Isn’t it the best when lab sustainability synergetically improves both science and environmental stewardship? Reviewing your method to estimate bacteria cell culture numbers is a great opportunity to reduce single-use lab plastic waste. It can also reduce your workload and support the accuracy of your experiments.
Conventional c.f.u./ml
Even though each research lab may have its own adaptation, microbiology protocols for ascertaining the viability of a bacterial culture are based on a century-old technique - updated with the convenience of modern plastic consumables.
Quantifying colony-forming units, or cfu/ml, is often used for monitoring bacterial culture viability and growth rates - a reproducibility factor in many microbiology applications. In one sense the conventional method is considered tried and true. Yet, it’s important not to take your c.f.u. protocol for granted since they are also somewhat notorious for variability.
It’s a task that requires plenty of hands-on time in most research labs. Samples are serially diluted 1:10 in triplicate, and then 50 to 100ul volumes of each dilution are spread onto individual agar plates. The unknown number of viable cells in the original sample can be calculated based on the number of colonies on the plate visible to the naked eye after incubating overnight. This is to say that conventional c.f.u. assays involve a lot of handing of single-use tubes, tips, and Petri dishes that end up in a biohazard bin.
Happily, there are alternative methods that save time and materials!
Less time, less plastic, less agar, and less biohazardous waste
While the Drop Plate method and the Track method have been around for a long time their greening potential was qualified recently within Joana Alves et al. A case report: insights into reducing plastic waste in a microbiology laboratory (2020) Access MIcrobiology. Research Fellow and Laboratory Manager Amy Pickering, Ph.D., and her colleagues at the University of Edinburgh, Roslin Institute performed an audit on plastic consumables use over many weeks and pursued multiple sustainability actions based on those results.
The Drop Plate method outcome was especially striking. One Petri plate took the place of thirty-six! It eliminates microcentrifuge tubes by performing serial dilutions in a 96 well plate or block with a multi-channel pipette. Instead of one 100ul sample dilution per plate, 5ul samples of bacterial culture dilutions are spotted on agar plates. The segmentation of your plate and arrangement of the spots should augment your visualization. Importantly the spots should be allowed to dry before moving the plate into an incubator.
Pikering shared that the biggest hurdle was ensuring that changing the method didn't alter experiment outcomes - that the method was just as accurate and sensitive. Trial experiments were done using the traditional and newer methods for comparison. “Once we were convinced that we hadn't lost accuracy, as seen by those that pioneered the track and drop techniques, it was really easy to make the transition” explained Pickering.
Once we were convinced that we hadn't lost accuracy, as seen by those that pioneered the track and drop techniques, it was really easy to make the transition.
- Amy Pickering, Ph.D., The Roslin Institute.
Testing the efficiency of the Drop Plate method for different microorganisms is best practice. With any microorganism dropping a ~5ul of suspension directly onto the agar eliminates an accuracy concern related to cell spreaders. Skipping the spreader step removes the risk of cell death from exposure to an ethanol flame sterilized spreader or loss due to adherence to any spreader type.
While you are maximizing each agar plate, it is also worth considering counting mini colonies at a size of 200–500 lm, using one of the fairly accessible plate counting apps and a cell phone. Counting early colonies, rather than colonies easily visible to the naked eye can save hours in incubation. It’s a neat trick employed by sophisticated robotics used in pharma and food labs. Rapid assessment of viable counts can be a real game-changer in lab resource efficiency.
Additional c.f.u. methods with enhanced accuracy and sustainability:
Jett et al. Simplified Agar Plate Method for Quantifying Viable Bacteria (1997) Biotechniques
Wang et al. Spot Plating Assay for the Determination of Survival and Plating efficiency of Escherichia coli in sub-MIC Levels of Antibiotics (2017) JEMI Methods
Sieuwerts et al. A simple and fast method for determining colony forming units (2008) Letters in Applied Microbiology
Dimitria T. Boukouvalas et al. Automatic segmentation method for CFU counting in single plate-serial dilution (2019) Chemometrics and Intelligent Laboratory Systems
P. Thomas et al. Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples (2015) Biotechnol. Reports
P. Thomas, A.C.Sekhar, M.M. Mujawar, Nonrecovery of varying proportions of viable bacteria during spread plating governed by the extent of spreader usage and proposal for an alternate spotting-spreading approach to maximize the CFU (2012) Jrnl. Applied Microbiology
