Chapter 3. Observing Microorganisms Through a Microscope

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1 Chapter 3 Observing Microorganisms Through a Microscope

3 Microbial Size Macroscopic organisms can be measured in the range from meters (m) to centimeters (cm) Microscopic organisms fall into the range from millimeters (mm) to micrometers (μm) to nanometers (nm) Viruses measure between nm Smallest bacteria measure around 200 nm Average bacteria measure 1 um Human cells average 10 to 15 um Protozoa and algae measure 3 4 mm

4 -6 1 micron = 1 x 10 meters = meters

5 The Size of Things Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Macroscopic View 1 mm Louse Range of human eye Microscopic View Reproductive structure of bread mold 100 µm Colonial alga (Pediastrum) Range of light microscope 10 µm 1 µm Red blood cell Most bacteria fall between 1 and 10 µm insize Escherichia coli bacteria 200 nm 100 nm Range 10 nm of electron microscope 1 nm Require special microscopes 0.1 nm (1 Angstrom) Mycoplasma bacteria AIDS virus Polio virus Large protein Diameter of DNA Amino acid (small molecule) Hydrogen atom Flagellum

6 Microscopes and Magnification. Unaided eye 200 μm 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 μ m E. coli bacteria T-even bacteriophages (viruses) Atomic force microscope 0.1 nm 10nm DNA double helix

8 Units of Measurement 1 µm = 10 6 m = 10 3 mm 1 nm = 10 9 m = 10 6 mm 1000 nm = 1 µm µm = 1 nm Note: the average human cell is 10 to 15 um and the average prokaryote is 1 um

9 A simple microscope has only one lens Anton van Leeuwenhoek s microscopic He made observations of pond water & fabric fibers Lens Location of specimen on pin Specimen-positioning screw Focusing control Stage-positioning screw Microscope replica

10 Light Microscopy The use of any kind of microscope that uses visible light to observe specimens // simple staining required to increase contrast Types of light microscopy Compound light microscopy Darkfield microscopy Phase-contrast microscopy Differential interference contrast microscopy Fluorescence microscopy Confocal microscopy

11 The compound light microscope. Ocular lens (eyepiece) Remagnifies the image formed by the objective lens Body tube Transmits the image from the objective lens to the ocular lens Arm Fine focusing knob Coarse focusing knob 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 Base Principal parts and functions

12 Compound Light Microscopy In a compound microscope, the image from the objective lens is magnified again by the ocular lens Total magnification = objective lens ocular lens

13 Principles of Light Microscopy Power of Objective Usual power of ocular Total magnification 4x scanning objective 10x 40x 10x low power objective 10x 100x 40x high dry objective 10x 400x 100x oil immersion objective 10x 1000x

14 The compound light microscope. Ocular lens Line of vision Path of light Prism The path of light (bottom to top) Body tube Objective lenses Specimen Condenser lenses Illuminator Base with source of illumination

15 Compound Light Microscopy Resolution is the ability of the lenses to distinguish two points A microscope with a resolving power of 0.4 nm can distinguish between two points 0.4 nm Shorter wavelengths of light provide greater resolution the human eye can resolve two objects that are no closer than 0.2 mm apart

16 The Effect of Wavelength on Resolution (a) Low resolution (b) High resolution

17 Compound Light Microscopy The refractive index is a measure of the light-bending ability of a medium The light may bend in air so much that it misses the small high-magnification lens Immersion oil is used to keep light from bending

$Refraction in the compound microscope using an oil immersion objective lens.$

18 Refraction in the compound microscope using an oil immersion objective lens. Unrefracted light Immersion oil Oil immersion objective lens Without immersion oil most light is refracted and lost Air Glass slide Condenser lenses Condenser Light source Iris diaphragm

19 Brightfield, darkfield, and phase-contrast microscopy. Eye Eye Eye Ocular lens Objective lens Specimen Condenser lens Only light reflected by the specimen is captured by the objective lens Unreflected light Opaque disk Ocular lens Diffraction plates Undiffracted light (unaltered by specimen) Objective lens Refracted or diffracted light (altered by specimen) Specimen Condenser lens Annular diaphragm Light Light Light Dark objects are visible against a bright background Brightfield Light reflected off the specimen does not enter the objective lens

