Revolution or Mere Evolution? Flow Cytometry and Verification of Cleaning and Disinfection in Food Manufacturing Facilities

Food safety depends in large part on the hygienic conditions in food manufacturing facilities. High levels of spoilage bacteria can affect the shelf life and quality of foodstuffs, while the presence of pathogens (such as Salmonella and Listeria) can lead to serious illness. Food manufacturers must be diligent in keeping their processing environment clean and free of pathogenic microorganisms to prevent the cross-contamination of the final product. But how is this currently being done?

Visual inspection
While visual inspection is a prerequisite, it is in and of itself not sufficient. It is a subjective and imprecise means of verifying proper cleaning. More importantly, even if a surface has no apparent residue, this does not mean it is immaculate. Visual inspection cannot ensure that all food residues from the previous run have been cleaned away or that a sanitizer has effectively reduced the microbial level on the surface.

Microbial enumeration with culture-based methods
These are the traditional methods for monitoring the hygiene of the processing environment. Generally, there are two ways to perform sampling: contact-based and swabbing-based. With contact-based methods, plates or dip-slides are placed on the surface to be sampled and then incubated. Swabbing-based sampling is carried out with swabsticks or sponges that are rinsed in a buffer solution which is then inoculated into sterile media and incubated. The main limitation of these traditional methods of microbial determination is the amount of time it takes to obtain results. Furthermore, most species of bacteria cannot be cultivated on agar, a phenomenon known as the great plate count anomaly.

ATP detection
Adenosine triphosphate (ATP) is a nucleotide that cells use to deliver energy. It can be thought of as the molecular “unit of currency" for energy within all living cells. Energy is transferred when ATP breaks down into its nucleoside and free phosphate. Hydrolyzing the covalent links of the phosphates liberates energy that is used for reactions. Commercial ATP test systems harness the luciferin/luciferase reaction, which is very common in nature, to generate light with the energy provided by ATP. The more light is emitted, the more ATP is present, which could indirectly indicate more food residues or (potentially) more microorganisms. Yet there is one important caveat: as these systems rely on an enzymatic reaction, potential inhibitors or less than optimal environmental conditions could elicit faulty results. Environmental temperature could increase reaction times, whereas light could make it difficult to obtain correct readings. Furthermore, disinfectants can interfere with the reaction, meaning that there may not be a real correlation between living bacteria present on the surface and the results of the ATP measurement. Hence, ATP-based methods are normally used to test surfaces before the application of the disinfectant.
ATP methods harbor a further disadvantage: they depend in their applicability on the nature of the food being processed. Most foods leave behind residue containing large amounts of ATP, which surpass by several orders of magnitude the amounts contained within bacterial cells. Practically, this means that ATP systems cannot be used to assess the microbial contamination of surfaces in most food processing facilities. Although no bacteria can be directly counted, ATP systems are widely employed because results are generated within seconds, a time-to-result available in no other commonly available technology until now. 
 

So, why can’t we have it all?
The holy grail of cleaning and disinfection verification
What food manufacturers need is a quick method that directly quantifies both bacteria and residue particles and is not influenced by disinfectants and temperature. Although this seems out of reach, the basic technology for making this happen already exists and is being applied today. 

Introducing Flow Cytometry
Flow cytometry (FCM) refers to a group of techniques that use optical or electrical signals to detect and measure certain physical or chemical properties of cells and particles suspended in a fluid. Nearly 300 studies conducted between 2000 and 2018 assessed FCM as a tool to characterize microbial water quality. This research was able to illustrate the value of FCM in water treatment, distribution and reuse. There is now a body of research documenting successful applications of FCM robust enough to suggest that it could reasonably and realistically see widespread adoption as a routine method for water quality assessment. 
What does all of this have to do with the assessment of cleaning and disinfection efficiency in food manufacturing facilities? Methods previously common in water quality determination were often limited by low sensitivity, high labor and time requirements, susceptibility to interference from inhibitory compounds, and difficulties in distinguishing between viable and non-viable cells. (These all sound familiar, don’t they?) But beware: fluorescence flow cytometers are generally unwieldy, expensive devices that require highly trained staff to operate. 

Getting the power of flow cytometry into a handheld device
To make FCM a viable solution for cleaning verification in food processing facilities, it needs to come in a portable format that is simple and easy to use, yet accurate enough to provide reliable counts of bacteria and residue particles in environmental samples. This has been made possible by the use of impedance flow cytometry. Impedance flow cytometry is a specific kind of flow cytometry: instead of optical systems such as laser technology, impedance flow cytometers employ an alternating current at varying frequencies which enable the device to detect and count cells and residue particles separately. While optical-based flow cytometers are only able to count cells labeled with dyes, impedance flow cytometers can perform the same operation without any need for labeling. Compared to other flow cytometric devices, they are compact, portable and battery-powered, enabling them to be used where the sample is taken. 

How can impedance flow cytometers distinguish between cells and residue particles?
The electro-magnetic properties of bacteria enable flow cytometers to distinguish them from other particles. The cytoplasm and the cell membrane of a bacterium change the electrical field in unique and identifiable ways. While the electrical current will move through metallic particles mostly unimpeded, non-conductive particles resist the field. Intact bacteria, however, resemble both non-conductive and conductive particles: the cell membrane prevents low frequencies from penetrating it, causing it to resemble non-conductive particles; at high frequencies, however, the electrical current penetrates the membrane. The microelectrodes in the impedance flow cytometer generate these electrical fields and enable the device to quantify the changes in conductivity and resistance in terms of separate measurements of intact cells and particles. 

Application of impedance flow cytometry to food safety: introducing CytoQuant®
As mentioned above, one advantage that impedance flow cytometers hold over other kinds of cytometric devices is their portability. Light, small, and battery-powered, they can operate in the field and at critical control points where hygiene is an overriding concern.
The CytoQuant® impedance flow cytometer is designed for use in just such areas, including food production facilities and clean rooms. Impedance flow cytometry brings considerable advantages to food producers looking to verify their food safety and cleaning programs: the fast and separate quantification of bacteria and residue particles (which can serve as an indicator for the cleaning efficacy), the sensitivity of the method, and the robustness of the swabbing kit and the cytometer itself. 
The CytoQuant® system is easy to use, as the device handles all the work except swabbing. A test run begins by swabbing a predefined area (e.g., 20 x 20 cm or 8 x 8 in) of the surface to be tested. The swab is placed in a tube containing a proprietary, conductive solution. After shaking the swab kit to suspend the bacteria, the user inserts it into the CytoQuant®. Two needles penetrate the bottom of the tube, connecting the liquid to the flow system in the device. Then, after the solution is introduced to the flow system, it is passed by the electrodes in the flow cell. After 30 seconds, the device registers separate results for bacteria and particles and displays them on the screen.

Revolution or Evolution?
The CytoQuant® mobile flow cytometer enables the immediate, on-site verification of cleaning and dis-infection procedures in food production facilities or other areas where hygiene is crucial. By directly quantifying bacteria and residue particles on surfaces without the negative influence of disinfectants or temperature, it provides substantial advantages over ATP devices, while the 30 second time-to-result makes it a perfect enhancement to hygiene programs that already use cultural methods. Considering the huge potential of impedance flow cytometry, it may at some point come to be regarded as equal to or even replace cultural methods as the standard in cleaning verification. This would amount to a true revolution in the field.  
 

This article was published in Spot On #15

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