Microscopy :

PRACTICAL 1 MICROSCOPY: Principles, Use and

Maintenance
PRINCIPLES, USE AND
MAINTENANCE
Structure
1.1 Introduction
1.2 The Microscope
1.2.1 Light Microscope
1.2.2 Electron Microscope

1.3 Parts of the Microscope

1.4 Principle of Microscopy
1.5 Use and Care of Microscope
1.6 Review Questions
Exercise 1: Use of Light Microscope

1.1 INTRODUCTION
We start the Practicals in the Food Microbiology and Safety Course with an orientation to
the microscope. This first Practical in the Manual will orient you to the different parts of
the microscope, their functions and maintenance. As a student of microbiology, it is very
important for us to know about the principle and working of microscopy, different types of
microscopes and correct use of microscopes for microbial observation.
Objectives
After studying this practical and undertaking the exercise given herewith, you will be able
to:

identify the different parts of the microscope and recognize their functions,

discuss the principle behind the working of a microscope,

recognize different types of microscope, and

use a microscope correctly in the laboratory.

1.2 THE MICROSCOPE

Microscope is a powerful and crucial basic tool in the field of microbiology. Microbiology,
as we have already studied in our theory Course, is the science dealing with the organisms
too small to be seen with unaided eye. The existence of microbial life to the world was
introduced first by Antony Van Leeuwenhoek in 1673, with the help of simple, crude,
self-made, single-lens microscope having a magnification of about 300. Over the years,
microscopes have evolved to increase the magnification several thousand fold. Modern
day microscopes are either light microscopes or electron microscopes.

Light microscopes use either visible light or ultraviolet rays to illuminate specimens. These
are generally used to look at intact cells. On the other hand, electron microscopes use
electron beams instead of light rays and electromagnets instead of lenses. These are
generally used to look at internal structures or details of cell surface.

Let us get to know about these different types of microscope.

1
Food Microbiology and 1.2.1 Light Microscope
Safety
Practical Manual Modern light microscopes are compound microscopes. Here the magnified
image formed by the objective lens is further enlarged by one or more additional
lenses. A variety of light microscopes are used now-a-days by microbiologists
for different purposes. These are enumerated next.
(a) Bright Field Microscopes – Figure 1.1 illustrates the compound bright
field microscope. This is the most commonly used microscope in biology
and microbiology courses. It is called so because it forms a dark image
against a brighter background. It contains 2 lens systems for magnification:
ocular lens in eyepiece and the objective lens located in nosepiece, as
can be seen in Figure 1.1. The specimen is illuminated by a light focused
on it by a sub-stage lens called a condenser. With this microscope,
specimens are made visible because of the differences in contrast that
exist between them and the surrounding medium. Contrast differences
arise because cells absorb or scatter light in varying degrees. Live cells,
however, are difficult to observe through this microscope due to absence
of contrast between specimen and the surrounding medium. It is therefore,
used to observe nonviable and stained preparations where contrast is
increased and variations in colour between cell structure is evident.

Figure 1.1: Compound bright field microscope

(b) Dark Field Microscope -- In this microscope, condenser system is

modified, so that only reflected and refracted light by specimen forms an
2
 image. Here, the object is brightly illuminated while the field surrounding the object Microscopy :
appears black. This microscope can be used to observe live unstained cells and Principles, Use and
Maintenance
organisms.
(c) Phase Contrast Microscope – Unpigmented and unstained living cells can be easily
observed by phase contrast microscope. It has a special objective and a condenser
that converts slight differences in refractive index and cell density into easily detected
variations in light intensity and produce visible image of the structure under study.
The image appears dark against a light background. Phase contrast microscopy is
used widely to study microbial motility, determining the shape of living cells and
detecting microbial structures like inclusion bodies and endospores etc.
(d) Fluorescent Microscope – In above said microscopes, image is produced from
light that passes through a specimen. In fluorescent microscope, however, specimen
image is formed because of the light emitted by the object itself. This microscope is
used more frequently to visualize specimens that are tagged with a fluorescent dye.
The source of illumination here is an ultraviolet light obtained from a high-pressure
mercury vapour arc lamp or hydrogen quartz lamp. Fluorescent dye absorbs the
ultraviolet light of desired wavelength and re-emits the energy in visible range.
Fluorescent portion of the dye becomes visible against a black background. The
fluorescent microscope has become an essential tool in medical microbiology and
microbial ecology for identifying the pathogens and other microbes.
The forms of light microscopy just considered, forms two-dimensional images. Besides,
certain new forms of light microscopy have been developed which gives three-dimensional
images. These are differential interference contrast microscopy, atomic force microscopy
and confocal scanning laser microscopy.
Having learnt about the light microscopes, let us get to know about the electron
microscopes.

