Fall 2020 Microbiology Lab

Microscopy and
Cell Morphology
Lab Manual:
PART 2 Introduction and
Exp. 5 Microscopic
Examination of Stained Cell
Preparation
Textbook:
Chapter 4 Microscopy
I. Review - Metric Units
Memorize the relationships of different metric units
and make sure that you can convert metric units without any assistance.
1 m = 100 cm = 1,000 mm = 106 µm = 109 nm

Fall 2020 Micro Lab - Microscopy

 Size of Microbial Cells
• Human red blood cell = 7.5 µm (top view) = 0.0075 mm
• Most bacterial cells = 0.5 - 8 µm = 500-8000 mm
• Most viral particles = 20-800 nm = µm= ____ mm
• Coronavirus = 80 nm = 0.08 µm = ________ mm
What can you see with your naked eyes?

Fall 2020 Micro Lab - Microscopy

 Units of Measurement
T-Phage
• 1 µm = 10 m = 10 mm
–6 –3

• 1 nm = 10–9 m = 10–6 mm
• 1000 nm = 1 µm
• 0.001 µm = 1 nm

Estimate the length and the width of a E. coli cell:_______________

Estimate the length and width of a T-Phage: __________________
If a microbe measures 10 μm in length, what is the length in nm? _____

Figure 3.2
 II. Microscopy
Learning Objectives:
• Identify name and function of all components of a
compound microscopes.
• Explain general principles of microscopy. (wavelength of
light, magnification, resolution and contract)
• Describe different types of microscopes and their
applications.
• Demonstrate the proper use and care a compound
microscope.

Fall 2020 Micro Lab - Microscopy

A. General Principles of Microscopy

Quality of enlarged images depend on

1. Magnification
2. Resolution (determined by wave length of light
source and NA of lenses)
RP = Wave Length of light (nm)
NA* condenser + NA objective
3. Illumination (Light source)
4. Contrast between the image and background
1. Magnification
• Magnification • Compound Microscope
– image enlargement – image is enlarged by
by a lens objective lens and
eyepiece

Fall 2020 Micro Lab - Microscopy

Magnification of a Compound
Microscope
Total Magnification = magnification of eye piece
(10X) x Magnification of objective lens
Oil lens High Dry Low Power Scan lens

What is the total magnification of the specimen if you place

the slide under the high dry objective lens?

Fall 2020 Micro Lab - Microscopy

Check your understanding
1. You place a slide under an oil lens and the image of a bacterial cell appears
to be 2 mm long. What is the true length of the cells without magnification?
2. A protozoan is 40 µm in length. If you want the image to be enlarged to 4
mm under a compound microscope, which objective lens you should use?
3, If a cell appears to be 5 mm long under low power lens, what is the real length of
this cell?
•Total magnification = ________100______
•Image of the cell = 5 mm = true length x total magnification
True length = _0.05_____ mm

Tip: Image under a microscope is always larger than the true size.

Fall 2020 Micro Lab - Microscopy

 2. Resolution (Resolving Power)
RP = ability to distinguish two points that
are close together
(ability to see fine details)
Determined by two factors:
• the property of the lens (numerical aperture)
• λ of light source)
Resolution = λ of light
2 x numerical aperture

Which microscope has a better resolution which allows you to see finer
details? A. RP = 25 B. RP = 2.5

Fall 2020 Micro Lab - Microscopy

3. Wavelength (λ) and Resolving Power
• Wavelength (λ) = distance between two
corresponding part of a wave

Fall 2020 Micro Lab - Microscopy

Explain why shorter wavelengths of light
provide greater resolution?
Wavelengths (λ) of Light Sources
Which light source will provide better resolving power?
λ of visible light = 400 – 700 nm = 0.4-0.7 µm
λ of UV light = 100 – 400 nm = 0.1-0.4 µm
Fig.4.1 The electronmagnetic spectrum

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 Numerical Aperture
• Function of the diameter of the lens in relation to
the focal length
• The value of numerical aperture is written on the
objective lens.

Calculate RP of each lens (using λ = 500 nm)

Which objective lens provides the best resolution (the
smallest RP value)?

