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Unit II - Microbiology
Sources:
• Pelczar M.J. and Chan E.C.S., Elements
of Microbiology, McGraw-Hill, Inc.
1981
• Paustian, T. ,
http://www.bact.wisc.edu/MicrotextBook/
University of Wisconsin-Madison 2002
(Textbook on the web)
Student Objectives:
• 1. To become familiar with the
ubiquitous nature of
microorganisms.
• 2. To learn some basic
bacteriological lab techniques.
• 3. To understand how
bacteria grows.
• 4. To become familiar with
major groups of
microorganisms.
.
• 5. To know how
microorganisms can be
controlled.
• 6. To be familiar with
diseases caused by
microorganisms.
Section One
•History of
Microbiology
The First Microscope.
• Invented in late 1500’s
• A simple microscope = like a
magnifying lens
The discovery of microorganisms.
• Leeuwenhoek = Dutch naturalist in late
1600’s
– Ground lenses and made microscopes.
– First to record observations of
microscopic organisms seen in
rainwater.
– Called the little organisms
“animalcules”.
• Basic kinds of bacteria as
observed by Leeuwenhoek
–spirilla – spiral-shape
• cocci – spherical
• bacilli – rod-shaped
Spontaneous Generation
• Belief that organisms arose
from non life
• Disproved by use of beef
broth experiments
• Louis Pasteur – the gooseneck
flask
Pasteur
Schulze acid base
Schwann’s flame
Schroeder’s
cotton plug
Pasteur’s
Fermentation
• Used for many years.
• Pasteur discovered
microbes were
responsible
Disease
• 1540’s – first theories that small organisms
caused disease.
Key Discoveries
• Pasteur isolated anthrax –
causing microbes
• Koch discovered anthrax was
caused by bacilli bacteria
• Koch developed culture
media
Koch’s postulates
• A specific microbe is associated with a
given disease.
• That microbe can be isolated and grown.
• The pure culture can be used to infect a
host organism.
• Microbes can be “recovered” from
infected organism
Koch
Immunization
• Any process that develops
resistance in a host to a
specific disease.
Pasteur discovered
immunization accidentally.
• By using old cultures, injected
chickens remained healthy.
• He injected a new culture in
those chickens and again they
remained healthy.
Source: http://www.tutorvista.com/content/biology/biology-iv/immunesystem/vaccination-and-immunisation.php
•
Source: http://www.bbc.co.uk/schools/gcsebitesize/history/shp/modern/indrevmethodsrev2.shtml This source says Charles Chamberland (France: 1880) discovered by chance (when he left bacteria
exposed to air) that injecting chickens with an attenuated (weakened) form of chicken cholera gave
them immunity to the disease (ie he discovered the principle of inoculation) . Pelczar and Chan
credit this discovery to Pasteur.
Other breakthroughs
• Jenner and smallpox
immunization
• Vaccinations (from vacca = cow)
• Rabies vaccines.
• Antisepsis
• Many more !!
Today’s Microscope.
• Bright field – dark objects in bright
area
• Compound – multiple lenses. Up to
2000 x
• Electron – maximum magnification
because of short wavelengths. Up to
400,000 x
The oil immersion lens.
• is 100 X
• Requires oil to prevent light
refraction
• Gives maximum
magnification for compound
light microscope.
Quiz – History of Microbiology
• COMING SOON
• GET READY !!!!
Section 2
• Laboratory Techniques
Microscopes
• Light microscopes – max 2000 X
• Electron microscopes – max
400,000 X
Bright-field Microscopy
• Light background and dark
objects.
The Compound Light Microscope
• Main components for
directing light.
–Eyepiece, objectives, stage,
condenser, iris diaphragm, fine
and course adjustment knobs,
base, mirror, etc.
Resolving Power
• Resolving Power = wavelength
NA objective + NA condenser
• NA = numerical aperature
• Wavelength rane is 400 to 700 nm
• NA for high dry is about 0.85
• NA for oil-immersion objective: 1.2 to 1.4
• Whatever the resolving
power, if two objects are
closer together than the
numerical resolving power,
they appear as one.
• “The resolving power of a microscope is the
ability of the devise to distinguish between
two objects. A light microscope can only
resolve two objects that are bigger than 250
nanometers because objects smaller than
this fall well below the size of a wavelength
of visible light (500 – 650 nanometers).
Non lens system can ever resolve two dots
that are closer together than half the
wavelength of the light that is used to view
them.” Source:
http://www.blurtit.com/q571512.html
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No doubt you have seen a sunrise and a sunset.
