Microscopy, Staining, and Classification

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1 PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 4 Microscopy, Staining, and Classification

2 Figure 3.4 Approximate size of various types of cells. ~10 um Red Blood Cells 1.5mm 1500 um Width of penny = 1500

3 Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes.

4 Official definitions Magnification, the ratio of an object s image size to its real size Resolution, the measure of the clarity of the image, or the minimum distance of two distinguishable points Contrast, visible differences in brightness between parts of the sample

5 Microscopy General Principles of Microscopy Magnification (enlarge) Resolution (tell apart 2 objects close together) Contrast (Differences in intensity between two objects)

6 General rule for any microscopy/detector Smaller the wavelength smaller the object you can see (small objects need small hands) Think about giant fingers and texting

7 Figure 4.1 The electromagnetic spectrum. Smaller the object = Use smaller wavelength VIBGYOR

8 Microscopy Microscopes are used to visualize cells In a light microscope (LM), visible light is passed through a specimen and then through glass lenses Glass lenses focuses light and enlarges and resolves objects

$Lenses refract (bend) the light, so that the image is magnified Light Air Glass Magnification Magnify$

9 Lenses refract (bend) the light, so that the image is magnified Light Air Glass Magnification Magnify using = 50/5 lenses = 10X Focal point 5 50 Specimen Convex lens Inverted, reversed, and Enlarged image

10 Microscopy Light Microscopy Bright-field microscopes Simple single lense Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms

11 Microscopy Light Microscopy Bright-field microscopes Compound multiple lenses More than one - Series of lenses for magnification Light passes through specimen into objective lens Have one or two ocular lenses Total magnification = magnification of objective lens X magnification of ocular lens E.g. 10 times X 10 times = 100 times

12 Figure 4.4 A bright-field, compound light microscope. occular lens Line of vision Ocular lens Remagnifies the image formed by the objective lens Body Transmits the image from the objective lens to the ocular lens using prisms Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base objective lens

$Special type of objective lens = oil immersion lens Microscope objective Lenses Microscope objective Refracted light rays lost to lens Glass cover slip More light enters lens Glass cover slip$

13 Figure 4.5 The effect of immersion oil on resolution. Special type of objective lens = oil immersion lens Microscope objective Lenses Microscope objective Refracted light rays lost to lens Glass cover slip More light enters lens Glass cover slip Immersion oil Slide Slide Specimen Light source Light source Without immersion oil With immersion oil Increases magnification and resolution

14 Microscopy General Principles of Microscopy Contrast Differences in intensity between two objects, or an object and its background Increase contrast by staining

15 Figure 6.3ab Brightfield (unstained specimen) 50 µm Brightfield (stained specimen) Staining increases contrast

16 Figure 4.16 Simple stains. Staining increases contrast

17 Staining Principles of Staining Dyes used as stains are usually salts Chromophore is the colored portion of the dye Acidic dyes (negatively charged) stain alkaline structures Basic dyes (positively charged) stain acidic structures Basic dyes are more commonly used since inside of most cells is negatively charged

18 Staining Simple stains Differential stains Gram stain Acid-fast stain Special stains Negative stain Flagellar stain Endospore stain

19 Staining Most microorganisms are difficult to view by brightfield microscopy Coloring specimen with stain increases contrast and resolution Specimens must be prepared for staining

20 Figure 4.15 Preparing a specimen for staining.

21 spirogyra

23 Figure 3.12 Bacterial shapes and arrangements. 3 Shapes 1) coccus 2) bacillus 3) spirillum 1) staphylo 2) strepto 2 main arrangements

24 X X Genus species Genus species X Genus species Microbe Naming Rules Binomial Nomenclature Genus species G. species Escherichia coli Escherichia coli E. coli Genus species G. species E. coli

25 Microbe Naming Rules Based on shape and arrangement Arrangement+shape species Staphylococcus species Streptobacillus species

26 cocci bacilli shape arrangement Staphylococci Streptococci Streptobacilli

28 Negative staining

Rhodospirillum rubrum This large (0.7-1.

29 Rhodospirillum rubrum This large ( µm), nonpathogenic gram negative spirillum exhibits polar amphitrichous flagellation and a characteristic red pigment.

30 Rhodospirillum rubrum

Lab 1, 2 and 3: Stain, Observe and Identify the Microbes. BIOHAZARD Rules. VIOLATORS will lose points. A) Lab Safety Rules Lab Safety Form Signup

Lab 1, 2 and 3: Stain, Observe and Identify the Microbes. BIOHAZARD Rules. VIOLATORS will lose points. A) Lab Safety Rules Lab Safety Form Signup MICROLAB PREPARATIONS A) Lab Safety Rules Lab Safety Form Signup B) Lab Participation Instructor Review Peer Review Form C) Biohazard Rules How to dispose Trash REQUIRED Items: 1) LAB Manual/Journal 2)