Ch 3: Observing Microorganisms Through a Microscope

Transcription

1 Ch 3: Observing Microorganisms Through a Microscope

2 SLOs Review the metric units of measurement Define total magnification and resolution Explain how electron and light microscopy differ Differentiate between acidic and basic dyes Compare simple, differential, negative, and special stains List the steps in preparing a Gram stain. Describe the appearance of Gram-positive and Gram-negative cells after each step Compare and contrast Gram stain and acid-fast stain Explain why endospore and capsule stains are used

3 SLOs cont.: Check Your Understanding If a microbe measures 10 μm in length, how long is it in nanometers? What does it mean when a microscope has a resolution of 0.2 nm? Why do electron microscopes have greater resolution than light microscopes? Why doesn t a negative stain color a cell? Why is the Gram stain so useful? Which stain would be used to identify microbes in the genera Mycobacterium and Nocardia? How do unstained endospores appear? Stained endospores?

4 Units of Measurement Review Table µm = m = mm 1 nm = m = mm 1000 nm = µm µm = nm

5 Figure 3.2 Microscopes and Magnification. Unaided eye 200 m Light microscope 200 nm 10 mm Scanning electron microscope 10 nm 1 mm Tick Actual size Red blood cells Transmission electron microscope 10 pm 100 m E. coli bacteria T-even bacteriophages (viruses) Atomic force microscope 0.1 nm 10nm DNA double helix Foundation Fig 3.2

6 Sizes Among Microorganisms Protozoa: 100 µm Yeasts: 8 µm Cells Alive How big is a...? Bacteria: 1-5 µm (some much longer than wide) Rickettsia: 0.4 µm = nm Chlamydia and Mycoplasma: 0.25 µm Viruses: nm

7 Principles of the Compound Light Microscope Magnification: Ocular and objective lenses of compound microscope (total mag.?) Resolution: Ability of lens to... Maximum resolving power depends on... For light microscope: m Contrast: Stains change refractive index contrast between bacteria and surrounding medium Fig 3.1

8 Refractive Index Measures light-bending ability of a medium Light may bend in air so much that it misses the small high-magnification lens. Immersion oil is used to keep light from bending. Fig 3.3

9 Microscopy: The Instruments Brightfield Microscopy Simplest of all the optical microscopy illumination. techniques Dark objects are visible against a bright background. Darkfield Microscopy Light objects visible against dark background. used to enhance the contrast in unstained samples. Instrument of choice for spirochetes Fig 3.4

10 Spirochetes (Treponema pallidum) viewed with darkfield microscope

11 Fluorescence Microscopy Uses UV light. Fluorescent substances absorb UV light and emit visible light. Cells may be stained with fluorescent chemicals (fluorochromes). Immunofluorescence Fig 3.6; T. pallidum

12 Figure 3.6a Fig 3.6 Principle of Immunofluorescence

13 Electron Microscopy: Detailed Images of Cell Parts Uses electrons, electromagnetic lenses, and fluorescent screens Electron wavelength ~ 100,000 x smaller than visible light wavelength Specimens may be stained with heavy metal salts Two types of EMs:?

14 SEM or TEM? Bacterial division Compare to Fig 3.10 Leaf surface

15 ? 10, ,000 ; resolution 2.5 nm.

16 Preparation of Specimens for Light Microscopy Staining Techniques Provide Contrast Smear air-dry heat-fix Basic dyes: cationic chromophore Acidic dyes: anionic chromophore negative staining (good for capsules) Three types of staining techniques: Simple, differential, and special

17 Simple Stains Use a single basic dye. A mordant may be used to hold the stain or coat the specimen to enlarge it. Differential Stains React differently with different bacteria Gram stain Acid fast stain

18 Figure 3.12 Gram staining. Gram-positive Gram-negative Application of crystal violet (purple dye) Application of iodine (mordant) Alcohol wash (decolorization) Application of safranin (counterstain) Rod (gram-negative) Cocci (gram-positive) Fig 3.12

19 Gram Stain Compare to Fig 3.12 crystal violet safranin

20

21 Gram Stains using Compound Light Microscope Streptococcus mutans Bacillus anthracis

22 Negative Stain Observe cell shape and size Fig 3.14 Used for bacteria with capsules

23 Acid Fast Stain Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. Non acid-fast cells lose the primary stain when rinsed with acid-alcohol, and are counterstained with a different color basic stain Fig 3.13

24 Special Stains Compare to Fig 3.14 Endospore stain: Heat is required to drive a stain into the endospore. Flagella staining: requires a mordant to make the flagella wide enough to see. Capsule stain uses basic stain and negative stain

25 Clinical Case: Microscopic Mayhem