The sky turns red, orange, yellow and even
purple because the colored wavelengths of light
travel farther through the air (atmosphere). When
the sun is low in the sky, this long journey
through the atmosphere means the colors with
shorter wavelengths, like blue, have already
scattered or bounced off in numerous directions.
Orange sunsets (yellow and red light waves)
appear when the air is clean. Sunsets that are the
most spectacular occur when red wavelengths
reflect off of overhead clouds.
Spectators continue to see light in the sky long
after it has turned dark on the ground. Why is
this? Because night doesn't "fall". It actually rises
from the ground as the sun goes farther below the
horizon.
Civil twilight occurs when the sun is 6° below the
horizon. This is from the time that the sun drops
below the horizon until artificial lights (street or
home) are needed.
Astronomical twilight occurs when the sun is 18°
below the horizon. This is when there is no
sunlight on the western horizon and stars can be
seen.
Twilight is shorter in the tropics because the sun's
path is more perpendicular to the Earth's plane
and it takes less time to go from 6° to 18° below
the horizon at this angle.
White nights occur in extreme northern latitudes
where the evening twilight merges with the
morning twilight.
http://sci.odu.edu/sci/Scire/05Edition/sunset.html
Preparation of Specimens
• Wet mount - short term
mount – high dry
• Hanging Drop – high dry
• Stained smears – best for
bacterial form - oil
immersion
Wet mount
source: http://www.greatscopes.com/act005.htm
Hanging Drop Slide
Source: http://homepage.smc.edu/wissmann_paul/microbiology/motility.html
Negative Staining
• Stains background and makes
bacteria stand out more
clearly.
–Nigrosine stain
Staining the Bacteria (positive)
• Stains bacteria and not the
background.
– Main stains are methylene blue,
gentian violet (crystal violet), and
carbol fuchsin.
The Staining Process
Three basic steps.
• Smearing
• Fixing
• Staining
Step One – the smear
• A very clean slide
• Draw circle on back of slide
and label back of slide.
• Sterilize your loop.
• Place very small drop of
sterile water on slide
• Sterilize loop.
• Obtain sample on loop
– Remember Bacteria are very small !!
• Smear bacteria in water
Step two – Fixing the smear
• Air dry the smear
• Pass slide through burner flame
up to 3 times
• Do not flame the slide until all
water has disappeared !!!
Step three - staining
• Cover the smear with stain
for a few seconds.
• Wash all the stain off the
slide.
• Dry using filter paper
Special Staining
• Gram Stain procedure–
divides bacteria into two
groups.
• Gram positive – organisms that
are stained purple.
• Gram negative – organisms that
retain counterstain color.
• Acid-fast staining – to classify
those bacteria which retain
stain when washed with acids
or alcohol.
Growing Bacteria in the Lab
• Culture and Media
Growth Requirements:
• energy source
• a source of carbon
• required nutrients,
• proper O2, pH, temp, etc.
Culture:
• A population of
microorganisms cultivated
in a medium
Culture media:
• are designed to provide
all the essential
nutrients in solution for
bacterial growth
.
Pure Culture
• Culture containing
only one species of
organism.
Types of Media
• Liquid – for pure batch
cultures
• Solid – isolation of pure
cultures. Agar.
Assignment:
• Read online textbook section:
“Culture Media for the
Growth of Bacteria” Page 16
of Word document on CD
Section 3
• Survey of the Microbial
World
Terminology:
• Microbiology – study of small
living organisms of microscopic
size.
• Protist – a microorganism in the
Kingdom Protista. (includes
most microorganisms)
• Prokaryote - a cell in which
the nuclear substance is not
enclosed in a membrane.
(includes the bacteria)
• Eukaryote – a cell that has a
definitive or true nucleus
(these cells make up the
bodies of all non protist
organisms)
• Virus – obligate intracellular
parasitic organism that is
smaller than bacteria. They
can only reproduce in cells of
host organisms.
The Cell – A review
• General Structure:
–Membrane –Nucleus – contains
nucleoproteins
–Cytoplasm (cyto= cell, plasm =
formed substance)
• Characteristics of all living things:
– Reproduce
– Energy needs and growth
– Elimination of waste
– Response to stimuli
– Susceptible to mutations
• Look for the 3 general
structures in each of the
following three organisms.
