Microscopy, Staining, and Classification

Transcription

1 CSLO CHECK CSLO1. Describe distinctive characteristics and diverse growth requirements of prokaryotic organisms compared to eukaryotic organisms. PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 4 PSLO3. Will demonstrate proficiency and safe practice sin the use of laboratory equipment and basic laboratory techniques. Microscopy, Staining, and Classification

2 CH4-section1 Principles behind Microscopy Types of Microscopes Electron Microscopes Light Microscopes Probe Microscopes Different staining methods for Microscopy

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. Light Microscopes Electron Microscopes Probes Microscopes

4 CONCEPT General Principles of Microscopy which influence observation of microbes FACT 4.1 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 CONCEPT General Principles of Microscopy FACT 4.1 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 Need both high Resolution and Magnification High Magnification but Low Resolution

7 To increase A) Magnification and B) Resolution Use smaller wavelength CONCEPT 4.2 VIBGYOR Smaller the object = Use smaller wavelength

8 Light vs Electron microscope CONCEPT 4.2 Visible Light Electron has very small wavelength compared to visible light Which microscope will let you see smaller things?

9 Light vs Electron microscope Uses visible light FACT 4.2 Uses electron glass lenses magnet 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 CONCEPT 4.3 Light changes direction (refracts) when it moves from one medium to another Light Air Air Glass

12 polish glass Magnification = 50/5 = 10X CONCEPT 4.4 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 10X, 40X, 100X Have one or two ocular lenses 10X 10X CONCEPT 4.5 FACT 4.3 Total magnification = magnification of objective lens X magnification of ocular lens e.g. 10 times X 10 times = 100 times 10X 40X 100X

15 CONCEPT 4.3 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 FACT 4.4 immersion oil reduces scattering of light 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 immersion oil Increases magnification and resolution

17 50 µm Staining increases contrast FACT 4.5 Brightfield (unstained specimen) Brightfield (stained specimen) Staining kills cell

18 Different types of Microscopy FACT 4.6 Light Microscopy Four kinds of light microscopy 1) Bright-field microscopes show bright background

19 FACT 4.7 2) Dark-field microscopy Specimen appears light against dark background Increases contrast and enables observation of more details Examine live specimens DarkBright-field microscopes show dark background Bacterium Bright field 1) 2) Dark field

20 3) Phase Microscopy 2 types 3A) Phase-contrast microscopy 3B) Differential interference contrast microscope aka Nomarski Phase contrast Nomarski FACT 4.8 Contrast is created by combining light waves in or out of phase Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining Shows 3D Light rays in phase produce brighter image, while light rays out of phase produce darker image.

21 4) Fluorescent microscopes Direct UV light source at specimen Fluorescent stains absorb UV and radiates energy back as a longer, visible wavelength UV FACT 4.9 UV light increases resolution and contrast

22 4A) Fluorescence microscopy 3 types Can help you look at object of interest only FACT 4.10

23 4B) Immunofluorescence FACT 4.11 help you detect specific pathogens by attaching fluorescent dyes to the antibody specific for that pathogen Antibodies Fluorescent dye Bacterium Cell-surface antigens Antibodies carrying dye Bacterial cell with bound antibodies carrying dye

24 4C) Confocal microscopes FACT 4.12 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 2 types of electron microscopes Transmission electron microscopes Scanning electron microscopes FACT 4.13

26 Figure 4.11 A transmission electron microscope (TEM) FACT 4.13 All electron microscopy needs vacuum - Why? Consequences = Only dead objects seen CONCEPT? Light microscope (upside down) Column of transmission electron microscope Lamp Electron gun Condenser lens Specimen Specimen Objective lens Objective lens (magnet) Eyepiece Final image seen by eye Projector lens (magnet) Final image on fluorescent screen TEM needs sections - why? TEM sees transmitted electrons

27 Figure 4.12 Scanning electron microscope (SEM). SEM sees scattered electrons SEM sees surface of microbes

28 Probe Microscopy 2 types Probe travels over specimen FACT 4.14 Scanning tunneling microscopes (highest magnification and resolution) Atomic force microscopes