Fall 2020 Micro Lab - Microscopy

 4. Contrast
• Differences in intensity
between two objects, or
an object and its
background
• Important in determining
resolution.
• Staining increases contrast
• Use of light that is in phase
increases contrast
Why do I need immersion oil?
• When you use oil lens, you need to add a drop of immersion oil
on your slide and soak the lens in the oil drop to enhance the
lighting of the image.

Attention: Do not contaminate any other three objective lenses with immersion
oil. These lenses can be easily ruined if you left oil on these lenses.
Fall 2020 Micro Lab - Microscopy
Why do I need immersion oil?
Why?
1.When light rays pass through glass slide and
air, due to different density of air and glass,
light rays will change their paths (refraction)
(Fig. a)
2.The opening of oil lens is very small. Without
oil (Fig. a), some light will not enter the
objective lens due to reflection not enough
light  dark image.
3.Oil has density similar to glass. Immerse
objective lens in oil (figure b) so that light rays
will travel from glass slide oil  objective
without any reflection  more light 
brighter image.

Attention: Do not contaminate any other three objective lenses with immersion
oil. These lenses can be easily ruined if you left oil on these lenses.
Fall 2020 Micro Lab - Microscopy
What is a parfocal microscope?
• Parfocal" refers to objectives that can be changed
with minimal or no refocusing.
What is a parfocal microscope?
• Why are these 4 objective lenses mounted
on tubes with different heights?
These tubes help position the 4 lenses at the right
working distance on top of your slides so that the images
will remain in the focus range as you switching lenses
from one to the other. This is called parfocal microscope.

• Tip: Once you focus the image with 10 X lens, you can turn
to other objective lenses and fine tune the focus with the
fine adjustment
(Do not use coarse adjustment to change the position of the
stage drastically when you switch objective lens.)
Fall 2020 Micro Lab - Microscopy
Objective, Working Distance, and Diaphragm
Read Figure 5.3 Objective, working distance, Diaphragm opening on your
lab manual and answer the following questions:
1. Arrange the 4 objective lens in order based on the diameter so the
lenses (From the largest to the smallest):______________________________
2. Which lens allow the largest amount of light to enter the lens
therefore the image will be the brightest? Which lens will allow the
least amount of light to enter?
3. How do you adjust the amount the light entering the objective lens
so that the image will not be too bright or too dim?
4. When you switch objective lens from low power to oil lens, you
should
A. open iris diaphragm to bring in more light
B. close iris diaphragm to dim light
C. not touch the iris diaphragm
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 B. Types of Light Microscopes

• Text p. 100-102 (6th ed)

1. Bright Field – background is bright
(lack of contrast if the objects are not
stained)
2. Dark Field – background is dark and
objects are bright (better contrast)
3. Phase Contrast – light ray path is
manipulated by a specific condenser for
better contrast between unstained
objects and background
4. Confocol

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 Two Basic Types of Bright-field
Microscopes
Simple Microscope Compound Microscope
1. Contain a single
magnifying lens
1. 2 lenses (ocular and objective)
2. Similar to magnifying
glass for magnification
3. Leeuwenhoek used 2. Total magnification =
simple microscope to
observe microorganisms magnification of objective lens ×
magnification of ocular lens
3. Light passes through specimen
into objective lens
4. Oil immersion lens ↑ resolution
5. Most have condenser lens
(direct light through specimen)
 Brightfield Illumination

• Dark objects are visible

against a bright background
• Light reflected off the
specimen does not enter the
objective lens

Figure 3.4a
 Darkfield Microscopes
• Light objects are visible
against a dark background
• Light reflected off the
specimen enters the
objective lens

Figure 3.4b
Phase-Contrast Microscope

• Accentuates diffraction of
the light that passes through
a specimen
• ↑contrast of the specimen
• Use: view live specimen (no
stain)

Give examples when you need to view

mos alive.

