Autonomous High-Throughput Colony Separator

Autonomous High-Throughput Cell Colony Separator

Within the past decade, advances in culturing methods have addressed the challenges of the “Great Plate Count Anomaly” a phenomenon where only approximately 1% of colony forming units (CFU) are successfully cultivated using conventional growth media in situ. The entire microbial population is is left undiscovered along with its trove of potential secondary metabolites, small molecules that could serve as useful drugs as therapeutics in roles of cancer treatment, immunosuppresives, and antibiotics.

Diffusion membrane systems have seen successes where up to 60% of the sample microbial population is cultivated in situ (Ferrari, 2005) and (Epstein, 2010). This has opened the door to a resurrection in a particular method of drug discovery using the Waksman platform with biotech firms such as Novobiotic discovering 25 compounds from 50,000 isolates.

The process of working with colonies barely visible to the naked eye for downstream analysis in the form of genomic sequencing, domestication (cultivating in vitro), and screening however, is a tedious and costly process.

The research problem

Once a small sample (less than 1mm in diameter) is cultivated in situ, they are typically sent for 1 downstream analysis process. This can be genomic sequencing (a destructive process), domestication (culture in vitro, which if only using 1 media can have low success), or screening.

To optimize the efforts of culturing microbes in situ in the first place, separating a culture with minimal stress to the microbes into equal parts would enable experiments in parallel as well as a greater chance of domestication and pure isolated culture for any future analysis.

The proposal

To address this, automating the process of separating colonies that have been cultivated in situ from diffusion chambers such as Ichip (Epstein, 2010) would open the door for numerous studies and experiments that could be performed on never before cultivated microbes (also known as the uncultivables).

Thus it is proposed that a machine provides the following functions

Extracts and separates colonies from a diffusion chamber at a rate of at least 10,000 samples a day while preserving extracellular structures such as biofilms that enable intracellular communication to increase the chance of successful cultivation in vitro enabling further analysis.

Further analysis includes the following:

Genomic sequencing

Optimization of growth media (petri dishes are hostile environments to many microbes since it resembles nothing like their natural environments. Identifying the key growth factors such that microbes could be grown in situ in pure culture would enable researchers to perform numerous studies).

Drug discovery. The golden age of antibiotic discovery ended in the 1960s when the 1% of bacteria that could be cultivated in vitro was exhausted. Now, since the number of bacteria that can be domesticated and cultured in vitro has increased by a magnitude, platforms such as the Waksman platform, can be used to screen for bacteria producing secondary metabolites with interesting bioactivity. For example, the antibiotic properties of Teixobactin (Lewis, Epstein, 2015), anticancer properties of Azurins, and immunosuppressives (to reduce organ transplant rejection, septic shock, or autoimmune diseases).


Ichip, or Isolation Chip, developed in the Epstein Lab at Northeastern University. Bottom is an Ichip produced with a small CNC router.

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Isolation chips with chambers 0.13″ or 3.2mm in diameter. The larger diameter enables for easier removable of agar plugs (which the bacteria is housed in) and quicker machining with the resources currently available.


Early validation of existing prior art (Ichips). Cultivation of soil samples in a diffusion chamber in situ.

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Examples of cultures extracted from diffusion chambers at 40x magnification.

The Current State of the Art

The current state of the art can be broken down into two categories.

1: Separation methods that destroy extracellular structures.

2: Separation methods that preserve extracellular structures.

*Extracellular structures being the polysaccharides that make up biofilms for example. (Donlan, 2002)

State of the art separation methods that destroy extracellular structures for example are resuspension in a solution and then plating an aliquot. The drawbacks of this method is that communication between cells is cut off and cells must then rely on diffusion.

State of the art separation methods that preserve extracellular structures for example, are laser capture microdissection. The drawbacks of this method is that preparing and separating a sample can take 1-1.5 hours and require a skilled technician. In addition, the machines start at $70,000.

The machine subsystems

Extraction of samples from a diffusion slide

Imaging to identify colonies as well as to generate a tool path

Separation of colonies into n number of equal parts, where n is an integer set by the user (most likely between 2 and 8).

Separation of colonies into containment chambers

Storage of colonies into containment chambers that can be readily plated or experiments performed (such as a microplate)