Lab Exercises Week 1:

#3 Microscope#5 Simple Staining#1 Ubiquity of Microorganisms

Pre lab due: NonePost lab due: 1/17/15 & 1/24/15

• Light passes through specimen and then series of magnifying lenses

• Bright-field microscope is most common type• Three key concepts

– Magnification: apparent increase in size• Modern compound microscopes have two lens types:

objective and ocular

Principles of Light Microscopy

• Magnification is product of objective (4x, 10x, 40x, and 100x) and ocular lens (10x)

• Condenser lens (betweenlight source and specimen) focuses light on specimen, does not magnify

Ocular lens(eyepiece)Magnifies theimage, usually10-fold (10×).

Specimenstage

CondenserFocusesthe light.

Iris diaphragmControls theamount of lightthat enters theobjective lens.

Objective lensA selection of lensoptions providedifferentmagnifications. Thetotal magnification isthe product of themagnifying power ofthe ocular lens andthe objective lens.

Light source

RheostatControls thebrightness of thelight.

Courtesy of Leica, Inc., Deerfield, FL

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Microscopy-Brightfield

1.5. Size in the Microbial World

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1 micron (µ) = 10–6 metermicrometer (µm) = 10–6 meter = .000001 meter

Nucleus

MitochondriaProteinsSmallmolecules

Atoms Lipids Ribosomes Smallestbacteria

Mostbacteria

Most eukaryotic cells Adult roundworm

Human height

10 m1 m0.1 m1 cm1 mm100 µm10 µm0.1 nm 1 nm 10 nm 100 nm 1 µm

The basic unit of length is the meter (m), and allother units are fractions of a meter.

These units of measurement correspond to unitsin an older but still widely used convention.

1 angstrom (Å) = 10–10 meternanometer (nm) = 10–9 meter = .000000001 meter

1 meter = 39.4 inchesmillimeter (mm) = 10–3 meter = .001 meter

Prion fibril

Viruses

Electron microscope

Light microscope

Unaided human eye

Oil has same refractive index as glass

Samples can be immobilized, stained to visualize Basic dyes (positive charge)

• Attracted to negatively charged cellular components

Acidic dyes (negative charge)• Negative staining: cells repel, so colors background• Can be done as wet mount

3.2. Microscopic Techniques: Dyes and Staining

Spread thin film ofspecimen over slide.

Allow to air dry. Flood the smear withstain, rinse, and dry.

Examine with microscope.

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Pass slidethroughflame toheat-fixspecimen.

Goldilocks technique

Using a sterile loop place three drops of water onto one slide.

Re-sterilize your loop and get some bacteria on your loop (enough that you can see it with your eye).

Mix with water to create a smear. 1st one you will be able to see the bacteria (very

cloudy). 2nd one you will only barely be able to see the

bacteria (slightly cloudy). 3rd one you should not be able to see bacteria in

water drop (clear).

Two types most common• Coccus: spherical• Rod: cylindrical

Variety of other shapes• Vibrio, spirillum, spirochete• Pleomorphic (many shapes)• Great diversity often found in low

nutrient environments

3.3. Morphology of Prokaryotic Cells

(e)

(c)

(a)

Vibrio

Spirochete

Coccus Rod (bacillus)

Spirillum

11.4 µm1 µm

15 µm15 µm

7.5 µm

(b)

(d)

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(a): © SciMAT/Photo Researchers, Inc.; (b, c, d, e): © Dennis Kunkel Microscopy inc.1 m(a) 1 m (b)

a: Courtesy of Walther Stoeckenius; b: Courtesy of James T. Staley

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Most prokaryotes divide by binary fission• Cells often stick together

following division• Form characteristic groupings

Groupings

Cell dividesin one plane.

Diplococcus

Chains

Chain of cocci

Packets

(a)

Packet

Clusters

(b)

Cell dividesin two or more planesperpendicular to oneanother.

Cluster

Cell dividesin several planes atrandom.

(c)

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(a): (top): © George Musil/Visuals Unlimited; (bottom): © David M. Phillips/Visuals Unlimited; (b): © R. Kessel & C. Shih/Visuals Unlimited; (c): © Oliver Mecks/Photo Researchers, Inc.

1. Algae

2. Fungi

3. Protozoa

4. Bacteria

5. Viruses

Which microbes are eukaryotes? Which are prokaryotes? Which can perform

photosynthesis? Which are classified based on

locomotion? Which have cell walls? Which have some type of

nucleic acid?

Major Groups of Microbial World

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Microbial World

Domain Bacteria EucaryaArchaea

Prokaryotes (unicellular) Eukaryotes

Algae(unicellular ormulticellular)

Protists

Helminths(multicellular

parasites)

Fungi(unicellular ormulticellular)

Protozoa(unicellular)

Viruses Viroids Prions

Organisms(living)

Infectious agents(non-living)

Prokaryotes found growing in severe conditions• Ocean depths, volcanic vents, polar regions all harbor

thriving prokaryotic species• Many scientists believe that if life exists on other

planets, it may resemble these microbes

Individual species have limited set of conditions Important to grow microbes in culture

• Medical significance• Nutritional, industrial uses

Microbial Ubiquity

Prokaryotes inhabit nearly all environments• Some live in comfortable habitats favored by humans• Some live in harsh environments

• Termed extremophiles; most are Archaea

Major conditions that influence growth

• Nutrient availability

• Temperature

• Atmosphere- Oxygen

• pH

• Water availability

4.5. Environmental Factors That Influence Microbial Growth