Step 1. Prepare media and controls

Prepare cation-adjusted Mueller-Hinton broth (CAMHB) or iron-depleted CAMHB for cefiderocol testing. Set up growth and sterility controls per CLSI M07 or EUCAST E.Dis.5.1 guidance.

Step 2. Serial dilution of antimicrobial compounds

Myra performs 2-fold serial dilutions across 96-well flat-bottom plates for standard MIC runs. For checkerboard assays, orthogonal dilution gradients are generated in a single automated run (one compound titrated across rows, the second across columns), producing the full concentration matrix without manual intermediate transfers.

Step 3. Inoculum dispensing

Bacterial inoculum is prepared to a target density of ~5 × 10⁵ CFU/mL (0.5 McFarland, 1:100 dilution) and dispensed consistently across all wells, which eliminates pipette-to-pipette variability that can shift apparent MIC endpoints by one or two dilution steps.

Step 4. Incubation

Plates are incubated at 35 ± 2°C for 18–20 hours under aerobic conditions, consistent with CLSI and EUCAST broth microdilution guidelines.

Step 5. Visual growth assessment

Growth inhibition is assessed by visual turbidity or spectrophotometric readout. Clear wells indicate inhibition, and the MIC is reported as the lowest concentration producing complete growth suppression. FICI values are calculated from the checkerboard matrix to characterize synergy (FICI ≤0.5), indifference, or antagonism.

VALIDATED SCRIPTS

Microbiology on Myra

Scripts are verified by BMS, by kit manufacturer partners, or by the Myra user community. All scripts include protocol documentation and direct links to the MyraScript page. Parameters, consumables, and deck layout are configurable through Workbench software.

Kit	Verified By

KEY CAPABILITIES

What Myra brings to automated antimicrobial susceptibility testing systems

Accurate liquid handling is essential for reliable antimicrobial susceptibility testing. Learn how a liquid handler can help standardize AST workflows while improving consistency and reproducibility.

Consistent 2-fold serial dilutions

Uniform transfer volumes at each dilution step reduce cumulative pipetting error that otherwise inflates MIC endpoint uncertainty.

Checkerboard matrix generation

Automated orthogonal dilution gradients make dual-drug interaction studies practical at throughputs that are not feasible manually.

CLSI and EUCAST compatible

Flexible plate layouts and dilution ranges accommodate both CLSI M07 broth microdilution and EUCAST E.Dis.5.1 methodologies.

BSC compatible footprint

Myra’s compact dimensions allow operation within many microbiological safety cabinets, maintaining containment integrity for work with high-risk isolates.

Rapidly reconfigured

Different organisms, antimicrobial compounds, dilution ranges, and plate layouts are accommodated through software reconfiguration — no hardware changes required.

Reduced inter-operator variability

Automation normalises pipetting performance across operators, making multi-user AST datasets meaningfully comparable rather than confounded by technique differences.

CASE STUDY

MIC and checkerboard assays for carbapenem-resistant Gram-negative pathogens

Center for Applied Biotechnology Studies, Department of Biological Sciences, California State University Fullerton, USA.

EXPERIMENTAL DESIGN

Clinically relevant isolates, challenging compounds

The study evaluated Myra’s performance for both standard MIC broth microdilution and checkerboard assay preparation using a panel of carbapenem-resistant Gram-negative clinical isolates, the organism category where reliable susceptibility data matters most and where manual throughput limitations are most acutely felt.

Organisms tested:
Acinetobacter baumannii (CRAB) | Klebsiella pneumoniae (CRKP) | Pseudomonas aeruginosa (CRPA)

Antimicrobial compounds evaluated:
Cefiderocol | Sulbactam | Meropenem | β-lactam / β-lactamase inhibitor combinations

MIC broth microdilution

2-fold serial dilutions prepared in 96-well flat-bottom plates with CAMHB (iron-depleted CAMHB for cefiderocol per CLSI guidance). Bacterial inoculum dispensed to a final density of ~5 × 10⁵ CFU/mL. Incubation at 35 ± 2°C for 18–20 h under aerobic conditions.

