1. The
University of
Lethbridge
BIOLOGY 3400
Principles of Microbiology
LABORATORY MANUAL
Spring, 2012
Written by: L. A. Pacarynuk and H.C. Danyk
Revised: December, 2011

2. TABLE
OF
CONTENTS
Exercise:
Page
Biology
3400
Laboratory
Schedule.............................................................................................................2
Grade
Distribution.....................................................................................................................................3
Occupational
Health
and
Safety
Guidelines...............................................................................................5
Guidelines
for
Safety
Procedures...............................................................................................................6
Exercise
1
–
Introduction
to
Microscopy....................................................................................................9
Exercise
2
–
General
Laboratory
Principles
and
Biosafety.......................................................................13
Exercise
3
–
Free-­‐Living
Nitrogen
Fixation...............................................................................................14
Exercise
4
–
Winogradsky
Column
..........................................................................................................21
Exercise
5
-­‐
Bacterial
and
Yeast
Morphology...........................................................................................23
Exercise
6
–
Bacterial
Reproduction.........................................................................................................28
Exercise
7
–
Ames
Test.............................................................................................................................31
Exercise
8
–
Biochemical
Tests.................................................................................................................34
Exercise
9
–
Yeast
Fermentation..............................................................................................................39
Exercise
10
-­‐
Virology...............................................................................................................................43
Appendix
1
–
The
Compound
Light
Microscope......................................................................................49
Appendix
2
–
Preparation
of
Scientific
Drawings.....................................................................................52
Appendix
3
–
Aseptic
Technique..............................................................................................................54
Appendix
4
–
The
Cultivation
of
Bacteria.................................................................................................59
Appendix
5
–
Bacterial
Observation.........................................................................................................64
Appendix
6
–
Laboratory
Reports...........................................................................................................
65
Appendix
7
–
Use
of
the
Spectrophotometer..........................................................................................67
Appendix
8
–
Media,
Reagents,
pH
Indicators.........................................................................................69
Appendix
9
–
Care
and
Feeding
of
the
Microscopes................................................................................76
1

3. BIOLOGY
3400
LAB
SCHEDULE
SPRING,
2012
Jan.
10
No
lab
Jan.
12
No
lab
Jan.
17
Introduction,
Microscopy
Jan.
19
General
Lab
Procedures,
Biosafety
Jan.
24
General
Lab
Procedures,
Biosafety
–
Complete;
N-­‐Fixation
Jan.
26
Winogradsky
Column
Jan.
31
Bacterial
Morphology;
N-­‐fixation
Feb.
2
Bacterial
Morphology
Feb.
7
Bacterial
Morphology;
N-­‐fixation
Feb.
9
Bacterial
Morphology
Feb.
14
Bacterial
Growth
Feb.
16
Bacterial
Morphology
–
Complete;
N-­‐fixation:
Polymerase
Chain
Reaction
Feb.
21
Reading
Week
Feb.
23
Reading
Week
Feb.
28
Ames
Test
Mar.
1
Ames
Test
–
Complete;
N-­‐fixation:
Agarose
Gel
Electrophoresis
Mar.
6
Biochemical
Tests
-­‐
Selective
and
Differential
Media,
IMViC
Tests
Mar.
8
Selective
and
Differential
Media,
IMViC
tests
–
Complete
Mar.
13
Yeast
Fermentation
Mar.
15
Winogradsky
Column
Mar.
20
Virology
(phage
isolation)
Mar.
22
Virology
(phage
elution)
Mar.
27
Virology
(amplification)
Mar.
29
Virology
(titre/host
range)
Apr.
3
Virology
-­‐
Complete
Apr.
5
no
lab
Apr.
10
Lab
report
due
2

4. Laboratory
Grade
Distribution:
The
laboratory
component
of
Biology
3400
is
worth
50%
of
your
course
mark.
It
is
distributed
as
follows:
• Skills
Tests
10%
• Assignments
20%
• Lab
Books
10%
(to
be
handed
in
three
times)
• Lab
Report
10%
th
On
Yeast
Fermentation;
due
Tuesday
April
10
at
the
beginning
of
lab
Performance:
Up
to
10%
of
laboratory
grade
(5
marks
out
of
50)
will
be
subtracted
for
poor
laboratory
performance.
This
includes
(but
is
not
limited
to)
failure
to
be
prepared
for
the
laboratory,
missing
lab
notebook
or
lab
manual,
poor
time
management
skills,
improper
handling
and
care
of
equipment
such
as
microscopes
and
micropipettors,
and
unsafe
practices
such
as
not
tying
hair
back,
chewing
gum,
applying
lipstick,
eating,
drinking,
or
chewing
on
pencils,
and
sloppy
technique
leading
to
poor
results.
As
we
are
working
with
potential
pathogens,
students
displaying
improper
or
careless
techniques
will
be
asked
to
leave
the
lab
and
will
have
at
least
5%
of
their
laboratory
grade
deducted
immediately.
Missing
a
lab
for
which
there
is
a
skills
test
or
assignment
requires
documentation.
Upon
presentation
of
this
documentation,
you
will
either
have
to
complete
the
assignment
or
skills
test
as
soon
as
possible
or,
if
this
is
not
possible,
your
lab
grade
will
be
recalculated.
The
lab
books
will
be
collected
and
graded
three
times
during
the
semester.
Although
most
exercises
are
completed
as
groups,
the
lab
books
are
to
be
completed
individually,
and
must
represent
individual
effort.
The
following
page
provides
you
with
tips
on
how
to
construct
your
books.
Unannounced
skills
tests
will
be
given
during
the
semester.
Students
are
expected
to
work
independently
on
some
technical
aspect
of
microbiology
and
will
be
graded
based
on
their
techniques
and
their
results.
As
proficiency
in
microbiological
techniques
is
considered
an
essential
component
of
the
course,
students
are
only
permitted
three
lab
period
absences
(you
do
not
require
any
documentation).
Missing
more
than
three
labs
will
result
in
a
grade
of
0
being
assigned
for
the
lab
(at
this
point,
it
is
recommended
that
students
consult
with
Arts
and
Science
Advising
for
the
option
of
completing
the
laboratory
the
following
year).
Students
are
still
responsible
for
the
material
missed
(and
their
assignments,
lab
reports
etc.
will
be
graded
as
such).
There
are
no
make-­‐up
laboratories.
Late
Assignments
will
be
penalized
as
follows:
For
Assignments
and
the
Lab
Report:
after
the
start
of
lab,
but
by
4:30
pm
on
the
due
date
–25%;
by
9:00
am
the
next
morning
-­‐50%,
and
after
9:00
am
the
following
day,
no
marks
will
be
given.
Extensions
for
the
lab
report
and
Assignments
will
only
be
granted
for
situations
involving
prolonged
illness
(documentation
is
required).
3

