PRACTICAL 1 MICROSCOPE STUDY Microscope Study and

Identification of

AND IDENTIFICATION OF Pathogenic Microbes

PATHOGENIC MICROBES

1.0 Objectives
1.1 Introduction
1.2 Light Microscopy
1.2.1 Compound Light Microscope: Bright Field Microscope

1.2.2 Parts of A Compound Light Microscope

1.2.3 Principle of Image Formation And Magnification In A Compound Microscope

1.2.4 Resolving Power of Microscope

1.2.5 Field Diameter

1.3 Adjustment And Use of The Various Lens Systems

1.3.1 Procedure For Focusing The Microscope Under Low Magnification By Using
Low Power Lenses

1.3.2 Procedure For Focusing The Dry, High, Magnification

1.3.3 Procedure For Focusing The Microscope Under High Power Magnification By
Using Oil Immersion Lens System

1.4 General Guidelines For The Care of A Microscope

1.5 Identification of Pathogenic Organisms
1.5.1 Pathogenic Bacteria

1.5.2 Pathogenic Fungi

1.5.3 Pathogenic Animal Like Protists (Protozoa)

1.5.4 Pathogenic Helminths

1.5.5 Exercise 1: To Study The Various Structures Of A Light Microscope And To

Observe A Few Microorganisms From Prepared, Permanent Slides

1.6 Safety Instructions For Working In The Laboratory

1.8 Let Us Sum Up
1.9 Answers To Check Your Progress

1.0 OBJECTIVES
After completing this practical exercise, you should be able to:
• demonstrate the correct use of the compound optical/light microscope;
• draw the path of light through a compound microscope;
• name the major parts of a compound microscope;
• demonstrate the correct handling (care) of the microscope during and
after its use; and 9
Mounting and • demonstrate skill in the use of low power lens, high power lens and oil-
Staining
Techniques immersion lens for viewing glass slides movement with biological
specimens and materials.

1.1 INTRODUCTION
In nursing practice, we come in close contact with people, both sick and
healthy, and also with equipments which are sterile, clean and contaminated.
We should therefore, prevent infection and control the spread of infection.
This is possible only when we are aware that to invisible microorganisms
are present everywhere.

Microbes are not visible without a microscope. Thus, the microscope is an

essential tool for the study of microbes.

The present practical is designed for you to become familiar with and
proficient in the use and care of a compound, light microscope. This
knowledge and skill with reference to the compound, light microscope will
be valuable to you in your work in the Primary Health Centres. You need
patience and practice in developing your skills of observation of specimens
and their internal structures through a microscope. Discovering microbes by
observing them under the microscope requires curiosity and patience.

In order to perform this practical successfully we advise you to review the

following theory units of the Course BNS-202(Block 2 “Bio-physics”): Unit
5- Light in the Block 2(Unit 5 Section 5.4) which among other aspects deals
with the properties of light and Block 3 ( Microbiology) which deals with
the theory and application of the light, compound microscope and also
focuses in detail on the important features of various microorganisms that
are pathogenic to h u m a n s .

1.2 LIGHT MICROSCOPY

The term microscope is derived from two Greek words namely “Micro”
meaning small and “Scope” meaning to view. The compound, light, optical
microscope is an instrument for viewing small objects invisible to naked eye.
Many kinds of microscopes come under the category of light microscopes,
as all of them use light to visualize images. Examples of light microscopes
include brightfield microscopes, dark field microscopes, phase-contrast
microscopes, differential interference contrast microscopes, fluorescence
microscopes, confocal scanning laser microscopes, and two-photon
microscopes. All these various types of light microscopes are often used to
complement each other in medical diagnosis and in research.

1.2.1 Compound light Microscope: Bright field Microscope

The most commonly is type of light microscope which is used the bright field
microscope, generally referred to as the compound, light microscope. The
10 Bright field microscope has two or more lenses which help in the production
of a dark magnified image on a bright background. Microscope Study and
Identification of
Pathogenic Microbes
A compound light microscope contains (i) a two-lens system, namely: (a) the
objective lens system and the (b) occular or eye lens system, (ii) a light
source and (iii) mechanical adjustable parts. The lens system of the occular
and also the objective contain a series of lenses, usually 8 to 10 in the
objective and 2 to 3 in the ocular/eye piece. The closely placed lens elements,
both in objective and ocular lens due to being placed, close to each other act
as a single lens and correct any aberration that may occur in the image of the
specimen/object which is to be magnified for viewing. The compound, light
microscopes typically provides magnification in the range of 40x-1000x.

