6 techniques for rapid sterility testing

21 September 2017

In the pharmaceutical and diagnostic industries, there is an ever growing demand for the rapid detection of bacterial and fungal cells. Faster detection means corrective actions or treatment can happen sooner.

Traditional sterility testing requires visual detection of contaminants incubated in culture media for up to 14 days. This delay incurs storage and diagnostic costs and could delay appropriate treatment for patients. The subjectivity of the visual examination of media can also result in incorrect interpretation. A demand exists for a faster, more reliable system.

Several rapid methods for detection have been developed, based on:

1. Impedance: the measurement of ionic change from metabolic by-products.

Impedance uses a pair of electrodes immersed in the growth medium. During the growth phase, bacterial metabolism transforms uncharged or weakly charged compounds in the medium into highly charged compounds that change the electrical properties of the medium. Some automated systems use this technique.

2. CO2 detection: using biosensors to measure CO2 production from respiring cells.

3. Flow cytometry: analysing the physical and chemical characteristics of particles or cells in a fluid as it passes through a laser.

Fluorescent dyes are used to detect viable cells, which differentially stain live and dead cells. The laser excites these dyes, causing them to emit light at varying wavelengths for detection. Dyes such as Propidium Iodide (PI), DAPI, DRAQ7, 7-AAD and TO-PRO-3 will only enter cells with compromised membranes. Once they do, they will bind to DNA and fluoresce so that these cells can easily be detected and eliminated as non-viable.

4. Endotoxin: uses Limulus amebocyte lysate (LAL).

These assays mimic the biology of the Horseshoe crab. This animal experiences coagulation in its haemolymph when exposed to bacterial endotoxins, protecting it from bacterial invasion. The main component of the haemolymph, LAL, is used to detect tiny quantities of the endotoxin Lipopolysaccharide (LPS) present in the membranes of gram-negative bacteria.

5. ATP Bioluminescence: measurements of adenosine triphosphate (ATP).

ATP is the principal energy carrier in all living organisms, including microorganisms. The luciferase enzyme uses ATP in the cell to oxidise luciferin, resulting in the emission of light. The microbial biomass is directly proportional to the amount of ATP in the sample, and hence the amount of light produced.

6. Nucleic acid-based technology: uses polymerase chain reaction (PCR).

These techniques can use primers designed for DNA regions in specific organisms, or they can target conserved regions of DNA common to a genus or all bacteria.

Traditional rapid detection systems require a threshold number of bacteria to function. Low-level detection needs a long incubation period, enabling bacteria proliferation to reach a detectable level. It can still take several days to detect contamination.

At Microgenetics, we aimed to develop a test that was not only rapid but able to detect a single bacterial cell within a few hours. SwiftDetect can isolate low-level contamination from a large volume of liquid. It uses qPCR to detect bacterial and fungal cells, setting it apart from other nucleic acid detection methods. As a PCR-based technology, as well as providing reliable evidence of contamination, SwiftDetect is also able to detect specific bacterial and fungal genera and species.