Lecture 3-1: Microbial cell biology - 上海交通大学微生物...

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

on

J. L

eb

ea

u

Mo

lec

ula

r P

oro

be

s, In

c.,

Eu

ge

ne

,

Ore

go

n

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

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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

Microbiology, Shanghai Jiao Tong University