1896 1920 1987 2006
Lecture 3-1:
Microbial cell biology
Chapter 3 inBROCK BIOLOGY OF MICROORGANISMS
Zhao Liping, Chen Feng
School of Life Science and Technology,
Shanghai Jiao Tong University
http://micro.sjtu.edu.cn
Microbiology, Shanghai Jiao Tong University
I. Microscopy and cell morphology
3.1 Light Microscopy
3.2 Three-Dimentsional Imaging: Interference Contrast,
Atomic Force, and Confocal Scanning
3.3 Electron Mciroscopy
3.4 Cell Morphology and the Significance of Being Small
1896 1920 1987 2006
3.1 Light Microscopy
光学显微镜
Figure 2.1a
Ocular
lenses
Objective lens
Stage
Condenser
Focusing knobs
Light source
Specimen on
glass slide
Figure 2.1b
Visualized
image
Eye
Ocular lens
Intermediate image
(inverted from that
of the specimen)
Objective lens
Specimen
Condenser lens
Light source
None
100, 400,
1000
10
10, 40, or
100 (oil)
Magnification Light path
1896 1920 1987 2006
Staining: Increasing Contrast
for Bright-Field Microscopy
染色: 增加明视野显微镜的对比度
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Staining: Increasing Contrast
for Bright-Field Microscopy
Staining: Increasing Contrast
for Bright-Field Microscopy
Staining: Increasing Contrast
for Bright-Field Microscopy
Figure 2.3I. Preparing a smear
II. Heat fixing and staining
III. Microscopy
Spread culture in thin
film over slide
Pass slide through
flame to heat fix
Dry in air
Flood slide with stain;
rinse and dry
Place drop of oil on slide;
examine with 100
objective lens
Slide Oil
Steps of
staining
cells for
microscopic
observation
1896 1920 1987 2006
Differential Stains: The
Gram Stain
鉴定染色: 革兰氏染色法
Figure 2.4Step 1
Step 2
Step 3
Step 4
Result:All cells purple
Result:All cells
remain purple
Result:Gram-positive
cells are purple;
gram-negative
cells are colorless
Result:Gram-positive
(G+) cells are purple;
gram-negative (G-) cells
are pink to red
Flood the heat-fixed
smear with crystal
violet for 1 min
Add iodine solution
for 1 min
Decolorize with
alcohol briefly
— about 20 sec
Counterstain with
safranin for 1–2 min
G+
G-
Le
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J. L
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Mo
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s, In
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,
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FLASH
Figure 2.4b
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Different Staining Method:
1896 1920 1987 2006
Phase-Contrast, Dark-Field,
and Fluorescence Microscopy
相差, 暗视野, 荧光显微技术
Dark-Field Microscopy
相差显微镜细胞各部分折射率和厚度不同,光线通过后光波的相位发生改变,通过环状光阑和相差板,利用光的干涉将相位差变变振幅差(明暗差)
Phase-Contrast MicroscopyDeveloped to improve contrast differences between cells and the
surrounding medium.
Principle: cells slow the speed of light passing through them, so
differ in refractive index (折射率)from their surroundings. This
results in a difference in “phase” (相)between cell and its
surroundings.the subtle difference is amplified by a special ring in
the objective lens of the phase-contrast microscope.
Figure 2.5
A) Bright-field B) Phase contrast C) Dark-field
1896 1920 1987 2006
Figure 2.6
Fluorescence microscopy 荧光显微技术
1896 1920 1987 2006
3.2 Three-Dimensional Imaging:
Interference Contrast, Atomic Force,
and Confocal Scanning Laser
Microscopy
三维成像技术
Figure 2.7a
Nucleus
Interference contrast microscopy 干涉相显微技术
Figure 2.7b
Atomic force microscopy 原子力显微技术
Figure 2.8
Confocal Scanning Laser Microscopy 激光共聚焦扫描显微技术
Confocal Scanning Laser Microscopy 激光共聚焦扫描显微技术
带荧光蛋白的线虫(普通杆状线虫 Caenorhabditis elegans).描述了线虫中一种可遗传的修饰突变体,该突变体中beta-integrin基因翻译的产物与GFP融合表达。
1896 1920 1987 2006
3.3 Electron Microscopy
电子显微镜
Figure 2.9
Electronsource
Evacuatedchamber
Sampleport
Viewingscreen
5. 透射电子显微镜;6. 扫描电子显微镜
Transmission electron micrograph 透射电子显微镜
Electron beams do not penetrate very well,even a single cell is
too thick to be viewed directly. So thin sectioning techniques are
needed to prepare specimens.
Figure 2.10a
Cytoplasmic membrane
DNA(nucleoid)
Septum Cell wall
Transmission electron micrograph 透射电子显微镜
Scanning electron micrograph 扫描电子显微镜
The specimen is coated with a thin film of a heave metal
such as gold. An electron beam is then directed onto the
specimen and scans back and forth across it. Electrons
scattered are collected and they activate a viewing screen.
Figure 2.10c
Scanning electron micrograph 扫描电子显微镜
5. 透射电子显微镜;6. 扫描电子显微镜
Transmission electron
micrographScanning electron
micrograph
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