Combining 2 Powerful Technologies to Enable
Further Discovery in Bacterial Studies
Study Flow
Direct Absolute Count (NovoCyte)
Number of Bacteria (Titer)
Infection of Cells of Interest with Certain Amount of
Bacteria
Monitoring of Cell Index Changes of Target Cells to Assess
Bacteria Mediated Cytotoxic Effects (xCelligence)
2
Bacterial Count with NovoCyte
Significance of Bacterial Count
• Food manufacturers
– Required by regulatory authorities e.g. FDA to monitor the number and type of
bacteria in their products
– Beer and wine companies monitor the growth of yeast in their distilling process
• Environmental concerns
– Water treatment plants monitor the effectiveness of their sterilization process
• Biotechnology firms
– Closely regulate bacterial growth to produce useful pharmaceutical products
• Clinical laboratories
– Monitor the growth rate of bacteria from patients to determine their antimicrobial
sensitivity
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How to Count Bacteria with Precision
• By dilution and plating
– Dead bacteria do not form colonies. Some bacteria occur as single cells while
other species hang together in chains or clumps of 2 or more baceteria
• Counting chambers
– Consist of a special microscope slide with a coverglass
– Can't tell which bacteria are alive thus this method is useless in disinfection
studies
• Membrane filters
– Huge volumes of liquid (e.g. water) can be filtered to show a few bacteria per liter
– Filter can be rinsed with sterile water to remove anything that could potentially
interfere with bacterial growth
• Photometers and spectrometers
– Efficient, no need to wait overnight for the colonies on the agar plates
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Direct Absolute Counting using NovoCyte
• Syringe Pump Fluidics
o Direct absolute cell/particle counts
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Direct Absolute Counting: NovoCyte vs Others
Competitor's Flow Cytometer with Reference
Beads
ACEA Novocyte (Volumetric)
Total
lymphocyte
CD3+
/ul
Sample 1
1937
1435
QC Blood Sample 2
1570
1160
404
Sample 3
1605
1175
408
Sample 4
846
634
Sample 5
886
605
297
Sample 6
1710
888
288
Fresh
Blood
CD3+CD8+ CD3+CD4+
Total
/ul
/ul
lymphocyte
CD3+
/ul
CD3+CD8+ CD3+CD4+
/ul
/ul
2057
1492
676
1563
1146
406
669
685
1558
1153
405
676
902
659
279
925
619
296
286
578
1779
913
294
597
Absolute Counting: NovoCyte vs Others
(Cont’d)
2300.00
2100.00
1900.00
1700.00
1500.00
1300.00
1100.00
900.00
700.00
500.00
500.00
Competitor’s Flow Cytometer
Competitor’s Flow Cytometer
1700.00
y = 1.0179x + 12.705
R² = 0.9843
1000.00
1500.00
2000.00
1500.00
1300.00
1100.00
900.00
700.00
500.00
500.00
2500.00
y = 1.0051x + 9.1898
R² = 0.9925
700.00
ACEA NovoCyte（Total lymphocyte）
900.00
1100.00
1300.00
1500.00
ACEA NovoCyte（CD3+）
430.00
410.00
390.00
370.00
350.00
330.00
310.00
290.00
270.00
250.00
250.00
Competitor’s Flow Cytometer
Competitor’s Flow Cytometer
800.00
y = 0.9709x + 11.179
R² = 0.9977
300.00
350.00
400.00
ACEA NovoCyte（CD3+CD8+）
450.00
y = 0.9662x + 21.106
R² = 0.9962
700.00
600.00
500.00
400.00
300.00
200.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
ACEA NovoCyte（CD3+CD4+）
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Direct Absolute Counting of E. Coli with NovoCyte
1:1000 Dilution
Experiment Settings:
FSC-H Threshold: 2000
Volume: 30uL
Sample Flow Rate: 14uL/min
1:10000 Dilution
Sample
Count in Gate P1
Abs. Count (/uL)
1:1000
1:10000
135,727
15,529
4,524
518
Advantages of Direct Absolute Bacterial Counting
with NovoCyte
o Low CV’s (±2%)
o High accuracy (±5%)
o Provides consistent results between sample runs
o Automatic cleaning (low carry over of <0.1%)
o “Plug-and-play” operation
o Efficient, up to 20,000 events/sec
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Bacteria Mediated Cytotoxicity (xCelligence)
Bacteria mediated toxicity
• Direct damage
– Results from the means of a bacteria utilizes to adhere to host, grow and
evade host defences
– Usually the more minor form of bacteria mediated toxicity
• Hypersensitivity reactions
– An immune response that is excessive to a point where it leads to
damage (as with endotoxins) or is potentially damaging to the individual
host
• Toxin-induced damage
– About 220 bacterial toxins are known of which 40% disrupt plasma
membranes
– Exotoxins, endotoxins
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Bacterial Assays
• Bacterial agglutination
– Commonly used to identify specific bacterial antigens, and in turn, the
identity of such bacteria
– Important technique in diagnosis
• Bacterial count assays (please refer to the previous section)
– Absolute direct count using our NovoCyte
• Conventional asssys for bacterial-mediated cytotoxicity (e.g. manual cell
count with Trypan blue, MTT, release of LDH, ATP assay, microscopic
analysis)
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Bacteria species validated on xCelligence system
• Clostridium difficile
• Bacillus
• Vibrio cholera
• Vibrio vulnificus
• Neisseria meningitidis
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Study One
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Responses of four cell lines to Vibrio cholerae toxin
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Analytical sensitivity of CT diluted with pooled
negative stool specimens
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Representative CT-RTCA results for isolates and
clinical specimens
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Study Two
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RtxA1 causes acute necrotic cell death
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Summary – Key Benefits of xCelligence
• A simple alternative to traditional methods for measuring bacteria mediated
cytotoxicity
• Sensitive readout on cell mophology and adhesion changes in response to
bacterial infection
• Quantitative monitoring of onset and kinetics of bacterial mediated effects in
real time for up to hundreds hours
• Indentify the optimal bacterial titer and assay time point for subsequent
screening of inhibitory compounds, neutralizing antibodies or neutralizing
serums.