5. Preparation
of
a
Lab
Book:
Your
lab
book
provides
you
with
a
detailed
record
of
your
experiments
performed.
This
record
proves
invaluable
when
preparing
manuscripts
for
publication,
or,
more
immediately,
when
preparing
lab
reports.
This
lab
book,
as
with
all
of
the
reports
and
assignments
is
an
individual
effort.
Choice
of
Lab
Book
Standard
black
lab
books
can
be
purchased
from
the
book
store
but
these
are
not
required
for
this
course.
The
only
required
features
are:
• Pages
are
non-­‐removable
(no
spiral
bindings)
• All
pages
must
be
numbered
in
the
top
outer
corner
• page
numbers
may
be
hand-­‐written
on
EVERY
page
in
INK
In
General
• all
entries
must
be
made
in
blue
or
black
ink
(except
drawings)
• date
EVERY
entry
• never
remove
a
page
or
use
white-­‐out
• if
an
entry
needs
to
be
deleted,
strike
out
the
entry
with
a
single
straight
line
(the
deleted
entry
must
be
readable)
• keep
up
to
date,
a
lab
book
is
meant
to
be
filled
out
as
the
experiments
are
carried
out
and
NOT
after
the
fact
• record
anything
that
may
be
useful
to
you
when
preparing
your
lab
reports
• leave
plenty
of
space
throughout
the
lab
book
to
add
comments
after
the
fact
Table
of
Contents
Designate
the
first
2
pages
as
the
Table
of
Contents
• record
information
and
pages
numbers
as
you
go
Lab
Entries
For
each
lab
be
sure
to
include
the
following;
1. Objective
2. Method
Summary
• do
not
rewrite
the
protocol
from
the
lab
manual
• highlight
any
specific
changes
to
the
lab
protocol
• include
times
and
dates
for
when
work
was
performed
• record
product
names
and
manufacturers
used
-­‐
enzymes,
chemicals,
equipment
(micropipettors,
baths)
• include
incubation
conditions
for
cultures
and
reaction
3. Observations
&
Results
• record
any
&
all
observations,
this
goes
beyond
number
results
• include
diagrams
and
any
other
form
of
raw
data
• include
calculations
as
appropriate
4. Conclusions
• did
you
achieve
your
objective?
Why
or
why
not?
• use
your
results
to
support
your
conclusions
5. Answer
the
thought
questions
at
the
end
of
the
lab
(as
applicable)
• use
reference
citations
as
needed
• these
may
be
graded
4

6.
THE
UNIVERSITY
OF
LETHBRIDGE
Policies
and
Procedures
Occupational
Health
and
Safety
Manual
SUBJECT:
CHEMICAL
SPILLS
PROCEDURE
Precaution
should
be
taken
when
approaching
any
chemical
spill.
1. UNKNOWN
SPILL
a. Clear
the
area
b. Call
Security
at
329-­‐2345
c. Secure
the
area
and
do
not
let
anyone
enter
d. Call
Utilities
at
329-­‐2600
and
request
air
be
turned
on
at
the
spill
site
e. Security
will
respond
and
determine
the
severity
of
the
spill
f. Security
will
immediately
notify
the
spill
team
as
follows:
• Chemical
Release
Officer:
331-­‐5201
• Risk
and
Safety
Services
(OHS
Officers):
329-­‐2350/329-­‐2190
(office)
or
394-­‐
8716/330-­‐4495
(cellular)
• Risk
and
Safety
Services
(Manager):
382-­‐7176
(office)
• DBS
Environmental
only
if
above
not
available
328-­‐4483
(24
hrs)
2. KNOWN
SPILL
a. Clear
the
area
b. Call
Security
at
329-­‐2345
c. Secure
the
area
d. Call
Utilities
at
329-­‐2600
and
request
air
be
turned
on
at
the
spill
site
e. Security
will
respond
and
determine
the
severity
of
the
spill
f. Security
will
immediately
notify
the
spill
team
as
follows:
• Chemical
Release
Officer:
331-­‐5201
• Risk
and
Safety
Services
(OHS
Officers):
329-­‐2350/329-­‐2190
(office)
or
332-­‐
2350/394-­‐8716
(cellular)
• Risk
and
Safety
Services
(Manager):
382-­‐7176
(office)
• DBS
Environmental
only
if
above
not
available
328-­‐4483
(24
hrs)
3. NOTIFICATION
a. Risk
and
Safety
Services
will
notify
the
appropriate
departments,
including
notification
of
appropriate
government
agency.
5

7. GUIDELINES
FOR
SAFETY
PROCEDURES
Students
enrolled
in
laboratories
in
the
Biological
Sciences
should
be
aware
that
there
are
risks
of
personal
injury
through
accidents
(fire,
explosion,
exposure
to
biohazardous
materials,
corrosive
chemicals,
fumes,
cuts,
etc).
The
guidelines
outlined
below
are
designed
to:
a)
minimize
the
risk
of
injury
by
emphasizing
safety
precautions
and
b)
clarify
emergency
procedures
should
an
accident
occur.
EMERGENCY
NUMBERS:
City
Emergency
911
Campus
Emergency
2345
Campus
Security
2603
Student
Health
Centre
2484
(Emergency
-­‐
2483)
THE
LABORATORY
INSTRUCTOR
MUST
BE
NOTIFIED
AS
SOON
AS
POSSIBLE
AFTER
THE
INCIDENT
OCCURS.
EMERGENCY
EQUIPMENT:
Your
lab
instructor
will
indicate
the
location
of
the
following
items
to
you
at
the
beginning
of
the
first
lab
period.
• Closest
emergency
exit
• Closest
emergency
telephone
and
emergency
phone
numbers
• Closest
fire
alarm
• Fire
extinguisher
and
explanation
of
use
• Safety
showers
and
explanation
of
operation
• Eyewash
facilities
and
explanation
of
operation
• First
aid
kit
GENERAL
SAFETY
REGULATIONS:
• Eating
and
drinking
is
prohibited
in
the
laboratory.
Keep
pencils,
fingers
and
other
objects
away
from
your
mouth.
These
measures
are
to
ensure
your
safety
and
prevent
accidental
ingestion
of
chemicals
or
microorganisms.
• Personal
protective
wear
is
mandatory.
Lab
coats,
safety
glasses
and
closed-­‐toed
shoes
must
be
worn
at
all
times
during
lab
exercises
which
involve
potential
for
chemical
or
biological
spills.
• Coats,
knapsacks,
briefcases,
etc.
are
to
be
hung
on
the
hooks
provided,
stowed
in
the
cupboards
beneath
the
countertops,
or
placed
along
a
side
designated
by
your
instructor.
Take
only
the
absolute
essentials
needed
to
complete
the
exercise*
with
you
to
your
laboratory
bench.
(*
e.g.
manual,
pen
or
pencil)
• Mouth
pipetting
is
NOT
permitted;
pipet
pumps
are
provided
and
must
be
used.
• Always
wash
your
hands
prior
to
leaving
the
laboratory.
• Students
are
not
allowed
access
to
the
central
Biology
Stores
area
for
any
reason.
Consult
your
instructor
if
you
require
additional
supplies.
• Report
any
equipment
problems
to
instructor
immediately.
Do
NOT
attempt
to
fix
any
of
the
equipment
that
malfunctions
during
the
course
of
the
lab.
• Use
caution
when
handling
chemical
solutions.
Consult
the
lab
instructor
for
instruction
regarding
the
clean-­‐up
of
corrosive
or
toxic
chemicals.
6

8. • Contain
and
wipe
up
any
spills
immediately
and
notify
your
lab
instructor
(see
SPILLS
below).
Heed
any
special
instructions
outlined
in
the
lab
manual,
those
given
by
the
instructor
or
those
written
on
reagent
bottles.
• Long
hair
must
be
restrained
to
prevent
it
from
being
caught
in
equipment,
Bunsen
burners,
chemicals,
etc.
• Dispose
of
broken
glass,
microscope
slides,
coverslips
and
pipets
in
the
specially
marked
white
and
blue
boxes.
There
will
be
NO
disposal
of
glassware
in
the
wastepaper
baskets.
• You
are
responsible
for
leaving
your
lab
bench
clean
and
tidy.
Glassware
must
be
thoroughly
rinsed
and
placed
on
paper
toweling
to
dry.
SPILLS:
• Spill
of
SOLUTION/CHEMICAL:
While
wearing
gloves,
wipe
up
the
spill
using
paper
towels
and
a
sponge
as
indicated
by
the
lab
instructor.
• Spill
of
ACID/BASE/TOXIN:
Contact
instructor
immediately.
DO
NOT
TOUCH.
• BACTERIA
SPILLS:
If
necessary,
remove
any
contaminated
clothing.
Prevent
anyone
from
going
near
the
spill.
Cover
the
spill
with
10%
bleach
and
leave
for
10
minutes
before
wiping
up.
Discard
paper
towels
in
biohazard
bag.
Discard
contaminated
broken
glass
in
designated
biohazard
sharps
container.
DISPOSAL:
•
Broken
glass,
microscope
slides,
coverslips
and
Pasteur
pipets
are
placed
in
the
upright
white
‘broken
glass’
cardboard
boxes.
NO
PAPER,
CHEMICAL,
BIOLOGICAL
OR
BACTERIAL
WASTE
MATERIALS
should
be
placed
in
this
container
•
Petri
plates,
microfuge
tubes,
pipet
tips
should
be
placed
in
the
orange
biohazard
bags.
The
material
in
this
bag
will
be
autoclaved
prior
to
disposal.
•
Bacterial
cultures
in
tubes
or
flasks
should
be
placed
in
marked
trays
for
autoclaving.
•
Liquid
chemicals
should
be
disposed
of
as
indicated
by
the
instructor.
DO
NOT
dispose
of
residual
solution
in
the
regent
bottles.
In
case
of
any
uncertainty
in
disposal
please
consult
the
lab
instructor.
•
Slides
of
bacteria
should
be
placed
in
the
trays
filled
with
10%
bleach
that
are
located
at
the
ends
of
the
laboratory
benches.
HEALTH
CONCERNS:
Students
who
have
allergies,
are
pregnant,
or
who
may
have
other
health
concerns
should
inform
their
lab
instructor
so
that
appropriate
precautions
may
be
taken
where
necessary.
7