1.2.2 Parts of a Compound, Light Microscope

Let us now study the various parts of a compound microscope with the help
of figure 1.1 and the functioning of its various parts with the help of figure
1.2.

• Base

The solid bottom of the microscope is called the base of the microscope. It
supports the rest of the structures of the microscope and gives stability to the
microscope.

• Anterior part of Microscope head

The head or head tube part or head of the compound light microscope has a
tubular body, the top end of which houses the eye piece which forms the
occular lens system.. At the other or lower end of the tube are a set of 2-3
objective lenses of varying magnification mounted on a rotating nosepiece.
The objective lens system is generally referred simply as the objective or
objective lens. The objective lenses are situated close to the object/specimen
to be viewed.

• Arm

An L-shaped arm connects the head end or anterior end of the microscope to
the base. The arm is a structure that supports the lens assembly and also the
stage assembly where the glass slide is placed for study.

• Objective lens system/Objective

The objective lens system is commonly referred to as the objective. The

function of the objective lens system is to collect the rays of light from object
and focus it beyond twice the focal length in order to form a real, inverted,
magnified image for viewing by the occular/eye lens. The objective lens
systems as mentioned earlier is a multi component lens systems usually
constructed of 8 to 10 in objectives lenses which are designed and and fitted
in such a way so as to minimize aberrations in the image of the
object/specimen to be viewed. Each objectives lens system is fitted within a
metal tube/casing which is mounted on a revolving nose-piece. The nose- 11
Mounting and piece has the provision for fixing 3or 4 objectives, each one of which has a
Staining
Techniques different magnifying power. Usually the objectives mounted on the nose
piece will have a magnifying power of 6 times or 10 times or 40 times or 100
times. Each objective will have its magnification written on its metal casing
as 6x or 10x or 40x or 100x.

Fig. 1.1: The compound light microscope its main parts.

The objectives can be of two types namely, dry objective or immersion

objective. An objective which is used to view images with the air present
between the object/specimen or coverslip covering the object/specimen to be
viewed and the objective lens is called as a dry objective. In contrast, an
objective that requires a liquid, usually in the form of transparent oil in order
to view the object/specimen is called as an immersion oil objective. The
immersion oil is placed on the coverslip, which covers the object/specimen
placed on the glass slide or directly on the object/specimen. The immersion
oil occupies the space between object/specimen or the coverslip and the
immersion oil objective

The focal length of the lens system of the objective determines its
magnification since the focal length of a lens is inversely proportional to the
magnification. This means that in an objective lens an increase in its
magnifying power would result in a decrease in its focal length.Thus
objective lens with higher magnification would have a shorter focal length.

• Nose piece /Objective Turret

The rotating structure on which the objective lens unit is mounted is called
the nose piece or objective turret or revolver as it can revolve. The position of
the three or four objectives fitted on the nose piece can be changed without
disturbing any adjustments.
12
 Microscope Study and
Identification of
Pathogenic Microbes
• Eyepiece/Occular/Ocular Lens

The ocular lens system also often referred to as ocular lens or eyepiece or
eyepiece lens system is fitted into a draw tube which as mentioned earlier is
located at the top or head end of the body tube of the microscope. The ocular
lens system is used to observe enlarged or magnified virtual image of the
object or specimen to be viewed. This is possible due to the arrangement of a
prism and a number of lenses within the body tube of the head of the
microscope as described below (Fig1.2).

A prism is present at the top of the body tube in such a way so it enables the
ray of light coming from objective lens system to bend by 45o. This bent
beam of light enters into the draw tube which is fitted with the eyepiece lens
system. The eyepiece lens system is a 2- component lens system (lower field
lens and upper eye lens) and may magnify the image formed by the
objective lens system(whose magnification power maybe 6 or 10 or 40 or
100 times depending on the magnifying power of the objective lens used).
The occular lens system or eyepiece may be of 10X or 15 X magnifications.
The magnification power of the occular lens/eyepiece, for example 10x or
15x is given on the metal casing containing the lenses of the eyepiece/
occular unit. Normally an eyepiece of 10x, that is, with 10 times
magnification is employed.

