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Bacteria can be classified into two groups based on their oxygen needs. Obligate aerobes and anaerobes. Aerobes require oxygen for growth because they perform respiratory metabolism which utilizes oxygen as a final electron acceptor. Anaerobes can be further divided into sub groups depending on their sensitivity to oxygen. Aerotolerant anaerobes can grow in the presence of oxygen and are not usually harmed by its presence. Their metabolism does not require oxygen but does however involve fermentation which uses different compounds as a final electron acceptor. Obligate anaerobes can’t tolerate oxygen and must be cultivated under conditions in which oxygen is removed ortherwise they are killed. Obligate anaerobes use fermentation and anaerobic respiration for energy production. Inorganic compounds such as nitrates or sulfate replace oxygen as the final electron acceptor. It is believed that most strict anaerobes contain enzymes and proteins that can be easily damaged by the presence of oxygen. One can culture anaerobic bacteria by utilizing the unique funcionality of the Gaspak anaerobic jar system.
Media can be made with different components that will promote the growth of certain bacteria while inhibiting the growth of others. This medium is known as a selective medium. Antibiotics, dyes, and various inhibitory compounds are often incorporated into media to create selective conditions for growing specific organisms. The dyes eosin and methylene blue when incororporatd into EMB media do not affect the growth of gram-negative bacteria however they do inhibit the growth of gram-positive bacteria. When sodium chloride is placed in mannitol-salt agar it promotes the growth of Staphyloccus aureus but inhibits the growth of other bacteria that can’t tolerate high salt concentrations. A differential medium has substances that cause bacteria to alter how they appear. When S. aureus grows on mannitol-salt agar, it ferments mannitol, changing a ph indicator from red to yellow around the colonies. Other staphylococci can’t ferment mannitol and their growth on this medium results in no change in the indicator. ( image from quizlet.com ). Using selective and differential medium becomes extremely important in quality control and quality assurance techniques.
Bacteria can be divided into groups based on their nutritional requirements and how they obtain nutrients. Organisms can be divided into two groups based on their carbon requirements: heterotrophs and autotrophs. Heterotrophs obtain their carbon from organic compounds like lipids, carbohydrates, amino acids, peptides, and proteins. Autotrophs obtain their carbon by fixing carbon dioxide. Autotrophs must make all of the compounds that make up the cell. In addition to obtaining carbon, organisms must produce energy to synthesize compounds such as proteins and nucleic acids. Chemoorganotrophs use fermentation and respiration for their energy needs and most bacteria belong to this group. Chemolithotrophs oxidize inorganic ions such as nitrate or iron to obtain energy to fix carbon dioxide. Photoautotrophs contain photosynthetic pigments such as chlorophyll or bacteriochlorophyll that convert light energy to chemical energy through photosynthesis. Energy produced during photosynthesis is then used to fix carbon dioxide. Photoheterotrophs obtain their energy from photosynthesis however they obtain their carbon from organic molecules such as glutamate or succinate.
The motility of bacteria can be determined by several methods. It can be determined microscopically by observing cells in a wet mount. In this method a drop of cells is placed on a microscope slide and covered with a cover glass. The slide is then observed with a phase contrast microscope. It is better to use a phase contrast microscope for this application due to transparent nature of the bacterial cells. The disadvantage of this procedure is that the cell dries out rather quickly which can cause problems if observations need to be made over a long period of time. The second method is called the hanging drop technique. In this procedure a drop of cells is placed on a cover slip which is then placed on a special slide with a concave depression in its center. The coverslip is held in place with petroleum jelly. This creates an enclosed glass chamber that prevents drying. It is important to distinguish between cells that are moving due to the vibrations of the table and microscope and cells that are actually motile.
The motility of bacteria is provided by the flagella, a rigid helical structure that extends up to 10 microns out from the cell. Flagella allow cells to move towards nutrients or away from harmful substances such as acids in a process known as chemotaxis. Flagella are less than .2 microns thick and therefore are below the resolution of the light microscope. In order for the flagella to be viewed they must be stained using a special procedure. A single flagellum is composed of a rigid filament that occurs in the form a single helix. This makes up the main body of the flagellum structure. The filament is connected to a hook that is attached to a shaft that is inserted into a series of rings. The shaft is inserted into a series of rings. The number of rings differs depending on whether the cell is gram-positive or gram-negative. The shaft rings along with other accessory proteins make up the basal body of the flagellum. Proteins associated with the basal body transport protons across the cell membrane which creates a charge differential. This charge differential forces the rings to rotate which results in the rotation of the shaft, hook, and filament.
