Isolation of Bdellovibrio bacteriovorus

I would love to start telling about it with this piece of writing I found online, by Laurel Crosby.

“A mild ocean breeze plays over the water surface, dispelling any notion that danger lurks in the murky depths. However, a gruesome event is about to occur as a silent attacker speeds forth toward an unsuspecting victim. In a furious collision, the savage meets its target and whittles its way into the body of the innocent prey. Once inside, the transformation begins - the predator ceases its frenzy and prepares to multiply. The host is reduced to a protective cocoon, supplying food and shelter for the growing parasite. Within hours, the nourishment is drained and the ghost-like shell of the host bursts open to release a new generation of deadly predators. And all the while, the waters remain still...”
This savage predator is none other than the bacteria Bdellovibrio bacteriovorus. With a length of about 1.4 microns, a comma shaped body and a corkscrew-like flagellum rotating at deadly speeds, the bdellovibrio is just one of its kind. It is the only bacteria that preys on other bacteria (bdellovibrio is gram-negative and it attacks only other gram-negative bacteria). Bdellovibrio spends most of its life between the cell membranes of its prey. It is generally found in sewage and soil. The life cycle of the bdellovibrio deserves to be elaborated upon.


The life cycle of a single bdellovibrio.

In the first step, it attacks the host cell. There is a short cognitive phase after which the bdellovibrio permanently attaches itself to the host. It then drills through the outer membrane of the host using its corkscrew flagellum. Once inside, it loses its flagellum. The hole it had drilled is now sealed. Many molecules inside the host are broken down or dissolved. The host cell assumes a roundish structure, called the bdelloplast. The bdellovibrio starts eating the host inside out, growing in size in the process. It then begins to reproduce, just like any other bacteria, via cell-division. Once the nutrients of the host get exhausted, the bdelloplast bursts open and all the new bdellovibrio produced is shot out in all directions, on their way to find new hosts. The phase of life spent inside the host cell is often referred to as the ‘growth phase’ and distinguished from the ‘attack phase’.

WHAT WE DID

Tirtashree, Pallavi, Sravani and I: We collected water samples from the sewage-rich Mula river (Pune). We mixed a bit of the water in a thick suspension of E.coli and spread it on a nutrient agar plate prepared beforehand (mix nutrient broth, agar agar,autoclave it, melt and fill the plates with requisite amount, allow it to solidify). We then kept our plates in the incubator for a day. We would come back and check the next day. If there bdellovibrio in our sample, we would be able to see plaques wherever the bdellovibrio attacked and killed E.coli.

RESULT
We observed plaques on our plates, characteristic of bdellovibrio. These were the clearings on our thick carpet of E.coli that we spread on the plates, wherever the bdellovibrio killed the E.coli. Unless there was a phage in our plates, we can say (with optimism) that we had managed to find some bdellovibrio!
Our attempts to isolate bdellovibrio by scraping out the parts around the clearings on the plate and culturing them again in other plates, along with a thick carpet of E.coli went in vain. We did not observe any plaques. We concluded that something must have gone wrong in our procedure; we might not have taken care of some crucial factors while making the plates, perhaps. Unfortunately, we could not identify our mistake. Nevertheless, we are still starting the isolation procedure afresh. There is nothing to write about it at the moment, but hopefully there will soon be.

FURTHER STUDY
We plan to extend our experiment once we succeed with the isolation process. We know it is a tough going because bdellovibrio are not easy to find. However, the fact that bdellovibrio is a species we do not know and understand much about excites me. There is always more scope for discovery. There’s one thing, for example: we know that bdellovibrio reproduces only when it is inside the host cell. However, in some experiments, bdellovibrio has reported growth even in controlled laboratory conditions in environments similar to those in host cells. What is it that the bdellovibrio actually needs in order to grow and reproduce? These are some of the mysteries that continue to elude scientists.