Checkerboard assay

Orthogonal dilution gradients of two compounds generated across the 96-well plate — one compound titrated across rows, the second across columns — producing a complete antimicrobial interaction matrix for FICI calculation in a single automated run.

RESULTS

Reproducible serial dilution patterns across replicate plates and operators

Myra generated consistent dilution series for both assay formats. Replicate MIC plates showed well defined growth inhibition patterns with clear endpoints, and checkerboard matrices produced interpretable FICI gradients across the full concentration range. Operator-to-operator variability (a persistent challenge in manual AST at scale) was substantially reduced compared to equivalent manual preparation.

Carbapenem-resistant species evaluated

Antimicrobial compounds including novel siderophore cephalosporin

96-well

Flat-bottom format, CLSI/EUCAST compatible plate layout

Orthogonal antimicrobial dilution gradients enable assessment of antimicrobial interactions across the concentration matrix.

The checkerboard setup — where even a single mispipetted transfer can render the interaction matrix uninterpretable — showed reproducible orthogonal gradients across replicate runs, validating Myra’s suitability for the precise multi-step transfers that dual-drug synergy studies demand.

“The main advantages of using Myra for these assays are improved consistency in liquid handling, reduced manual pipetting, better reproducibility of serial dilutions, and increased efficiency when setting up checkerboard plates, that can be time-consuming and prone to variability.”

María Soledad Ramírez, Ph.D. – Center of Applied Biotechnology Studies, Department of Biological Sciences, California State University Fullerton, USA

CASE STUDY PDF

MYRASCRIPT

Frequently Asked Questions

Can Myra automate an antimicrobial susceptibility test using CLSI M07 or EUCAST methodology?

Yes. Myra’s flexible scripting accommodates both CLSI M07 and EUCAST E.Dis.5.1 plate layouts, dilution ranges, and broth formulations. Iron-depleted CAMHB for cefiderocol testing and standard CAMHB for broader compound panels are both supported. You configure the chemistry; Myra handles the liquid handling.

How does Myra handle the orthogonal dilution gradients required for checkerboard assays?

Myra generates the full checkerboard interaction matrix in a single automated run. One compound is serially diluted across rows while the second is diluted across columns, producing the complete concentration grid needed for FICI calculation without requiring manual intermediate transfers between dilution series.

Can Myra be used inside a biological safety cabinet or laminar flow hood?

Myra’s compact footprint is designed to fit within many Class II BSCs and laminar flow hoods. This is particularly relevant for AST work involving ESKAPE pathogens, carbapenem-resistant Enterobacterales, or other isolates where containment is required throughout assay preparation, not just during inoculum handling.

How does automated preparation affect inter-operator and inter-run reproducibility?

Manual broth microdilution is subject to pipetting technique differences between operators. Variation in tip angle, speed, and timing can shift apparent MIC endpoints by a dilution step or more. Myra normalizes these variables, making multi-operator datasets meaningfully comparable, and reducing the need to run in-house calibration controls just to account for technician-to-technician noise.

Can the same system handle both MIC dilution series and checkerboard matrix preparation?

Yes. Both workflows run on the same Myra hardware, reconfigured through software. Labs running standard MIC panels can add checkerboard capability without additional equipment investment, and the same scripts can be adapted for different organisms or compound classes as your research focus changes.

Is Myra suitable for evaluating novel antimicrobials like siderophore cephalosporins that require modified broth conditions?

Yes. Myra is media-agnostic, meaning you prepare the correct broth formulation off-deck (e.g. iron-depleted CAMHB for cefiderocol) and Myra handles all subsequent liquid transfers. There is no constraint on the compound class or broth composition, provided the liquid is within standard viscosity parameters for automated pipetting.

Automated MIC and Checkerboard Assays on Myra