Now, over the years the microscope has evolved and scientists have developed different kinds of microscopes to adequately examine different organisms/specimens.
Dark field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual water-borne single-celled organisms. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.
Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. A phase contrast microscope does not require staining to view the slide. This type of microscope made it possible to study the cell cycle . Phase contrast images have a characteristic grey background with light and dark features found across the sample. Light and dark fringes appear around regions with a change in optical density, for example the boundary between water and a cell. This normally manifests as a light halo around a dark object.
is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples . DIC works on the principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features. A relatively complex lighting scheme produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo.
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption
Direct fluorescent antibody ( DFA or dFA ) (also known as &quot;Direct immunofluorescence&quot;  ) is a laboratory test that uses antibodies tagged with fluorescent dye that can be used to detect the presence of microorganisms . This method offers straight-forward detection of antigens using fluorescently labeled antigen-specific antibodies. Because detection of the antigen in a substrate of patient sample (cellular smear, fluid or patient- inoculated culture medium) is the goal, DFA is seldom quantitative.
An electron microscope is a type of microscope that uses a particle beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope , because electrons have wavelengths about 100,000 times shorter than visible light ( photons ), and can achieve better than 50 pm resolution  and magnifications of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes
In staining for microscopic examination for diagnosis or research acid dyes are used to color basic tissue proteins in contrast to basic dyes, which are used to stain cell nuclei and some other acidic components of tissues
A simple stain consists of a solution of a single dye. Some of the most commonly used dyes are methylene blue, basic fuchsin, and crystal violet. Simple stains allow one to distinguish the shape (morphology) of the bacteria. For example, E. coli and Bacillus Subtillus are bacilli or rod-shaped bacteria. Many bacilli occur singularly, but chains may also be observed. Bacilli very greatly in length and diameter. Staphylococcus aureus and Streptococcus pneumoniae are cocci or spherical bacteria. Cocci may occur singularly, in pairs (as in Streptococcus pneumoniae) , or in clusters (as in Staphylococcus aureus) . R. rubrum is a spirillum or curved bacterium, Spirilla always occur singularly.
Differential stains are more complex than simple ones and use more than one stain to differentiate cellular components. They are used to examine structural differences between bacterial groups or to provide contrast to different structures within the same organism
As mentioned, Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). Most bacterial phyla are Gram-negative, namely the cyanobacteria , spirochaetes and green sulfur , most Proteobacteria (the exceptions in the Proteobateria are some members of the Rhickettsiales and the insect-endosymbionts of the Enterobacteriales ) and many other phyla.   [ edit ] Gram-positive bacteria Main article: Gram-positive bacteria On the other hand, Gram-positive bacteria have generally a single membrane (monoderm) surrounded by a thick peptidoglycan The Gram stain procedure uses 3 different stains. These are crystal violet, Gram’s iodine, and safranin. The cells are first stained with crystal violet, then Gram’s iodine. Following a rinse in alcohol, to de-colorize the cells, the cells are then stained with safranin. The Gram stain procedure separates almost all bacteria into two large groups: the Gram-positive bacteria that stain blue and the Gram-negative bacteria that stain pink. Bacteria take up the Gram stain differently because they differ in cell wall composition. Gram-positive bacteria have a thick cell wall layer. Alcohol does not readily penetrate to decolorize the cell wall of the previously applied crystal violet stain. Gram-negative cells have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is removed from these cell walls that are then stained with the safranin counterstain
It is helpful in diagnosing Mycobacterium tuberculosis since its lipid rich cell wall makes it resistant to Gram stain . It can also be used to stain few other bacteria like Nocardia . The reagents used are Ziehl–Neelsen carbolfuchsin , acid alcohol and methylene blue . Acid-fast bacilli will be bright red after staining
Negative staining is an established method, often used in diagnostic microscopy , for contrasting a thin specimen with an optically opaque fluid. For bright field microscopy , negative staining is typically performed using a black ink fluid such as nigrosin . The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores , appear light against the dark surrounding background
Yasmine Kanaan, Ph.D. Tel. 202 806 9540 Cancer Center, Rm # 410 Observing Microorganisms
Microorganisms? Much too small to be seen with unaided eyes
Mordant - intensifies the stain or coats a structure to make it thicker and easier to see after it is stained Example: Flagella - can not normally be seen, but a mordant can be used to increase the diameter of the flagella before it is stained Salmonella typhosa Counterstain: a second stain applied to a smear, provides contrast to the primary stain.