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A ten-year search for synchronous cells: obstacles, solutions, and practical applications.

Helmstetter CE - Front Microbiol (2015)

Bottom Line: My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end.In this essay, I recount my personal journey in this obsessive experimental pursuit.That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida Institute of Technology Melbourne, FL, USA.

ABSTRACT
My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end. In this essay, I recount my personal journey in this obsessive experimental pursuit. That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells. Subsequent studies with this methodology led to an understanding of the basic properties of the relationship between chromosome replication and cell division. Accordingly, I end this reminiscence with a simple, fool-proof graphical strategy for deducing the pattern of chromosome replication during the division cycle of cells growing at any rate.

No MeSH data available.


Related in: MedlinePlus

Buchner funnel version of a cellular baby machine. (A) Funnel set-up for binding cells to a membrane filter surface. (B) Orientation and appearance of the funnel during elution of newborn cells from the membrane filter surface.
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Figure 4: Buchner funnel version of a cellular baby machine. (A) Funnel set-up for binding cells to a membrane filter surface. (B) Orientation and appearance of the funnel during elution of newborn cells from the membrane filter surface.

Mentions: Figure 4 shows the basic set-up and procedure using a Buchner funnel with a 150-mm diameter perforated base plate (Scientific Equipment of Houston). To prepare the funnel as shown in Figure 4A, the membrane filter must be sealed to the bottom plate of the funnel. This is most easily accomplished by first running a narrow bead of biologically safe silicone sealant (such as Factor II A-4100 or aquarium-safe silicone) around the bottom of the funnel just inside the 142-mm circumference of a membrane filter. Next, place the mesh screen loosely inside the bead. I recommend using a 120-mm diameter screen cut from a Buchner polyethylene disc (Avogadro's Lab Supply, Inc.). Lastly, place the membrane filter on top of the bead and press down on the edge. I have used the top portion of 130-mm two-piece polypropylene Buchner funnel for this purpose (Avogadro's Lab Supply, Inc.), but any ring of that approximate diameter should work well. After curing, this procedure yields a perfect seal, produces a surface of about 120 mm in diameter for attachment of the cells, and enables culture medium to flow uniformly across the membrane after inversion of the apparatus. The seal is easily removed with a razor blade for the next experiment.


A ten-year search for synchronous cells: obstacles, solutions, and practical applications.

Helmstetter CE - Front Microbiol (2015)

Buchner funnel version of a cellular baby machine. (A) Funnel set-up for binding cells to a membrane filter surface. (B) Orientation and appearance of the funnel during elution of newborn cells from the membrane filter surface.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4373376&req=5

Figure 4: Buchner funnel version of a cellular baby machine. (A) Funnel set-up for binding cells to a membrane filter surface. (B) Orientation and appearance of the funnel during elution of newborn cells from the membrane filter surface.
Mentions: Figure 4 shows the basic set-up and procedure using a Buchner funnel with a 150-mm diameter perforated base plate (Scientific Equipment of Houston). To prepare the funnel as shown in Figure 4A, the membrane filter must be sealed to the bottom plate of the funnel. This is most easily accomplished by first running a narrow bead of biologically safe silicone sealant (such as Factor II A-4100 or aquarium-safe silicone) around the bottom of the funnel just inside the 142-mm circumference of a membrane filter. Next, place the mesh screen loosely inside the bead. I recommend using a 120-mm diameter screen cut from a Buchner polyethylene disc (Avogadro's Lab Supply, Inc.). Lastly, place the membrane filter on top of the bead and press down on the edge. I have used the top portion of 130-mm two-piece polypropylene Buchner funnel for this purpose (Avogadro's Lab Supply, Inc.), but any ring of that approximate diameter should work well. After curing, this procedure yields a perfect seal, produces a surface of about 120 mm in diameter for attachment of the cells, and enables culture medium to flow uniformly across the membrane after inversion of the apparatus. The seal is easily removed with a razor blade for the next experiment.

Bottom Line: My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end.In this essay, I recount my personal journey in this obsessive experimental pursuit.That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida Institute of Technology Melbourne, FL, USA.

ABSTRACT
My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end. In this essay, I recount my personal journey in this obsessive experimental pursuit. That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells. Subsequent studies with this methodology led to an understanding of the basic properties of the relationship between chromosome replication and cell division. Accordingly, I end this reminiscence with a simple, fool-proof graphical strategy for deducing the pattern of chromosome replication during the division cycle of cells growing at any rate.

No MeSH data available.


Related in: MedlinePlus