1. The Size of It All Types of Microscopes

2. The Size of It All Remember that 1 inch = 2.54 cm and that 1 meter contains 1000000 micrometers (µm) or 1000000000 nanometers (nm) Microorganisms are measured in µm or nm.

3. Question?? If a typical E.coli is 1 µm in length, how many inches would that be? 1 µm x 1m x 100 cm x 1 in. = ? 1000000µm 1 m 2.54cm 0.00003937 inches!!

4. Types of Microscopes Light Microscopes: 1.Compound Light Microscope (LM) 2.Dissection or Stereoscope 3.Darkfield Microscope 4.Phase-Contrast Microscope 5.Differential Interference Contrast (DIC) 6.Fluorescence Microscopy 7.Confocal Microscopy

5. Types of Microscopes Electron Microscopes: 1.Transmission Electron Microscope (TEM) 2.Scanning Electron Microscope (SEM) Scanned Probe Microscopes: 1.Scanning Tunneling Microscope (STM) 2.Atomic Force Microscope (AFM)

6. Compound Light Microscope Uses visible light Magnifies up to 2000x, but generally only 1000x Good for: magnification resolution refractive index bright field illumination can examine live organisms

7. Darkfield Microscope Good for examining live organisms Phase-Contrast Microscope Can be used to visualize internal organelles of a cell Uses one beam of light Differential Interference Contrast Similar to phase-contrast except it uses two beams of light

8. Fluorescence Microscope Used to rapidly detect and identify microbes in tissues or clinical specimens Technique: fluorescent antibody (FA) or immunofluorescence. Specific antibodies are treated so that they are fluorescent. The antibodies are then applied to the specimen, if the antigens are present the specimen will appear to fluoresce.

9. Confocal Microscopes Similar to fluorescent microscopy except it uses laser light Transmission Electron Microscope An ultra thin section of specimen is placed on a copper mesh grid where a beam of electrons pass through to an electromagnetic objective lens, which magnifies the image. The final image is seen as light and dark areas, referred to as a transmission electron micrograph. Disadvantages: 1. requires a very thin slice of the specimen 2. no 3-D aspect 3. specimens must be fixed, stained and placed under a vaccum which can cause distortion and shrinkage.

10. Scanning Electron Microscope Provides a 3-D image, the stream of electrons are provided by an electron “gun” The image is referred to as a scanning electron micrograph Advantages: intact cells and viruses can be seen Disadvantages: surface structure only

11. Scanned Probe Microscopy Uses a probe to map atomic and molecular shapes (surface). Can be used to determine magnetic, chemical and temperature characteristics of the cell.

12. Making Things Visible - Stains Staining: A technique used in which color (dye) is added to a specimen to aid in visualizing structures. Stains (Dyes): Basic dyes adhere to anions. Acidic dyes adhere to cations. *Bacteria have a slightly negative charge at neutral pH so they require basic dyes.

13. Simple Stains: A single stain is used to high light basic cellular shape and structure. (Mordants maybe required to intensify the stain.) Differential Stains: A series of dyes are used to distinguish between various organisms and structures. Generally a primary stain is applied followed by a rinse or decolorizer and then finished with a counterstain to color structures that did not retain the primary stain.

14. Making A Slide Label the slide Apply the specimen to the slide Fixing the specimen to the slide – Heat fixing – Spray fixative (generally alcohol based) – Wax setting – Frozen

15. The “How” of the Gram Stain 1.Crystal Violet (primary stain) enters the cytoplasm of all bacteria. 2.Gram’s Iodine (mordant) forms a large chemical complex with the crystal violet. 3.Decolorizer dehydrates the peptidoglycan layer of the gram positive cells making it harder for the crystal violet- iodine complex to leave the cell. This will cause the gram positive bacteria to be seen as bluish-purple. In gram negative cells the decolorizer dissolves the outer membrane leaving small holes that the crystal violet-iodine complex to wash out – leaving the cell “colorless”. 4. Safranin (counterstain) enters the cytoplasm of the bacteria allowing the gram negative bacteria to be seen as reddish- pink.

16. It’s About the Cell Wall Gram Positive Cells: the cell walls have a thick layer of peptidoglycan and lower lipid content. The cell walls will dehydrate when exposed to the decolorizer retaining the crystal violet- iodine complex. Gram Negative Cells: the cell walls have a thinner peptidoglycan layer and a layer of lipopolysaccharides. The outer layer will dissolve when exposed to decolorizer leaving small holes that will allow the crystal violet- iodine complex to diffuse out of the cell.

17. Types of Differential Stains Gram’s Stain: (1884) Hans Christian Gram 1.Crystal Violet (primary stain) – up to 1 minute 2.Gram’s Iodine (mordant) – 30 seconds 3.Decolorizer – 50/50 alcohol acetone – 10 seconds 4.Safranin (counterstain) – 30 seconds **Remember which stain the cell retains depends on the cell wall structure.

18. Gram Positive Cocci Gram Positive Rods Gram Negative CocciGram Negative Rods

19. ACID FAST STAIN Used for bacteria with a waxy coat, primarily used for mycobacterium and nocardia. Technique: 1.Carbol fuchsin (primary stain) 2.Decolorizer 3.Methylene Blue (counter stain)

20. Wright’s Stain Also called Wright’s Giemsa Stain Used for differential staining of blood cells. Technique: Methylene Blue (primary stain) - basic Phosphate buffer rinse Eosin (counter stain) - acidic

21. Special Stains Special stains are used to visualize specific parts of the cell. 1.Capsule stain – generally done as a negative staining technique where the background is colored not the specimen. India Ink is used to visualize the Capsules around the yeast.

22. 2. Endospore stain: stains the spore portion of the bacteria. Fulton Endospore Stain is the most commonly used. Technique: Malachite Green (primary) Water Rinse Safranin (counter stain)

23. 3. Flagella stain: staining technique developed to visualize the flagellum. It is a combination of carbol fuschin and a mordant. 4. Trichrome Stain: often used for the identification of parasites. It is a combination of acid fuschin and picric acid.