1.2.2 Electron Microscope

Electron microscopes have higher magnification and resolving power than light
microscopes. What do we mean by magnification and resolving power? We shall get to
know about this later in section 1.4. But for now, it is important to note that in electron
microscope, the limit of resolution is 0.2 nm and magnification can be up to more than one
million. These microscopes can be used to visualize submicroscopic cellular particles,
viruses etc. Electron microscopes are of two types:
(a) Transmission Electron Microscope – Here, thin and dehydrated specimen is used.
Electrons pass through the specimen and form image on to photographic film. These
are used to observe internal cell structure.
(b) Scanning Electron Microscope – It can be used to observe intact cells or cell
components directly. Then sections are not necessary. It is used for visualizing
surface characteristics rather than intracellular structures. A narrow beam of electrons
scan back and forth, producing a three-dimensional image as electrons are reflected
off the specimen’s surface.
Though a variety of optical instruments are available for routine laboratory work, compound
bright field microscope is the one commonly found in all biological laboratories. Let us get
to know the different parts of this microscope.

1.3 PARTS OF THE MICROSCOPE

Microscope, as you may have noticed in Figure 1.1 or for that matter even seen in a
laboratory, is a metal body composed of a base and an arm to which remaining parts are
attached. The components of the microscope are:
3
Food Microbiology and i) Light Source – It is either mirror or electric illuminator present at the base. Identify
Safety the mirror in Figure 1.1. Some microscopes have reversible mirror with one side flat
Practical Manual
and other concave. Concave side of the mirror is used for sunlight and its flat side is
used for artificial light, e.g., tungsten lamp. Other microscopes have built in light
source.
ii) Abbe Condenser – It is present beneath the stage, as shown in Figure 1.1. It
collects and focuses a cone of light on the slide. Its position can be adjusted vertically
to regulate the light passing on to the specimen. It also has iris diaphragm, a shutter
that is also used to adjust the amount of light entering into the lens system.
iii) Stage – It is a fixed platform positioned about halfway up the arm and provide
surface for keeping the slide. Slide is held in position either by simple slide clip or by
a mechanical stage clip, as you can see in the Figure 1.1. Latter helps the viewer in
moving the slide around during viewing by use of stage control knobs. Stage has an
opening in the center through which light can pass from an illuminating source.
iv) Body Tube – Above the stage and attached to the arm is the body tube, to which
eye piece(s) or ocular lens and revolving nosepiece is attached. Microscopes having
eyepieces for both eyes are called binocular microscopes. Nosepiece contains
three to five objective lenses of different magnifying power, which can be rotated at
will. Microscope should be parfocal, i.e., the image should remain in focus when
objectives are changed. The objectives are nearer the specimen and it produces the
real magnified image of the specimen at focal plane. This image is further magnified
by ocular lens to produce the final image.
From our discussion above, you would have got a clear idea about the parts of a microscope.
Next, how does the microscope works? What is the principle behind the working of a
microscope? Read the next section and find out.

1.4 PRINCIPLE OF MICROSCOPY

When the light passes from one medium to another, refraction occurs, i.e., the ray is bent
at the interface. The direction and the magnitude of bending are determined by the
refractive indices of the two media forming the interface. The refractive index is a measure
of how greatly a substance slows the velocity of light when light passes from air to glass
or vice versa.
When the light rays strikes the lens, a convex lens will focus these rays at specific point
called focal point as illustrated in Figure 1.2. The distance between the center of the lens
and the focal point is called the focal length. Convex lens act as a magnifier. It provides
a clear magnifying image at a much closer range. Lens strength is related to focal length.
A lens with a short focal length has a more magnification power than a lens having a
longer focal length.

4 Figure 1.2: Lens function

Magnification means enlargement. In compound microscope, it is carried out by two-lens Microscopy :
system – objective lens and ocular lens. Objective lens produces the real image of the Principles, Use and
Maintenance
specimen, which is projected up into focal plane and then magnified by the ocular lens to
produce the final image, as illustrated through the light pathway of compound bright field
microscope in Figure 1.3. Though magnification is important, it has limits. Unlimited
enlargement by increasing magnifying power of the lenses is not possible because of the
limitations of resolving power.
Microscope should not only produce the enlarged image but also the clear image.
Resolution or resolving power of the lens is its ability to separate two close points as
separate entities.

