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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.
In natural environments bacteria exist as mixed populations. This presents a challenge when trying to study a single species. It is very rare to find a single species living in natural environments. Robert Koch, a pioneer in medical microbiology, knew that he would have to find a way to isolate bacteria from other species if he wanted to prove that a specific agent caused a disease. His lab contributed many techniques to microbiology including the method designed to obtain a pure culture of bacteria. Pure cultures contain only a single bacteria. Pure cultures allow one to study the morphological and physiological characterisitics of an individual organism. Two common methods are the streak plate and the pour plate.
Aseptic technique ensures that no contaminating organisms are introduced into culture materials when these materials are innoculated. It also prevents the handler of the organisms from being contaminated. Aseptic technique can be used to transfer broth culture to a plate or to transfer a colony from a plate to a slant culture for example. First the work area is treated with disinfectant to kill any microorganisms. This may not kill endospores if they are present. Cultures are transferred using innoculating loops and needles. These items are sterilized by inserting them into the flame of a bunsen burner until they are red hot. Tubes are sterilized as well by removing the cap and flaming the mouth. After innoculation the loop or needles is flamed in the bunsen burner to destroy any organisms that remain. The loop or needle can then be placed back into its holder.
Fungi are heterotrophic eukaryotic organisms that produce exoenzymes and absorb their nutrients. Fungi may be saprophytic or parasitic and can be unicellular or filamentous. The features that separate fungi from other organisms are: eukaryotic, heterotrophic, lack tissue differentiation, have cell walls of chitin or other polysaccharides and propagate by spores. Organisms such as water molds, mushrooms, puffballs, yeasts, and molds. These organisms do not have a consistent genetic background and are thought to have evolved from two different ancestral lines. Traditional methods of classification rely on morphological characteristics however more modern techniques use genetic analysis to classify fungi. Data collected relatively rececently from genetic analysis suggests that classification based on morphology does not reflect evolutionary relationships.
The shape and structure of bacteria make it easy to identify them. The structure of bacteria is simple. They have supercoiled DNA in the cytoplasm and are able to carry out photosynthesis and respiration. Their cell wall is made of a molecule called peptidogylan that is not found in any other kind of cell. Bacteria range from .5 microns to 10 microns. The shapes of bacteria can be grouped into three morphological types: rods (bacilli), cocci (spherical) and spirals or curved rods. The rods or bacilli can have rounded, flat, or tapered ends and they can be motile or nonmotile. Cocci may occur singly, in chains, in tetrad, or in irregular masses. Most cocci are nonmotile because they lack flagella, the organelles of motility. The spiral bacteria can exist as slender spirochaetes, as a spirillum, or as a comma-shaped curved rod (vibrio).
A disease casued by microorganisms (such as bacteria, viruses) that enter the body and multiply in the tissues at the expesne of the host is said to be an infectious disease. Infectious diseases that are transmissible to other people are called communicable. The transfer of communicable infectious diseases between individuals can be accomplished by direct contact such as handshaking, kissing, and sexual contact. They can also be spread indirectly through food, water, objects and animals. Epidemiology is the study of how, when, where, what, and who are involved in the spread of disease in human populations. Epidemiologists determine the classification of the disease. If the number of newly reported cases in a given period of time in a specific area is excessive, an epidemic is in progress. If the disease spreads to one or more continents, a pandemic is in progress.