Introduction

All living things are made of cells. They are fundamental units of life and act as the building blocks for advanced biological systems. All cells have nucleic acids, lipids, carbohydrates, and proteins. These essential components enable a cell to reproduce and function as an independent unit.

Figure 1: Diagram of prokaryotic and eukaryotic cells. Prokaryotes lack a nucleus and individual organelles. Prokaryotes also have a cell wall surrounding the plasma membrane. Eukaryotes have a true nucleus and many individual organelles which perform distinct functions for the cell.

Cells are generally separated into prokaryotic and eukaryotic cells. “Karyose” is a Greek word meaning “kernel” and in biological terms represents the nucleus. “Pro-” means before and “Eu-” mean true; therefore, prokaryotic means before nucleus; prokaryotes do not have a distinct nucleus. Eukaryotic means true nucleus; eukaryotes do have a distinct nucleus.

Figure 2: Schematic displaying the basic structure of a bacterial cell (prokaryote). This image shows how the bacteria lacks many of the internal organelles that eukaryotes have. It also displays how the DNA is organized within the nucleoid, and that the cell wall is surrounding the cell membrane.

Microbiology is the study of microscopic organisms that exist singly (unicellular) or in cell clusters of various forms. Microorganisms can be either prokaryotic and eukaryotic (protists and fungi). Microbiology also includes the study of viruses, which are acellular organisms that contain hereditary material that can be either DNA or RNA, but viruses lack the ability to reproduce independently. Viruses, therefore, require a host cell to replicate.

Prokaryotic Cells

Prokaryotes inhabit all possible environments on earth, are extremely adaptable, and are the oldest known organisms on the earth (some bacteria are 3.5 billion years old). Prokaryotes (bacteria) are simple, unicellular organisms enclosed by a bilayer membrane with a surrounding semi-rigid cell wall, and no internal membrane bound structures. Additionally, prokaryotes are divided into two domains (groups): Domain Bacteria and Domain Archaea. These domains are divided on the basis of cell wall composition, sensitivity to antibiotics, and ribosomal RNA composition. Prokaryotes of the Domain Archaea have cell walls that lack peptidoglycan, frequently perform atypical metabolic processes, and live in extreme environments (e.g., very high temperatures, extreme acidity, high salt concentrations, etc.).

Prokaryotic cell structure is overall much more simple that than of eukaryotes, with the exception that the cell membrane surrounding prokaryotes which is much more complex. Internally, prokaryotes do not have a true nucleus; rather, prokaryotic DNA is circular and is surrounded by cytoplasm. Prokaryotic DNA is bound by special proteins that allow it to be packaged within the cell. There is no nuclear membrane and DNA exists as a single, continuous, circular molecule that varies in size among different organisms. Some microbes have multiple circular DNA molecules that encode different genes.

The prokaryotic cell membrane serves a number of critical functions: it protects the microbe from harsh environments, prevents excess water from leaking into the cell and causing it to swell and burst (osmotic lysis), and protects against chemicals and even drugs (antibiotics). Cell membranes also allow for selective transport of important factors into (nutrients) and out (wastes) of the cell, provide an electrical differential to allow oxidative respiration in aerobic organisms, contain enzymes important for DNA, lipid and cell wall material synthesis; and, it is essential for cell movement and sensory systems. It differs from the eukaryotic cell membrane in it has a much higher percentage (by mass) of proteins embedded within the phospholipids.

Figure 3: Schematic demonstrating various bacterial shapes and examples of each.

Additionally, bacteria (with the exception of mycoplasmas) have a unique layer surrounding the cell membrane called the cell wall. It is composed of peptidoglycan, which is formed by repeating units of 2 sugars with 4 amino acid side arms interconnected so that the repeating units form an unified molecule that resembles a chain-link fence. The 2 sugars are constant in the peptidoglycan wall of bacteria; however, the amino acid side arms and type of cross-bridges does change among different microbes. Differences in the amino acid side chains and type of cross-links affects whether the bacteria will take up different staining compounds. The cross-linking of the repeating sugar-amino acid groups leads to a very strong and semi-rigid, 3-dimensional structure that further serves to protect the bacteria and provide support, especially against osmotic lysis. The amount of peptidoglycan material in the bacterial cell wall largely determines how a bacteria reacts to specific stains that are applied to it.