29 Table 4.2 Comparison of Types of Microscopes (1 of 3)

30 Table 4.2 Comparison of Types of Microscopes (2 of 3)

31 Table 4.2 Comparison of Types of Microscopes (3 of 3)

32 Staining for Light Microscopy Principles of Staining Simple Staining = one stain/dye Differential Staining = more than one stain to differentiate between different types of microbes CONCEPT 4.6 FACT Basic dyes Basic dyes (positively charged) stain acidic (negatively charged) structures Acidic dyes (negatively charged) stain alkaline structures

33 Inside of most bacterial cells is negatively charged + Basic dyes CONCEPT 4.6 Therefore Basic dyes are more commonly used to stain bacterial cells Positive Staining

34 Inside of most bacterial cells is negatively charged CONCEPT 4.6 Acidic dyes Therefore Acidic dyes are more commonly used to stain background Negative Staining

35 FACT 4.16 Gram Staining Mechanism

36 FACT 4.16 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 Table 4.3 Some Stains Used for Light Microscopy

41 Staining for Electron Microscopy FACT 4.17 CONCEPT 4.7 Chemicals containing heavy metals are used as stains for electron microscopy transmission electron microscopy

42 CH4-section2 Different Methods to identify microbes 1 Staining Methods How to name microbes How to classify microbes 2 3 Other Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids Taxonomy

43 Microbe Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids

44 Physical characteristics CONCEPT 4.8 Protozoa, fungi, algae, and parasitic worms can often be identified based only on their morphology Some bacterial colonies have distinct appearance used for identification

45 Gas bubble Inverted tubes to trap gas CONCEPT 4.9 Biochemical tests Acid with gas Acid with no gas Inert Hydrogen sulfide produced No hydrogen sulfide

46 Serological (Blood) Tests CONCEPT 4.10 An agglutination test Antibody-Antigen Complex causes agglutination ZIKA virus Ebola virus Patient Blood (contains antibodies) plus add virus on slide

47 Phage typing Identifying bacteria based on which phage infects and lyses the bacteria Phage T4 Phage T7 Bacteria A Plaques No Plaques CONCEPT 4.11 Bacterial lawn Plaques (holes) Bacteria B No Plaques Plaques

48 Analysis of nucleic acids CONCEPT 4.12 Nucleic acid sequence can be used to classify and identify microbes Prokaryotic taxonomy now includes the G + C content of an organism's DNA

49 Taxonomic Keys Dichotomous keys CONCEPT 4.13 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

50 Different Methods to identify microbes 1 Staining Methods How to name microbes How to classify microbes 2 3 Other Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids Taxonomy

51 Binomial nomenclature CONCEPT 4.14 naming species of living things by giving each species a name composed of two parts Each species has a unique name humans belong to the genus Homo species Homo sapiens. Homo sapiens noun-genus Capitalized adjective-specific epithet

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

53 X X Y Genus specific epithet Genus specific epithet Genus specific epithet Y Genus specific epithet Escherichia coli Escherichia coli E. coli

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

55 Microbe Naming Rules CONCEPT 4.14 Genus is based on shape and arrangement specific epithet is based on unique characteristic (Genus) (specific epithet) (Arrangement+shape) (unique characteristic) Streptobacillus pnemoniae Escherichia coli

56 cocci bacilli shape arrangement Streptococci Streptobacilli Staphylococci

57 Lets name this microbe Genus specific epithet Staphylococcus partyanimalia

58 Different Methods to identify microbes 1 Staining Methods How to name microbes How to classify microbes 2 3 Other Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids Taxonomy consists of Identification, Nomenclature and classification

59 CONCEPT 4.15 Taxonomy consists of identification, nomenclature, and classification Why do Taxonomy? Organize large amounts of information about organisms Make predictions based on knowledge of similar organisms

60 FACT 4.19 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

61 Domains are highest taxonomic units Carl Woese compared nucleotide sequences of rrna subunits Proposal of three domains as determined by ribosomal nucleotide sequences genetic information Eukarya, Bacteria, and Archaea FACT 4.20 CONCEPT 4.16 Goal of modern taxonomy is to reflect phylogenetic hierarchy not just morphology but understanding genetic relationships among organisms build phylogenetic or evolutionary trees Bat vs Bird