Automated bacteria count
Automated Bacteria Counts - Unique imaging solution
BioSense Solutions presents a new one-of-a-kind imaging solution to automate and count total bacteria in a pure liquid sample. To the right is image of an E. coli sample run in the µCount3D instrument. Cells are found in suspension and counted in 23 z-stacks. Bacteria found are presented in one layer using 3D segmentation.
The µCount3D can assist you, if you need a fast and precise total count of your bacteria concentration prior to experimenting or need to add a certain concentration.
Note that the µCount3D is not designed for counting bacteria in aggregates or to count filamentous bacteria.
How we image
The patented FluidScope technology is a tilted camera technology. When images are taken, we get to image a volume instead of a plane. Every image has a height of 150µm and since images overlap we get to create both a vertical and horizontal z-stack. All objects present in this volume, is captured in focus.
For bacteria counts we use volume imaging and count bacteria present in the entire imaged volume. Bacteria settle very slowly and are present throughout the imaged volume. Using our µCassetteB with a defined inner height of 800µm we find the bottom and elevate 300µm to image a free volume away from top and bottom. We capture all bacteria down to our pixel size of 0,5µm.
The µCassetteB is developed with triplicate sample chambers the Count3D software will provide total bacteria/ml and supply images for documentation.
µCount 3D Specifications
- Species: Bacteria, Fungi, Yeast and Algae
- Size: H: 20cm W: 10cm D: 20cm
- Weight: 3kg
- Power consumption: Standby 7W, Running 16,8W
- Microbe size: from 0,5µm
- Countable Range: 1 x 104 – 1 x 107
- 3 chamber time to result: ~8 minutes
- Output: Organisms/ml, images for documentation, PDF Report
- Sample containers: BioSense Solutions triplicate µCassetteF & µCassetteB
- Create your own specialized algorithms
Linearity response for 1 µm QC beads
Linearity response is a critical parameter in evaluating the performance of counting systems. It refers to the ability of a counting system to produce results that are directly proportional to the actual number over a specified range of concentrations.
A 2-fold dilution series with 1µm QC beads, (Bangs Laboratories Inc, Small Bead Calibration Kits, 833) have been made. Starting concentration ~2 x 106 have been diluted in sterile filtered (pore size 0.20µm) demineralized water. The samples have been vortexed and 3 x 80 µL pipetted into a µCount cassette. Triplicates of all 8 samples in the range concentration from 2 x 106 to 1.6 x 104, have been measured in µCount3D and analyzed with the µCount software.
The plot (Figure 1) shows a linearity response of R2 = 0.9992.
Linearity response for Bacillus spores
A 2-fold dilution series using a commercial nematicide product containing 3 different Bacillus spores (Bacillus subtilis, 2 x Bacillus paralicheniformis strains) has been made. Commercial product concentration ~ 1.67 x 1010. Product was diluted in sterile filtered 0.9% NaCl buffer (pore size 0.2µm) to a starting concentration of 1.2 x 107. Starting concentration was adjusted using the µCount3D instrument. 2-fold dilutions were vortexed and pipetted in triplicate (3 x 80µl) into triplicate chambered µCassetteB (BioSense Solution). Triplicate of 10 dilutions with concentrations from 1.2 x 107 to 2.3 x 104 was measured in the µCount3D and analyzed with the Count3D software. Algorithm used: Bacteria.
The plot (Figure 2) shows a linearity response of R2 = 0.9972
µCount3D Vs. Colony counts on agar
A validation test was set up to compare counts of E. coli using the µCount3D instrument and colony counts on agar. For precision colony counting, the IntuGrow fast CFU/ml assay was used (IntuBio, Farum, Denmark, www.intubio.dk). Protocol: E. coli (K12) was grown overnight in TSB. 10ml of 0.9% NaCl buffer was sterile filtered using a 0.2µm filter to remove particles in buffer. Overnight E. coli was diluted 100X in an Eppendorf tube to reach a desired starting concentration ~ 1 x 107. E. coli concentration was measured in triplicate using a µCassetteB, 65µl/chamber (BioSense Solutions, Farum, Denmark). µCount3D algorithm used: Bacteria. Experimental starting concentration was measured to be 6,97 x 106 and the sample centrifuge tube was labelled “1”. For IntuGrow plate count, tube “1” was further diluted to reach an expected colony count of 20-100 colonies. For plate counts,10µl of diluted sample “1” was applied in triplicate on mini agar disks (BioSense Solutions, Farum, Denmark) with TSA. Mini agar disks were placed in a 12-well microtiter plate and agar surface was imaged using an oCelloScope™. Emerging colonies were counted using the IntuGrow software (IntuBio, Farum, Denmark). From original tube “1”, A 2-fold dilution series containing 500µl 0.9% NaCl and 500µl sample was prepared. Rack with tubes was placed under cold conditions to reduce new divisions of E. coli. A total of 8 x 2-fold dilutions was made. Procedure from tube “1” was repeated to count and plate all samples in triplicate.