Some compound light microscope are called monocular as they have only a
single eyepiece, though most newer ones are binocular as they have two
eyepieces as you can see in Figure 1.1.In case of a binocular microscope the
magnification of both the eyepiece should be the same while being used.

• Magnification

The ocular and objective lenses work together to create a magnified

image. The total magnification is the product of the ocular magnification
times and the objective magnification times: It can thus be calculated by
multiplying the ocular magnification with objective magnification. For
example, if a 10x objective lens is selected and the ocular lens is 10x, then
the total magnification would be (10x) X(10x) =100x.

• Coarse and Fine Adjustment Knobs

Two types of adjustments knobs are present behind the body tube of the
microscope. They are positioned on the arm of the microscope. A large knob
is used for coarse control. It is used for the vertical movement of the lens
tube. A smaller knob is used for fine adjustment control. It is used to bring
the image of the specimen into sharp focus, while viewing specimens under
higher magnification( at 40x or 100 x objective magnification).

• Stage

The stage is a flat platform with clips. The stage is attached to the lower part 13
Mounting and of the arm of the microscope and is placed below the objectives and above
Staining
Techniques the mirror. The stage supports the glass slide on which the object called the
specimen which is to be viewed by the microscope is placed. The glass slide,
with the specimen is clipped into place by the clips of the stage of the
microscope. The stage on which the glass slide containing the specimen is
placed can be moved by the mechanical stage which is composed of a
movable rack and pinion system.The movement of the mechanical stage is
by means of two knobs. One knob moves the slide left and right, the other
moves it forward and backwards.

• Condenser Lens

A condenser lens usually an Abbe’s condenser lens system is present just

below the specimen stage and focuses the beam of light on the specimen from
any illuminated source. The condenser lens is also formed of several lenses.
The condenser lens is useful in getting sharp images of magnification 400X
and above. The amount of light let into the condenser lens can be controlled
by an iris diaphragm placed beneath it.

• Diaphragm or Iris

The diaphragm or iris regulates the amount of light entering the condenser
i.e. helps in adjusting the intensity of light. It is located under the stage,
between the light source and condenser.

• Mirror: Illuminator

In the light, compound microscope a steady and effective light source is used
for illumination. For illumination, an illuminator in newer models of light,
compound microscope is used while in the older light, compound microscope
a mirror which is concave on one side and plain on the other side is attached
to the base of the microscope. The illuminator or mirror can be adjusted to
reflect the light upwards. In older models of the light, compound microscope
an external table lamp is used as the light source. In newer, models of the
light, compound microscope a permanent built in light source in the form of a
tungsten lamp is present.

1.2.3 Principle of Image Formation and Magnification in a

Compound, Light Microscope
Compound, light microscopes create an image of the specimen by the
following steps: (Figure 1.2a & 1.2b)

1. When, a specimen is placed between the objective and condenser lens.

2. Then the light emitted from the light source is focused over the specimen
with the help of a condenser lens.
3. After that, the light passes through the specimen and goes upwards,
towards the objective lens.

14
4. The objective lens captures the light coming from the specimen and Microscope Study and
Identification of
creates a magnified image of the specimen, which is called the primary Pathogenic Microbes
image (shown in fig 1.2b as image I).Then this image passes through the
body tube of the objective lens to the ocular lens or eyepiece which
further magnifies the image (shown in fig 1.2b as image II).
5. At last, the viewer can see a clear and magnified image of the specimen
through the eyepiece.

Fig. 1.2(a)

Fig. 1.2(b)

Fig 1.2. The compound light microscope: its main parts and their
function: a)The pathway of light in the compound light microscope is
shown in the form of dashed lines which starts from the light source and
moves through the specimen, then the ocular lens towards the occular
lens .b) The light pathway and the principle of using the lens systems
in the compound light microscope for viewing a magnified image of a
specimen by it - When, a specimen on a glass slide to be viewed is placed
between the objective and condenser lens then a light source is used to
provide light. The light emitted from the light source is focused by the
condenser lens onto the specimen to be viewed. This light passes through 15
Mounting and the specimen and is collected by the the objective lens which then focuses
Staining
Techniques a real image of the specimen inside the microscope (image 1). Image I is
further magnified by a second lens or group of lenses (called
the eyepiece) giving the viewer an enlarged, inverted image of the
specimen (image 2).

