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Antimicrobial screening tests provide information on antimicrobial efficacy rapidly and cost-effectively.

Screening methods vary widely in principle and practice, with each having different strengths and weaknesses.

This page provides information on a variety of antimicrobial screening methods, separated into the following three categories:

  • Conventional screening methods
  • Technology-driven assays
  • Scaled-down versions of standard methods

Note that there is an important difference between inhibiting microbial growth and killing a microbes in a short period of time.  Many of the screening techniques listed below measure inhibition of microbial growth, not kill rate. Inhibition of growth tends to be easier to measure, but it is not always a reliable proxy for kill rate. Consider alcohol, bleach, and the antibiotic ciprofloxacin as examples:  Alcohol and bleach are both biocides, but in an inhibition-focused screening tests, they will rank second and third place to the antibiotic ciprofloxacin, which exerts extraordinarily strong inhibitory effects but relatively little killing effect over short timeframes. Thus, screening for inhibition would “miss” these important biocides. If microbial inhibition is the goal, then inhibition-based methods should be used. If microbial kill is the goal then screening methods with biocidal efficacy endpoints are superior.

Conventional Screening Methods

Zone of Inhibition or Disk-Diffusion Test (ZOI), also known as the Kirby-Bauer Test

This technique utilizes paper disks soaked with antimicrobial agent, which are placed on agar plates that have been seeded to grow a “lawn” of bacteria or fungi. This method is commonly used to assess resistance of bacteria and fungi to antibiotics. The larger the zone of growth inhibition, the greater potency the antibiotic is known to have.

Zone of inhibition testing can be used to screen antimicrobial products for efficacy.  It is fast, relatively inexpensive, and provides a dramatic visual endpoint in the form of  a clear circle around the antimicrobial-containing disk. The diameter of a zone of inhibition can be measured, usually in millimeters, to produce quantitative data for comparison with other antimicrobial agents.

Variability in the method is generally low, but the dryness of the agar, the loading rate of the disk, and the water-solubility of the antimicrobial agent can all contribute to variance in the size of the zone of inhibition.

Minimum Inhibitory Concentration (MIC)

Arguably the most used antimicrobial screening technique of all time is the Minimum Inhibitory Concentration test, or MIC.  The method utilizes microtiter plates, which are plastic dishes with either 96 wells, 384 wells, or 1536 wells.  Most microbiology labs work with 96 well microtiter plates, organizing the products in each of 8 rows across 12 columns using 8 or 12-channel micropipettes.

For MIC testing, the product is serially diluted in inoculated wells, usually from by column from left to right, with a couple terminal columns saved for positive and negative controls. After dilution, a growth broth seeded with the target microorganism (or set of microorganisms) is added. Mueller-Hinton Broth is the most common broth used for MICs. The dilution series creates a gradient of antimicrobial product concentrations and each well is inoculated with the same population of microbes for the test. After incubation, turbidity, which is cloudiness of the broth, a normal sign of microbial growth, is seen at more dilute wells. Clear broth, indicating prevention of growth, is observed in the more concentrated wells.  The lowest concentration at which growth is prevented is called the Minimum Inhibitory Concentration, or MIC for that particular microorganism and antimicrobial agent.

Minimum Bactericidal Concentration (MBC)
The Minimum Bactericidal Concentration Test, or MBC, is used to determine the concentration at which an antimicrobial agent exerts a biocidal effect on bacteria. The test is conducted in the same fashion as the MIC test described above, but after a period of time at the researcher’s discretion, wells are diluted through neutralizing media and plated to agar to quantify surviving cells. If the contact time is long enough for non-inhibited wells to demonstrate cloudiness due growth of bacteria, then only those wells above the MIC need to be plated to agar. Conventionally a 99.9% reduction in bacterial concentration is considered biocidal. Thus, the MBC is the greatest dilution at which a 99.9% reduction in bacterial levels is observed.