Figure 1.3: Light pathway of compound bright field microscope

The minimum distance (d) between two objects that reveals them as separate
entities or resolving power of a lens can be calculated by Abbe’s equation as:

0.5 λ
d =
ηsinφ

Where λ is the wavelength of the light used and ηsin φ is the numerical aperture (NA) as
highlighted in Figure 1.4.
5
Food Microbiology and
Safety
Practical Manual

Figure 1.4: Numerical Aperture of a Lens (η sin φ)

Numerical aperture is a characteristic of each lens and is printed on the lens. It can be
defined as a function of the diameter of the objective lens in relation to its focal
length. It depends on the refractive index (η) of the medium in which the lens works and
also upon the objective itself. Theta (φ) is defined as half of the angle of the cone of
light entering an objective. Sin φ cannot be more than 1. Therefore, the lens working
in air with refractive index 1 can have N.A.1. η will have to be increased for increasing
the resolution. This can be done by using the mineral oil. Wavelength is also an important
factor in resolution. With shorter wavelength, the resolving power increases. Just like
magnification, resolution also has a limit. By decreasing wavelength, resolving power of
the lens cannot be increased indefinitely because –

(a) the visible portion of the electromagnetic spectrum is very narrow and borders of
very short wavelengths are found in ultraviolet range of spectrum.

(b) This relationship of Resolving Power with λ is valid only when light rays are parallel.
As such ‘Resolving Power’ is also dependent on another factor, i.e., refractive
index. When the light passes from air to glass slide, and from glass slide to air, there
is a loss of light due to bending of rays as can be seen in Figure 1.5. This reduces the
numerical aperture and thus the resolving power of the lens. This loss in light can be
compensated by using mineral oil in between glass slide and objective lens. Mineral
oil is a colourless liquid having same refractive index as glass. This does decrease
the bending of ray, as shown in Figure 1.5, so that more light rays enter the objective
lens thus increasing the resolving power. Proper specimen illumination is also important
in determining resolution.

Figure 1.5: Immersion lens operating in air and immersion oil

Resolving Power of a microscope depends upon the numerical aperture of both condenser
and objective, i.e.,
6
 λ Microscopy :
d microscope = Principles, Use and
NA + NA Maintenance
Objective Condenser

It was found that limit of resolving power of a microscope at best be about 0.2 µm and
limit of magnification is about 1000 times the numerical aperture of the objective lens.
With an understanding about the principle of the microscope, we are now ready to use the
microscope.
To ensure good results and observations, it is important that we use and take good care of
our microscope. This aspect is highlighted in the next section.

1.5 USE AND CARE OF MICROSCOPE

Proper care and maintenance of microscope is needed. Following points should be kept in
mind while handling the microscope:
(i) Instrument should be kept in special cabinets while not in use.
(ii) Microscope should be held firmly by holding the arm with right hand and base with
left arm.
(iii) All the lens systems should be cleaned with lens tissue to remove dust, oil, etc.,
which may decrease the efficiency of the microscope. Blotting paper, cloth or towel
should not be used for cleaning.
(iv) If the lens is sticky or oily, the lens can be cleaned with xylol followed by 95%
alcohol. The lens is wiped dry with lens paper. This should be performed only if
necessary as consistent use of xylol may loosen the lens.
Now answer the questions given in the next section and judge your understanding on this
topic.

1.6 REVIEW QUESTIONS

1. Name different types of microscopes used in microbiology.
.................................................................................................................................
................................................................................................................................
2. Define the following terms:
Magnification ..........................................................................................................
................................................................................................................................
Resolution ...............................................................................................................
................................................................................................................................
.................................................................................................................................
Numerical aperture .................................................................................................
................................................................................................................................
................................................................................................................................
Parfocal ...................................................................................................................
3. What is the use of immersion oil? How it increases the magnification?
................................................................................................................................
................................................................................................................................

................................................................................................................................ 7
Food Microbiology and 4. Label the diagram of compound light microscope.
Safety
Practical Manual

5. What is the function of following parts of microscope?

- Iris diaphragm ................................................................................................

- Condenser .....................................................................................................

- Coarse Adjustment Knob ..............................................................................

- Fine Adjustment Knob ...................................................................................

- Stage Clip ......................................................................................................

- Mirror .............................................................................................................

- Objective Lens ..............................................................................................

- Ocular Lens ....................................................................................................

6. Why the body tube of the microscope should not be lowered while looking through
the ocular lens?
................................................................................................................................
................................................................................................................................
................................................................................................................................
7. What precautions should be taken while working with light microscope?
................................................................................................................................
................................................................................................................................
Now conduct the following exercise.