1.2.4 Resolving Power of Microscope

Resolving power of a microscope refers to the smallest detail that a
microscope can resolve (clearly show) when imaging (producing an
image) a specimen. Lens have the ability to show two adjacent objects as
distinct units. The smallest distance by which two points can be separated and
distinguished as individual objects by a microscope is called its resolution.

In simple terms this means that if two objects are placed close together on the
stage of a microscope and can be seen and distinguished as two distinct
objects, then the microscope can be said to have a high resolving power.In
microscopes with low resolving power, the image of the two specimens on
the stage of the microscope will appear as a single image.

The resolving power of a microscope is calculated by the formula given by

Reyleigh which is as follows
Resolving power = d = 0.61λ
--------
n sinα

where:

λ is the wavelength of visible light,

n is the refractive index(n also called R.I.) of the medium surrounding the
specimen.

α is the half angle of light entering the objective lens from the specimen

0.61 is the constant

n sinα is also referred to as the numerical aperture of the objective lens (NA).
The numerical aperture of a microscope objective is a measure of its ability
to gather light and resolve fine details of specimen at a fixed object
distance.

Resolution or resolving power is expressed in micrometres (μm) (which

is one millionth of a meter).

The resolving power of the microscope is highest when λ is smaller and α has
a higher value. In other words, resolving power is inversely proportional to
wavelength. In a light microscope, from the visible spectrum one can obtain a
wavelength of 0.5 µm (0.5 x 10-6 m). In a light microscope with the best
resolution, one can distinguish images between two points which are 0.2 µm
(0.2 x 10-6 m) apart whereas by way of comparison an electron microscope
16 can easily resolve images which are about 1 x 10-10 m apart.
The resolving power of microscope as you know also depends on the Microscope Study and
Identification of
refractive index (n: also called R.I.) also called the index of refraction of the Pathogenic Microbes
medium surrounding the specimen. Refractive index, is the measure which
determines how much the path of a light ray is bent, or refracted when
passing from one medium into another. In light microscope the refractive
index is the measure of how much the light ray is bent while passing through
the air medium from the glass slide to the objective lens. The refractive index
of air is lower than that of glass so when the light rays pass from the glass
into the air, they bend or refract due to which all the light rays do not all pass
into the objective lens. Loss of refracted light can be compensated by putting
mineral oil on the cover ship of glass slide or the biological specimen to be
viewed which has the same refractive index as glass.

1.2.5 Field Diameter

In a light microscope, the field of view (FOV) is the diameter of the circle of
light that you see when looking through eyepiece of the light microscope.
Thus FOV = diameter of circle of light. We earlier referred to objective and
occular lenses of different magnifications. As the magnification power
increases, the FOV gets smaller. You can verify this fact by changing the
ocular lens of different magnifications in the microscope.

1.3 ADJUSTMENTS AND USE OF THE

VARIOUS LENS SYSTEMS
Once you are familiar with the various parts of a microscope, you can start
using it. In order to achieve the best results with microscopic observations it
is important to learn how to adjust light and how to focus the various lens
systems.

Let us now study step-by-step the various procedures for the correct use of a
compound, light microscope. You need to read beforehand the procedure for
using the microscope properly and be able to identify the parts of a
microscope before you actually handle it in the laboratory.

1.3.1 Procedure for Focusing the Microscope under Low

Magnification by Using Low Power dry objective
Lenses
Since the compound light microscope as the name suggests is an optical
instrument, the source of illumination is very essential to magnify and
observe an object (specimens/cells/tissues) which is mounted on a glass
slide, is placed under the dry objective lens and is basically invisible to the
naked eye. Let us now learn how to use the microscopes for observing
specimens/objects under low power and high power magnification

