Lesson 2 - Yeast Morphology and Evaluation

Mitochondria

Mitochondria are membrane-bound organelles which vary in shape, size and number depending on growth conditions. They have internal structures called cristae. Mitochondria are the powerhouses of the cell and when yeast is growing under fully aerobic conditions with a low concentration of sugar they contain the pathways responsible for generating energy in the form of adenosine triphosphate (ATP). The pathway is the electron transport chain and the process is called oxidative phosphorylation. A lot to take in a first sight but do not worry this will be described properly in the lesson dealing with yeast biochemistry. Mitochondria contain their own DNA and other pathways important in brewing are also located here; for example, the pathway responsible for the synthesis of isoleucine and valine (ilv pathway) which is indirectly the source of diacetyl. Under brewery or distillery fermentation conditions mitochondria do not develop fully and are called pro-mitochondria. These are still important in brewery and distillery fermentations but are not used to produce energy.

Cell wall

Like plants yeast cells are surrounded by a rigid thick wall made up of mainly carbohydrates with various proteins embedded within it. It protects the cell against physical damage and gives it its shape. It is relatively porous to all but very large molecules. It has a lot of other functions. It contains the components responsible for flocculation, the process where most brewing yeast strains clump together towards the end of fermentation forming large “flocs”. The cell wall contains the visible signs of budding, the process used by brewing yeast to reproduce. Where the new bud separates from the mother cell a circular mark is left on each, the birth scar in the case of the bud and simply bud scar in the case of the mother. You can visualise the scars with dyes such as calcofluor and so if you count the number of scars present on a cell it indicates how old it is.

Plasmamembrane (cell membrane)

This structure forms the true barrier between the inside of the cell and the external medium. It acts as a doorway and controls what enters and leaves the cell. Many tests which measure whether or not a cell is living or dead, such as the methylene blue viability stain, actually check whether or not the membrane is intact (live) or not (dead). The plasmamembrane is a double layered structure made up of roughly equal quantities of fatty molecules (lipids, mainly phospholipids and sterols) and proteins. Most of these proteins have specific functions such as controlling transport of nutrients and products into and out of cells, the enzymes involved in the synthesis of the wall and sensing changes in the external environment. The space between the plasmamembrane and the wall is called the periplasm. This has a gel-like structure which probably protects the delicate plasmamembrane. The periplasm also contains some enzymes of importance such as invertase which converts sucrose into glucose and fructose.

Vacuole

This usually the most visible organelle. It is separated from the cytoplasm by a membrane called the tonoplast. Vacuole size, number and shape varies greatly with the growth conditions. This is easy to understand because vacuoles are principally recycling organelles. They breakdown large molecules such as redundant proteins into their component amino acids where they can be stored until needed to make new proteins. Vacuoles often contain other storage in the form of bodies; for example, polyphosphate, often called volutin granules.

Nucleus

This is where most of the cell’s DNA, or genome, is located. In brewing yeast this takes the form of 16 chromosomes. Although normally not visible the chromosomes are duplicated when the cells multiply and a new copy passes into the new one. The nucleus is bound by a membrane and this contains pores which allow communication with the rest of the cell. Within the nucleus is a body called the nucleolus. This is responsible for the synthesis of ribosomes, bodies which are involved in reading the genetic code determined by the sequence of DNA found in the chromosomes.

Ribosomes

Ribosomes are the organelles responsible for translating the cell’s genetic code. They are found in very large numbers located in free form in the cytoplasm, attached to the rough endoplasmic reticulum and also within mitochondria. They consist of two subunits both made up of RNA and proteins. They are made by the nucleolus. Messenger RNA (mRNA) is found in the ribosomes and is made when the DNA code in the chromosomes is transcribed and translated. In response to reading the code via mRNA, transfer RNA (tRNA) delivers selected amino acids to the ribosomes where they are used to construct proteins.

Endoplasmic reticulum (ER)

This is a system of hollow membranes which extend throughout the cytoplasm from the nucleus to the plasmamembrane. Two types are seen; smooth ER mainly takes the form of tubes and rough ER, usually in the form of flattened sacs to which on the outer surface ribosomes are attached, which explains the name. The ER is the system of highways used by the cells to transport proteins, made by the ribosomes, to the location within the cells where they are required, or to the membrane for transport out of the cell. The smooth ER is also involved in the synthesis of a whole range of other compounds such as carbohydrates and lipids.

Golgi body

This organelle consists of a large number of flattened discs each bound by a membrane. Its role is to label and move proteins around the cell. The proteins are made in the endoplasmic reticulum and sent to the Golgi body as a packet surrounded by a membrane to form a vesicle. The protein enters the Golgi body where its structure is modified, a process called post-translational modification. The modified proteins are then placed into new vesicles and sent to the destinations in the cell where they are needed. As a result of these functions the Golgi body is often referred to as the cell’s “post office” responsible for taking in parcels of proteins and delivering them to their final homes.

Storage particles

When nutrients are plentiful cells will store the excess in the form of particles. These can be seen by staining the cells with appropriate dyes. Commonly seen storage particles in the cytoplasm are lipids such as sterols and fatty acids and also the reserve carbohydrate, glycogen. As you are all very well-aware you can monitor starch breakdown in a mash by staining a sample with iodine – if it goes black there is still some starch present. Barley grains store sugar in the form of starch, yeast do the same thing but in the form of glycogen. If you mix yeast cells with iodine the glycogen takes up the stain and gives a dark brown colour. This is useful quick test for pitching yeast. If there is not much staining then the yeast has used up most of its glycogen stores to help it survive any prolonged storage. It still needs some of this glycogen when it is pitched so that it can make use of oxygen and make the sterols it needs for good membrane function. If it is not there because the yeast has been stored for too long a sluggish fermentation would be expected – not good!

Cytoplasm

The intracellular organelles, storage bodies and other structures are together called the cytoplasm. The semi-liquid material in which all of these are suspended is called the cytosol. It consists of many dissolved salts, cellular metabolites and many enzymes. For example, glycolysis, the pathway responsible for breaking down fermentable sugars to form pyruvate, the precursor of alcohol and CO₂, is located in the cytosol.

Bud scars

Each time a yeast cell undergoes budding a characteristic bud scar is left on the surface of the cell wall. The scar is permanent and is a circular structure which with a raised border and is rich in the polysaccharide chitin. The new daughter cell also bears a mark where it was attached to the mother cell, in this case known as the birth scar. This also contains chitin but birth scars are smaller and less persistent than bud scars. Chitin is a polymer made up of long chains of linear β-1,4 linked molecules of N-acetyl-D-glucosamine. This material which also forms the exoskeleton of insects and crustaceans is the second most abundant biopolymer in nature after cellulose.
The chitin in the scars binds to fluorescent dyes such as calcofluor and this can be used to visualise the scars using a suitable microscope. Since a new scar is formed each time a yeast cell buds staining with fluorescent dyes provides a convenient method of determining the age of a cell; more buds, more fluorescence, the older the cell.