MICROSCOPY AND STAINING CHAPTER 3

2 Metric Units

3 Light Properties Wavelength

4 polarity Light is a wave Filters can block waves in off axis planes

5 Waves can be added ++ ==

6 Light Properties Resolution

7 Wavelength/Resolution Interaction

8 Light Properties Reflection Transmission

9 Light Properties Absorption Refraction bending

10 Light Microscopy Types Compound Bright Field

11 Oil immersion With oil without Some info lost Oil with intermediate refractive index

12 Microscopy — Dark Field

13 Microscopy — Phase Contrast Dual Beam

14 Phase contrast Phase-contrast microscopy was invented in 1936 by Frits Zernike, a Dutch mathematical physicist. It is based on the principle that cells differ in refractive index (a factor by which light is slowed as it passes through a material) from their surroundings. Light passing through a cell thus differs in phase from light passing through its surroundings. This subtle difference is amplified by a device in the objective lens of the phase-contrast microscope called the phase ring, resulting in a dark image on a light background (Figure 2.5b). The ring consists of a phase plate—the key discovery of Zernike—that amplifies the minute variation in phase. Zernike’s discovery of differences in contrast between cells and their background stimulated other innovations in microscopy, such as fluorescence and confocal microscopy (discussed below). For his invention of phase-contrast microscopy, Zernike was awarded the 1953 Nobel Prize in Physics.

15 Microscopy — DIC Differential Interference Contrast

16 DIC “differential interference contrast” Similar to phase contrast, but input light is polarized

17 Microscopy — Fluorescence Ultraviolet light flourescein

18 Advantages of fluorescence Can use specialized chemical probes that target specific features and then tag with fluorescent dyes Downside: must use expensive filters and excitory frequencies

19 Microscopy — Confocal Confocal Allows 3 dimensional viewing Allows multiple dyes to be overlaid

20 Confocal microscopy Allows 3 dimensions

21 Combined confocal and fluorescence Antibody labeling

22 Microscopy Imaging Digital

23 Fig. 2-15

24 Electron Microscopy Transmission (TEM) Scanning (SEM) Scanning Tunneling (STM)

25 TEM Most popular for bacteria. Allows imaging internal features, but requires heavy metal staining.

26 Electron Microscopy Images

27 Microscopy Techniques Wet Mounts Smears Staining

Fig  Slide Dry in air Flood slide with stain; rinse and dry Place drop of oil on slide; examine with 100  objective lens Spread culture in thin film over slide Pass slide through flame to heat fix Oil I. Preparing a smear III. Microscopy II. Heat fixing and staining

29 Staining cells - increases contrast Simple stain - one dye - shows size, shape, and arrangement Methylene blue - yeast Cheek cell

30 Common stains Safranin (*basic, + charge) Crystal violet red

31 Differential stains Use multiple dyes or dyes that interact with organisms differently. Primary stain / counterstain

32 Gram Stain Gram Stain The single most important stain in microbiology. Set the initial taxonomy of bacteria. Crystal violet *(basic stain)

33 Gram Stain

34 Acid Fast Stain Carbol-fuchsin stains acid fast organisms

35 Acid Fast The Ziehl-Neelsen stain, also known as the acid-fast stain, was first described by two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1894), a pathologist. It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis (TB). 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.

36 Acid Fast of Mycobacterium tuberculosis

37 Negative Stain Sometimes referred to as capsular stain India ink or nigrosin

38 Flagellar Stain Salmonella typhimurium

39 Endospore Stain Used on spore forming bacteria such as Bacillus sp. Malachite green stains spores