The low power lens system of the objective lens of the compound light
microscope is used to scan or visualize larger objects such as insects,
17
Mounting and parasites, ova or colonies of bacteria, pus cells, etc. It is also used to focus in
Staining
Techniques position the object which is to be studied under higher magnification. It is
thus essential to first learn the technique of focusing an object under low
magnification which is described below:

i) Place the microscope in a desired orientation on a plane surface. Do not

change the position thereafter.
ii) It is important that the ocular and objective lens are clean. Thus, before
using the microscope, gently clean both the lenses by using a lens
cleaning paper and lens cleaning fluid.
iii) Select the suitable objective lens system. It is better to start with a low
power lens system. Rotate the revolving nose-piece and bring the 10X
objective (low power) to the path of light and click it into the position.
The distance between the stage and the objective should be
approximately equal to the focal length of the objective.
iv) Place the glass slide with the object/specimen mounted on it onto the
mechanical stage assembly. Adjust the screws of the mechanical stage in
order to bring the desired part of the object/specimen to be viewed in the
centre of the stage aperture.
v) Now look through the ocular/eyepiece of the microscope. If the
microscope has an external light source in the form of an external lamp
then it is provided with a mirror whose one surface is plane and other is
concave surface. In a microscope provided with a mirror the mirror is
manually oriented so that the light from external lamp gets focused in
the centre of mechanical stage assembly in the form of a bright, uniform
circular field that gets maximum amount of light. In microscopes with
built in lamps/ illuminators the light from the built in lamp is adjusted
with the knob of the illuminator so that a bright, uniform, circular field
that gets maximum amount of light can similarly be formed in the centre
of mechanical stage assembly.
vi) After this, view the image of the specimen/object by means of the
eyepiece/occular, for this purpose move the movable rack and pinion
system by turning its adjustment knob so that the specimen or part of the
specimen to be viewed is placed in the centre of the circular bright, field.
vii) Now raise the condenser to its top position and slightly open the iris
diaphragm because the specimen/object or its part is clearly seen when
the iris is barely open. The aperture of the iris diaphragm is opened
wider while using objectives of high magnification as more light is
needed at higher magnification.
viii) In order to bring the image of object/specimen into sharp focus, slowly
open and close the diaphragm so that a better contrast and clearer image
is obtained of the specimen
ix) To further bring the image of the specimen/object or its desired part into
sharp focus viewing by the eyepiece use the coarse adjustment control.
18
 You can also raise or lower the condenser in order to adjust the light in Microscope Study and
Identification of
order to get a clear image of the specimen/object or its part is to be while Pathogenic Microbes
viewing it with help of eye piece.
x) Record your observations in the lines given below: and later copy your
observations in your practical note book.
xi) Finally, after using the microscope, remove the slide from the stage,
clean it with a lens paper and put it back in the slide box. Also clean the
occular/eye piece and objectives lenses of the microscope and return the
microscope to its box.

1.3.2 Procedure for focusing the microscope under the Dry,

High, Power Magnification Objective Lenses
High power lenses are used for viewing stained and unstained specimens/
objects and their various parts under high magnification. If you need to view
a larger image of the specimen/object or part of the specimen/object then, you
need to view it by using an objective that has higher magnification power.
Specimen/object or its parts can be viewed at higher magnification by either
dry, high magnification objective lenses of 40 x or when further
magnification is required by means of objective of lenses of higher
magnification of namely 100x. For viewing objects/specimens or their part
under 100x objective lenses the coverslip of the mounted objects/specimens
or their part is covered by immersion oil (Refer again to subsection 1.2.1)
Parafocal: The
In this subsection we will describe the procedure of viewing microscope is said to
objects/specimens or their parts under high, dry, objective lens with 40X be parafocal if an
magnification. Objective lenses with 40x magnification as mentioned earlier image brought into
sharp focus under low
do not require immersion oil for viewing objects/specimens or their parts. power remains in focus
High dry objective lens with 40x magnification are used to view intracellular when the high power
details like motility of bacteria, structure of pus cells, RBCs, WBCs, ova, objective replaces the
low objective.
fungi, cysts, crystals etc. The procedure for viewing an object/specimen or its
part by the dry, high power objective lenses is as follows

