Instead of using a stain to achieve the contrast necessary for observing microorganisms, altering microscopic techniques to enhance contrast offers another approach. Phase contrast microscopy is one such contrast-enhancing technique. By this method, beams of light pass through the specimen and are partially deflected by the different densities or thicknesses (i,e., refractive indices) of the microbial cells or cell structures in the spedmen. The greater the refractive index of an object, the more the beam of light is slowed, which results in decreased light intensity These differences in light intensity translate into differences that provide contrast. Therefore, phase microscopy translates differences in phases within the specimen into differences in light intensities that result in contrast among objects within the spedmen being observed.
Microscopy that depends on staining microorganisms only allows dead organisms to be observed. Because staining is not part of phase contrast microscopy, this method offers the advantage of allowing observation of viable microorganisms. The method is not commonly used in most aspects of diagnostic microbiology, but it is used to identify medically important fungi grown in culture (for more information regarding the use of phase contrast microscopy for fungal identification see Chapter 50).
Counterstain (methylene blue)
Figure 6-9 The Ziehl-Neelsen acid-fast stain procedures and principles. A, Add-fast positive bacilli. B, Add-fast negative badlli. (Modified from Atlas KM: Principles of microbiology, St Louis, 1995, Mosby.)
Primary stain (carbolfuchsin red)
Decolorizer (HCI, alcohol) <
Counterstain (methylene blue)
Steps for staining
Cells on slide
1 Rx smears on heated surface (60° C for at least 10 minutes).
2 Flood smears with carbolfuchsin (primary stain) and heat to almost boiling by performing the procedure on an electrically heated platform or by passing the flame of a Bunsen burner underneath the slides on a metal rack.The stain on the slides should steam. Allow slides to sit for 5 minutes after heating; do not allow them to dry out. Wash the slides in distilled water (note: tap water may contain acid-test bacilli). Drain off excess liquid.
3 Rood slides with 3% HCI in 95% ethanol (decolorizer) for approximately 1 minute. Check to see that no more red color runs off the surface when the slide is tipped. Add a bit more decolorizer for very thick slides or those that continue to "bleed" red dye. Wash thoroughly with water and remove the excess.
4 Flood slides with methylene blue (counterstain) and allow to remain on surface of slides for 1 minute. Wash with distilled water and stand slides upright on paper towels to air dry. Do not blot dry.
5 Examine microscopically, screening at 400x magnification and confirm all suspicious (i.e„ red) organisms at 1000x magnification using an oil-immersion lens.
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