Download INTRODUCTION TO THE MICRSCOPE Introduction to microscopy S

INTRODUCTION TO THE MICRSCOPE
Introduction to microscopy
Severall ti
S
times during
d i the
th semester
t you will
ill be
b required
i d to
t utilize
tili
the light microscope to observe cells and tissues. To be successful you
will need to have a working knowledge of how to use the microscope.
Improper use of the microscope is not only frustrating to the student, but
may result in damage to the microscope or the specimen being observed.
Goals
By the end of this laboratory,
laboratory you will have the ability to:
1)
Name the parts of the microscope and their functions.
2)
S
Successfully
f ll use the
th microscope
i
to
t observe
b
biological
bi l i l specimens
i
.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure for basic microscopy
A
A.
I
Important
t t parts
t off the
th microscope.
i
1.
The microscopes used in lab are called compound
microscopes because they have two magnifying lens.
lens
a.
The ocular lens is the lens that you look through.
bb.
The
h objective
bj i lens
l
i the
is
h lens
l
that
h is
i immediately
i
di l
above the specimen.
There are three objective
objecti e lens.
lens
- the scanning lens with the 4X label
- the low power lens with the 10X label
- the high power lens with the 43X or 45X label.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure for basic microscopy (cont.)
A
A.
I
Important
t t parts
t off the
th microsocpe
i
(cont.).
( t)
c.
To calculate the total magnification, the
magnifying power of the ocular lens is multiplied by the
magnifying power of the objective lens.
Example: When using the scanning power lens (4X) the total
magnification is 40X.
Ocular lens =
10X
X
X
Objective lens =
4X
Total magnification
40X
MICROSCOPY AND CELL STRUCTURE
Experimental procedure for basic microscopy (cont.)
A
A.
I
Important
t t parts
t off the
th microsocpe
i
(cont.).
( t)
2.
There are two different focusing knobs that will be used.
a.
The course focus knob which is the outer larger
portion of the focus knob allows for dramatic or gross
movement of the objective lens.This
lens This focus knob should
not be used when focusing the high power objective
lens.
b.
The fine focus knob which is the inner small
portion of the focus knob allows for very slight
movement off the
h objective
bj i lens.
l
This
Thi lens
l
should
h ld only
l be
b
used for focusing the low and high power lens.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure for basic microscopy (cont.)
A
A.
I
Important
t t parts
t off the
th microsocpe
i
(cont.).
( t)
3.
The stage has two metal clips to hold the microscope slide
in place.
place A grease interface between the stage and the stage
platform allows for slow precise movement of the stage.
4.
Below the stage is a condenser with an iris diaphragm that
allows light to be focused onto the specimen through a hole in the
stage.
a.
As the magnification is increased, you may have
to open the iris diaphragm to allow more light onto the
specimen.
specimen
MICROSCOPY AND CELL STRUCTURE
Experimental procedure for basic microscopy
B
B.
Ob
Observation
ti off the
th letter
l tt “e”.
“ ”
1.
You will place a slide with a typed letter “e” on the stage
of your microscope.
microscope
2.
You will then observe the letter “e” under the scanning,
low power, and finally high power objective lens.
Remember: If you cannot find the specimen/object you are looking for
j
lens. As the magnification
g f
increases, the
do not ggo to the next objective
depth of field and the diameter of the field decrease making it harder, not
easier to find what you are looking for.
3.
Rotate the objective lens back to scanning, raise the lens
with the coarse adjustment knob, and carefully remove the slide.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure basic microscopy (cont.)
C
C.
Di
Diameter
t off fi
field.
ld
1.
You will place a measuring stick on the stage of the
microscope to measure the diameter of the field.
field
2.
You will align the measuring stick so that it passes
through the middle of the circular field and count the number of
millimeters. (See Figure 4.2 in the lab manual).
3.
Since microbes are usuallyy measured in units of
micrometer rather than millimeters, you will need to convert
your millimeter measurement into micrometers. There are 1,000
micrometers in 1 millimeter.
millimeter
MICROSCOPY AND CELL STRUCTURE
Experimental procedure basic microscopy (cont.)
D.
Focal plane/depth of field
1.
