Make a 1:1 mixture of sample and negative stain (eg.Hold a coated grid flim side up in a pair of self clamping forceps.Procedure to view in Transmission Electron Microscope (TEM) Focus a thin area under oil immersion and observe the unstained cells surrounded by the gray stain. Allow the smear to dry without heating.Ħ. Maintaining a small acute angle between the slides, push the spreader slide toward the clean end of the slide being stained dragging the drop behind the spreader slide and producing a broad, even, thin smear.ĥ. Tilt the clean slide toward the drop forming an acute angle and draw that slide toward the drop until it touches the drop and causes it to spread along the edge of the spreader slide. Rest one end of the clean slide on the center of the slide with the stain. Use another clean slide to spread the drop of stain containing the organism using the following technique.Ĥ. Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop.ģ.
#INDIA INK STAINING PROTOCOL LUNGS FREE#
Place a very small drop (more than a loop full, less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.Ģ. Nigrosin 100 gm/L, Formalin 5 ml/L in water Procedure of Negative Stainingġ. The bacteria will show up as clear spots against a dark background. The glass of the slide will stain, but the bacterial cells will not. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. This means that the stain readily gives up a hydrogen ion (proton) and the chromophore of the dye becomes negatively charged. India Ink or Nigrosin is an acidic stain. Negative staining requires an acidic dye such as India Ink or Nigrosin.
It is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power. It is also used for the study and identification of aqueous lipid aggregates like lamellar liposomes (le), inverted spherical micelles (M) and inverted hexagonal HII cylindrical (H) phases by Negative staining transmission electron microscopy. It is also used to prepare biological samples for electron microscopy. eg: Spirilla. It can also be used to stain cells that are too delicate to be heat-fixed. The main purpose of Negative staining is to study the morphological shape, size and arrangement of the bacteria cells that is difficult to stain.
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