i) First focus the object/specimen which is placed on a glass slide under

low power of the microscope by following each and every step from i to
vii given earlier in sub-section 1.3.1.
ii) After following the steps i to vii listed in sub-section 1.3.1. use the
mechanical stage movement to bring the desired specimen/object or part
of the object/specimen to be viewed under the microscope in the centre
of the field of vision.
iii) Replace the low power objective with the dry, high power objective lens
with 40X magnification. The low power objective lens is replaced by the
40X high power, dry objective lens by rotating the, revolving the nose-
piece and bringing the high power (40X) dry objective lens into
position ie, into the path of light. If the microscope is parfocal you
should be able to clearly see the sharp and magnified image of the
object/specimen by the occular/eye piece. As in all compound light 19
Mounting and microscopes the lens systems of low power objective and the lens system
Staining
Techniques of dry, high power objective are matched, i.e. parfocal, the high power
dry lens is almost in focus when replaced by the low power objective
lens.
iv) However the amount of light for viewing the image under high
magnification needs to be increased. In order to do achieve this increase
the aperture of the iris diaphragm.
v) Now observe with the help of the eyepiece/occular lens system the image
of the object/specimen or part of the object/specimen placed under the
high power objective(40X). It will be very slightly out of focus. Bring it
into sharp focus by using the fine adjustment knob which is usually done
by turning the knob forward.
vi) With the help of the eyepiece view the clear image of the
object/specimen on mounted on the glass slide and placed under the dry,
40x objective lens of the microscope. Record your observations in the
given lines below and later copy them in your practical note book.

1.3.3 Procedure for Focusing the Microscope under High

Power Magnification by using Oil Immersion objective
Lens System
Oil immersion lens system is used when specimens/objects or their parts are
very small and the magnification power of the high, dry, objective lens is
inadequate in magnifying them suitably for visualization by the eyepiece. Oil
immersion objective lens system of the compound light microscope is used
for viewing tiny, stained and unstained organisms such as bacteria and yeasts,
parasites, animal-like protists such as malarial parasitic stages, etc. and their
parts.

The oil immersion lens gives better resolution because the oil has the same
refractive index as glass, thus forming a homogeneous medium for light to
pass from the specimen to the lens. The procedure to use an oil immersion
lens system (Fig.1.3) for viewing a specimen/object or its part is given below
as follows:

i) Follow each and every step from i to vii given in sub-section 1.3.1 and
steps i to v in subsection 1.3.2 so that the object/specimen mounted on
the slide and covered by a coverslip is well focused first under low(10x)
power(steps i to vii in subsection 1.3.1) and then under high(40x),dry
power(steps i to v in subsection 1.3.2).
ii) Adjust the light for oil immersion lens system by increasing the size of
the iris diaphragm so that the amount of light is increased.

20
 Microscope Study and
Identification of
Pathogenic Microbes

(a) Move the high-dry lens out of (b) Place a drop of immersion oil in the
postion of the slide centre

c) Move the oil immersion lens into position

Fig. 1.3: Procedure to use immersion lens
iii) Remove the high power 40x objective lens and place a single drop of
suitable oil (e.g. cedar wood oil) on the surface of the coverslip covering
the object/specimen placed on the glass slide.
iv) Move the oil immersion power 100x objective lens over the immersion
oil which is is placed on the coverslip under which the object/ specimen
is placed.
v) Once the oil immersion (100x) objective lens is in position over the
specimen, view the image with the eyepiece/ocular lens and focus to get
a clear image by only using the fine adjustment knob. The focusing
should not take more than two turns in either direction to bring the
specimen into sharp focus. To do this observe the spherical field and
slowly rotate the fine adjustment first in the forward direction and if
necessary then in backward direction till the object comes in sharp focus.
vi) Adjust the light intensity. Select the suitable field region to be viewed
and record your observations in the given lines below and copy the
observations later in your practical file.
vii) After observing the object/specimen, lower the stage to break contact
between the oil lens system and remove the slide.
viii) Before replacing the microscope in its box, wipe out the residual oil on
the objective lens system and the coverslip of the glass slide with a xylol
swab. Follow this by wiping the lens of the objective lens system and the
coverslip of the glass slide with an alcohol swab and then immediately
dry up the objective lens surface of the objective lens system and the
coverslip by means of a cleaning lens paper/clean silk cloth.
21
Mounting and ...........................................................................................................................
Staining
Techniques ...........................................................................................................................
...........................................................................................................................
...........................................................................................................................
...........................................................................................................................
...........................................................................................................................
...........................................................................................................................