9. This
form
must
be
completed,
signed,
and
submitted
to
the
laboratory
instructor
before
any
laboratory
work
is
begun.
*
*
*
*
*
*
*
*
I
have
read
and
I
understand
the
safety
rules
that
appear
in
this
manual.
I
recognize
that
it
is
my
responsibility
to
observe
them,
and
agree
to
abide
by
them
throughout
this
course.
Name
(please
print)
Date
Signature
Course:
Biology
3400
Semester:
Spring
2012
8

10. EXERCISE
1
INTRODUCTION
TO
MICROSCOPY
MICROSCOPY
To
view
microscopic
organisms,
their
magnification
is
essential.
The
microscope
is
the
instrument
used
to
magnify
microscopic
images.
Its
function
and
some
aspects
of
design
are
similar
to
those
of
telescopes
although
the
microscope
is
designed
to
visualize
very
small
close
objects
while
telescopes
magnify
distant
objects.
Please
review
Appendices
1
and
9.
Magnification
is
achieved
by
the
refraction
of
light
travelling
though
lenses,
transparent
devices
with
curved
surfaces.
In
general,
the
degree
of
refraction,
and
hence,
magnification,
is
determined
by
the
degree
of
curvature.
However,
rather
than
using
a
single,
severely-­‐curved
biconvex
lens
such
as
that
of
Leeuwenhoek's
simple
microscopes,
Hooke
determined
that
image
clarity
was
improved
through
the
use
of
a
compound
microscope,
involving
two
(or
more)
separate
lenses.
Operation
of
the
Compound
Microscope
Students
should
be
familiar
with
all
names
and
functions
of
the
components
of
their
compound
light
microscopes
as
demonstrated
in
Appendix
1.
Properties
of
the
Objective
Lenses
1.
Magnification
Magnification
is
a
measure
of
how
big
an
object
looks
to
your
eye.
The
number
of
times
that
an
object
is
magnified
by
the
microscope
is
the
product
of
the
magnification
of
both
the
objective
and
ocular
lenses.
The
magnification
of
the
individual
lenses
is
engraved
on
them.
Your
microscope
is
equipped
with
ocular
lenses
that
magnify
the
specimen
ten
times
(10X),
and
four
objectives
which
magnify
the
specimen
4X,
10X,
40X,
and
100X.
Each
lens
system
magnifies
the
object
being
viewed
the
same
number
of
times
in
each
dimension
as
the
number
engraved
on
the
lens.
When
using
a
10X
objective,
for
instance,
the
specimen
is
magnified
ten
times
in
each
dimension
to
give
a
primary
or
"aerial"
image
inside
the
body
tube
of
the
microscope.
This
image
is
then
magnified
an
additional
ten
times
by
the
ocular
to
give
a
virtual
image
that
is
100
times
larger
than
the
object
being
viewed.
2.
Resolution
Resolution
is
a
measure
of
how
clearly
details
can
be
seen
and
is
distinct
from
magnification.
The
resolving
power
of
a
lens
system
is
its
capacity
for
separating
to
the
eye
two
points
that
are
very
close
together.
It
is
dependent
upon
the
quality
of
the
lens
system
and
the
wavelength
of
light
employed
in
illumination.
The
white
light
(a
combination
of
different
wavelengths
of
visible
light)
used
as
the
light
source
in
the
lab
limits
the
resolving
power
of
the
100X
objective
lens
to
about
0.25
µm.
Objects
smaller
than
0.25
µm
cannot
be
resolved
even
if
magnification
is
increased.
Spherical
aberration
(distortion
9

11. caused
by
differential
bending
of
light
passing
through
different
thicknesses
of
the
lens
center
versus
the
margin)
results
from
the
air
gap
between
the
specimen
and
the
objective
lens.
This
problem
can
be
eliminated
by
filling
the
air
gap
with
immersion
oil,
formulated
to
have
a
refractive
index
similar
to
the
glass
used
for
cover
slips
and
the
microscope's
objective
lens.
Use
of
immersion
oil
with
a
100X
special
oil
immersion
objective
lens
can
increase
resolution
to
about
0.18
µm.
Resolving
power
can
be
increased
further
to
0.17
µm
if
only
the
shorter
(violet)
wavelengths
of
visible
light
are
used
as
the
light
source.
This
is
the
limit
of
resolution
of
the
light
microscope.
The
resolving
power
of
each
objective
lens
is
described
by
a
number
engraved
on
the
objective
called
the
numerical
aperture.
Numerical
aperture
(NA)
is
calculated
from
physical
properties
of
the
lens
and
the
angles
from
which
light
enters
and
leaves.
Examine
the
three
objective
lenses.
The
NA
of
the
10X
objective
lens
is
0.25.
Which
objective
lens
is
capable
of
the
greatest
resolving
power?
3.
Working
Distance
The
working
distance
is
measured
as
the
distance
between
the
lowest
part
of
the
objective
lens
and
the
top
of
the
coverslip
when
the
microscope
is
focused
on
a
thin
preparation.
This
distance
is
related
to
the
individual
properties
of
each
objective.
4.
Parfocal
Objectives
Most
microscope
objectives
when
firmly
screwed
in
place
are
positioned
so
the
microscope
requires
only
fine
adjustments
for
focusing
when
the
magnification
is
changed.
Objectives
installed
in
this
manner
are
said
to
be
parfocal.
5.
Depth
of
Focus
The
vertical
distance
of
a
specimen
being
viewed
that
remains
in
focus
at
any
one
time
is
called
the
depth
of
focus
or
depth
of
field.
It
is
a
different
value
for
each
of
the
objectives.
As
the
microscope
is
focused
up
and
down
on
a
specimen,
only
a
thin
layer
of
the
specimen
is
in
focus
at
one
time.
To
see
details
in
a
specimen
that
is
thicker
than
the
depth
of
focus
of
a
particular
objective
you
must
continuously
focus
up
and
down.
Observing
Bacteria
Three
fundamental
properties
of
bacteria
are
size,
shape
and
association.
Bacteria
generally
occur
in
three
shapes:
coccus
(round),
bacillus
(rod-­‐shaped),
and
spirillum
(spiral-­‐
shaped).
Size
of
bacterial
cells
used
in
these
labs
varies
from
0.5
µm
to
1.0
µm
in
width
and
from
1.0
µm
to
5.0
µm
in
length,
although
there
is
a
range
of
sizes
which
bacteria
demonstrate.
Association
refers
to
the
organization
of
the
numerous
bacterial
cells
within
a
culture.
Cells
may
occur
singly
with
10

