CSLO CHECK PowerPoint Lecture Presentations prepared by ...

PowerPoint® Lecture

Presentations prepared by

Mindy Miller-Kittrell,

North Carolina State

University

C H A P T E R

© 2017 Pearson Education, Inc.

Microscopy,

Staining, and

Classification

4

CSLO1. Describe distinctive characteristics and diverse

growth requirements of prokaryotic organisms compared

to eukaryotic organisms.

PSLO3. Will demonstrate proficiency and safe practice

sin the use of laboratory equipment and basic

laboratory techniques.

CSLO CHECK


Principles behind Microscopy

Types of Microscopes

Light Microscopes

Electron Microscopes

Different staining methods for Microscopy

CH4-section1

Probe Microscopes


Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of

various types of microscopes.

Electron Microscopes

Probes Microscopes

Light Microscopes


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

FACT 4.1CONCEPT 4.1


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?

FACT 4.1CONCEPT 4.1


A) Magnification vs B) Resolution

High Magnification but Low Resolution

Need both high Resolution

and Magnification


VIBGYOR

Smaller the object = Use smaller

wavelength

To increase A) Magnification and B) Resolution

Use smaller wavelength CONCEPT 4.2


Light vs Electron microscope

Visible Light

Electron

has very

small

wavelength

compared

to visible

light

Which microscope will let you see

smaller things ?

CONCEPT 4.2


Light vs Electron microscope

Uses visible light Uses electron

Magnifies objects 100,000XMagnifies objects 1000X

FACT 4.2

glass lenses magnet


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


Light

Air

Glass

Light changes direction (refracts) when

it moves from one medium to another

Air

CONCEPT 4.3


Focal point

Specimen Convexlens

Inverted,

reversed, and

Enlarged

image

5 50

Magnification = 50/5 = 10X

Magnify using lenses

polish glass

CONCEPT 4.4


• 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


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

Total magnification

= magnification of objective lens

X magnification of ocular lens

e.g. 10 times X 10 times

= 100 times

FACT 4.3CONCEPT 4.5

10X

40X

100X

10X


Light

Air

Glass

Light changes direction (refracts) when

it moves from one medium to another

Air

CONCEPT 4.3


Figure 4.5 The effect of immersion oil on resolution.

Glass cover slip

Slide

Specimen Light source

Without immersion oil

Lenses

Immersion oilGlass cover slip

Slide

Light source

With immersion oil

Microscope

objective

Refracted light

rays lost to lens

Microscope

objective

More light

enters lens

immersion oil

Increases magnification

and resolution

FACT 4.4

Oil immersion lens (100X) is a

special type of objective lensimmersion oil reduces

scattering of light


Brightfield(stained specimen)

50

µm

Staining increases contrast

Brightfield(unstained specimen)

FACT 4.5

Staining kills cell


Different types of Microscopy

Light Microscopy

1) Bright-field microscopes show bright background

FACT 4.6

Four kinds of light microscopy


Bacterium

Bright field Dark field

2) Dark-field microscopy

• Specimen appears light against dark background

• Increases contrast and enables observation of more

details

• Examine live specimens

1) 2)

DarkBright-field microscopes show dark background

FACT 4.7


Phase contrast Nomarski

3A) Phase-contrast microscopy

3B) Differential interference contrast microscope aka Nomarski

3) Phase Microscopy – 2 types

Used to examine living organisms or specimens that would

be damaged/altered by attaching them to slides or staining

Light rays in phase produce brighter image, while light rays out

of phase produce darker image.

FACT 4.8

Contrast is created by combining light waves in or out of

phase

Shows 3D


4) Fluorescent microscopes

• Direct UV light source at specimen

• Fluorescent stains absorb UV and radiates energy back

as a longer, visible wavelength

UV light increases resolution and contrast

UV

FACT 4.9


4A) Fluorescence microscopy – 3 types

Can help you look at object of interest only

FACT 4.10


4B) Immunofluorescence

Antibodies

Bacterium

Cell-surfaceantigens

Bacterial cell withbound antibodiescarrying dye

Fluorescent dye

Antibodiescarrying dye

help you detect specific pathogens

by attaching fluorescent dyes to the antibody

specific for that pathogen

FACT 4.11


4C) Confocal microscopes

Use UV lasers to illuminate fluorescent chemicals in a single plane

Computer constructs 3-D image from digitized images

FACT 4.12


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


Figure 4.11 A transmission electron microscope (TEM)

Lamp

Light microscope(upside down)

Column of transmissionelectron microscope

Lamp

Condenser

lens

Specimen

Objective lens

Eyepiece

Final image

seen by eye

Electron gun

Specimen

Objective lens

(magnet)

Projector lens

(magnet)

Final image on

fluorescent screen

All electron microscopy needs vacuum - Why ?

TEM needs sections - why ?