Checkerboard Screening
So-called checkerboard screening assays are useful tools for evaluating the effects of combinations of various antimicrobial agents. They are used to evaluate both synergistic and antagonistic effects. They are typically conducted as part of MIC assays, but instead of antimicrobial concentration gradients running left to right in rows or top to bottom in columns, they are run with different antimicrobial agents in both directions (such as with 8 different agents loaded by row and diluted, then one agent loaded in all columns and diluted). This enables the researcher to observe unexpected, pronounced antimicrobial effects at very low concentrations as well as antagonistic effects at higher concentrations.

Agar Dilution
Agar Dilution Assays are useful for screening one or a few antimicrobial agents across a range of bacterial species. For these assays, agars are prepared containing various concentrations of antimicrobial agent, then different bacteria are struck to agar and incubated to assess growth. This technique provides a fast and easy way to screen dozens or even hundreds of different bacteria for inhibition by a given antimicrobial at one or a few concentrations.

Liquid-Based Time-Kill Tests
Liquid time-kill assays are relatively easy to conduct in the laboratory and yield high-quality, quantitative data on the biocidal effects of antimicrobial agents. For these tests, the antimicrobial agent is loaded into a small tube or vial, usually as 1 ml or 10 ml, to which a relatively small volume of microbial inoculum is added, usually 0.01 ml or 0.1 ml. The vial is then mixed, a contact time is allowed to elapse, then a portion of the vial is transferred to a neutralizing medium and enumerated by plating to agar. Standardized versions of such tests are available, such as ASTM E2315.

Surface-Based Time-Kill Tests
Surface time-kill assays are similar to liquid time-kill assays, but the reaction of the biocide and microorganisms takes place on the top of non-porous surface, upon which a thin layer of microorganisms has been dried for a brief period of time, usually between 20 and 40 minutes. These tests generally provide a greater challenge to antimicrobial agents, since the agents must penetrate the microbial film in addition to killing the microorganisms. The advantage of this sort of screening test is its natural correlation to the tests required by EPA and FDA for product registration or pre-market approval.

Technology-driven assays

E-Tests utilize a strip of material embedded with a gradient of antimicrobial agent, usually antibiotics. The test is conducted in the same fashion as a Zone of Inhibition test, but instead of a circular zone, an ellipsoid zone is seen. The point at which the zone intersects the antibiotic-containing strip is a useful measurement of potency.

ATP Swabs
Adenosine triphosphate (ATP) is sometimes referred to as the “currency of energy” in living things, because all cells, whether from humans, animals, plants, or individual bacterial cells, rely on ATP to power cellular processes. So ATP is found in all living things except viruses, and has therefore found some use in antimicrobial screening. ATP assays are usually coupled with swabs, and are conducted by swabbing a surface that either has or has not been treated with an antimicrobial agent, then comparing the amount of ATP detected. ATP tests use the reaction of the organism’s ATP with a light-producing molecule in the kit to produce a light signal in the test. This signal is detected by a sensor, which converts the amount of light detected to a unit called a Relative Light Unit (RLU). RLU varies by ATP Test device manufacturer, so these screens are relative in nature and best conducted using a single ATP test platform. ATP tests are considered a “rough” screen for antimicrobial activity because microbes contain relatively little ATP compared to animal and food cells, and they are subject to interference from other sources of ATP in the environment (such as grape juice on a conveyor belt in a food manufacturing environment). It should also be kept in mind that destruction of ATP is not necessarily indicative of the killing of a microorganism because ATP screening tests are subject to confounders, such as biocides inactivating the enzyme responsible for the signal, which can appear in the test as destruction of microbes. A main advantage of ATP testing for screening is it’s fast, inexpensive, and requires little training. It is critical for people using this technique to periodically confirm results using more traditional microbiological methods to confirm the validity of the results.

