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In Chapter 4 and Lab 1, 2 and 3 you

will learn the basic skill sets

necessary to

Stain, Observe and Identify the Microbes

ACGM/CSLO goals:

1. Use and comply with laboratory safety rules, procedures, and

universal precautions.

2. Demonstrate proficient use of a compound light microscope.

3. Describe and prepare widely used stains and wet mounts, and

discuss their significance in identification of microorganisms.

4. Perform basic microbiology procedures using aseptic techniques for

transfer, isolation and observation of commonly encountered, clinically

significant bacteria.

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MICRO CLASS/LAB PREPARATIONS

Required Item:

TEXTBOOK,

MASTERING BIO,

ACCESS CODE,

LAB Manual/Journal

Scantrons 882-E, Scantron No.2 pencil

USEFUL Items: RED PEN

Colored Pencils

Purple

Pink

Red

Blue

Green

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BIOHAZARD Rules

All laboratory supplies (which touches specimen) and specimen must be placed in the BIO HAZARD

bin.

Please inform students that NO TRASH items should be placed in the BIOHAZARD bins

NO EXCEPTIONS. Do not put soda cans , regular trash in the biohazard bin (NO FOOD ALLOWED).

Very important

VIOLATORS will lose points

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MICRO LAB PREPARATIONS

Lab Performance

My Review

Peer Review Form

Lab Safety Form Signup

PowerPoint® Lecture

Presentations prepared by

Mindy Miller-Kittrell,

North Carolina

State University

C H A P T E R

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Microscopy,

Staining, and

Classification

4

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Figure 3.4 Approximate size of various types of cells.

Red Blood Cells

~10 um

15001.5mm

1500 um

Width of penny

=

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

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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

Official definitions

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Microscopy

General Principles of Microscopy

Magnification (enlarge)

Resolution (tell apart 2 objects close together)

Contrast (Differences in intensity between two

objects)

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Smaller the wavelength

smaller the object you can see

(small objects need small hands)

Think about giant fingers and texting

General rule for any microscopy/detector

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Figure 4.1 The electromagnetic spectrum.

VIBGYOR

Smaller the object = Use smaller wavelength

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Microscopy

Microscopes are used to visualize cells

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

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Light

Air Glass

Focal point

Specimen Convexlens

Inverted,

reversed, and

Enlarged

image

5 50

Magnification = 50/5 = 10X

Lenses refract (bend) the light, so that the image is magnified

Magnify using lenses

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Microscopy

Light Microscopy

Bright-field microscopes

Simple – single lense

Contain a single magnifying lens

Similar to magnifying glass

Leeuwenhoek used simple microscope to observe

microorganisms

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Microscopy

Light Microscopy

Bright-field microscopes

Compound – multiple lenses

More than one - Series of lenses for magnification

Light passes through specimen into objective lens

Have one or two ocular lenses

Total magnification = magnification of objective lens X

magnification of ocular lens

E.g. 10 times X 10 times = 100 times

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Figure 4.4 A bright-field, compound light microscope.

Coarse focusing knob

Moves the stage up and

down to focus the image

Illuminator

Light source

Diaphragm

Controls the amount of

light entering the condenser

Condenser

Focuses light

through specimen

Stage

Holds the microscope

slide in position

Objective lenses

Primary lenses that

magnify the specimen

Body

Transmits the image from the

objective lens to the ocular lens

using prisms

Ocular lens

Remagnifies the image formed by

the objective lens

Line of vision

Ocular lens

Path of light

Prism

Body

Objective

lenses

Specimen

Condenser

lenses

Illuminator

Fine focusing knob

Base

Arm

objective lens

occular lens

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Figure 4.5 The effect of immersion oil on resolution.

Glass cover slip

Slide

Specimen Light source

Without immersion oil

Lenses

Immersion oil

Glass cover slip

Slide

Light source

With immersion oil

Microscope

objective

Refracted light

rays lost to lens

Microscope

objective

More light

enters lens

Special type of objective lens = oil immersion lens

Increases magnification and resolution

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Microscopy

General Principles of Microscopy

Contrast

Differences in intensity between two objects, or an

object and its background

Increase contrast by staining

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Figure 6.3ab

Brightfield(stained specimen)

50 µ

m

Staining increases contrast

Brightfield(unstained specimen)

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Figure 4.16 Simple stains.

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Staining

Principles of Staining

Dyes used as stains are usually salts

Chromophore is the colored portion of the dye

Acidic dyes (negatively charged) stain alkaline

structures

Basic dyes (positively charged) stain acidic

structures

Basic dyes are more commonly used since inside of

most cells is negatively charged

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Staining

Simple stains

Differential stains

Gram stain

Acid-fast stain

Endospore stain

Special stains

Negative stain

Flagellar stain

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Staining

Most microorganisms are difficult to view by bright-

field microscopy

Coloring specimen with stain increases contrast

and resolution

Specimens must be prepared for staining

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Figure 4.15 Preparing a specimen for staining.

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paramecium

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penicillium

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spirogyra

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Figure 3.12 Bacterial shapes and arrangements.

2) strepto

1) coccus 2) bacillus

3 Shapes

3) spirillum

2 main arrangements

1) staphylo

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Microbe Naming Rules

Genus speciesGenus species

Genus species

Genus species

X

X Escherichia coli

Escherichia coli

E. coli

E. coliG. species

G. species

Genus speciesX

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Microbe Naming Rules

Based on shape and arrangement

Arrangement+shape species

Staphylococcus species

Streptobacillus species

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cocci bacilli

arrangement

shape

Streptococci

Staphylococci

Streptobacilli

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Negative staining

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This large (0.7-1.0 mm), nonpathogenic gram

negative spirillum exhibits polar amphitrichous

flagellation and a characteristic red pigment.

Rhodospirillum rubrum

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Rhodospirillum rubrum

Negative staining

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Rhodospirillum rubrum

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Next time we meet there will

be a short pre-lecture quiz based

on lab 1,2 and 3