1.4 GENERAL GUIDELINES FOR THE USE

AND CARE OF A MICROSCOPE
1) Carry the compound light microscope, consisting of two oculars (eye
pieces) by placing one hand beneath the base and by holding the arm of
the microscope with the other hand and place it on the lab table/bench.
2) Do not tilt the microscope but instead adjust your lab stool so that you
can comfortably use your microscope and view the desired
object/specimen or its part through the eye piece.
3) Keep the mechanical stage clean and free of oil. Keep all lenses free of
oil except when using it, to view the object specimen/tissue under
immersion oil. Use only lens paper to clean the objective lens with clean
silk cloth.
4) Keep all occular and objective lenses clean. Do not touch the lenses with
your hands. Before putting away the microscope in its box or under the
dust cover, clean off the oil from the objective lens if you have used
immersion oil. For cleaning the immersion oil from the objective lens
first carefully wipe off the residual oil immersion from its surface with a
xylol swab. Follow this by wiping the lens with an alcohol swab and then
immediately dry the lens by means of a lens cleaning paper/clean silk
cloth. Return your microscope to the designated area and put it back in
its box or under the dust cover.
5) Also clean the coverslip of the glass slide which has been viewed under
immersion oil, by first wiping off the immersion oil on the cover slip
with a xylol swab and then with an alcohol swab. Immediately dry up the
coverslip with lens cleaning paper and keep it in the slide box.
6) When a problem does arise with your microscope, obtain help from the
instructor. Do not use another microscope unless yours is declared “out
of action”.

1.5 IDENTIFICATION OF PATHOGENIC

ORGANISMS
1.5.1 Pathogenic Bacteria
22
In this subsection of the present laboratory exercises, permanent glass slides Microscope Study and
Identification of
of human pathogenic bacteria of the Domain Bacteria: (i) the gram positive Pathogenic Microbes
stained cocci that occur in clusters (Staphylococci) and in chains
(streptococci) and (ii) the, gram negative bacilli (eg. Escherichia coli) will be
provided to you by the counselor for viewing the arrangement, shape, size
and internal structures of their cells. The slides provided by the counselor
should first be focused under low power objective lens and later examined
under dry or oil immersion objective lens. Hanging drop preparation for
studying motility of organism is studied under oil immersion high power
objective.

You should also view permanent the slides that you will prepare in Exercise
2, when you will learn the techniques for preparing glass slides of bacterial
and blood films and staining them in order to be seen under the microscope.

1.5.2 Pathogenic Fungi

Yeasts are eukaryotic, single-celled microorganisms classified as members of
the Fungi Kingdom. Yeast is scientifically called Candida and causes the
human fungal infection called candidiasis The study and observation of
yeast by means of the light, compound microscope is only possible by using
its high power dry objective (40x)or oil immersion objective(100x) .

Filamentous fungi, can be studied by means of the light, compound

microscope initially under low power 10x.However,if more details of the
filamentous fungi has to be studied then it is viewed by the occular/eyepiece
under dry, high power objective (40X). Stained slides are better because
structures can be visualized better.

1.5.3 Pathogenic Animal like Protists (Protozoa)

Human pathogenic, intestinal animal-like protists (protozoa) like parasites
(Giardia, Entamoeba) should be seen under high, dry power objective (40X)
whereas blood borne animal-like protist (protozoan) parasites should be seen
under the oil immersion (100X).

1.5.4 Pathogenic Helminths

Helminths are big enough to be seen by the naked eye (e.g. round worm).
However, their ova are best visualized/identified u n d e r high, dry power of
the objective lens (40x) of the microscope.

1.5.5 Exercise 1:

• AIM: To Study the Various Structures of a Light Microscope and to

Observe a Few Microorganisms From Prepared, Permanent glass
Slides

• Material Required

— Light, compound, binocular, Microscope 23

Mounting and — Prepared, permanent glass slides of, stained bacteria/protozoa/algae
Staining
Techniques or fungi
— Oil immersion
— Tissue paper
— Lens paper
— Xylol swab
— Alcohol swab

• Procedure

1) Place your microscope on the lab bench in front of you.

2) First study the various structures of the microscope with the help of
subsection 1.2.2(Parts of a compound, light microscope) of this practical.
Draw a well labelled diagram of the microscope.

3) In order to study the cells/structure of bacteria/protozoa/algae or fungi

take a permanent glass slide of stained, bacteria/protozoa/algae or fungi
provided by your counselor. Clean the slide with tissue paper and then
place it on the stage of the light microscope.

