Introduction

In previous labs, you explored the abundance of microorganisms that co-exist with us (in or on our bodies and surrounding environment) by growing them on media that provided the nutrients required for their survival. The streak technique used to spread the sampled areas on the surface of the solid media (agar) plates dilutes the bacteria such that a single bacterial cell is isolated. This cell then begins to divide exponentially (1 to 2, 2 to 4, 4 to 8, etc.) mainly by binary fission. In binary fission, the bacterial chromosome (DNA) is reproduced, separates to two sides of the bacteria, and the cell elongates. The cell wall and membrane invaginate until it forms a new wall that completely divides the newly formed chromosomes. The bacteria then separates to form two cells.

The formula for exponential growth is N_f = N₀ x 2ⁿ, where N_f is the final number of bacteria; N₀ is the initial number of bacteria; and n is the number of cell divisions. This exponential growth from a single cell becomes a colony of identical daughter bacteria on an agar plate. By this process, pure isolates from a mixed bacterial source can be grown; this greatly aids the microbiologist in identifying specific bacteria from, for example, a throat swab. In Figure 1, the blue line demonstrates the four basic bacterial growth phases.

Figure 1: Examples of ideal exponential (green) and actual (blue) bacterial growth. Under ideal conditions(green line), bacteria will grow unchecked at an exponential rate. In theory, 1 bacteria, at a weight of 1 picogram (1x10-12 grams), could generate a population of bacteria that weighs as much as a blue whale after only 68 divisions! However, bacterial growth in liquid media typically follows the blue line, demonstrating various phases of growth. Exponential growth is usually halted by lack of nutrients, build up of harmful waste products, or pH changes.

Phase 1 is the lag phase wherein there is a small change in the number of cells; the bacteria are intensely metabolic during this phase. Phase 2 is the exponential, or log, phase of growth, where reproduction (binary fission) is most active. It is during this phase that the exponential growth equation holds true. Bacteria are highly sensitive to any growth inhibiting substances (e.g., drugs, radiation, temperature fluctuations, etc.) during this phase. Phase 3 is the stationary, or plateau, phase, wherein exponential growth ceases as the number of newly formed bacteria is balanced by the number of dead and dying bacteria. The metabolic activity of the culture decreases as well. Phase 4 is the death, or log decline, phase. In phase 4 the number of dead bacteria exceeds the number of live bacteria, and this phase may continue until the entire population is eliminated.

Physical Requirements for Growth

Bacteria in culture (either in liquid media or on agar plates) have certain physical and chemical requirements for effective growth. Physical requirements include proper temperature, pH, and osmotic pressure. Bacteria, when considered as a whole, can survive and live at great temperature extremes: from -10 °Celsius to over 110 °Celsius. Individual species, however, demonstrate a much more narrow range for optimal growth. Most bacteria that live in or on our bodies grow best between 15 and 45 °Celsius; these types of bacteria are called mesophiles. See Table 1 for a thermal classification of bacteria.

The acidity or alkalinity (pH) range of media also can affect bacterial growth. Most bacteria grow best when the pH is near 7.0 (neutral pH), although some can grow in much more acidic conditions (pH 4). Bacteria that generate acids are used in some methods of food preservation as the acidic conditions prevent the growth of unwanted (i.e., food spoiling) bacteria. Media used in the lab typically contains a buffer that prevents pH changes to ensure efficient bacterial growth.

Osmotic pressure refers to any difference in solute concentration across a semi-permeable membrane. The osmotic pressure gradient is measured by tonicity, the physical distribution of solutes in two solutions separated by a semi-permeable membrane. A hypotonic solution has a greater number of solutes inside the membrane, an isotonic solution has the same number of solutes on each side of the membrane, and a hypertonic solution has greater number of solutes outside of the membrane. Excess solutes outside of a bacteria (a hypertonic solution) cause water to leak out of the cell. This results in dehydrating the bacteria andinhibition of growth. A solute-poor (hypotonic) solution causes the reverse to occur: water floods into the bacteria, causing the bacteria to swell, potentially lysing the cell due to disruption of the cell wall. Therefore, solutes in the media must be in equilibrium with the bacteria (isotonic solution) for optimal growth.