Figure 3.4c
Check your understanding:

Choose an appropriate light microscopes for observing the

following specimens:
1. Observe bacterial cell morphology after staining
2. Exam a clinical specimen for the presence of live Treponema
pallidium (a spirochete that cannot be stained easily and live cells
are motile.)
3. Live cells (semi-transparent) – watch motility and cell division
4. Observe internal structures of cells such as nucleus without
staining

A. Light microscope
B. Dark microscope
C. Phase contrast microscope

Fall 2020 Micro Lab - Microscopy

 Fluorescence Microscope
Fluoresence: Some molecules can absorb invisible
radiation such as UV and emit
light with a visible λ
Fluorescence Microscope:
• Use UV as light source
• Specimens are stained with fluorescent dye or
• Specimens may be attached by antibody conjugated
with fluorescent dye (immunofluorescence)

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Fluorescence Microscope
What is the advantage using fluorescent microscope?

What are the applications of immunofluorescence.

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Confocal Microscope
• Use UV lasers to illuminate fluorescent target in a
single plan (1.0 um thickness)
• Image of each plan of the specimen is taken.
• These images are assembled by a computer to
generate a 3 D image.
 Electron Microscope
• Use electron beams (λ = 0.001 nm) to resolve fine details of
small specimens. (smaller than the λ of light)
• High resolution  allow high magnification
(10,000 -100,000 X)

Fall 2020 Micro Lab - Microscopy

 Two Types of Electron Microscopes
Transmission EM (TEM)
• Electrons pass through a thin
slice of a specimen  then pass
through “objective” and
“project lens” e- beam reach a
fluorescent screen  generate
an image on fluorescent screen
• Dense area blocks e-  dark
Thin area blocks less e- 
bright
Fall 2020 Micro Lab - Microscopy
Scanning EM (SEM)
• Specimen is coated with gold or
platinum
• e- beams scan the target back and
forth
• e- bounce off from the target
surface are detected by a detector
and create a 3D image
YouTube: How a Scanning Electron
Microscope Works (5:25)
https://www.youtube.com/?gl=US
Check your understanding:
1. What is the magnification of EM?
A. 1000x B. 10,000x C. 100,000x D. both B and C
2. Which type of EM can produce 3D pictures?
3. Can you watch live cell activities using EM?
4. Which of the following must be observed under EM?
A. bacterial cells such as E. coli
B. red blood cells
C. protozoa
D. viruses
(Tip: Check size of each on the size chart in a previous slide.)

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C. Microscope Tutorial
Watch this YouTube demonstration (26:58)
https://www.youtube.com/watch?v=oMASHb4VyyU
• This tutorial teaches you how to properly operate and care a typical
compound microscope with tips to focus your slides easily.

Fall 2020 Micro Lab - Microscopy

Check your understanding
1. Name the function of the following parts:
1) Condenser
2) Iris diaphragm
3) Stage control
4) Coarse focus adjustment
5) Fine focus adjustment
2. Name two factors that change resolution of your microscope:_____, _____
3. Resolution can be improved by using a light source with a long wavelength.
A. True B. False
4. Resolution can be improved by using a lens with a large numerical aperture.
A. True B. False
5. Refraction of light occurs when light pass through two materials with different
A. weight B. density C. water content
6. Why is immersion oil required when use oil lens?
7. How do you clean your microscopes body and lenses?
8. What is a parfocal microscope? Do you need to drop the sage to its lowest position when
you switch high dry objective lens to oil lens?

Fall 2020 Micro Lab - Microscopy

 Swift Microscopes with a LCD display

• Most microscopes in Microbiology Lab are equipped with

a LCD screen. However it needs to be charged with a
charger in your drawer. The LCD screen will show
whatever you see under the objective lens (You may need
to finetune focus to make image sharp. )

Fall 2020 Micro Lab - Microscopy

 Swift Microscope with a Tablet

• Six of the microscopes in microbiology lab

is equipped with a tablet.
• The tablet is stored in a closet. If you wish
to use it, make sure to get the tablet from
your instructor and charge the tablet.
• Instruction is in the tablet kit.
• Make sure to put all tablet accessories back
to the box at the end of the lab.
• Using tablet is optional. You can use the
microscope without the tablet.

Fall 2020 Micro Lab - Microscopy

Swift Microscope - Tablet Accessory Kit

Instruction

Fall 2020 Micro Lab - Microscopy

Demonstration Videos:
1. Practice the correct procedure for use and care microscope.
Demo: Basic Microscope Set Up and Use (4:23)
2.
Smartphone Microscopic Photography - The Morrison Technique: F
ree Hand, No Adapter
(1:27)