Morphology

Bacteria are classified also by their morphology and this is often reflected in how they are named. Bacteria occur in numerous shapes, including but not limited to rods (bacilli), spheres (cocci), spiral (spirilla), comma-shaped (vibrio) and filamentous forms. Spherical bacteria are called cocci and can occur singly or in clusters of 2 to many cells. Common examples of spherical bacteria are Streptococcus (which form chains) and Staphylococcus (which assume a “cluster of grapes” shape).

Rod-shaped bacteria are called bacilli; some examples are E. coli (a common gut-inhabiting microbe), Bacillus cereus (may cause food poisoning) and Bacillus anthracis (produces the causative agent of anthrax, which has been used as a biological weapon). Spiral (spirilla) bacteria include Treponema species (responsible for the sexually transmitted disease syphilis) and Campylobacter (food poisoning); comma-shaped bacteria include Vibrio cholerae (causative agent of cholera).

Figure 4: Many bacteria are spherical, or cocci, in shape.	Figure 5: E. coli bacteria are rod-shaped, or bacilli.	Figure 6: Some bacteria may be spiral, or spirillia, in shape.

Eukaryotic Cells

Eukaryotes are much more complex than prokaryotes and evolved from their simpler ancestors. Eukaryotic cells have a phospholipid and protein cell membrane enclosing a cytoplasm containing cellular organelles of specific and diverse function and the nucleus, which contains highly packaged DNA. Organelles are membrane-bound compartments that allow independent biochemical processes to occur simultaneously.

Organelles in eukaryotes include:

Endoplasmic Reticulum (ER): Extensive internal membranous structure that provides for protein production and transport within the cell. It also contains various enzymes for metabolic reactions.

Golgi Apparatus: Contain various digestive enzymes used for breaking down internal components.

Lyosomes: Extensive internal membranous structure that provides for protein

Peroxisomes: Contain enzymes used for cell protection and metabolism.

Mitochondria: Bacteria-like organelles that metabolize oxygen to produce energy for the cell. It also contains circular DNA and can divide (replicate) to produce new mitochondria during cell division.

Nucleus: Contains all the genetic information that governs all cell activities. It contains a Nucleolus (which is rich in RNA), ribosomal proteins, and ribosomal RNA. This is also the site of RNA synthesis.

Eukaryotic cells also have a protein-rich cytoskeleton that provides 3-dimensional structural support, tensile strength, and cell-associated movements (gliding, contraction and cytokinesis).

Staining

Even with the use of a microscopes, the majority of cells are devoid of color and appear colorless or transparent when viewed through a microscope. This makes it difficult to locate the identifiable internal structures. For this reason, biological stains are used to facilitate visualization. Stains increase the contrast between the microorganism and the surrounding tissue (or slide). Bacteria are typically negatively charged so positively charged dyes (such as crystal violet) are used to stain the cells.

Different stains and staining methods are used depending on what organisms are being studied. To begin the staining process, a bacterial sample is placed on a slide and then “fixed” to the slide by gentle heating. Fixing the sample accomplishes three things:

1. It kills the bacteria.
2. It securely attaches the bacteria to the slide so that the sample is not lost during the staining procedure.
3. It makes the bacteria easier to stain.

Simple and Differential Staining

Some bacterial stains are simple, meaning only one type of stain is used. Simple stains are typically easy to perform and provide basic information about morphology. However, simple stains can't typically identify the type of bacteria in the sample. Differential stains are often used to identify bacteria. Differential stains contain two or more different stains and can distinguish between different bacteria through their chemical or structural features.

Negative Staining

Negative staining provides a more detailed assessment of a microbe's morphology than simple staining does because the background (rather than the microbe) is stained. This prevents the staining procedure from causing any distortion to the microbe's ultrastructure. It also makes the outline of the cells highly visible. Bacterial cells are negatively charged, as are some stains (such as nigrosin). Negative staining works because “like” charges repel each