12. cells
separating
after
division;
showing
random
association.
Cells
may
remain
together
after
division
for
some
interval
resulting
in
the
presence
of
pairs
of
cells.
When
cells
remain
together
after
more
than
a
single
division,
clusters
result.
Cell
divisions
in
a
single
plane
result
in
chains
of
cells.
If
the
plane
of
cell
division
of
bacilli
is
longitudinal,
a
palisade
results,
resembling
a
picket
fence.
Both
bacterial
cell
shape
and
association
are
usually
constant
for
bacteria
and
hence,
can
be
used
for
taxonomic
identification.
However,
both
properties
may
be
influenced
by
culture
condition
and
age.
Further,
some
bacteria
are
quite
variable
in
shape
and
association
and
this
may
also
be
diagnostic.
Micrometry
When
studying
bacteria
or
other
microorganisms,
it
is
often
essential
to
evaluate
the
size
of
the
organism.
By
tradition,
the
longest
dimension
(length)
is
generally
stressed,
although
width
is
sometimes
useful
for
identification
or
other
study.
Use
of
an
Ocular
Micrometer
(Figure
1)
An
ocular
micrometer
can
be
used
to
measure
the
size
of
objects
within
the
field
of
view.
Unfortunately,
the
distance
between
the
graduations
of
the
ocular
micrometer
is
an
arbitrary
measurement
that
only
has
meaning
if
the
ocular
micrometer
is
calibrated
for
the
objective
being
used.
1) Place
a
micrometer
slide
on
the
stage
and
focus
the
scale
using
the
40x
objective.
2) Turn
the
eyepiece
until
the
graduations
on
the
ocular
scale
are
parallel
with
those
on
the
micrometer
slide
scale
and
superimpose
the
micrometer
scale.
3) Move
the
micrometer
slide
so
that
the
first
graduation
on
each
scale
coincides.
4) Look
for
another
graduation
on
the
ocular
scale
that
exactly
coincides
with
a
graduation
on
the
micrometer
scale.
5) Count
the
number
of
graduations
on
the
ocular
scale
and
the
number
of
graduations
on
the
micrometer
slide
scale
between
and
including
the
graduations
that
coincide.
6) Calibrate
the
ocular
divisions
for
the
40x
and
the
100x
objective
lenses.
Note
that
immersion
oil
is
not
necessary
for
calibration.
Figure
1.
Calibration
of
an
ocular
micrometer
using
a
stage
micrometer.
The
mark
on
the
stage
micrometer
corresponding
to
0.06
mm
(60
µ m)
is
equal
to
5
ocular
divisions
(o.d.)
on
the
ocular
micrometer.
∴
1
ocular
division
equals
60
µ m/5
ocular
divisions
or
12
µ m.
11

13. Once
an
ocular
micrometer
has
been
calibrated,
objects
may
be
measured
in
ocular
divisions
and
this
number
converted
to
µm
using
the
conversion
factor
determined.
Bacterial
size
is
generally
a
highly
heritable
trait.
Consequently,
size
is
a
key
factor
used
in
the
identification
of
bacterial
taxa.
However,
for
some
bacteria,
cell
size
can
be
modified
by
nutritional
factors
such
as
culture
media
composition,
environmental
factors
such
as
temperature,
or
other
factors
such
as
age.
METHODS:
For
each
student:
• Compound
light
microscope
• Various
prepared
slides
of
bacteria.
• Stage
micrometer
• Ocular
micrometer
• Immersion
oil
1) Use
the
diagram
in
Figure
1
to
calibrate
the
40x
and
the
100x
objectives
on
your
compound
microscopes.
Record
these
values
in
your
lab
book
as
you
will
then
use
these
values
when
measuring
cells
and
structures
for
the
rest
of
the
lab.
Note:
Do
NOT
use
immersion
oil
when
calibrating
the
100x
objective.
This
is
the
ONLY
time
during
the
term
that
you
will
not
use
immersion
oil
with
this
objective.
2) Use
the
compound
microscope
to
observe
the
prepared
slides
of
bacteria
using
the
10x
and
40x
objective
lenses.
Observe
the
same
slides
under
the
100x
objective
using
immersion
oil.
3) Diagram
two
of
the
organisms
viewed
following
the
instructions
found
in
Appendix
2.
12

14. EXERCISE
2
GENERAL
LABORATORY
PROCEDURES
AND
BIOSAFETY
A
primary
feature
of
the
microbiology
laboratory
is
that
living
organisms
are
employed
as
part
of
the
experiment.
Most
of
the
microorganisms
are
harmless;
however,
whether
they
are
non-­‐pathogenic
or
pathogenic,
the
microorganisms
are
treated
with
the
same
respect
to
assure
that
personal
safety
in
the
laboratory
is
maintained.
Careful
attention
to
technique
is
essential
at
all
times.
Care
must
always
be
taken
to
prevent
the
contamination
of
the
environment
from
the
cultures
used
in
the
exercises
and
to
prevent
the
possibility
of
the
people
working
in
the
laboratory
from
becoming
contaminated.
Ensure
that
you
have
read
over
the
guidelines
on
Safety,
and
those
on
Aseptic
technique
(Appendix
3).
As
well,
you
should
be
familiar
with
the
contents
of
the
University
of
Lethbridge
Biosafety
web
site:
www.uleth.ca/artsci/biological-­‐sciences/bio-­‐safety
METHODS
Part
1:
General
Laboratory
Procedures
Work
individually
to
prepare
a
streak
plate
and
a
broth
culture
using
the
E.
coli
cultures
provided.
Refer
to
Appendix
3
as
necessary.
Part
2:
Biosafety
You
will
be
provided
with
the
following:
• Sterile
swabs
• Sterile
water
• Potato
Dextrose
Agar
(PDA)
plates
and
Luria
Bertani
(LB)
plates
Work
in
pairs
to
complete
the
following
exercise:
1) Draw
a
line
on
the
back
of
each
plate
to
divide
the
plates
in
half.
Label
one
half
of
the
plate
with
the
name
of
the
surface
to
be
tested.
Label
the
other
half
of
the
plate
as
“after
disinfection”.
2) Moisten
the
swabs
provided
with
a
small
amount
of
sterile
water.
Brush
the
surface
to
be
tested
with
the
swab,
and
then
use
the
swab
to
inoculate
one-­‐half
of
each
of
your
two
plates.
3) Disinfect
the
surface,
moisten
another
swab,
and
repeat
using
the
other
half
of
both
plates.
Wrap
the
plates
with
parafilm.
4) Your
plates
will
be
incubated
for
16-­‐20
hours
at
30oC,
and
then
refrigerated
at
4oC.
During
the
next
laboratory
period,
evaluate
your
plate
results
and
record
the
number
of
colonies
present
on
each
half
of
both
plates.
Make
observations
of
colony
morphology.
Thought
Questions:
(Use
the
Biosafety
Web
Site
as
a
reference)
• Were
differences
in
colony
morphology
and
number
observed
on
the
two
types
of
media?
Why?
• Does
disinfection
of
work
surfaces
completely
eliminate
all
microbial
organisms?
What
evidence
do
you
have?
• What
is
an
MSDS
and
where
can
you
find
one?
• In
Canada,
the
Laboratory
Centre
for
Disease
Control
has
classified
infectious
agents
into
4
Risk
Groups
using
pathogenicity,
virulence
and
mode
of
transmission
(among
others)
as
criteria.
What
do
these
terms
mean?
• What
criteria
would
characterize
an
organism
classified
in
Risk
Group
1,
2
3
or
4?
• There
are
many
“Golden
Rules”
for
Biosafety.
Identify
4
common
sense
practices
that
will
protect
you
in
your
microbiology
labs.
13