FACT 4.13

TEM sees transmitted electrons

Consequences = Only dead objects seen

CONCEPT ?


Figure 4.12 Scanning electron microscope (SEM).

SEM sees scattered electrons

SEM sees surface of microbes


• Probe Microscopy – 2 types

• Probe travels over specimen

Scanning tunneling

microscopes

(highest magnification

and resolution)

Atomic force

microscopes

FACT 4.14


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


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

• Basic dyes (positively charged) stain acidic (negatively

charged) structures

• Acidic dyes (negatively charged) stain alkaline

structures

++

+ +++++++ +

++ ----

--+++ +

++ --- --

++

+ ++++

Basic dyes

CONCEPT 4.6

FACT 4.15


Inside of most bacterial cells is

negatively charged

- --- --++

+ ++++

Basic dyes

Positive Staining

----- -

Therefore Basic dyes are more commonly used

to stain bacterial cells

CONCEPT 4.6


Inside of most bacterial cells is

negatively charged

- --- ---

Acidic dyes

----- -

Therefore Acidic dyes

are more commonly used to stain background

---

--

- -

-- --

Negative Staining

CONCEPT 4.6


Gram Staining MechanismFACT 4.16


Gram Staining Method

Crystal Violet

Primary Stain Step

Gram positive cell wall

Gram negative cell wall

FACT 4.16



Gram’s Iodine

Mordant Step



Alcohol

Decolorizing Step



Safranin

Counter Stain Step


Table 4.3 Some Stains Used for Light Microscopy


Staining for Electron Microscopy

Chemicals

containing heavy

metals

are used as stains

for electron

microscopy

FACT 4.17

CONCEPT 4.7

transmission electron microscopy


Other Identifying

Characteristics

Physical characteristics

Biochemical tests

Serological tests

Phage typing

Analysis of nucleic acids

How to classify microbes

Different Methods to identify microbes

Staining MethodsHow to name microbes

Taxonomy

1

2

3

CH4-section2


• Microbe Identifying Characteristics

• Physical characteristics

• Biochemical tests

• Serological tests

• Phage typing

• Analysis of nucleic acids



Protozoa, fungi, algae, and parasitic worms can often be

identified based only on their morphology

Some bacterial

colonies have distinct

appearance used for

identification

CONCEPT 4.8


No

hydrogen

sulfide

Hydrogen

sulfide

producedAcid with gas Acid with no gas Inert

Gas bubble Inverted tubes to trap gas

Biochemical tests

CONCEPT 4.9


An agglutination test

Serological (Blood) Tests CONCEPT 4.10

Antibody-Antigen

Complex causes

agglutination

ZIKA virus Ebola virus

Patient Blood

(contains antibodies)

plus add virus on slide


Bacterial lawn

Plaques

(holes)

Phage typing

Identifying bacteria based on which

phage infects and lyses the bacteria

CONCEPT 4.11

Phage T4 Phage T7

Bacteria A Plaques No

Plaques

Bacteria B No

Plaques

Plaques



Nucleic acid sequence can be used to classify and identify

microbes

Prokaryotic taxonomy now includes the G + C content of

an organism's DNA

CONCEPT 4.12


• 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

CONCEPT 4.13


Other Identifying

Characteristics


Biochemical tests

Serological tests

Phage typing





Taxonomy

1

2

3


humans belong to the genus Homo

species Homo sapiens.

naming species of living things by giving

each species a name composed of two parts

noun-genus

Capitalizedadjective-specific epithet

Binomial nomenclature

Homo sapiens

CONCEPT 4.14

Each species has a unique name


Homo naledi (2015)

Homo sapiens

members of Genus are very different


Genus specific epithet


X

XGenus specific epithetYGenus specific epithetY

Escherichia coliEscherichia coliE. coli


Figure 3.12 Bacterial shapes and arrangements.

2) strepto

1) coccus 2) bacillus

3 major shapes/morphology

3) spirillum

2 major arrangements

1) staphylo

FACT 4.18


Microbe Naming Rules

Genus is based on shape and arrangement

specific epithet is based on unique characteristic

CONCEPT 4.14

(Arrangement+shape) (unique characteristic)

(specific epithet)(Genus)

Streptobacillus pnemoniae

Escherichia coli


cocci bacilli

arrangement

shape

Streptococci

Staphylococci

Streptobacilli


Staphylococcus partyanimalia

Lets name this microbe



Other Identifying

Characteristics


Biochemical tests

Serological tests

Phage typing





Taxonomy

1

2

3

consists of

Identification,

Nomenclature and

classification


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

CONCEPT 4.15


His system

classified organisms

based on

characteristics in

common

Grouped organisms

that can

successfully

interbreed into

categories called

species

Used binomial

nomenclature

FACT 4.19Carolus Linnaeus and Taxonomic Categories


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

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

CONCEPT 4.16

FACT 4.20

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