Acid fast staining is used to stain bacteria with mycolic acid in their cell walls. The Ziehl-Neelsen staining method is used which involves mixing carbolfuchsin with phenol and heating the cells for 5 minutes. Phenol allows the penetration of carbolfuchsin into the cell wall and the heat further fixes the stain. The cells are then treated with acid-alcohol which acts as a decolorizer however it does not remove the stain from the mycolic acid-carbolfuchsin complex. These cells are referred to as acid-fast. Cells that do not contain mycolic acid in their cell walls are decolorized by the acid-alcohol and referred to as non-acid-fast. Non-acid-fast bacteria are counterstained with methylene blue and appear blue under the microscope. Acid-fast cells appear red to pink in stained preparations. Heating the cells with phenol can create fumes that may damage mucus membranes and eyes. The staining method may be modified to prevent these fumes. The concentrations of phenol and carbolfuchsin may be increased however no heat is used.
Bacteria such as mycobacterium have cell walls that contain a high lipid content. One of the cell wall lipids is a waxy material called mycolic acid. Mycolic acid is a complex lipid that is made of fatty acids and fatty alcohols and have carbon chains up to 80 carbons in length. Mycolic acid significantly affects the staining properties of these bacteria and prevents them from being stained by many of the stains routinely used in microbiology. The acid fast stain is an important tool in the identification of Mycobacterium tuberculosis, the organism that causes tuberculosis and Mycobacterium leprae, the bacterium that causes leprosy in humans. Mycolic acid makes the cell wall impermeable to many stains. In order to stain bacteria with high lipid content one must use a technique that makes them more permeable to stains. The Ziehl-Neelsen method and the Kinyoun acid-fast method will be discussed in next week’s post.
In the previous post we discussed the unique properties of endospores. These unique properties mean that they are not easily penetrated by stains. If endospore containing cells are stained by crystal violet, the spores appear as unstained areas in the vegetative cell. If heat is applied while staining with malachite green, the stain penetrates the endospore and green is not removed by subsequent washing with decolorizing agents or water. In this situation heat is acting as a mordant much like iodine in a gram stain procedure. There are two methods for spore staining. The Schaeffer-Fulton method uses malachite green to stain the endospore and safranin to stain the vegetative portion of the cell. A properly stained spore forming cell will have a green endospore contained in a pink sporangium. The Dorner method produces a red spore with a colorless sporangium. Nigrosin is used to provide a dark background. Both the sporangium and endospore are stained however the safranin diffuses out of the sporangium and into the nigrosin leaving a colorless sporangium.
When bacteria belonging to the genus Bacillus or Clostridium run out of essential nutrients, they produce endospores. Endospores allow the bacteria to survive extreme conditions that are not optimal for growth. If conditions become favorable, the endospore can go through a process called germination and form a new vegetative cell. Endospores are dehydrated structures and are not actively metabolizing. Endospores are also resistant to heat, radiation, acids, and many chemicals such as disinfectants that normally harm and kill vegetative cells. Endospores are resistant due to their protein coat or exosporium that forms a protective barrier around the spore. Heat resistance is directly correlated with water content. The higher the water content of the cell the less resistant to heat it will be. The water content of the spore is 10-30%. The water content is low because calcium ions form a complex with proteins and a dipicolinic acid. The complex forms a gel that controls how much water can enter the cell. The only way to destroy endospores is to expose them to steam under pressure which generates temperatures of 121 degrees celsius. An autoclave can create such conditions.
In 1884 the Danish physician Christian Gram developed a new type of stain known today as the gram stain. He attempted to design a stain that differentiated bacterial cells from eukaryotic cells however he failed. What resulted from his work is one of the most important stains in microbiology. The Gram stain is an example of a differential stain. This type of stain takes advantage of the fact that different cells or different structures within cells react differently to various dyes. Gram positive bacteria retain the crystal violet-iodine complex after decoloring with alcohol or acetone and appear purple under the microsope. Gram negative bacteria lose the crystal violet-iodine complex and must be counter-stained with a red dye such as safranin. Gram negative bacteria appear pink under the microscope.