15. EXERCISE
3
FREE-­LIVING
NITROGEN
FIXATION
PART
A:
ISOLATION
OF
FREE-­‐LIVING
NITROGEN
FIXING
MICROORGANISMS
Nitrogen
is
an
important
component
of
amino
acids,
cell
walls
and
other
cofactors
present
in
all
cells.
Nitrogen
gas
comprises
greater
than
75%
of
our
atmosphere,
but
it
is
one
of
the
most
stable
bonds
in
nature,
and
is
unavailable
for
use
in
this
form.
At
one
time
early
in
the
evolutionary
history
of
life
on
earth,
all
cells
may
have
had
the
ability
to
fix
N2
gas
into
a
more
usable
form
(nitrate,
nitrite
or
ammonia).
Today
however,
only
a
few
species
of
bacteria
and
archaea
are
capable
of
converting
N2;
all
other
organisms
rely
on
N2-­‐fixing
prokaryotes
for
their
fixed
nitrogen
requirements.
M.W.
Beijerinck,
a
Dutch
microbiologist,
successfully
isolated
free
living
nitrogen
fixing
bacteria
in
1901.
He
inoculated
soil
samples
into
enrichment
media
containing
glucose
and
mineral
salts,
but
lacking
any
source
of
nitrogen
other
than
atmospheric
nitrogen.
He
observed
cells
that
are
today
identified
as
members
of
the
genus
Azotobacter.
Subsequently,
other
aerobic,
free-­‐living
nitrogen
fixing
genera
of
bacteria
have
been
isolated
and
identified,
including
Azomonas,
Azospirillum
and
Beijerinckia.
Nitrogen
fixation
occurs
only
when
an
enzyme
called
nitrogenase
is
present.
The
enzyme
consists
of
two
distinct
proteins
(i)
dinitrogenase,
which
reacts
with
N2,
and
(ii)
dinitrogenase
reductase,
which
reduces
nitrogen
gas
to
ammonia.
The
dinitrogenase
reductase
component
is
irreversibly
inactivated
by
the
presence
of
oxygen.
Several
strategies
have
evolved
to
enable
free-­‐living,
aerobic
organisms
like
Azotobacter
to
fix
nitrogen.
Azotobacter
has
a
very
high
respiratory
rate,
which
is
thought
to
prevent
any
stray
oxygen
from
coming
into
contact
with
the
nitrogenase
enzyme.
Additionally,
free-­‐living
nitrogen
fixers
often
secrete
copious
amounts
of
slime
which
may
prevent
extra
oxygen
from
entering
the
cells.
There
is
also
evidence
suggesting
that
in
the
presence
of
oxygen
nitrogenase
can
combine
with
a
specific
protein
inside
the
cell
that
shields
the
oxygen
sensitive
site
and
prevents
it
from
interacting
from
oxygen.
When
oxygen
levels
drop,
nitrogenase
can
resume
its
activity.
Over
the
course
of
the
semester
we
will
isolate
free
living
nitrogen
fixing
bacteria
from
prairie
soil,
establish
pure
cultures
and
attempt
to
identify
cultures
using
modern
day
molecular
techniques.
METHODS:
For
each
lab:
• 5,
250
mL
flasks
containing
N-­‐free
medium;
10
plates
N-­‐free
medium
• Balance,
weigh
boats
and
spatula
• N-­‐free
soil
sample
Work
in
groups
of
4
to
inoculate
your
flasks.
• Weigh
out
1
g
of
the
soil
sample
provided,
and
add
it
to
an
Erlenmeyer
flask
containing
100
mL
of
N-­‐
free
medium.
14

16. • Swirl
gently
to
mix.
Label
the
flask
with
your
lab,
bench
number,
and
date.
Make
sure
that
the
cap
or
foil
is
loosened
sufficiently
to
allow
air
to
enter
the
culture.
• After
7
days,
remove
the
flask
and
look
for
the
presence
of
a
thin
film
of
growth
on
the
surface
of
the
medium.
Use
a
sterile
inoculating
loop
to
remove
some
of
this
film
and
prepare
a
streak
plate.
The
streak
o
plate
will
be
incubated
for
a
further
7
days
at
30 C.
• Examine
wet
mounts
from
your
broth
culture
using
the
phase
contrast
microscope.
Prepare
Gram
stains
of
the
film
and
look
for
large
Gram
negative
cells
that
may
be
bacillus
or
coccoid
in
shape.
They
may
occur
singly,
or
in
arrowhead-­‐shaped
pairs.
Record
observations
in
your
lab
book.
• After
the
incubation
period
is
complete,
examine
your
streak
plate.
Look
for
large,
translucent,
mucoid
colonies.
Prepare
a
wet
mount
from
an
isolated
colony
and
view
it
using
a
phase-­‐contrast
microscope.
Prepare
another
streak
plate
using
cells
from
the
same
colony.
This
plate
will
be
incubated
again,
and
observations
will
be
made
later
in
the
term.
Additionally,
this
culture
will
be
used
in
Part
B
of
this
exercise.
Thought
Questions:
• Define
enrichment.
What
aspect(s)
of
the
medium
used
in
this
exercise
made
it
an
enrichment
medium?
Why
did
we
use
the
same
medium
for
plating
after
free-­‐living
nitrogen
fixers
were
isolated?
What
term
would
we
use
to
describe
the
medium
in
this
case?
• Why
did
we
sample
the
film
on
top
of
the
culture,
rather
than
the
sediment
on
the
bottom
of
the
flask?
• When
you
viewed
your
Gram
stains,
you
may
have
observed
cells
on
your
slides
that
didn’t
appear
to
be
Azotobacter.
Why
might
these
other
genera
be
present?
PART
B:
IDENTIFICATION
OF
MICROORGANISMS
USING
PCR
OF
16s
rDNA
The
DNA
from
microbes
can
be
isolated
and
may
be
studied
via
construction
of
BAC
(Bacterial
Artificial
Chromosome)
libraries
(for
an
example,
see
Rondon,
et
al.,
2000).
More
simply,
an
appreciation
of
diversity
may
be
obtained
by
using
universal
primers
for
PCR
amplification
of
rDNA
genes
from
the
Bacterial
domain
on
a
preparation
of
total
DNA
from
an
environmental
sample.
The
resulting
pool
of
nucleotide
fragments
may
then
be
cloned,
unique
clones
sequenced,
and
the
resulting
sequences
analyzed
in
order
to
characterize
and
potentially
identify
the
microbes
present.
In
Part
B
of
this
exercise
you
will
perform
PCR
using
primers
specific
for
prokaryotic
16s
rDNA
to
isolate
ribosomal
DNA
from
putative
Azotobacter
cultures
and
then
visualise
this
DNA
using
Agarose
Gel
Electrophoresis.
In
addition,
DNA
from
successful
PCRs
will
be
sent
for
sequencing
and
you
will
then
be
using
online
tools
to
perform
sequence
analysis
to
confirm
the
identity
of
your
cultures.
PCR
of
Soil
Bacteria
Two
different
primer
sets
will
be
employed.
Each
group
will
only
be
using
one
set
on
their
particular
culture.
Note
that
the
primer
designations
refer
to
location
of
primer
binding
site
on
the
16s
rDNA
molecule.
Given
this
information,
predict
the
sizes
of
your
PCR
products
for
both
primer
sets.
15

