MCB 11

Biology and
Applications of
Microorganisms
Content Contributors

Prof. John Daniel P. Ong

Dr. Renard M. Jamora
Prof. Domini Theresa M. Pecundo
 Exercise 1A: The
Compound Microscope
Prepared by: John Daniel P. Ong, Renard M.
Jamora, and Domini Theresa M. Pecundo
Expected Learning Outcomes
✓ Handle and care for the microscope properly
✓ Identify and describe the function of each part
of the compound microscope
✓ Demonstrate how to operate a compound
microscope
 Microbiology

• Study of organisms and

agents that are too small to
be seen by the naked eye
(<1mm).

Source: Source: https://www.pinterest.ph/pin/489485053242270366/?lp=true

https://www.pinterest.ph/pin/568368415444900163/?nic_v2=1a4k
xDEL9
 Microbiology
• understanding the nature and functioning of the
microbial world
• applying our understanding of the microbial world for
the benefit of humankind and planet Earth

• As a basic science – uses microbial cells to probe the

fundamental processes of life.
• As an applied biological science – forefront of many
important breakthroughs
 Relative sizes of microbes:
Size Relationships Among Microbes

Source: Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., & Stahl, D. A. (2015). Brock biology of microorganisms (Fourteenth edition.). Boston: Pearson.
Five major groups of organisms/agents:

Bacteria Algae Fungi

Viruses Protozoa
Bacteria

Bacillus subtilis swimming at 400X magnification.

Source: https://www.youtube.com/watch?v=n2nVrfgZ45s
Algae

Green alga at 400X magnification.

Source: https://www.golias.net/akvaristika/experiments-algae-sugars.php
Virus

An image of the new coronavirus taken with an electron microscope.

Credit: U.S. National Institutes of Health/AP/Shutterstock
Source: https://www.nature.com/articles/d41586-020-00660-x
Protozoa

Paramecium showing contractile vacuole and ciliary motion using DIC (350x-1000x)
Credit: Mr. Ralph Grimm Jimboomba Queensland, Australia
Source: https://www.youtube.com/watch?v=mqhwlUowGl4
Fungi

Saccharomyces cerevisiae at 1000x.

Source: https://wineserver.ucdavis.edu/industry-info/enology/wine-microbiology/microscopy
 Microscope
• Came from the Greek words
mikros (small) and skopein
(to watch/see).
• Anton Van Leeuwenhoek
a.
was the first to observe Source:
microorganisms (i.e. https://museumboerhaave.wordpress.com/2011/07/29/r
eplica-van-leeuwenhoek-microscope-english-version/

bacteria)
b.
Figure 9. (a) A replica of Antoni van
Source: Madigan, M. T., Martinko, J.
Leeuwenhoek’s microscope (b) Van M., Bender, K. S., Buckley, D. H., &
Leeuwenhoek’s drawings of Stahl, D. A. (2015). Brock biology of
microorganisms (Fourteenth
bacteria, published in 1684. edition.). Boston: Pearson.
Source:
https://www.sapaviva.com/antoni
e-van-leeuwenhoek/
Parts of a Microscope

Source:
https://microbenotes.co
m/parts-of-a-
microscope/
 Concepts of Microscopy
Magnification (M) = IS/AS
• Linear M
▪Eyepiece – 10X
▪LPO – 10X
▪HPO – 40X
▪OIO – 100X
• Total M
▪(Linear M of eyepiece)(Linear of objective)
▪Ex. Total M of OIO = (10X)(100X) = 1,000X
 Important Features of Objectives
1. Focal Point (F)
Focal Length:
LPO = 16 mm
HPO = 4 mm
OIO = 1.8 mm

Relationship of M
and FL?

Inverse
Source: https://www.letstalkacademy.com/publication/read/MICROSCOPY
 Important Features of Objectives
2. Working Distance – distance between the front
surface of the lens and the surface of the cover glass
or specimen when it is in sharp focus.
Source: http://helid.digicollection.org/en/d/Jwho02e/8.html

WD: 4-8 mm 0.5-0.7 mm 0.1 mm

Source: https://www.microscopyu.com/tutorials/workingdistance
 Important Features of Objectives
3. Resolving Power/ Resolution (R)
d = limit of resolution

A B A B

Unresolved Image Resolved Image

Relationship of d
and R?
 Important Features of Objectives
Limit of resolution

Source: https://static.sciencelearn.org.nz/documents/files/000/000/723/original/Micro-v1-F.pdf?1544407653
 Important Features of Objectives
Example:
Distance = 0.2µm
Microscope Limit of
resolution
A B
1 0.1 µm
2 1 µm

