Microscope 3-1

Compound Light Microscope

• In a compound microscope, the image from the
objective lens is magnified again by the ocular lens
• lens system: ocular and objective
10X
• light source: visible light

• Total magnification
 Magnification
• Magnification: To enlarge an image only in appearance,
not in Physical size
• Each lens has its own magnification.
Figure 3.1a The Compound Light
Microscope

d m
O
puragh
Eisa
Figure 3.1b The Compound Light
Microscope

inverts
image
Figure 7.5 The Radiant Energy
Spectrum

Kills bacteria

0
I
blue filter 450hm
wavelength _better resolution
shorter
Compound Light Microscopy
• The refractive index is a measure of the light-
bending ability of a medium
• Light may refract after passing through a specimen to
an extent that it does not pass through the objective
lens
refractive index
oil Glass
1 5 1.5

air glass
1.0 l s
Figure 3.3 Refraction in the Compound
Microscope Using an Oil Immersion Objective
Lens

must b down
 Microscope Resolution
• Resolution is the ability of the lenses to distinguish
two points
resolution
• A microscope with a resolving power of 0.4 nm can
distinguish between two points at least 0.4 nm apart
• Shorter wavelengths of light provide greater resolution
Y
blue Ex 6nm o s o
m
fireater

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short great
u
n m sunt
m2 8hm
 Depth of field
• Depth of field is the thickness of the specimen that
is in focus at one time.

Rmag _b DOF LOX

tox

Lab tip add oil bit 4 4100 x

RECORDING DATA FOR THE LAB
NOTEBOOK
• Results: Create similar tables as shown in Data sheet 3-1.
Ignore the numerical aperture and calibration of ocular
micrometer columns. We will determine the calibration in a
different lab. For the thread slides make sure that you
record the number that is on the slide next to your data.
• Discussion questions: Answer questions 2 , 5b and 11
from Data sheet 3-1. Use the answer from question 5a to
help you answer 5b (Answer to question 5a is 347nm).
 Calibration of the ocular micrometer
3-2
Ocular lens

g
•Purpose: To calibrate the ocular micrometer so that it can
be used to take measurements of the specimen under the
microscope.

• The ocular scale is UNKNOWN ocular units

ou
• The stage micrometer scale is KNOWN mm
• Calibration done by superimposing both scales and
determining how many ocular graduations coincide with 1
gradation on the stage micrometer.
on the slide

OUI
 Stage Micrometer
up
line zeros
the
TWO

our
wanton

i r

e
Ocular micrometer

80Ocular
units
I
 Superimposing both scales
write what is
what 1mm4000µm
convert 1.0mm 35 ou
micrometers
MM 1 0µm 1000µm 3son
1000µm
28.614
Tou
Divide µm
Th I
I
1.7mm 60ou

1700µm 6Oou
pick 3 lines tht matchup 167
1 28.33 u
u
find average
 Task
• Calibrate the ocular micrometer at each objective lens except
for the oil immersion lens (100X).
The calibration of the oil immersion lens is 1 μm/ou.

• Follow the procedure in your lab manual on how to calibrate

the ocular micrometer.
• You will use the average calibration to calculate the sizes of
the cells and the letter e to be observed in the next activity.
• Example:
HX Objective Lens
– Scanning

Stage Micrometer (μm) Ocular Micrometer (ou) Calibration

Measuring the size of different types of cells and the
letter e using the ocular micrometer
1. Measuring the letter e:
a) Using prepared slides and your scanning objective, determine the size of the letter e. Measure the
length of the letter e.
b) Use the ocular micrometer calibration of the 4X objective for your calculations. Record the size in
ocular units (OU) and micrometers ( m) in the table provided.

2. Measuring a protozoan cell:

a) Using prepared slides and your low power objective, determine the size of an Amoeba or Paramecium.
Indicate in your table which protozoa you measured. Measure the length of the protozoa.
b) Use the ocular micrometer calibration of the 10X objective for your calculations. Record the size in
ocular units (OU) and micrometers ( m) in the table provided.

3. Measuring red blood cells:

a) Using blood smear prepared slides, determine the size of a red blood cell (RBC) using the 40X
objective lens.
b) Use the ocular micrometer calibration of the 40X objective for your calculations. Record the size in
ocular units (OU) and micrometers ( m) in the table provided.

4. Measuring bacterial cells:

a)
of Item love
Using the bacterial types prepared slides, determine the size of a bacterial cell that is rod shaped using
the oil immersion objective (100X). Measure the length of the rod.
b) Use the ocular micrometer calibration of the 100X objective for your calculations. Record the size in
ocular units (OU) and micrometers ( m) in the table provided.
 a
lo ou

look x 2
q
1
47
20
 Example:
What is the size of the red blood cell at the 40X
objective lens?
calibration 5Mmlon
20 ou x
Stone_100µm
What is the calibration?

2004 0.02 mm
0.02mm 20µm

20µm l
I i
Toon w
100 x
 Nutrient agar recipe
factor
1 Beef extract growth
amino acids
2 peptone
solidify
3 Agar

complex
media

Agar
from red algea
bacteria
its not a food for
melt able 100 C
solid 42 C

Pour able 55 C 60 C
Autoclauing
sterilize glassware Medea
Steed no living microorganism
Wurst be 1210C and 15ps pressure

15 minutes

States of Media
liquid w nutrients
1 Broth
4