17. For
preparation
of
your
reaction
mixtures:
Benches
1,
3,
and
5:
Working
with
the
people
at
your
bench,
each
group
will
be
setting
up
3x
reactions
as
outlined
below:
Primer
Template
Source
FP1/1492R
Unknown
Culture
FP1/1492R
E.
coli
FP1/1492R
No
template
Benches
2/4:
Working
with
the
people
at
your
bench,
each
group
will
be
setting
up
3x
reactions
as
outlined
below:
Primer
Set
Template
Source
27F/805R
Unknown
Culture
27F/805R
E.
coli
27F/805R
No
template
METHODS:
Reagents:
• Taq
(Invitrogen)
• 10x
PCR
buffer
• 50
mM
MgCl2
Primers
(*Y
=
C
or
T)
• FP1
(AGAGTTYGATYCTGGCT)*1
(10
pmol/µL)
• RP1492
(TACGGYTACCTTGTTACGACT)*1
(10
pmol/µL)
• 27F
(AGAGTTTGATCCTGGCTCAG)2
(10
pmol/µL)
• 805R
(GACTACCAGGGTATCTAATCC)2
(10
pmol/µL)
• dNTP
mix
(8
mM)
• Optima
Water
(Fisher
Scientific)
Cultures:
• Pure
culture
of
organism
isolated
from
soil
• Plate
culture
of
E.
coli
Equipment:
• Thermocyclers
(BioRad)
• Micropipettors
and
sterile
tips
• Parafilm
• Ice
buckets
and
ice
• Sterile
0.5
mL
tubes
• Sterile
0.2
mL
PCR
tubes
• Biohazard
bags
• Permanent
markers
Note:
Use
aseptic
technique
throughout.
Keep
your
tubes
on
ice
at
all
times!
• Obtain
three
0.2
mL
PCR
tubes
from
the
sterile
container
at
the
side
of
the
lab.
Decide
on
appropriate
16

18. codes
for
labeling
the
tubes
(keeping
in
mind
that
other
groups
are
carrying
out
the
same
reactions).
Label
the
tubes
on
the
tops
and
on
the
sides
using
permanent
marker.
Place
the
tubes
on
ice.
• Obtain
a
0.5
mL
tube
for
your
Master
Mix.
Keep
this
tube
on
ice.
Use
the
information
outlined
in
Table
1
to
set
up
your
Master
Mix.
This
mix
contains
everything
required
in
order
for
DNA
replication
to
occur.
Generally,
Master
Mixes
contain
enough
volume
to
set
up
the
number
of
reactions
+
1.
In
your
case,
you
will
be
preparing
enough
mix
for
4
reactions.
Work
carefully.
Table
1.
Components,
starting
concentrations
and
volumes
for
set-­‐up
of
PCRs.
Component
and
Starting
Final
Amount
to
add
Master
Mix
vol.
(for
Concentration
Concentration
for
ONE
reaction
total
#
Reactions
+
(µL)
1)
(µL)
Optima-­‐Water
32
128
10x
PCR
buffer
1x
5
20
50
mM
MgCl2
1.5
mM
2
8
dNTP
mix
(8
mM
of
all
4)
40
nmoles
(0.8
mM
5
20
of
all
4)
Primer
1
0.4
µ M
2
8
Primer
2
0.4
µ M
2
8
Taq
DNA
polymerase
(5
U/µL)
5
U
(units)
1
4
Template
DNA
1
Leave
Template
out
of
Master
Mix!
Final
Volume
50
µ L
50
µ L
Note:
One
primer
set
per
reaction
mixture!
• While
the
Master
Mix
is
being
set
up,
other
group
members
should
be
setting
up
template
preparations.
Obtain
a
small
square
of
parafilm.
For
each
bacterial
culture
(soil
bacteria
and
E.
coli
–
what
is
the
role
of
E.
coli?),
use
a
micropipettor
with
a
sterile
tip
to
pipette
20
µ L
of
sterile
Optima-­‐water
(Fisher
Scientific)
onto
the
parafilm.
• Take
a
10
–
100
µL
micropipettor
and
put
on
a
sterile
tip.
Touch
the
tip
to
a
single
colony
from
your
soil
bacterial
culture
plate.
Pipette
up
and
down
into
the
Optima
water
on
the
parafilm.
This
mixture
will
be
used
as
your
template
source.
• Mix
E.
coli
in
the
same
fashion
with
your
second
drop
of
Optima
water
on
the
parafilm.
Again,
1
µL
of
this
mixture
will
be
used
as
template
in
your
second
reaction.
• For
your
third
reaction,
you
will
be
leaving
out
template
and
replacing
it
with
an
equal
volume
of
sterile
Optima
water.
What
is
the
purpose
of
this
reaction?
• After
preparation
of
Master
Mix,
add
the
appropriate
volume
of
template
(1
µL)
to
each
tube,
then
check
with
the
Instructor
to
see
where
everyone
else
is
at.
When
all
of
the
groups
are
at
the
same
stage,
add
the
appropriate
volume
of
Master
Mix
(49
µL
)
to
each
tube.
Keep
your
tubes
on
ice
until
in
the
PCR
machine.
GENTLY
tap
tubes
to
mix.
When
everyone
is
ready,
the
instructor
will
then
show
you
how
to
operate
the
thermocycler.
The
parameters
you
are
using
for
the
PCR
are:
17

19. o
12
minutes
at
95
C
(used
not
only
in
initial
DNA
denaturation,
but
also
to
lyse
the
bacterial
cells)
30
cycles
of:
o
• 1
minute
at
94
C
o
• 45
seconds
at
55
C
o
• 90
seconds
at
72
C
A
final
elongation
of:
o
• 20
minutes
at
72
C
o
The
samples
will
be
stored
at
-­‐20
C
upon
completion.
Thought
Questions:
• What
are
the
purposes
of
the
primers
in
PCR?
• What
happens
at
each
temperature?
• How
is
annealing
temperature
determined?
• What
is
meant
by
stringency?
How
can
you
ensure
high
stringency?
• If
you
left
out
the
forward
primer,
would
you
expect
to
see
a
band
resulting
on
the
gel?
If
you
did,
explain
what
this
would
mean.
• Is
it
possible
to
design
PCRs
given
only
an
isolatable
protein?
Why
or
why
not?
What
are
some
of
the
problems
associated
with
such
an
experiment?
How
might
you
adapt
the
reaction
conditions
to
optimise
yield
of
desired
product?
Suggested
Background
Reading:
Amann
et.
al.,
1995.
Phylogenetic
identification
and
in
situ
detection
of
individual
microbial
cells
without
cultivation.
Microbiol.
Rev.
59
(1):
143-­‐169.
Aas,
J.
A.,
Paster,
B.
J.,
Stokes,
L.
N.,
Olsen,
I.,
and
Dewhirst,
F.
E.
2005.
Defining
the
Normal
Bacterial
Flora
of
the
Oral
Cavity.
J.
Clin.
Microbiol.
43:
5721-­‐5732.
Cole,
J.
R.,
Chai,
B.,
Farris,
R.
J.,
Wang,
Q.,
Kulam,
S.
A.,
McGarrell,
D.
M.,
Bandela,
A.
M.,
Cardenas,
E.,
Garrity,
G.
M.,
and
Tiedje,
J.
M.
2007.
The
ribosomal
database
project
(RDPII):
introducing
myRDP
space
and
quality
controlled
public
data.
Nuc.
A.
Res.
35:
D169-­‐D172.
DeLong
and
Pace,
2001.
Environmental
diversity
of
bacteria
and
archaea.
Syst.
Biol.
50(4):
470-­‐478.
Gabor,
E.
M.,
deVries,
E.
J.,
and
Janssen,
D.
B.
2003.
Efficient
recovery
of
environmental
DNA
for
expression
cloning
by
indirect
extraction
methods.
FEMS.
44(2):
153-­‐163.
Kelley,
S.T.,
Theisen
U.,
Angenent,
L.T.,
Amand,
A.S.,
and
Pace,
N.R.
Molecular
Analysis
of
Shower
Curtain
Biofilm
Microbes.
Appl.
Environ.
Microbiol.
70:
4187-­‐4192.
Pace,
1997.
A
molecular
view
of
microbial
diversity
and
the
biosphere.
Science.
276:
734-­‐740.
Whitford,
M.
F.,
Forster,
R.
J.,
Beard,
C.
E.,
Gong,
J.,
and
Teather,
R.
M.
1998.
Phylogenetic
analysis
of
rumen
bacteria
by
comparative
sequence
analysis
of
cloned
16S
rRNA
genes.
Anaerobe.
4:
153-­‐163.
18