8
 Microscopy :
Principles, Use and
EXERCISE Maintenance

USE OF LIGHT MICROSCOPE 1

Aim : To study different parts of light microscope.
Date : ..........................
Requirement : Compound light microscope
Slide of Bacillus sp. and Aspergillus sp. or any other bacterial and
fungal specimen.
Before you begin this exercise, study different parts of the microscope, as described in
section 1.4.
Procedure:
Now carry out the exercise following the steps enumerated herewith.
1. Place the microscopic slide with any specimen on the stage with the help of stage
clips. Move the slide to keep the specimen above the opening in the stage.

2. Adjust the light with the help of mirror and by adjusting the height of the condenser
with the condenser knob. Iris diaphragm can be opened or closed to regulate the
amount of light entering the condenser.

3. Rotate the scanning or low power lens into position. Lower the body tube to its
lowest position and then raised slowly to bring specimen into focus with the coarse
adjustment knob. Sharp focusing can be done with fine-adjustment knob.

4. Rotate the nosepiece to bring high power or oil immersion lens into position. For
viewing fungal slide (i.e., Aspergillus), use high power lens (40x) and for bacteria
(i.e., Bacillus) use oil immersion lens (100x). Because of parfocal nature of
microscopes, minor focus adjustment can be done by using fine-adjustment knob.
As the magnification of lens increases, the distance between the objective lens and
slide, called working distance decreases. For oil immersion lens, a drop of oil is
placed over the specimen before being used.

5. Scan the slide without the application of additional immersion oil.

6. After using microscope, clear all lenses with dry lens paper and use xylol to remove
oil from stage.

7. Lower the body tube completely and place the low power objective in position.

8. Put the microscope back in cabinet.

Precautions:
1. Clean the lens system properly.
2. Use xylol only when necessary.
3. Never lower the body tube while looking through the ocular lens.
4. Use only a drop of immersion oil for oil immersion lens.
5. Focus the slide first at low-power lens using coarse adjustment knob. Then change
to other lens in position and focusing should be done only with fine adjustment knob.
6. High power objective lens should not touch the oil drop on slide.
9
Food Microbiology and Observations and Results:
Safety
Practical Manual
Record your observations in the space provided herewith. Comment on the process or
adjustments (if any) you had to make in observing the slide. Give description of what you
see in the slide.

........................................................................................................................................

........................................................................................................................................

........................................................................................................................................

........................................................................................................................................

........................................................................................................................................

Submit your responses for evaluation.

..................................................
Counsellor Signature

10
 Microscopy :
Principles, Use and
Maintenance

11
 LIST OF EXERCISES
S. No. Exercises Page No.
1. Use of Light Microscope 17
2. Functioning and Use of various Microbiological Equipments Laboratory 34
3. Preparation of General Purpose Media 45
4. Preparation of Selective and Differential Media 48
5. Preparation of Culture for Yeasts and Moulds 51
6 Sub-culturing of a Given Culture 58
7. Isolation of Pure Culture of Bacteria by Performing Streak Plate Method 67
8. Estimation of Amount of Bacteria by Pour Plate Technique 78
9. Quantitative Determination of Viable Microbes of Spread Plate Method 81
10. Preparation of Bacterial Smear 98
11. Simple Staining of Bacterial Cultures 100
12. Gram Staining of Bacterial Cultures 103
13. Acid fast Staining of the Given Culture(s) 108
14. Spore staining in a Given Bacterial Culture 112
15. Capsule staining in a Given Culture 116
16. Preparation and Identification of Temporary Mounts of Fungal Culture 136
17. Morphological Characteristic of Yeast Cells 139
18. Morphological Study of Fungi by Slide Culture Technique 141
19. Performing Biochemical Tests on a Given Bacterial Culture 158
20. Evaluation of Carbohydrate Fermentation ability of Microorganisms 161
21. Differentiation Between Bacteria using IMViC test 164
22. Determine the Ability of Bacteria to Reduce Nitrates 175
23. Determine the Ability of Microorganism to produce Urease 178
24. Catalase Activity Test 181
25. Cytochrome Oxidase Activity 184
26. Testing Quality of Water using Presumptive Test 193
27. Confirmation of the presence of Coliform Bacteria in Positive Presumptive Test 196
28. Performing the Completed Coliform Test 198
29. Gram Staining of Bacterial Cultures 209
30. Determination of Fungal and Yeast Count in a Given Food Sample 212
31. Determination of Coliforms in the Given Food Samples by Presumptive Testing 215
32. Determination of the Quality of Milk Sample by Methylene Blue Reductase Test 219
33. Detection of Number of Bacteria in Milk by Breed Count 221
34. Assessing Sanitary Quality of Contact Surface by Swabbing Method 228
35. Analysis of Air of Processing Facility for Microbial Load 230
36. Determination of Metanil Yellow in Food Sample 234
37. Check the Presence of Rhodamine B in Given Food Sample 236
38. Test the Presence of Sugar in Honey 238
39. Detection of NaHCO3 (chalk) in Flour 240
40. Check for the Presence of Vanaspati and Rancidity in the Ghee 242
41. Check the Milk for the Presence of Proteins, Urea, Sugar and Starch 244
NOTE
Programme Incharges
Mrs. Bhavana Yogeshchandra Chauhan Dr. (Ms.) K. Prafulla Reddy
Programme I\c Programme I/ c
IGNOU Programme Study Centre IGNOU Programme Study Centre
SM Patel College of Home Science St. Mary’s College,
Vallabh Vidya Nagar, Laitumkhrah, Shillong
Dist. Anand-388120 Dist. East Khasi Hills
Gujarat Meghalaya-793 003