20 Brightfield, darkfield, and phase-contrast microscopy. Eye Eye Eye Ocular lens Objective lens Specimen Condenser lens Only light reflected by the specimen is captured by the objective lens Unreflected light Opaque disk Ocular lens Diffraction plates Undiffracted light (unaltered by specimen) Objective lens Refracted or diffracted light (altered by specimen) Specimen Condenser lens Annular diaphragm Light Light Light Light objects are visible against a dark background Light reflected off the specimen enters the objective lens Darkfield

$Eye Eye Eye Ocular lens Objective lens Specimen Condenser lens Only light reflected by the specimen is captured by the objective lens Unreflected light Opaque disk Ocular lens Diffraction plates$

21 Brightfield, darkfield, and phase-contrast microscopy. Eye Eye Eye Ocular lens Objective lens Specimen Condenser lens Only light reflected by the specimen is captured by the objective lens Unreflected light Opaque disk Ocular lens Diffraction plates Undiffracted light (unaltered by specimen) Objective lens Refracted or diffracted light (altered by specimen) Specimen Condenser lens Annular diaphragm Light Light Light Accentuates diffraction of the light that passes through a specimen Phase-contrast

$Microscopy Accentuates diffraction$ of the light that passes through a

22 Differential Interference Contrast Microscopy Accentuates diffraction of the light that passes through a specimen; uses two beams of light

23 Fluorescence Microscopy Uses UV light Fluorescent substances absorb UV light and emit visible light Cells may be stained with fluorescent dyes (fluorochromes) The principle of immunofluorescence.

the dyes The light illuminates each plane in a specimen

24 Confocal Microscopy Cells are stained with fluorochrome dyes Short-wavelength (blue) light is used to excite the dyes The light illuminates each plane in a specimen to produce a three-dimensional image // Up to 100 µm deep

25 Two-Photon Microscopy Cells are stained with fluorochrome dyes Two photons of long-wavelength light are used to excite the dyes Used to study cells attached to a surface // Up to 1 mm deep

study cells attached to a surface Resolution 1 µm

26 Scanning Acoustic Microscopy (SAM) Measures sound waves that are reflected back from an object Used to study cells attached to a surface Resolution 1 µm Scanning acoustic microscopy (SAM) of a bacterial biofilm on glass.

27 Electron Microscopy Uses electrons instead of light The shorter wavelength of electrons gives greater resolution Transmission Electron Microscopy (TEM) Used to observe viruses and internal structures of cells Ultrathin sections of specimens Light passes through specimen, then an electromagnetic lens, to a screen or film Specimens may be stained with heavy-metal salts 10, ,000 ; resolution 2.5 nm

28 Transmission and scanning electron microscopy. Electron beam Electromagnetic condenser lens Specimen Electromagnetic objective lens Electromagnetic projector lens Fluorescent screen or photographic plate Electron gun Viewing eyepiece Secondary electrons Specimen Primary electron beam Electromagnetic lenses Viewing screen Electron collector Amplifier Transmission

29 Scanning Electron Microscopy (SEM) An electron gun produces a beam of electrons that scans the surface of a whole specimen Secondary electrons emitted from the specimen produce the image 1,000 10,000 /// resolution 20 nm

30 Transmission and scanning electron microscopy. Electron beam Electromagnetic condenser lens Specimen Electromagnetic objective lens Electromagnetic projector lens Fluorescent screen or photographic plate Electron gun Viewing eyepiece Secondary electrons Specimen Primary electron beam Electromagnetic lenses Viewing screen Electron collector Amplifier Scanning

31 Scanned-Probe Microscopy Scanning tunneling microscopy (STM) uses a metal probe to scan a specimen Resolution 1/100 of an atom

32 Scanned-Probe Microscopy Atomic force microscopy (AFM) uses a metal-anddiamond probe inserted into the specimen Produces threedimensional images

33 Preparing Specimens for the Microscope Specimens are usually prepared by mounting a sample on a suitable glass slide that sits on the stage between the condenser and the objective lens The manner in which it is prepared depends on the condition of the specimen, either living or preserved the aims of the examiner: to observe overall structure, identify microorganisms, or see movement the type of microscopy available: bright field, dark field, phase contrast, or fluorescence

34 Preparing Smears for Staining Smear: a thin film of a solution of microbes on a slide A smear is usually fixed to attach the microbes to the slide /// Note: Gently heating slide will kill and fix (attach) bacteria to the slide Staining: coloring the microbe with a dye that emphasizes certain structures

35 Preparing Smears for Staining Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens. Stains (are salts) consisting of a positive and negative ion basic dyes have a positive charge // In a basic dye, the chromophore is a cation (cells have mostly negative charges associated with their structures) acidic dyes have a negative charge // In an acidic dye, the chromophore is an anion Staining the background instead of the cell is called negative staining

