Welcome to Microscopes Blog
Microscopes Blog is dedicated entirely to microscopes, industry-related news and product reviews. We try to do our best in providing our readers with the most adequate and truthful reviews on all microscopes products and their accessories. And we tend to share our thoughts on anything related to the fascinating field of microscopy. So if you share our passion of exploring the tiny objects that are too small for the naked eye to see, this blog is for you!
Paul’s Favorite Posts
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.
Capsular staining is a technique used to stain species of bacteria that are surrounded by an extracellular slime layer called a capsule or glycolayx. This structure provides protection for bacteria such as streptococcus pneumoniae. The capsule prevents phagocytic white blood cells from destroying the bacteria. This allows the pathogen to enter the lungs and cause pneumonia. The capsule also allows the bacteria to attach itself to solid surfaces. Streptococcus mutans can attach to the surface of teeth which can cause dental plaque. Most capsules are composed of polysaccharides but in some cases the capsule can consist of polypeptides with unique amino acids. Staining of the bacterial capsule cannot be accomplished by ordinary staining procedures. The capsule can be viewed by combining the simple stain with the negative stain. The negative stain is performed first followed by slight heat fixing. Crystal violet is then used to stain the bacterial cell. The capsule appears as a halo around the cell.
Negative staining can be used to study the morphology of bacterial cells and characterize some of the external structures, such as capsules, that are associated with bacterial cells. Negative stains are acidic and have a negatively charged chromophore that does not penetrate the cell. The stain is repelled by the cell because the overall charge of a bacterial cell is negative. The background surrounding the cell is colored by the stain which results in an indirect stain of the cell. Cells will appear transparent against a dark background. Examples of negative stains are india ink and nigrosin. To stain cells using this method one would mix the organism with a small amount of the stain and spread a thin film over the surface of the microscope slide. The cells are not usually heat fixed prior to staining. Once the smear has air dried it can be examined with a microscope under the 100x oil immersion objective.
Using a single stain to color a bacterial cell is commonly referred to as a simple stain. The most common dyes for this type of stain are methylene blue, fuchsin, and crystal violet. These dyes work well because they have positively charged color bearing ions or chromophores. Bacteria are negatively charged which creates a natural attraction with positively charged dyes. Basic or cationic dyes have a positive charge making them practical for bacterial staining whereas acidic dyes like eosin have a negative charge. Dyes such as eosin will not stain bacterial cells due to their repelling forces. Staining times for most simple stains are relatively short, usually from 30 seconds to 2 minutes, depending on the affinity of the dye. After a smear has been stained for the required time, it is washed off gently, blotted dry, and examined directly under oil immersion. This type of slide is useful in determining basic morphology and the presence or absence of certain kinds of granules.
Bacterial smear preparation is an integral technique in microbiology laboratories. There are several objectives when preparing a smear. The first is to make sure that the cells adhere to the microscope slide so that they do not get washed off during the staining and washing procedures. It is also important to prevent the cells from shrinking. Finally, it is very important to prepare thin smears. If a smear is too thick one will not be able to see individual cells, their arrangement, or the details of their microstructures. Thick smears with large clumps of cells can entrap the stain preventing it from being washed. The first step in preparing a smear depends on whether the organism has been growing in liquid or solid media. Two or more loopfuls of liquid media containing the organism can be placed on the slide. One can use an inoculating loop to disperse organisms from solid media into a drop of water on the slide.