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 4. Discuss how microscopy has revealed the structure and function of microorganisms.

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 3 General Principles of Microscopy which influence observation of microbes Magnification Resolution Contrast 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 3 General Principles of Microscopy A) Magnification (enlarge) B) Resolution (tell apart 2 objects close together) C) Contrast (Differences in intensity between two objects) How to increase all 3 of these to achieve optimal observation of microbes?

6 A) Magnification vs B) Resolution High Magnification but Low Resolution

7 How to increase A) Magnification and B) Resolution Use smaller wavelength VIBGYOR Smaller the object = Use smaller wavelength

8 General rule for any microscopy/detector Smaller the wavelength smaller the object you can see Light vs Electron microscope Visible Light Electron has very small wavelength compared to visible light

9 Light vs Electron microscope Visible Light Electron Magnifies objects 1000X Magnifies objects 100,000X

10 Light Microscopy 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

11 Light changes direction (refracts) when it moves from one medium to another Light Air Air Glass

12 polish glass Magnification = 50/5 = 10X Focal point 5 50 Specimen Convex lens Magnify using lenses Inverted, reversed, and Enlarged image

13 Light Microscopy Bright-field microscopes Simple Microscopes Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms for the first time in history Fresh pond water

14 Light Microscopy (Bright-field microscopes) Compound multiple lenses 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

15 Light changes direction (refracts) when it moves from one medium to another Light Air Air Glass

16 Figure 4.5 The effect of immersion oil on resolution. Oil immersion lens (100X) is a special type of objective 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 Without immersion oil Light source With immersion oil Oil immersion lens Increases magnification and resolution

17 50 µm Staining increases contrast Brightfield (unstained specimen) Brightfield (stained specimen)

18 Microscopy Light Microscopy 1) Bright-field microscopes Simple/ Compound

19 Figure 4.8 Four kinds of light microscopy. Bacterium Bright field 1) 2) Dark field 2) Dark-field microscopes Specimen appears light against dark background Best for observing pale objects Increases contrast and enables observation of more details

20 3) Phase Microscopy 2 types A) Phase-contrast microscopy B) Differential interference contrast microscope aka Nomarski Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining Phase contrast Nomarski Light rays in phase produce brighter image, while light rays out of phase produce darker image. Contrast is created because light waves are out of phase

21 4) Fluorescent microscopes Direct UV light source at specimen Fluorescent stains absorb UV and radiates energy back as a longer, visible wavelength UV UV light increases resolution and contrast

22 4A) Fluorescence microscopy. Can help you look at object of interest and avoid clutter

23 4B) Immunofluorescence. Can help you detect specific pathogens/objects Antibodies Fluorescent dye Bacterium Cell-surface antigens Antibodies carrying dye Bacterial cell with bound antibodies carrying dye

24 4C) Confocal microscopes Use UV lasers to illuminate fluorescent chemicals in a single plane Computer constructs 3-D image from digitized images

25 Microscopy Electron Microscopy Light microscopes cannot resolve structures closer than 200 nm Electron microscopes have greater resolving power and magnification than light microscopy Magnifies objects 10,000X to 100,000X Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms Two types Transmission electron microscopes Scanning electron microscopes

26 Figure 4.11 A transmission electron microscope (TEM). needs vacuum Light microscope (upside down) Column of transmission electron microscope Live? Lamp Electron gun Condenser lens Specimen Specimen Objective lens Objective lens (magnet) Eyepiece Projector lens (magnet) Final image seen by eye Final image on fluorescent screen need sections

27 Figure 4.12 Scanning electron microscope (SEM).

28 Probe Microscopy Magnifies more than 100,000,000 times Scanning tunneling microscopes Atomic force microscopes

29 Light Microscopy Staining Principles of Staining Dyes used as stains are usually salts Chromophore is the colored portion of the dye Basic dyes Basic dyes (positively charged) stain acidic (negatively charged) structures Acidic dyes (negatively charged) stain alkaline structures

30 Inside of most bacterial cells is negatively charged + Basic dyes Therefore Basic dyes are more commonly used to stain bacterial cells Positive Staining

31 Inside of most bacterial cells is negatively charged Acidic dyes Therefore Acidic dyes are more commonly used to stain background Negative Staining

32 Simple/Differential Staining? Positive/Negative Staining?

33

34 Gram Staining

35 Gram Staining Mechanism

36 Gram Staining Method Primary Stain Step Gram positive cell wall Crystal Violet Gram negative cell wall

37 Gram Staining Method Mordant Step Gram s Iodine

38 Gram Staining Method Decolorizing Step Alcohol

39 Gram Staining Method Counter Stain Step Safranin

40 Staining for Electron Microscopy Chemicals containing heavy metals used for transmission electron microscopy

41 Classification and Identification of Microorganisms Taxonomy consists of classification, nomenclature, and identification Organize large amounts of information about organisms Make predictions based on knowledge of similar organisms

42 Carolus Linnaeus and Taxonomic Categories His system classified organisms based on characteristics in common Grouped organisms that can successfully interbreed into categories called species Used binomial nomenclature

43 Binomial nomenclature naming species of living things by giving each species a name composed of two parts humans belong to the genus Homo species Homo sapiens. Homo sapiens noun-genus Capitalized adjective-specific epithet

44 members of Genus are very different Homo sapiens Homo naledi (2015)

45 Microbe Naming Rules Each species has a unique name Genus specific epithet G. specific epithet Genus specific epithet G. specific epithet Escherichia coli Escherichia coli E. coli E. coli

46 Microbe Naming Rules X X Y Genus specific epithet Genus specific epithet Genus specific epithet Y Genus specific epithet

47 Bacteria have shapes/morphology

48 Figure 3.12 Bacterial shapes and arrangements. 3 major shapes/morphology 1) coccus 2) bacillus 3) spirillum 1) staphylo 2) strepto 2 major arrangements

49 cocci bacilli shape arrangement Streptococci Streptobacilli Staphylococci

50 Microbe Naming Rules based on shape and arrangement (Arrangement+shape) (specific epithet) Streptobacillus (specific epithet) Staphylococcus (specific epithet)

51 Domains Carl Woese compared nucleotide sequences of rrna subunits Proposal of three domains as determined by ribosomal nucleotide sequences genetic information Eukarya, Bacteria, and Archaea Goal of modern taxonomy is to reflect phylogenetic hierarchy not just morphology but understanding genetic relationships among organisms Bat vs Bird

52 Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids

53 Physical characteristics Protozoa, fungi, algae, and parasitic worms can often be identified based only on their morphology Some bacterial colonies have distinct appearance used for identification

54 Gas bubble Inverted tubes to trap gasbiochemical tests Acid with gas Acid with no gas Inert Hydrogen sulfide produced No hydrogen sulfide

55 Serological Tests An agglutination test

56 Phage typing Bacterial lawn Plaques

57 Analysis of nucleic acids Nucleic acid sequence can be used to classify and identify microbes Prokaryotic taxonomy now includes the G + C content of an organism's DNA

58 Taxonomic Keys Dichotomous keys Series of paired statements where only one of two "either/or" choices applies to any particular organism Key directs user to another pair of statements, or provides name of organism

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