The references for the QUANTOM Tx™ Microbial Cell Counter and Centrifuge User Manuals are listed in the references.
1. Preparation of total microbial cell counting
1.1. Bacteria are cultured depending on the species as below for this protocol.
Listeria monocytogens in Brain and Heart Fusion (BHI) agar or broth, E. coli in nutrient agar or broth, and Campylobacter jejuni in Muller Hinton agar or broth are cultured for approximately 18 - 20 hours at 37 ºC.
The strains of Brucella abortus, B. melitensis and B. suis are cultured on a serum dextrose agar (SDA) plate at 37°C with 10% CO2 for 72 hours, followed by subculture on an SDA slant with incubation at 37 °C with 10% CO2 for 72 hours.
1.2. Prepare microbial cell suspensions. in or sterile physiological saline. The cells from Listeria, E. coli and Campylobacter are resuspened in sterile phosphate buffered saline (PBS, pH7.4, 0.01 mM) unless specified later, and the cells from the Brucella species from SDA slant are resuspended in 5 mL of sterile physiological saline. The bacteria are kept alive, or heat-killed at 80 °C for 20 – 30 minutes. The sterility is confirmed by culture. The initial approximate bacterial concentrations are determined by using either McFarland turbidity standards or optical density using a spectrometer. An optimal range between approximately 106 to 108 cells or CFU per milliliter (mL) is obtained by further dilutions in relevant buffers.
Note: The QUANTOM Tx™ can count samples with concentrations in the range of 1 x 105 to 1 x 109 cells/mL. The optimal range is 2 x 106 to 5 x 108 cells/mL (FAQs in the reference).
Note: Heat-killed L. monocytogenes in BHI enrichment broth, was compared with those in PBS in our studies, and there were no significant difference observed.
Note: Any calculated concentration with the order of magnitude 105 or lower were unreliable. Our study revealed that bacterial concentrations between 101 to 105 CFU/mL showed similar counting results of 105 CFU/mL.
1.2. Mix 10 µL cell sample with 1 µL QUANTOM Total Cell Staining Dye and 1 µL QUANTOM Total Cell Staining Enhancer. Mix thoroughly with gentle force.
1.3. Incubate at 37°C for 30 minutes in the dark. Mix thoroughly with gentle force.
1.4. Add 8 µL QUANTOM Cell Loading Buffer I to the mixture. Mix gently to avoid creating bubbles.
1.5. Load 5.5 µL (per side) into a QUANTOM M50 Cell Counting slide.
Note: based on our study, it is possible to reuse the slides after soaking the slides in disinfectant, thoroughly washing with distilled water, and air drying.
2. Preparation of viable microbial cell counting
2.1. Prepare a viable microbial cell suspension with an optimal range between 2x106 to 5x108 CFU/ml by diluting cell suspensions as necessary with QUANTOM Viable Cell Dilution Buffer (see note 1).
NOTE 1: Stain cells after dilution or resuspension with QUANTOM Viable Cell Dilution Buffer. PBS or water will decrease labeling efficiency. Culture media or sera may have esterase activity and lead to decreased viable cell staining and high background fluorescence (User Manual). In our study, viable L. monocytogenes in Brain and Heart Fusion (BHI) enrichment broth was compared with those in PBS, there were no significant difference
2.2. Mix 2 μL QUANTOM Viable Cell Staining Dye with 10 μL cell sample.
Note: Alternative dyes, such as fluorescent calcein AM, for viable cell staining can also be used (FAQs in the reference). Our study tested calcein AM cell-permeant dye from Thermo-Fisher Scientific (Cat# C3099) with similar results as those from Logos.
2.3. Incubate at 37°C for 30 minutes – 4 hours (depending on the bacterial species and strains) in the dark. Mix again (User’s Manual; and Time Taken section).
2.4. Add 8 μL QUANTOM Cell Loading Buffer I. Mix gently so as not to create bubbles.
2.5. Load 5.5 μL (per side) into a QUANTOM M50 Cell Counting Slide or chamber.
The operation details are referred to QUANTOM TxTM Centrifuge User’s Manual. The centrifugation is to immobilize and evenly distribute the cells throughout the counting chamber to ensure accurate cell counts.
3.1. Load slides vertically in the numbered slots of the QUANTOMTM Centrifuge with the sample facing the rotor. Balance rotor symmetrically with another slide in the opposite slot.
3.2. Centrifuge at 300 RCF for 10 minutes. Centrifugation force, time and other recommended parameters may need to be optimized according to cell size to distribute cells evenly along one focal plane (Centrifuge User’s manual).
The operation details are referred to QUANTOM TxTM User’s Manual. The QUANTOM Tx™ Microbial cell Counter captures up to 10 high resolution images and counts the cells in each. The software in the system can distinguish individual cells in various arrangements such as tight clusters or in sequence to produce accurate and reliable total bacterial cell counts. The creation and editing of protocols and settings for parameters such as faster focus and declustering level which leads to a higher sensitivity to clusters are referred to in the user’s manual.
4.1. After the machine has been turned on and the initialization has completed, insert the slide face up and sample side first into the slide port.
4.2. Adjust light level as needed (normally 5 for total, and 9 for viable cell). Note that this can affect counting results.
4.5. Adjust the focus manually using the focus bar and arrows, or press Autofocus. The autofocus further reduces variability due to human error.
4.6. Select the areas using arrows under the COUNT button based on the user’s instruction.
4.7. Press COUNT. Counting times can vary, about 30 seconds for a sample with a concentration of ~1 x 107
cell/mL and 10 images. The results will appear on the screen automatically and the slide will be ejected from the instrument.
Note: Because the counting is based on the random selection of ten spots, in order to obtain more accurate counting results, the same slide can be read more than once, and the average can be calculated.
4.8. View a graphical representation of the data in the Histogram and Gating tab.
4.9. Calculate subsequent dilutions using the dilution calculator under Dilution.
4.10. Save the data by quick save or by naming the file and manually selecting the save location.
5. Review data and the captured images with corresponding data. Transfer the data to computer using a USB drive or the Wi-Fi data transfer.