Bioluminescence Assays
Microorganisms can be genetically engineered to express bioluminescence proteins during growth and metabolism. Sensitive cameras can be used to detect the faint light that is produced during growth, indicating a lack of inhibition by antimicrobial agents. These assays are difficult to engineer and interpret, but have the advantage of a physical detection endpoint and easy observation of effect over time.

Stratix Labs ReadyNow Test Surfaces
Antimicrobial testing is inherently variable, and many of the microbes commonly used for surface-based antimicrobial screening begin to die as soon as they are inoculated onto the surface. This represents a point of variability and inconvenience in testing.

A company called Stratix Labs has developed a range of patented products intended to address this problem by providing researchers with pre-inoculated, microbiologically stable test surfaces for testing and screening. Their line of products incorporating stable microbial populations is called “ReadyNow.” These products can also eliminate some of the complexity involved with in-house screening, especially for smaller or less experienced labs.  ReadyNow pre-inoculated test surfaces can be utilized and enumerated in-house by companies with some microbiology expertise, or utilized at client request by labs like Microchem as a way to drive consistency in results, especially across different labs or testing sites. More information about Stratix Labs and its products is available here.

Scaled-Down or Modified Standard Antimicrobial Test Methods

Most antimicrobial product developers are familiar with standardized methods used to measure the efficacy of germicidal products, such as the ASTM E1153 sanitizer test method, the AOAC Use-Dilution Method for disinfectants, or the ASTM E1052 method for testing virucidal efficacy. These methods are often run in scaled-down form as “screens,” or small tests to gauge the efficacy of larger future studies, usually studies for submission to EPA or FDA.

Scaled-Down Versions of Standard Methods

The ultimate goal of much antimicrobial screening is to identify products that can pass the standardized tests used by EPA, FDA, and other regulatory agencies for product registration and pre-market approval. As a general rule, the closer in test design a method is to the standardized method required by regulators, the more accurate the screening data, but the more labor-intensive the study. Thus, scaled down versions of many standard methods are often used by antimicrobial product formulators to gauge the likelihood of passing the ultimate series of tests, which are usually conducted under GLP compliant test conditions.

The list below provides examples of common ways to scale down such methods

  • AOAC Use-Dilution Test – 10 or 30 carriers (test surfaces) are used in place of the typical 60 required per lot, per microorganism.
  • AOAC Germicidal Spray Products Test – 10 or 30 carriers (test surfaces) are used in place of the typical 60 required per lot, per microorganism.
  • Disinfectant Towelettes Test – 10 or 30 carriers (test surfaces) are used in place of the typical 60 required per lot, per microorganism.
  • Non-Food Contact Sanitizer Test – 1 or 3 carriers (test surfaces) are used in place of the five specified by the ASTM E1153 method.
  • Food Contact Sanitizer Test – There are two methods used for these sorts of claims, neither of which is readily scaled down. Therefore, product formulators typically run a non-GLP version of the test that will be conducted for registration.

Screening Using “Worst Case” Test Conditions

Another way to reduce the labor involved in antimicrobial testing while still generating valuable information is to conduct standard test methods in a so-called “worst case” fashion. Examples of changes that can be made to methods to render them “worst case” are listed below:

  • Use of the most resistant microorganism among the panel required for testing – The most resistant microorganism for a given germicide will often vary by active ingredient. For common disinfectants, Staphylococcus aureus and Pseudomonas aeruginosa are often more difficult to pass than Salmonella enterica. Therefore, screening can be done on one of those microorganisms to provide useful efficacy data for all.
  • Reducing the contact time – The extent of disinfection is a product of both the concentration of the antimicrobial agent and its contact time, so screening a product that is intended to have a contact time of 5 minutes at 3 minutes provides useful information, and a hedge against failures in testing for regulatory agencies brought about by the greater number of replicates and batches tested in the ultimate test series.

As you can see, there are dozens of antimicrobial screening techniques available, each with different strengths and weaknesses. If your company is interested in antimicrobial product screening, please contact Microchem for more information, and free consultation on the method that will provide the most value to your project.

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