How Oxygen Sensing Technology Can Deliver Faster Test Results for Food and Beverage Microbiology

Article by Colin Fricker1, Dilidaer Yusufu2 and Andrew Mills2

Oculer Ltd, Ballina, Ireland1 and School of Chemistry and Chemical Engineering, Queens University, Belfast, UK2

Why use O2 sensors?
O2 sensors are widely used in many areas of biology and they have been applied for monitoring the activity and growth of bacteria in the environment. There are a small number of reports of their use for the specific detection of microbial growth in the laboratory. A combination of appropriate culture media, temperature and an immobilised O2 depletion sensor can consistently and accurately detect the growth of microorganisms including bacteria, yeast and fungi. As the organisms grow, they deplete the level of O2 in the culture medium and, at a certain point in time, this depletion impacts the sensor resulting in a very rapid increase in the lifetime of the dye, which can be detected by an appropriate instrument.

Using an appropriate O2 sensor together with a suitable immobilising polymer results in a detection system which has a long shelf life, and is heat resistant, not impacted by pH or ionic strength of the medium. It is important to note that because the polymer that is used to immobilise the sensor is impermeable to most molecules other than gases, the sensor is not impacted by different food matrices or ionic species. The lack of impact of pH means that media can be buffered to a pH >7.0 to allow faster growth and recovery of certain organisms, something which is detrimental to the detection of carbon dioxide as, under such pH conditions, CO2 is initially converted to bicarbonate, resulting in longer detection times.

The theory behind O2 sensors
Almost all commercial O2 optical indicators are based on the ability of O2 to quench (i.e. suppress) the luminescence exhibited by a dye, D. The range of dyes for this purpose are limited and most contain platinum or ruthenium. D will luminesce when it is exposed to light it absorbs; the excitation light. The wavelengths, i.e. colours, of the excitation and resulting luminescence light are usually very different, which makes monitoring the latter easy. If the excitation light is delivered as a constant stream to the indicator, then it will luminesce continuously.

However, if, as in O2 sensing, it is delivered in the form of a very short pulse, i.e. a flash, the luminescence intensity will instantly peak and then decay to zero. In practice, the time it takes for this decay to end is difficult to determine and so, instead, the time is measured for the peak luminescence intensity to decrease by a defined fraction. In photochemistry in general and O2 sensor technology specifically, this fraction is always the reciprocal of the natural number, e, (2.71828) and so the fraction is 0.368. The time is referred to as the luminescence lifetime, Τ, of the dye.

For all luminescent dyes, the value for Τ in the absence of O2, i.e., Τo, is characteristic of the dye, and so is different for every dye. For O2-sensitive dyes, Τo is typically 40-100 ms and Τ decreases with the increasing partial pressure of O2, PO2, according to the Stern-Volmer equation,

Τo/Τ = 1 + KSVPO2

where KSV is the Stern-Volmer constant, which depends both on Τo and how quickly the O2 moves to the dye and quenches its luminescence.

In most commercial O2 optical indicators, the luminescent dye is embedded in an O2-permeable plastic, to form a coloured ‘dot’ – often green due to the usual Pt porphyrin dye used in this work. The sensor dot is usually deposited on the wall of a transparent bottle or sample tube, that contains the medium, aqueous or gaseous, under test. The lifetime of the dye in the dot is then monitored using a sensor probe pressed against the sample tube, opposite the O2 ‘dot’, as illustrated in Figure 1. The sensor probe provides the excitation light and collects the luminscence light and measures its lifetime.

When applied in microbiology to determine the log (cfu) in a test sample, a growth medium is placed in a vial and inoculated with the sample under investigation. As the bacteria grow in the medium, the level of dissolved O2 will decrease from its initial value associated with an air-saturated solution, i.e. 0.21 atm, to zero and so the measured value of Τ will increase from its value in air saturated solution to Τo. A typical set of Τ vs incubation time profiles for a range of different cfu inoculation values is illustrated in Figure 1. These profiles can be used to create a simple, straight line calibration graph relating log (cfu) to the time taken to reach a set value of Τ, i.e. a specific level of O2. This calibration graph can then be used to determine the value of the log (cfu) in a test sample for the bacteria.

Figure 1

Simplicity of testing
The use of systems which allow the direct detection of changes in culture media due to microbial growth allow for extremely simple detection in a far shorter period of time. Thus, typically, the sensor is immobilised in a culture vessel and the culture media added. The sample under test is then added directly to the vessel and incubated in a combined incubator/reader for the duration of the test. The sensor is interrogated at frequent intervals and when significant O2 depletion is detected, a change in reading is obtained, alerting the user that the sample contains microorganisms of interest. Because there is no need to wait for colonies to form, as required in the traditional methods used to determine microbial load, the results are available quickly and the higher the microbial load, the faster a positive signal is obtained.

The Oculer Rapid 930 Series
The Oculer Rapid 930 Series, marketed by Oculer Ltd is a self-contained benchtop instrument incorporating an incubator and luminescence detector, the use of which requires no specific microbiological expertise. Sample vials, containing appropriate culture media are supplied after gamma irradiation. There are two versions of the instrument suitable for incubation of 48 x 2 ml vials or 24 x 15 ml vials. If samples are incubated offline, the total number of samples that can be run increases to 432 and 216 respectively. When used in quantitative mode the Oculer instrument calculates the concentration of target bacteria and the results are expressed as colony forming units.

Widely used in the dairy industry for the detection of yeast and mould in finished products, particularly yoghurt, the Oculer Rapid 930 Series Y&M assay can deliver automated results within 48 hours without the need for specific microbiological expertise.

The Oculer Series 930 system uses sensitive oxygen sensors to detect the growth of yeast and moulds in a proprietary selective medium specially designed to suppress growth of other microorganisms.

Features and Benefits include:

Fast - yeast and mould results within 48 hours
Sensitive - can use large volume size up to 10mls
Fully Automated - collect real-time data without the need for highly skilled staff
High throughput - run up to 212 samples at once
Reduced risk - only release product after test results are available
Flexible - can be used for both qualitative (presence/absence) and quantitative (numerical) testing

To get more information or book a live system demonstration click here

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