20. Woese,
C.
R.,
Kandler,
O.,
and
Wheelis,
M.
L.,
1990.
Towards
a
natural
system
of
organisms:
Proposal
for
the
domains
Archaea,
Bacteria,
and
Eucarya.
Proc.
Natl.
Acad.
Sci.
USA.
87:
4576-­‐4579.
Agarose
Gel
Electrophoresis
METHODS
Reagents:
• 1x
TBE
buffer
• 0.8%
agarose
gels
(1
per
2
benches)
• 10x
loading
dye
• 2-­‐log
NEB
ladder
premixed
with
loading
dye
• Ethidium
bromide
bath
• PCR
samples
from
last
lab
Equipment
• Power
supplies
(1
per
2
benches)
• Micropipettors
• Sterile
tips
• Parafilm
• Transilluminator/camera
• Biohazard
bags
• Gloves
Note:
Two
groups
will
load
their
samples
(6
tubes
total)
onto
one
gel.
We
will
be
using
0.8%
agarose
prepared
in
1x
TBE.
• Obtain
and
completely
thaw
your
PCR
samples.
• Using
a
micropipettor,
'dot'
out
1
µL
aliquots
of
10x
loading
dye
in
a
line
on
a
thin
strip
of
parafilm.
Remove
a
7.5
µL
aliquot
of
your
first
sample,
mix
gently
with
the
loading
dye
on
the
parafilm,
and
proceed
with
loading.
Aim
for
approximately
1-­‐2x
final
concentration
of
loading
dye
per
sample
loaded
(and
recognise
that
this
is
NOT
exact).
Loading
Dye
–
1)
increases
the
density
of
the
sample
ensuring
that
it
drops
evenly
into
the
well;
2)
adds
colour
to
the
sample
to
simplify
loading;
and
3)
contains
dyes
that
in
an
electric
field
move
toward
the
anode
at
predictable
rates.
In
this
laboratory,
we
are
making
use
of
mixtures
containing
xylene
cyanol
FF.
This
dye
migrates
in
0.5x
TBE
at
approximately
the
same
rate
as
linear
DNA
of
4000
bp
in
size.
Often,
bromophenol
blue
is
used
in
conjunction
with
xylene
cyanol,
or
separately.
Bromophenol
blue
migrates
at
approximately
the
same
rate
as
linear
DNA
of
300
bp
in
size
in
0.5x
TBE
(2.2
fold
faster
than
xylene
cyanol
FF,
independent
of
agarose
concentration).
• Load
the
remainder
of
the
samples
in
the
same
manner,
leaving
at
least
one
well
empty
(to
be
used
for
a
DNA
ladder).
Be
sure
to
RECORD
the
order
in
which
the
samples
were
loaded.
• Load
10
µL
of
the
ladder.
One
type
of
size
standard
is
produced
by
ligating
a
monomer
DNA
fragment
of
known
size
into
a
ladder
of
polymeric
forms.
The
2-­‐log
DNA
ladder
from
New
England
Biolabs
consists
of
a
mixture
of
a
number
of
proprietary
plasmids
digested
to
completion
with
different
restriction
enzymes.
Ladders
tend
to
be
19

21. purchased
as
commercial
preparations.
For
an
example
please
see:
http://www.neb.com/nebecomm/products/productn3200.asp
• Turn
on
the
power
supply
and
set
the
voltage
to
100
V.
Place
the
lid
on
the
gel
and
start
the
run.
The
gel
will
run
for
30
minutes,
then
shut
off
automatically.
• After
the
run
is
complete,
turn
off
the
power.
Designate
one
group
member
to
put
on
gloves,
scoop
up
the
gel,
and
gently
slide
the
gel
into
the
ethidium
bromide
bath.
Caution:
Ethidium
bromide
is
a
mutagen
and
a
suspected
carcinogen.
At
very
dilute
concentrations
and
with
responsible
handling,
this
risk
is
minimised.
• Stain
the
gel
with
gentle
shaking
for
approximately
10
minutes.
One
group
member
again
should
put
on
gloves,
and
transfer
the
gel
to
the
gel
documentation
system.
View
using
the
UV
transilluminator.
Photographs
will
be
taken.
Please
ensure
that
you
bring
a
USB
memory
stick
so
that
you
can
obtain
the
photograph
of
your
gel
(these
will
NOT
be
emailed
out).
Caution:
Ultraviolet
light
is
damaging
to
naked
eyes
and
exposed
skin.
Always
view
through
filter
or
safety
glasses
that
absorb
harmful
wavelengths.
• Based
on
gel
results
and
quantification
of
your
DNA,
a
selection
of
samples
will
be
sent
off
for
sequencing.
In
order
to
facilitate
this,
use
a
piece
of
tape
to
completely
label
your
PCR
products
ensuring
that
the
label
corresponds
with
that
from
the
gel.
Thought
Questions
• What
factors
influence
DNA
migration
through
agarose?
Explain.
• Why
are
we
using
0.8%
agarose
for
resolution
of
our
PCR
products?
• Evaluate
your
gel
results
with
respect
to:
expected
fragment
sizes
and
reasoning,
and
control
results.
Do
we
have
evidence
to
suggest
that
we
were
successful
in
amplifying
16s
rDNA?
Explain
your
reasoning.
• What
are
some
of
the
advantages
and
disadvantages
of
molecular
techniques
for
identification
of
bacteria?
Compare
and
contrast
with
conventional
culturing
techniques.
20

22. EXERCISE
4
WINOGRADSKY
COLUMNS
All
life
on
earth
can
be
categorized
based
on
what
carbon
and
energy
sources
they
utilize.
Phototrophs
obtain
energy
from
light
reactions,
while
chemotrophs
obtain
energy
from
chemical
oxidations
of
organic
or
inorganic
substances.
The
carbon
used
for
synthesis
can
be
obtained
directly
from
CO2
(autotrophs),
or
from
previously
existing
organic
compounds
(heterotrophs).
Combinations
of
these
categories
give
rise
to
the
four
basic
strategies
of
life:
photoautotrophs
(plants),
chemoheterotrophs
(animals
and
fungi),
photoheterotrophs
and
chemoautotrophs.
The
prokaryotic
bacteria
and
archaea
are
the
only
forms
of
life
where
all
four
life
strategies
can
be
observed.
Winogradsky
columns,
named
for
the
Russian
microbiologist
Sergei
Winogradsky
(1856-­‐1953)
are
model
ecosystems
that
can
be
used
to
study
the
diversity
of
life
strategies
employed
by
bacteria
and
archaea.
Columns
are
prepared
by
filling
glass
tubes
mostly
full
of
mud
supplemented
with
cellulose
(shredded
newspaper),
calcium
carbonate
and
calcium
sulphate.
Initially
there
are
low
numbers
of
organisms
present
in
the
column,
but
after
two
to
three
months
of
incubation,
many
different
types
of
organisms
proliferate
and
occupy
distinct
zones
within
the
column
where
environmental
conditions
favour
their
growth.
After
the
column
is
constructed,
it
is
sealed
and
left
in
the
dark
for
several
days
to
promote
the
growth
of
aerobic
heterotrophs,
which
will
utilize
the
cellulose
in
the
column
and
deplete
the
oxygen.
This
is
the
first
of
a
succession
of
organisms
that
will
inhabit
the
column.
The
column
is
then
placed
in
indirect
light.
Cyanobacteria
and
algae
may
appear
in
the
water
at
the
top
of
the
column,
providing
aerobic
conditions
resulting
from
the
production
of
oxygen
from
photosynthesis.
Large
populations
of
chemoautotrophic
bacteria
may
also
appear
in
this
region
(Thiobacillus,
Beggiatoa).
These
organisms
fix
carbon
dioxide
and
obtain
energy
by
oxidizing
H2S.
Conversely,
if
the
water
at
the
top
of
the
column
contains
only
small
amounts
of
oxygen,
it
may
appear
to
be
red
due
to
the
presence
of
purple
non-­‐sulphur
bacteria
(Rhodobacter,
Rhodospirillum).
The
anaerobic
mud
at
the
bottom
of
the
column
may
be
home
to
species
like
Cellulomonas,
which
degrades
cellulose
to
component
monosaccharides,
and
Clostridium
and
other
species
which
degrade
the
monosaccharides
to
organic
acids
such
as
lactacte
and
acetate.
Lactate,
along
with
the
sulphate
in
the
column,
is
utilized
by
sulphate-­‐reducing
bacteria
(Desulfovibrio),
producing
H2S.
The
H2S
may
react
with
metals
in
the
mud
to
produce
a
black
precipitate.
H2S
also
diffuses
up
through
the
column,
and
may
be
used
by
other
bacterial
populations,
including
the
phototrophic
purple
sulphur
bacteria
(Chromatium)
and
green
sulphur
bacteria
(Chlorobium).
METHODS:
For
each
lab:
• 100
mL
graduated
cylinders
• Mud
samples
• Source
of
cellulose
• CaCO3,
CaSO4,
K2HPO4
• Balance,
weigh
boats
and
spatulas
• Stirring
rods
• Aluminium
foil
• 250
mL
beakers
21

