Bacterial Morphology and Staining Reading

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Utah Valley University *

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2065

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Biology

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Dec 6, 2023

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Bacterial Morphology and Staining OBJECTIVES: This lab exercise is designed to illustrate twhat bacterial morphology looks like under the microscope and the types of stains used to help identify bacteria.Read and study the following paragraphs. BACTERIAL MORPHOLOGY When a bacterial cell is deposited on the surface of a culture medium, it will begin to divide if the physical environment is favorable and the proper nutrients are present. As it divides, the number of cells increases exponentially. This will result in a mass of cells that is referred to as a colony . If the cell was a large enough distance from other cells that were originally present and the culture medium remains solid, all cells present in the colony will be descendants of the original cell and are genetically identical. Genetically identical cells are known as clones . If all cells in a colony belong to the same species and strain of bacteria, they can be used to create a pure culture . Different types of microbes have different, but characteristic, shapes. Under suitable conditions, the shape and size of microbes are relatively stable. It is important to know the morphological structure of microbes, as it provides us with a better understanding of microbial physiology, pathogenic mechanisms, antigenic features, and allows us to identify them by species. In addition, knowledge of microbial morphology can be helpful in diagnosing disease and in preventing microbial infections. Bacteria are complex and highly variable microbes. They come in four basic shapes: spherical ( cocci ), rod-shaped ( bacilli ), arc-shaped ( vibrio ), and spiral ( spirochete ). In addition to shape, bacterial arrangement is also a measure used to help with identification. Cocci bacteria can exist singly, in pairs (as diplococci ), in groups of four (as tetrads ), in chains (as streptococci ), in clusters (as stapylococci ), or in cubes consisting of eight cells (as sarcinae). Most bacilli appear as single rods. Diplobacilli appear in pairs after division, while streptobacilli are arranged in chains.

MICROSCOPY STAINING In last week’s reading, we discussed three important features of microscopy: magnification, resolution, and contrast. Contrast is often needed to readily see microscopic specimens, so stains or dyes are used to increase contrast in the specimens. Bacteria are transparent or translucent when observed with a microscope and can only be observed when the contrast between the cells and the background is increased. An easy way for increasing contrast is to stain the smear using one of the two types of dyes. Basic dyes contain ions that are positively charged and will be attracted to and color the bacterial cells, because the bacterial cell surfaces tend to have a negative charge. Acidic dyes contain ions that are negatively charged and are repelled by the bacterial cells. As a result, acidic dyes stain the background and are referred to as negative stains . The type of dye used is determined by the structure of the cell that is to be observed. In general, stains can be classified as simple, differential, or special. A simple stain involves the use of a basic dye, such as methylene blue, safranin, or crystal violet, which stains all cells the same color. A simple stain will primarily allow you to identify the shape of the bacterial cells and their arrangement. The second type of stain is a differential stain . In a differential stain, the smear of bacteria is first stained with a basic dye known as the primary stain . The smear is then washed with alcohol or some other chemical that will remove the primary stain from certain types of bacteria. Finally, the smear is flooded with a second basic dye referred to as the counterstain . Because of differences in their cellular structure, some types of bacteria will retain primary stain while others will be decolorized by the alcohol and will be colored by the counterstain. After the completion of the staining process, bacteria can be divided into two groups based on their color. Differential stains are very useful in identifying and classifying microbes. A special stain is the third type of stain. This stain causes only specific parts of the cell, such as capsules, endospores, or flagella, to be visible. During this lab exercise, we will stain two unknowns that were isolated last week. This will help us to determine their cellular morphology and to gain information that will aid us in identifying them. TYPES OF MICROSCOPY STAINS The most widely used differential stain is the Gram stain . This stain was developed in 1884 by the Danish bacteriologist Christian Gram. This procedure will cause bacteria to either be colored purple or pink based on their cell wall structure. The Gram stain consists of exposing the bacterial cells to four different reagents: a primary stain ( crystal violet ), a mordant that binds the dye (iodine), a decolorizer (acetone or ethyl alcohol) and a counterstain ( safranin ). Bacteria with many layers of peptidoglycan in their cell walls will retain crystal violet and will be colored purple at the completion of the Gram stain; these bacteria are said to be

Gram positive. Other bacteria with less peptidoglycan in their cell walls will have crystal violet washed out by the alcohol and will take on the color of the counterstain safranin. The basic dye safranin is red in color, so bacteria which are red or pink after the Gram stain are considered to be Gram negative. Endospores are visible in the Gram stain as spherical or oval structures that are colorless. The endospores do not stain, because their thick, tough coat, which makes them resistant to heat and other physical and chemical agents, also prevents them from taking up the crystal violet and safranin used during the staining process. Often other intracellular structures, such as vacuoles or inclusions, will not absorb the stain. To definitively identify colorless structures in cells as endospores, a special stain is required. The Schaeffer-Fulton method of staining endospores is similar to the Gram stain in that it involves two basic dyes. The primary dye is malachite green , which colors the cells and endospores a dark green color. Next the bacterial smear is exposed to heat to drive the stain into the endospores. The smear is then washed with water, which will decolorize the cells but not the endospores. The cells are then counterstained with safranin. If done correctly, the vegetative cells will be red and the endospores will be green in color. Only seven genera of bacteria, which are mostly Gram-positive bacilli, produce endospores. The most common spore-producing bacteria belong to the genera Bacillus and Clostridium . Members of these taxa are responsible for diseases such as botulism, anthrax, and gas gangrene. Because only relatively small number of bacterial species produce endospores,

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