36 Three Staining Procedures: Simple Stains, Differential, and Special Simple stain: use of a single basic dye A mordant may be used to hold the stain or coat the specimen to enlarge it

37 Simple Stains Simple Stains (a) Crystal violet stain of Escherichia coli (b) Methylene blue stain of Corynebacterium

38 Differential Stains Used to distinguish different types of bacteria Two different stains used in these procedures Used in the identification of bacteria E.g. Gram stain (distinguishes the type of cell wall // gram positive or negative) Note: Gram stain most useful in medicine to determine which antibiotic to prescribe! E.g. Acid-fast stain (detects unique lipid common in pathogenic bacteria // tuberculosis and lepersy)

39 Differential Stains Differential Stains (a) Gram stain. Purple cells are gram-positive. Pink cells are gram-negative. (b) Acid-fast stain. Red cells are acid-fast. Blue cells are non-acidfast. (c) Spore stain, showing endospores (red) and vegetative cells (blue)

40 Gram Stain Classifies bacteria into gram-positive or gram-negative Gram-positive bacteria tend to be killed by penicillin and detergents Gram-negative bacteria are more resistant to antibiotics Tells us something about structure of the bacteria s cell wall

41 Gram Stain Primary Stain: Crystal Violet Mordant: Iodine Decolorizing Agent: Alcohol-Acetone Counterstain: Safranin Color of Gram-Positive Cells Purple Purple Purple Purple Color of Gram-Negative Cells Purple Purple Colorless Red

The Gram Stain: Developed by Hans Christian Gram (1884) Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Step 1.

42 The Gram Stain: Developed by Hans Christian Gram (1884) Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Step 1. Crystal violet First, crystal violet is added to the cells in a smear. It stains them all the same purple color. Microscopic Appearance of Cell Gram (+) Gram ( ) Chemical Reaction in Cell Wall (very magnified view) Gram (+) Gram ( ) Both cell walls affix the dye 2. Gram s iodine Then, the mordant, Gram s iodine, is added. This is a stabilizer that causes the dye to form large complexes in the peptidoglycan meshwork of the cell wall. The thicker gram-positive cell walls are able to more firmly trap the large complexes than those of the gram-negative cells. Dye complex trapped in wall No effect of iodine 3. Alcohol Application of alcohol dissolves lipids in the outer membrane and removes the dye from the peptidoglycan layer only in the gram-negative cells. 4. Safranin (red dye) Because gram-negative bacteria are colorless after decolorization, their presence is demonstrated by applying the counterstain safranin in the final step. McGraw-Hill Companies, Inc. Crystals remain in cell wall Red dye masked by violet Outer membrane weakened; wall loses dye Red dye stains the colorless cell

43 Gram Staining Rod (gram-negative) Cocci (gram-positive)

44 Acid-Fast Stain Originated as a method to detect Mycobacterium tuberculosis // Also used to identify Leprosy Differentiates acid fast bacteria (pink) from non acid fast bacteria (blue) These bacteria have cell walls that are particularly impervious that holds fast (tightly or tenaciously) to the dye (carbol fuschin pink color) when washed with an acid alcohol decolorizer Stained waxy cell wall is not decolorized by acid-alcohol Also used in identification of other medically important bacteria, fungi, and protozoa

45 Acid-Fast Stain Color of Acid-Fast Color of Non Acid-Fast Primary Stain: Carbolfuchsin Red Red Decolorizing Agent: Acid-alcohol Red Colorless Counterstain: Methylene Blue Red Blue

46 Acid-fast bacteria. M. bovis Pink cells are acid fast bacteria // Cells counter stained with blue dye are not acid fast bacteria.

47 Special Stains Used to distinguish parts of cells Capsule stain Endospore stain Flagella stain

48 Special Stain / Negative Staining Illustrates Capsules Capsules Capsules

49 Special Staining // Endospore Staining Primary stain: malachite green, usually with heat Decolorize cells: water Counterstain: safranin

50 Special Staining // Endospore Staining Endospore Endospore staining

51 Special Staining // Flagella Staining Mordant used to attach stain to flagella so it appears thicker Carbolfuchsin attached to mordant stains flagella

52 Special Staining // Flagella Staining Flagellum Flagella staining

Observing Microorganisms through a Microscope

2016/2/19 PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College CHAPTER 3 Observing Microorganisms through a Microscope 1 Figure 3.2 Microscopes and Magnification.