Pellicle formation in a hay infusion
Cultures growing on the surface of liquid media often form a more or less continuous film called a pellicle. This film consists of micrbial cells and their exracellular products. Pellicles, therefore represent liquid/air interface biofilms. Complex pellicles can be formed on the surface of an aqueous culture in which organic matter is decomposing. Infusions made with hay in water quickly generate pellicles which are easy to harvest and study. One of the most spectacular pellicles we have observed was formed on the surface of a finger bowl of water being used to produce onion root tips for a mitosis lab.
One onion, suspended in the water, failed to produce roots or stalks. As it decomposed, a rich, not too smelly, pellicle formed on the surface of the water. This film contained numerous gram negative and gram positive rods, the latter including a large number of endospore forming cells.
By suspending a clean microscope slide below the surface of the liquid in the hay infusion culture, (see microfishing exercise) one can also sample the biofilm formed in a less aerobic portion of the same microbial environment.
In a beaker have students cut short lengths of hay (about 2 inches long) sufficient to fill about one fourth of the beaker.
- Fill the beaker with water. Cover the beaker to retard evaporation.
The hay segments will absorb water and sink to the bottom of the beaker. The nutrient leaching from the hay will support the growth of a rich population of bacteria including many gram positive spore-formers. Many of bacteria will grow at the air water interface forming a thick pellicle.
- Portions of this pellicle can be harvested by inserting a clean glass slide into the beaker through the pellicle and then carefully withdrawing the slide so that a portion of the film adhers to the slide.
- This biofilm can be observed directly under a cover slip with brightfield or phase contrast microscopy or it may be air dried and stained.
- Gram stains, spore stains and capsule stains are all instructive.