Resolved Image
Question: Which of the two microscopes will be
Ans: Microscope 1
able to distinguish the two objects as separate?
 Important Features of Objectives
3. Resolving Power/ Resolution (R)
d = limit of resolution

A B A B

Unresolved Image Resolved Image

Relationship of d
Inverse
and R?
 Important Features of Objectives

d = 0.5λ / nsinϴ

• d = limit of resolution
• λ = wavelength of light used (blue-green)
•n = refractive index of the medium
•ϴ = ½ the angle of the cone of λ entering
the objective
 Important Features of Objectives
n = ability of the medium to scatter light

Source: Bauman Ph.D., Robert W. 2012. Microbiology with Diseases by Body System. 3rd ed. Benjamin Cummings.

nair = 1.0 nglass = 1.5 noil = 1.5

 Important Features of Objectives
Source: http://microscopy.berkeley.edu/courses/tlm/objectives/objectives.html

ϴ = angle of the
cone of light
entering the
objective
ϴ
ϴ
WDLPO=4-8 mm
WDHPO=0.5-0.7 mm

Light rays that Spread out inside the obj.; more Does not spread out much; few light rays entering
pass through light rays are entering
Angle broad narrow
Resolution high low
 Important Features of Objectives

NA
d = 0.5λ / nsinϴ

• d = limit of resolution
• λ = wavelength of light used (blue-green)
•n = refractive index of the medium
•ϴ = ½ the angle of the cone of λ entering
the objective
 Important Features of Objectives
4. Numerical Aperture – measure of the resolving
power of an objective

N.A.
What relationship
LPO = 0.25
can we derive from
HPO = 0.65
M, FL and N.A.?
OIO = 1.25

M FL N.A.
 Important Features of Objectives
5. Parfocal – objectives are optically and
mechanically designed so that the distance between
the specimen and the aerial image is always constant

Source: https://www.olympus-ims.com/en/microscope/terms/parfocal_distance/
Video: Different Parts of the Microscope
Video: Proper handling of microscopes
Video: How to use the compound microscope/OIO
Copyright © 2020. John Daniel P. Ong.
All rights reserved.
No part of this video may be published without the
creator’s consent
Exercise 1B: Calibration of
the Microscope
Prepared by: John Daniel P. Ong
Expected Learning Outcomes

✓Calibrate the ocular micrometer (OM)

✓Determine the area of the field of vision (FOV)

✓Determine the size of a microscopic specimen

through the use of an ocular micrometer (OM)
 Two kinds of micrometers
Source: https://www.alibaba.com/product-detail/High-Precision-
CAT920-Microscope-Accessories-Micrometer_60648203526.html

Source: http://www.mecanusa.com/microscope/micrometer/Stage-Micrometer.htm

Ocular Micrometer Stage Micrometer

Comparison of OM and SM
Comparison OM SM
Placed Eyepiece Stage
Presence of # Yes No
Length of 1 Needs to be 0.01 mm or 10
small division calibrated 𝛍m

Function Determine the Calibrate OM

size of the and Determine
specimen area of FOV
Calibration
Calibration
Calibration

Under HPO
Calibration
OM division subtended by SM = 10
Area of proper line coincidence
SM division subtended by OM = 1

Second coinciding line

Under HPO
First coinciding line
 Calibration Factor
• Use the formula below to determine the CF for a particular objective
CF (value of 1 OM div.) = (SM div. subtended by OM div.) ( value of 1 SM div. (𝛍m)
OM div. subtended by SM div.

CF (value of 1 OM div.) = (1 division) (10 𝛍m)

10 divisions
CF (value of 1 OM div.) = 10 𝛍m
10

CF under HPO (value of 1 OM div.) = 1 𝛍m

 Calibration Factor
• Use the formula below to determine the CF for a particular objective
CF (value of 1 OM div.) = (SM div. subtended by OM div.) ( value of 1 SM div. (𝛍m)
OM div. subtended by SM div.
Area of proper line coincidence

OM division
subtended by SM = 10
SM division
subtended by OM = 1
Size of the specimen

Given: CF under HPO = 1 µm

OM divisions
subtended by the
specimen = 25 divisions

Size of Specimen = # of div. subtended by

Under HPO
the specimen x CF

Size of Specimen = 25 divisions x 1 µm

= 25 µm
Area of the Field of Vision

diameter = 75 divisions x (10 µm/ 1 division)

Number of SM divisions = 75
= 750 µm

𝐴 = 𝜋𝑟 2
= 𝜋(3752 )
= 𝜋(3752 )
= 4.41 𝑥 105 µm2
Video: Calibration of the microscope
Copyright © 2020. John Daniel P. Ong,
Renard M. Jamora, and
Domini Theresa M. Pecundo.
All rights reserved.
No part of this video may be published without the
creator’s consent