The plot (Figure 4) shows a linearity response of R2 = 0.991
Comments on results. The comparison test between the µCount3D and plate counts show a very good correlation. Experiment was carried out 4 times with equal correlation. In all experiments plate counts were slightly higher than what was counted on the µCount3D. We speculate that difference might be caused by pipetting. The samples used in the µCount3D instrument was used directly from the dilution sample tube. The samples used for plate counts was further diluted 100 – 1000 times and from this 10µl was inoculated on agar. A fair assumption would be that pipette inaccuracy could be cause of difference.
Figure 3: 2-fold dilutions of overnight E. coli culture culture in TSB. Blue line represents automated counts using µCount3D. Orange line represents colony counts on TSA using the automated IntuGrow assay.
Figure 4: Correlation of counting results from µCount3D and colony counts on TSA. Same data used as in figure 3.
Figure 5: Software view from the µCount3D showing the first triplicate count of E-coli used in experiment (tube 1).
Figure 6: Overview image of mini-agar disks with E-coli colonies from the IntuGrow software.
FAQ
- What is the volume used in µCassetteB? 65µl is pipetted into each chamber of the µCassetteB
- Can you ship all over the world? The µCount3D complies with international standards and is certified for use in Asia, Europe and the US and the Americas.
- Can I use a Macbook? No, the µCount3D is developed for PC only and minimum requirements are as follows:
Minimum Requirements
Processor: Intel i7 1800 Mhz
Hard disk: 512 GB, solid-state (an external SSD can be used)
Network card: 1 Gb/s
GPU: NVIDIA RTX series
Memory: 16 GB
OS: Windows 11
Tested on:
Dell Precision 3580
Processor: 13th Gen Intel(R) Core(TM) i7-1370P, 1900 Mhz, 14 Core(s), 20 Logical Processor(s)
Hard disk: 1 TB, solid-state
Network card: 1 Gb/s
Memory: 32GB
OS: Windows 11 pro
- Do you have re-usable µCassettes? No, currently we do not sell re-usable µCassettes.
- Can I count co-culture sample? The µCount3D is developed for pure cultures of bacteria and buffer should be sterile filtrated using a 0.2µm filter. However, we have seen examples of bacteria and yeast in Co-culture. Bacteria was counted in suspension and yeast on the bottom using the “Yeast algorithm”.
- Do I need to sterile filtrate my medium or buffer if I have MilliQ water? We recommend to sterile filtrate using a 0.2µm filter. We have seen that expected clear water contains particles. If particles are the size of bacteria, they will be counted as such. We typically test the water/buffer/medium using 1 chamber in a µCassette. Result should be “TFTC” (Too few to count).
- Can I use any medium? You have to use transparent buffer or medium.
- Is the µCount3D compatible with the oCelloScope platform? Yes, you can export counting jobs and images to UniExplorer.
- Can I use flourescent dyes in the µCount3D? The µCount3D is based on Bright Field Microscopy and will not excite or pick up flourescent signals.
Counting Bacteria
Counting the number of bacteria in a sample is a fundamental task in microbiology, providing valuable information about microbial populations and their abundance.
While manual counting methods such as hemocytometer or colony counting on agar plates are traditional approaches, automated methods have gained popularity due to their efficiency and accuracy.
Bacteria can be counted as total bacteria, total viable bacteria (TVC) and Colony Forming Units (CFU).
Automation technologies can be based on: Imaging, Laser light scattering and Electrical impedance.
- Imaging counters. A camera will image individual bacteria or colonies in a sample. Algorithms will count bacteria or colonies presents and based on dilution factor counts will be calculated. In addition, images are supplied to support counts. Example from the Count 3D software on the right, showing bacteria and concentration in a triplicate chamber cassettes.
- Laser light scattering. Bacteria are passed by a light beam one by one, and the scattering of light will reveal information about morphological features and sample will be counted. Laser light scattering is used in flow cytometry and bacteria can be counted and analyzed using dyes and stains.
- Electrical impedance. Bacteria are passed by two electrodes one by one and the difference in electrical current (impedance of objects) will reveal information about concentration of bacterial cells in a suspension.
All three technologies have advantages and disadvantages, and it is up to the user to decide what is the best solution for their lab. Most common of the three technologies for automating bacterial counts is probably the use of electrical impedance. This is due to the higher price and complexity using a flow cytometer. Flow cytometers are typically found in core facilities of universities and in the industry.
Bacteria is been a challenge to count using imaging technology due to their small size and the fact that not all will settle on a surface. BioSense Solutions have solved this challenge by imaging bacteria in a volume instead of a plane. All bacteria present in a defined volume will be counted.
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