A slide with three colored overlapping threads will be
used to illustrate the difference in the depth
p of field between the
three objective lens.
2.
By focusing carefully, it may be determined which of the
three threads is on the top,
top which is in the middle,
middle and which is
on the bottom.
MICROSCOPY AND CELL STRUCTURE
Introduction to cell structure
The cell
Th
ll is
i the
th smallest
ll t unit
it off life
lif andd all
ll living
li i organisms
i
are
composed of cells or cell products. There are two basic types of cells
found on our planet, the eukaryotic cells and the prokaryotic cells.
cells
The names of these two cell types indicate the condition of their
nuclear material. The eukaryotic cells have a true (eu
eu) nucleus (karyo).
The
h prokaryotic
k
i cells
ll on the
h other
h hand
h d are though
h h to be
b more primitive
i ii
(pro
pro or pre
pre) nucleus (karyo).
All living
li ing cells whether
hether eukaryotic
e kar otic or prokaryotic
prokar otic have
ha e several
se eral
common characteristics. This list includes a cell membrane, cytoplasm,
ribosomes, and DNA.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure observing cells
A.
Observation of prokaryotic cells.
1.
Bacteria will observed on a prepared slide containing
three different morphological types.
bacillus - rod
spirillium - spiral
coccus - round
MICROSCOPY AND CELL STRUCTURE
Experimental procedure observing cells (cont.)
A.
Observation of prokaryotic cells (cont.).
2.
Cyanobacteria (Blue-green algae) will be available as
live specimens.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure observing cells (cont.)
B.
Observation of eukaryotic cells.
1.
Amoeba will be available as live organisms. A wet
mount will be prepared from a culture dish. For best results take
your sample
l from
f
the
th bottom
b tt
off the
th dish.
di h The
Th amoeba
b will
ill be
b
observed as a gray granular non-distinct object. After a few
minutes of adaptation to the microscope slide the amoeba will
begin to move using its pseudopods.
This projection
is a pseudopod.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure for observing cells (cont.)
B.
Observation of eukaryotic cells (cont.).
2.
Elodea will be available as live organisms. A wet mount
of a leaf will be prepared. The individual cells will appear
rectangular
t
l with
ith distinct
di ti t cell
ll walls
ll andd internal
i t
l chloroplasts.
hl
l t
The small
green spheres
are the
chloroplasts ==>
This distinct
Thi
di i
line is a cell
wall.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure observing cells (cont.)
B.
Observation of eukaryotic cells (cont.).
3.
Onion cells will be observed from the skin taken from a
piece of onion which has been prepared with Janus green and
sucrose. The
Th cells
ll will
ill be
b observed
b
d as irregular
i
l cells
ll that
th t remind
i d
me of chicken wire.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure observing cells (cont.)
B.
Observation of eukaryotic cells (cont.).
4.
Cheek epithelial cells will be acquired from the lining of
your own cheek. The blunt end of a toothpick will be used to
scrape cell
ll from
f
the
th inside
i id off the
th cheek.
h k The
Th cells
ll will
ill be
b
dispersed in iodine on the microscope slide to make the nucleus
more apparent.
The distinct
structure in
i the
h
center is the
nucleus.
The outer
edge
d off the
h cell
ll
is the cell
membrane.
MICROSCOPY AND CELL STRUCTURE
Introduction electron microscopy
An electron
A
l t
microscope
i
has
h the
th capability
bilit off magnifying
if i a
specimen many thousands of times thus allowing observation of
organelles that cannot be seen with light microscopy. An electron
micrograph may be use to determine the true size of a cell or organelle.
Experimental procedure for electron microscopy
A.
Calculation of actual size from a micrograph.
Step1:
p Measure the cell or organelle
g
and convert the units to
micrometers
Step2: Divide the measurement in micrometers by the
magnification.
MICROSCOPY AND CELL STRUCTURE
Experimental procedure for electron microscopy (cont.)
B
B.
Ob
Observation
ti off organelles.
ll
You should be able to observe the following organelles in the
electron micrographs provided: nucleus,
nucleus mitochondria
mitochondria, endoplasmic
reticulum, ribosomes, golgi complex, cell membrane, and in addition to
these structures cell wall, central vacuole, and chloroplasts in plant cells.