Dark-field microscopy is ideally used to illuminate unstained samples causing them to appear brightly lit against a dark background. This type of microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle.

In a standard bright field microscope, light travels from the source of illumination through the condenser, through the specimen, through the objective lens, and through the eyepiece to the eye of the observer. Light thus gets transmitted through the specimen and it appears against an illuminated background.

Bright-field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample.

In a bright-field microscope, only the scattered lights are able to enter the objective lens and transmitted lights or unscattered light rays are omitted, that’s why the viewer sees a dark image against the brightfield. The only light that has been reflected or reflected by the specimen forms an image. Appear as dark.

An image that is produced by the transmitted wave (the wave that undergoes no diffraction) in a diffraction pattern formed on the back focal plane of the objective lens, using the objective aperture.

0.2 micrometers

The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample.

Since fixation and staining would kill the cells, darkfield microscopy is typically used for observing live specimens and viewing their movements. However, other approaches can also be used. For example, the cells can be thickened with silver particles (in tissue sections) and observed using a light microscope.

Principle of Negative Staining The acidic stain, with its negatively charged chromogen, will not penetrate the cells because of the negative charge on the surface of bacteria. Therefore, the unstained cells are easily discernible against the colored background. The practical application of negative staining is twofold.

microscopic observation is that it helps in the identification of the cell by the color change. A disadvantage of staining a specimen is that the stain can kill off the live specimen fairly.

The staining methods we will use kill the bacteria, reducing the risk of infection by pathogenic organisms. Since two dyes are used to distinguish types of bacteria, Gram staining is called a differential staining method. The Gram stain is a direct method, since the cells themselves retain dye.

Disadvantages: You have to use a kill stain to create contrast; you are unable to see life processes (motility) due to using a kill stain.

Disadvantages: Some bacteria are Gram stain variable (positive or negative results) Some bacteria are resistant to Gram stain (i.e. acid-fast bacteria) False results may occur if over-decolorized.

Gram devised his method that used Crystal Violet (Gentian Violet) as the primary stain, an iodine solution as a mordant followed by treatment with ethanol as a decolorizer.

What are the limitations of a simple stain? Can only determine cell shape and size. The preparation of a bacterial smear can be difficult to learn.

Discuss the reasons for heat fixing bacterial smears prior to staining.

• To kill bacteria on slide;
• to make dye penetrate cells;
• to coagulate proteins of cells thus making cells stick to slide.

Individual bacteria can assume one of three basic shapes: spherical (coccus), rodlike (bacillus), or curved (vibrio, spirillum, or spirochete). Considerable variation is seen in the actual shapes of bacteria, and cells can be stretched or compressed in one dimension.