• No labelling of cells or bacteria required
• No post-experiment cell handling, sample preparation, or data collection
(infect and walk away!)
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Publication list
1. Real-time cellular analysis coupled with a specimen enrichment accurately detects and quantifies Clostridium difficile
toxins in stool. Huang, B., Jin, D., Zhang, J., Sun, J. Y., Wang, X., Stiles, J., Xu, X., et al. (2014).Journal of clinical microbiology,
1(January). doi:10.1128/JCM.02601-13
2. In vitro assessment of marine bacillus for use as livestock probiotics. Prieto, M. L., O’Sullivan, L., Tan, S. P., McLoughlin, P.,
Hughes, H., Gutierrez, M., Lane, J. a, et al. (2014).Marine drugs, 12(5), 2422–45. doi:10.3390/md12052422
3. Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cell Cytotoxicity Monitoring. Jin, D., Luo, Y.,
Zheng, M., Li, H., Zhang, J., Stampfl, M., Xu, X., et al. (2013).Journal of clinical microbiology. doi:10.1128/JCM.01959-13
4. A bacterial RTX toxin causes programmed necrotic cell death through calcium-mediated mitochondrial dysfunction. Kim,
Y. R., Lee, S. E., Kang, I.-C., Nam, K. Il, Choy, H. E., & Rhee, J. H. (2013).The Journal of infectious diseases, 207(9), 1406–15.
doi:10.1093/infdis/jis746
5. Real-time impedance analysis of host cell response to meningococcal infection. Slanina, H., König, a, Claus, H., Frosch, M.,
& Schubert-Unkmeir, a. (2011).Journal of microbiological methods, 84(1), 101–8. doi:10.1016/j.mimet.2010.11.004
6. Assessment of Clostridium difficile infections by quantitative detection of tcdB toxin by use of a real-time cell analysis
system. Ryder, A. B., Huang, Y., Li, H., Zheng, M., Wang, X., Stratton, C. W., Xu, X., et al. (2010).Journal of clinical microbiology,
48(11), 4129–34. doi:10.1128/JCM.01104-10
7. Neisseria meningitidis induces brain microvascular endothelial cell detachment from the matrix and cleavage of
occludin: a role for MMP-8. Schubert-Unkmeir, A., Konrad, C., Slanina, H., Czapek, F., Hebling, S., & Frosch, M. (2010).PLoS
pathogens, 6(4), e1000874. doi:10.1371/journal.ppat.1000874
8. An ultrasensitive rapid immunocytotoxicity assay for detecting Clostridium difficile toxins. He, X., Wang, J., Steele, J.,
Sun, X., Nie, W., Tzipori, S., & Feng, H. (2009).Journal of microbiological methods, 78(1), 97–100. doi:10.1016/j.mimet.2009.04.007
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Appendix: Facts About Bacteria
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Bacteria - Basic Facts
• Prokaryotic microorganisms typically a few microns in length
• 40 million bacterial cells in a gram of soil and a million in a millilitre of fresh
water
• Have a number of shapes, ranging from spheres (cocci) to rods (bacilli or
vibrio for slightly curved rods or comma-shaped) and spirals (spirilla or
spirocchaetes)
• Many exist as single cells, others associate in characteristic patterns
– Neisseria form diploids (pairs)
– Staphylococcus group together in “bunch of grapes” clusters
– Actinobacteria can be elongated to form filaments and are often
surrounded by a sheath that contains many individual cells. E.g. Nocardia
form complex branched filaments similar in appearance to some fungal
mycelia
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Bacteria – Cellular Structures
• Extracellular
– Cell wall present on the outside of the cytoplasmic membrane
– Consists of peptidoglycan
– Essential to survival and the antibiotic penicillin kills the bacteria by
inhibiting the synthesis of peptidoglycan
– Gram-positive (thick cell wall) vs Gram-negative (thin cell wall)
• Intracellular
– Usually no membrane-bound organelles (e.g lack of true nucleus,
mitochondria, chloroplasts)
– A single circular chromosome located in the cytoplasm in an irregularly
shaped body called the nucleoid
– Micro-compartments (e.g. carboxysomes, magnetosomes)
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Bacteria - Biofilms & Quorum Sensing
• Often attach to surfaces and form biofilms.
• Bacteria living in biofilms form secondary structures such as micocolonies to
enable better diffusion of nutrients.
• Common in natural environments (e.g. soil, surfaces of plants) and during
chronic bacterial infections or infections of implanted medical devices
• Bacteria protected within biofilms are much harder to kill than individual
isolated baceteria
• In more harsh conditions (e.g. starved of amino acids), they would detect
surrounding cells and migrate toward each other (quorum sensing), and
aggregate to form fruiting bodies where they cooperate to perform separate
tasks
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