Paramecium – a representative cell
A Typical Bacterial Cell
Euglena – another typical cellular organism
Prokaryotic cells
• No internal membranes
• Division by fission not by
mitosis
• Cell wall with mucopeptide
material.
Some Prokaryotic cells
• have capsule or slime layer
covering cell wall
• have flagella (“whip”) for
motility
Prokaryotic cytoplasm
May contain:
• Ribosomes – proteins and RNA
• Granules - chemicals
• Nuclear material - DNA
• Mesosomes - folds of
Cytoplasmic membrane
Eukaryotic cells
• Contain internal
membranes - called
Endoplasmic Reticulum
(ER) – distinguishing it from
the prokaryotic cells.
Eucaryotic cell structures
• ER
•
•
•
•
•
Nucleus
Golgi apparatus
Mitochondria
Chloroplasts
Vacuole
• Microtubules
• Microfilaments
• Flagella
• Cilia
• Cell walls
Groups of Microorganisms
• Prokaryotic protists
• Eucaryotic protists
• Viruses
Please note:
• Classification schemes often change.
• Some literature lists Bacteria as a
member of Kingdom Monera and
not Protista.
• Others split bacteria into two
kingdoms called Archaebacteria and
Eubacteria.
Prokaryotic Protists
• Bacteria –
• 3 basic shapes
• Size range from 0.5 mm to 2.5 mm
• Cyanobacteria –
• Contain chlorophyll and
photosynthesize
Eucaryotic Protists
• Fungi
• Rigid cell walls, no chlorophyll
• Protozoa
• Include flagellates, amebas, ciliates, and
sporozoa – more on that later !
• Algae
• Contain chlorophyll
Paramecium – a protozoan
Viruses
• Not cellular
• Obligate parasites
• 20 nm to 300 nm in size
• Host specific
Section 4
• - Protozoans
Protozoa introduced
• Classified by locomotion
–Flagellates
–Amebas
–Ciliates
–Sporozoa
Flagellates
• Cell is surrounded by
pellicle – gives the
organism shape.
Disease-causing Flagellates
• Giardia lablia that nasty
intestinal parasite
• Trypanosoma (African sleeping
sickness – spread by tsetse flies)
• Trichomonas - vaginitis, spread as
venereal disease
Amebas
• Name comes from Greek word
meaning “change”
• Pseudopodia – “false feet” are
protoplasmic extensions
Disease-causing Amebas
• Entamoeba histolytica – cause
of amebic dysentery.
• (yes there are non disease
causing Amebas like Ent.
Gingivalis and Ent. Coli)
Ciliates
• Contain at least one macronucleus
and one micronuclei per cell.
• May have partial covering of cilia or
complete covering like Paramecium
Disease-causing Ciliates
• Only one !!
• Balantidium coli – causes “bloody
diarrhea” which is transmitted by
infected food or water from swine
feces.
Sporozoans
• All are parasitic.
• Feed on host’s cells or body
fluids.
• All produce spores – a
resistant or dormant cell.
Disease-causing Sporozoans
• Plasmodium - Malaria-causing
sporozoans
• Toxoplasma gondii - mild
infection usually requiring no
treatment but can cause birth
defects if mother is infected.
The changing face of Protozoans
• Cyst formation • Trophozoites - organism in stage
of active growth, also called
vegetative stage.
Section 5
•Algae
Algae Shapes
• Single cells may be:
–Spherical
–Rod-shaped
–Club-shaped
–Spindle-shaped
Algae Growth
• May grow as single cells.
• May grow in multicellular colonies.
• Colonies may be made of identical
cells.
• Colonies may be made of different
cells that specialize.
Algae Cellular
Structure
• Algae is Eukaryotic
• Contain chloroplasts.
• If motile, they have flagella.
• May have stalks or spines
for anchoring.
Classification of Algae
• Based on:
– Pigment
– Chemistry
– Flagella
– Cell wall features
– Cell organization (single or colony)
– Life cycles and reproduction.
Some major Divisions
• Chlorophycophyta – green algae
• Rhodophycophyta – red algae
• Chrysophycophyta – golden
algae
• Bacillariophycophyta - diatoms
Diatoms
Blue-green Bacteria/algae
Reproduction
• Assexual
– By binary fission (cell
division producing two
daughter cells)
– Spore formation.
• Sexual
–Gametes
–Zygote
Growth Requirements
• Algae are aerobic – need oxygen
• Algae are photosynthetic – need
light
• Algae require moisture – need
water
• Algae require nutrients