Dr Pranati Das Dr. Indira Chakroborty

Programme I/c Programme Incharge
IGNOU Programme Study Centre IGNOU Programme Study Centre
College of Home Science All India Institute of Hygiene and Public Health 110
OUAT, Bhubaneswar -151 0003 Orissa Chittranjan Avenue, Kolkata -700073

Dr. A. Jyothi Dr. K. D. Chandel

IGNOU Programme Study Centre Programme Incharge
Sri Padmavathi Mahila Visvavidyalayam (SPMVV) IGNOU Programme Study Centre
Tirupati-511502 Chitoor District Andhra Pradesh St. Bede’s College Shimla -2

Dr. Ha Joshi Dr. A. Sundravalli Programme Incharge (PIC)

Programme I/c IGNOU Programme Study Centre Mount Carmel
IGNOU Programme Study Centre College 58, Palace Road Bangalore -52
International College for Girls
Gurukul Marg, S.F.S. Dr. Naheed Vaidya
Mansarovar Progamme Incharge
Jaipur-302020 Rajasthan IGNOU Programme Study Centre
Institute of Home Science University of Kashmir
Dr. Molly Joshi Programme I/c Hazratbal -190006
IGNOU Programme Study Centre Srinagar J&K
Christian Medical College, Ludhiana -141008
Punjab Mrs. Sudha Handa
Programme Incharge
Mrs. Divya Rani Singh IGNOU Programme Study Centre
Programme I/c Sorogini Naidu Government Girls PG College
IGNOU Programme Study Centre Shivaji Nagar, Bhopal
D.D.U. Gorakhpur University
Gorakhpur, Uttar Pradesh Dr. Nilambari Dave
Programme Incharge (PIC)
Smt. Neelam Kumari IGNOU Programme Study Centre
Programme I/c Smt. S.B. Gardhi
IGNOU Programme Study Centre Institute of Home Science
Isabella Thoburn College Saurashtra University, Rajkot -360005
7, Faizabad Road
Lucknow-226020 Uttar Pradesh Dr. Hernla Aggarwal
Programme Incharge
Dr. Aruna Palta IGNOU Programme Study Centre
Programme I/c Govt’s Women College
IGNOU Programme Study Centre Gandhi Nagar, Jammu
D.B. Girls’ Autonomous PG College
Raipur-492001 Chattisgarh Dr. Kumud Khanna
Programme I/c
Smt. Archana Dixit IGNOU Programme Study Centre
Programme I\c Institute of Home Economics
IGNOU Programme Study Centre F-4, Hauz Khas Enclave
Govt. Girls P.G. College New Delhi -110016
Link Road, Bilaspur,
Dt. Bilaspur Yamuna Nagar
Chattisgarh-495001
Govt. K.R.G. P.G. College Sl. No. RSD/D/2676/23/9
Smt. M Merythilda Gwalior 4227 (RSD Director)
Programme I/c RD Cochin 23/9/05
IGNOU Programme Study Centre RD Kerala
Holy Cross Home Science College Dr. Jyoti Prasad
52, New Colony, Programme Incharge (P/C)
Thoothukudi -628003 IGNOU Programme Study Centre
Dt. Thoothukudi , Tamil Nadu Govt. KRG PG College, Gwalior