23.
Work
in
groups
of
four
to
set
up
your
Winogradsky
columns.
• Prepare
a
thick
slurry
in
the
beaker
using
your
source
of
cellulose.
If
using
cellulose
powder,
weigh
out
1-­‐2
g
of
powder
and
add
to
a
small
amount
of
water.
Add
more
water
as
necessary
to
make
a
thick
slurry
(still
needs
to
be
runny;
a
slurry
is
not
a
paste).
If
using
newspaper,
tear
it
in
small
pieces,
and
macerate
it
in
a
small
volume
of
water.
• Fill
the
graduated
cylinder
to
about
the
30
mL
mark
with
your
cellulose
slurry.
• Add
1.64
g
CaSO4
and
1.3
g
each
of
CaCO3
and
K2HPO4
to
200
g
of
mud
sample.
• Add
some
of
the
water
collected
with
your
mud
(“self”
water)
to
your
mud-­‐chemical
mixture,
and
mix
well.
• Slowly
pour
some
mud
into
the
column,
mixing
it
with
the
cellulose
slurry.
Your
column
will
begin
to
pack.
As
you
pack
the
column,
you
may
need
to
add
more
“self”
water
to
the
mixture.
The
slurry-­‐
mud-­‐water
mixture
should
occupy
about
2/3
of
the
graduated
cylinder
when
you
are
finished.
• Top
off
the
column
with
more
“self”
water
until
it
is
about
90%
full.
Note
the
appearance
of
the
column
in
your
lab
books.
Cover
the
top
with
aluminium
foil,
and
label
with
your
source
of
mud,
group
and
lab
number.
Wrap
the
sides
of
the
column
with
aluminium
foil,
and
apply
another
label
to
the
outside.
• Columns
will
be
incubated
at
room
temperature
for
2
weeks.
Remove
the
aluminium
foil
from
the
sides
of
the
column
and
make
observations
in
your
lab
books.
Place
your
column
near
the
window,
and
continue
to
make
observations
at
regular
intervals
during
the
remainder
of
the
semester.
Look
for
development
of
red,
brown,
purple,
black
or
green
regions
in
the
mud
or
water.
• We
will
occasionally
sample
regions
of
the
Winogradsky
column
and
examine
them
by
phase
contrast
microscopy
to
observe
microorganisms
that
are
proliferating.
Thought
Questions:
• What
is
the
function
of
each
chemical
(including
the
cellulose)
added
to
the
Winogradsky
column?
• What
may
have
happened
if
the
column
was
not
wrapped
in
aluminium
foil
for
the
first
two
weeks?
• Prepare
a
composite
sketch
of
your
column,
and
name
the
groups
of
bacteria
appearing
in
each
region.
Provide
an
explanation
as
to
why
each
group
appears
where
it
does
in
the
column.
• Describe
how
Winogradsky
columns
may
be
used
to
enrich
various
prokaryotes.
• How
is
a
Winogradsky
column
similar
to
a
real
ecosystem?
How
does
it
differ?
22

24. EXERCISE
5
BACTERIAL
and
YEAST
MORPHOLOGY
Bacteria
cells
are
very
difficult
to
observe
using
compound
light
microscopes
because
the
cells
appear
transparent
in
the
aqueous
medium
in
which
they
are
suspended.
Staining
the
cells
prior
to
observation
increases
the
contrast
between
the
cell
and
the
medium,
which
allows
for
the
visualization
of
cell
structures.
However,
the
application
of
stains
usually
leads
to
cell
death.
Phase
contrast
microscopes
enhance
the
contrast
between
cells
and
their
environment
without
the
use
of
stains,
meaning
that
living
cells
and
their
activities
can
be
observed.
We
will
use
both
approaches
to
study
the
morphology
of
microorganisms
in
this
exercise.
Staining
In
general,
prior
to
any
staining
procedure,
fixation
occurs.
Fixation
performs
two
functions:
(i)
immobilizes
(kills)
the
bacteria;
and
(ii)
affixes
them
to
the
slide.
Any
procedure
that
results
in
the
staining
of
whole
cells
or
cell
parts
is
referred
to
as
positive
staining.
Most
positive
stains
used
involve
basic
dyes
where
basic
means
that
they
owe
their
coloured
properties
to
a
cation
(positively
charged
molecule).
When
all
that
is
required
is
a
general
bacterial
stain
to
show
morphology,
basic
stains
such
as
methylene
blue
or
carbol
fuchsin
result
in
the
staining
of
the
entire
bacterial
cell.
Differential
stains
are
used
to
distinguish
bacteria
based
on
certain
properties
such
as
cell
wall
structure.
Differential
stains
are
useful
for
bacterial
identification,
contributing
to
information
based
on
bacterial
size,
shape,
and
association.
Differential
staining
relies
on
biochemical
or
structural
differences
between
the
groups
that
result
in
different
affinities
by
various
chromophores.
Gram
staining
behaviour
relies
on
differences
in
cell
wall
structure
and
biochemical
composition.
Some
bacteria
when
treated
with
para-­‐rosaniline
dyes
and
iodine
retain
the
stain
when
subsequently
treated
with
a
decolourising
agent
such
as
alcohol
or
acetone.
Other
bacteria
lose
the
stain.
Based
on
this
property,
a
contemporary
of
Pasteur,
Hans
Christian
Gram,
developed
a
rapid
and
extremely
useful
differential
stain,
which
subsequently
bears
his
name
-­‐
the
Gram
stain
used
to
distinguish
two
types
of
bacteria,
Gram
positive
and
Gram
negative.
Gram
negative
forms,
which
are
those
that
lose
the
stain
on
decolourization,
can
be
made
visible
by
using
a
suitable
counterstain.
The
strength
of
the
Gram
stain
rests
on
its
relatively
unambiguous
separation
of
bacterial
types
into
two
groups.
However,
variables
such
as
culture
condition,
age
or
environmental
condition,
can
influence
Gram
staining
of
some
bacteria.
The
bacterial
cell
wall
is
very
important
for
many
aspects
of
bacterial
function
and
hence,
the
Gram
stain
also
provides
valuable
information
about
the
physiological,
medicinal
and
even
ecological
aspects
of
the
bacteria.
Negative
staining
is
used
to
characterize
external
structures,
like
capsules,
that
are
associated
with
living
bacterial
cells.
Negative
stains
make
use
of
acidic
dyes
where
acidic
means
that
they
owe
their
coloured
properties
to
an
anion
(negatively
charged
molecule),
so
they
are
repelled
by
the
negatively
charged
cell
wall.
Hence,
the
cell
is
transparent
and
its
surroundings
are
coloured.
Negative
staining
is
useful
for
determining
cell
dimensions
and
visualizing
capsules,
as
heat
fixation
shrinks
both
cells
and
capsules.
23