Chemical Requirements for Growth

All living organisms contain proteins, nucleic acids (DNA and RNA), carbohydrates, and lipids. The chemical composition of these biochemicals includes carbon, nitrogen, phosphorus, and sulfur. Therefore, media must provide these essential elements for optimal growth. Carbon provides the backbone for all organic molecules and constitutes approximately half of the dry weight of a cell. Carbon can be supplied by plant or animal extracts, or by the energy source (carbohydrate) present in the media. Nitrogen is essential for nucleic acid, protein, and ATP synthesis. It can be provided by proteins present in the media. Sulfur is required for protein synthesis and phosphorus is needed for nucleic acid, lipid, and ATP synthesis. These elements can be provided by extracts or specific compounds added to the media. Water is also critical as it serves to solubilize the nutrients so that microbes can ingest them

Aerobic and Anaerobic Growth

Oxygen is necessary for those microorganisms that rely on aerobic respiration and that require oxygen for survival (obligate aerobes). Some bacteria, called facultative anaerobes, can survive either with or without oxygen. Facultative anaerobes perform aerobic respiration when oxygen is present but perform fermentation or anaerobic respiration in its absence. Bacteria that do not require oxygen for growth and may actually be killed by oxygen are called obligate anaerobes. They can perform either fermentation or anaerobic respiration but use a final inorganic molecule (such as sulfur or nitrogen) as the last step in respiration. Other types of oxygen-restricted bacteria include aerotolerant anaerobes, bacteria that cannot use oxygen but are not killed by it, and microaerophiles, bacteria that do require oxygen, but at lower concentrations than that in normal atmospheric air. Respiration and fermentation will be covered in more detail in a future lab.

Figure 2: Patterns of aerobic and anaerobic growth (green) in media (yellow). A) Obligate aerobes grow only in the presence of high oxygen. B) Microaerophiles require oxygen, but at lower concentrations than obligate aerobes. C) Obligate anaerobes grow only in the absence of oxygen. D) Facultative anaerobes grow best in high oxygen concentrations but can survive at all oxygen amounts. E) Aerotolerant anaerobes grow anaerobically regardless of oxygen concentration.

Environmental Factors and Growth

Alterations in oxygen content, temperature, pH, osmotic pressure, and the nutrient composition of media all can be considered environmental factors that affect bacterial growth. For example, attempts to grow an obligate anaerobic bacteria (e.g., Clostridium botulinum) on a streaked plate in atmospheric air will likely fail. This is because obligate anaerobes prefer environments that are devoid of oxygen. In fact, oxygen can even kill certain obligate anaerobes. For obligate anaerobes, a microbiologist would prepare two identical plates streaked with a sample thought to contain Clostridium. One plate would be incubated in atmospheric air while the other one would be incubated in an anaerobic jar (in which oxygen is removed by chemical means). The presence of colonies on the plate grown in anaerobic conditions and the absence of colonies on the plate grown in air would be strong evidence that an obligate anaerobe was present in the sample. Similarly, changing any of these factors during growth can be helpful in identifying which bacterial species are present.

Morphology and Growth Patterns

Figure 3: Sample image of a petri dish with multiple species growing on it. As demonstrated, different species can exhibit different colors, shapes, and other morphological qualities.

Bacterial colonies that arise from a single bacterium on an agar plate can grow to display a wide diversity of patterns. Colonies can be round, irregular, or grow like filaments with smooth, wavy, or root-like edges. They can lie flat on the surface of the agar, or be raised up from the surface or extend down into the agar. Colonies can have a dry, wet, or mucus-like appearance and may display different colors, especially depending on the type of growth media. Colony appearance is often an important first step in the identification of bacteria; carefully observe all cultures you prepare throughout these labs and make note of their morphology and patterns.

Growth Control via Physical and Chemical Means

Dispersal of microorganisms is a major cause of disease spread. In previous labs, you experienced how easily a microorganism can be spread. You even took a preventative measure to limit its spread and growth (hand washing). Hand washing is an example of a physical means that removes bacteria from a surface and the soap may aid in disrupting cell walls, which can perturb bacterial growth. Physical and chemical methods can be categorized as decontamination, disinfection, and sterilization. Decontamination is often the first line of control and involves physical cleaning of surfaces, often with soaps or detergents. Disinfection frequently targets specific microorganisms and commonly uses chemical substances, but can also include heat or ultraviolet light (radiation). Decontamination and disinfection usually do not eliminate all microorganisms but rather reduce their numbers to a level that is less likely to result in infection. Sterilization, on the other hand, is defined as “the process of destroying all microorganisms and their pathogenic products”. Sterilization is therefore the most stringent control and usually involves gases, ionizing radiation, or heat. Sterilization kills all bacteria and spores, which are difficult to eliminate by decontamination and disinfection methods.