The Coulter Principle, also called the electrical sensing zone (ESZ) method, can be used to determine the percentage of aggregates in bacteria cultures. This method is described in the ISO International Standard 13319 guidelines. In order to perform the test, an appropriate electrolyte solution is needed.
In most applications, phosphate buffered saline (PBS) solution can be used as the electrolyte. Sample preparation and analysis is then performed using Beckman Coulter’s Multisizer™ 3 to establish the size distribution of the cells. The results obtained provide the number percent of aggregates.
Multisizer™ 3 vs. Hemocytometer
The Multisizer™ 3 Coulter Counter allows fast, easy and precise determination of cell aggregation, and also provides consistent results independent of the operator’s judgment when compared to those obtained with a hemocytometer.
Evaluating Efficacy of Contact Lens Disinfection
Safety and effectiveness are standard concerns in any consumer products. The same measures apply to contact lens care disinfecting products as well. To this end, a number of procedures are employed to assess the efficacy of lens care disinfection solutions. In these procedures, a panel of pathogenic challenge organism suspensions is often used.
Microscopic analysis of such organism suspensions has revealed the presence of cell clumps. These cell aggregates inhibit consistent exposure to the test disinfectant, which result in wrong estimation of the actual number of challenge organisms present.
Therefore, in order to create a uniform cell suspension and reduce the number of cell clumps, filtration of pathogenic challenge organism suspensions can be performed before using as test inoculate. This technique also makes it possible to utilize suspension mediums comprising 0.05% Tween 80 as a dispersant.
Here, the Multisizer™ 3 can be used to perform cell suspension analysis before and after filtration to assess the effectiveness of filtration. This efficacy is evaluated by determining the number and size distribution of cell clumps in the bacterial cell suspension. The efficacy of the Tween 80 can also be verified.
Instrument Set Up and Calibration
In this analysis, 100μm and 30μm aperture tubes were used. A 100μm aperture tube can be used to study the particle size distribution and concentration from 2μm to 60μm, while a 30μm aperture from 0.6μm to 18μm.
Together, both apertures give an analysis range from 0.6μm to 60μm. Next, the instrument should be set and calibrated in accordance with the Multisizer™ 3 Operator’s Manual. In order to determine the number percent size distribution, the instrument’s control mode can serve as Time Mode, choosing 60 seconds as the run time.
Beckman Coulter has developed a balanced electrolytic solution called Isoton® II, which has roughly 1% of total salt concentration. This solution can be utilized to suspend the cells for testing on the Multisizer™ 3 Coulter Counter. In case a different saline solution is employed, the following possibilities will occur:
- The customized saline solution has 1% total salt concentration: In this condition, the electrolyte may either be the customized saline solution or the Isoton® II electrolytic solution.
- The total salt concentration of the customized saline solution is different from 1%: In this situation, the electrolyte will be the customized saline solution.
In both instances, the cells can be studied by using the customized saline solution for the sample transferred in a beaker.
A small and round beaker can be used to examine the cells through a 100µm aperture. Sample analysis is performed as follows:
- The beaker containing the sample is placed in the analyzer
- A multisizer stirrer is used to ensure that the sample is evenly distributed
- The aperture tube is flushed prior to each analysis
- Following each run, the electrode and aperture is washed before moving to the subsequent sample
- After sample analysis, each sample is filtered using a 10µm filter
- Finally, the filtrates are finally run using a 30µm aperture tube
The results obtained for each sample through the 100µm and 30µm apertures can be reported as a single size distribution covering the collective range for both types of apertures.
Reporting the Results
By utilizing the Interpolation function in the Multisizer™ 3 Software, results can be presented as the number percentage of cells above the preferred size categories in the size distribution range (Figures 1 and 2).
Figure 1. Results using a 30µm aperture.
Figure 2. Combined results from the 30µm and 100µm aperture.
The percentage of agglomerates can be determined by differentiating which one has the larger size for distribution of normal cell population. In the following case, it was assumed that above 3µm, the cells exist in agglomerates (Figure 3). Based on this assumption, the cell aggregation is deemed to be 1.37% of the total population.
Figure 3. Results using a 100µm aperture.
When the percentage of agglomerates in a sample is compared, the filtration efficiency and the possible effectiveness of the antimicrobial agent can be easily controlled. In addition, both the filtration process and the effectiveness of detergents can be improved to prevent cell clumps. In this analysis, the Multisizer™ 3 from Beckman Coulter was effectively used to quantify the size distribution of cells and the results thus obtained are presented as number percent of aggregates.
About Beckman Coulter
Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.
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