4) Adjust the eyepieces on a binocular microscope to your own focal length

by the two following methods:

a) Switch on the light of the microscope. Then first look through the
right eyepiece with your right eye and left eyepiece with your left
eyepiece and adjust the distance between the two eyepieces until a
circle of light appears..

b) For adjusting the right and left eyepieces of the binocular

compound, set light microscope to your focal length first cover the
left eyepiece with a small card or your hand and looking through the
right eyepiece and focus on the specimen/object mounted on the
glass slide and placed under the low-power (10 x) objective. You
can use the coarse adjustment knob to focus on the specimen/object.
When the right eyepiece has been focused according to your right
eye focal length then cover your right eyepiece with your hand or
small card and looking through the left eyepiece focus on the same
specimen/object mounted on the glass slide and placed under the
low-power (10 X) objective with the help of coarse adjustment knob.
Thus, the left eyepiece has been focused according to your left eye
focal length Now both the eyepieces/occular lenses have been
adjusted according to the focal lengths of your eyes so that you can
use both eyepieces to view a specimen/object properly.

5) Raise the condenser of the microscope up to the level of the mechanical

stage. For viewing objects/specimens or their parts under low power with
objective of 4x or10x you need to decrease the aperture of the iris
24 diaphragm so that only a minimum amount of light enters the objective
 lens. (Refer also to subsection 1.3.1). Microscope Study and
Identification of
Pathogenic Microbes
6) View the image of the microorganism mounted on the glass slide under
low power with the help of the ocular/eye piece. For this first use a
minimum of light by adjusting the condenser and iris diaphragm then
properly focus the occular/eyepiece and so that you obtain as clear
image. Record your observations and draw the cell structures of the
organism under low power.

7) When you have completed your observations under low power and
further want to view a larger the image of the mounted organism and its
cells under dry, high magnification then replace the low power objective
(10x) with the higher magnification (40x) objective by swinging it into
position. The image of the organism when the low power objective is
replaced by the higher power objective will still remain almost in focus.
This is because all of the objectives (with the possible exception of the
4x) are parfocal. That is, when an object is in focus with one lens, it will
also be in focus with all of the lenses. Only a slight adjustment will be
required. You only need the fine adjustment knob to bring the image in
focus. However, an increase in the amount of light will be needed which
can be achieved with the help of the condenser and the iris diaphragm.
Now again, record your observations draw the general size , shape and
structural components of the cells of the microorganism being viewed
under dry, high, power of magnification by means of the occular/eye
piece. (Refer also to subsection 1.3.2) .
8) In order to study the microorganism and its cells under greater
magnification than that of dry, high, power of magnification you need to
use the immersion oil objective lens which has a 100x power of
magnification. For this, move the high power objective lens (40x) out of
position and place a drop of immersion oil on the area of the cover slip
of the permanent, stained ,glass slide you are observing with ocular/eye
piece. Carefully click the oil immersion objective lens into position. The
100x objective lens should be immersed in the oil (Fig. 1.3). View the
image of the cells of the organisms with occular lens/ eyepiece and very
carefully focus it by mean of the fine adjustment knob so that a clear
image is obtained. You may need to increase the amount of light with the
help of the condenser and the iris diaphragm.
Now again, record your observations and draw the general size ,
shape and internal structures of the cells of the microorganism
being viewed under oil immersion. (Refer also to subsection 1.3.1) .
9) When your observations are complete, lower the stage and remove the
microscope slide. After this bring back the low-power objective into
position. Do not rotate the high-dry (40x) objective through the
immersion oil. Clean the oil off the objective lens first with xylol swab
and then with alcohol swab and dry the lens with lens paper/clean silk
cloth. Objective lens of low power and high power in which immersion
25
Mounting and oil has not been used can be cleaned by lens paper or tissue paper.
Staining
Techniques 10) Once the microscope has been cleaned place it back in its cabinet or
cover it with its dust cover.
11) After viewing a glass slide under immersion oil, the oil on the coverslip
of the glass slide has to be cleaned. For this purpose remove the
immersion oil on the coverslip of the glass slide first with a xylol swab
and then with an alcohol swab. After this dry the coverslip with lens
paper/ tissue paper. You need to repeat this procedure for all the glass
slides that you observe under immersion oil. Glass slides containing
objects or specimens to be viewed under low and high power
magnification (not under immersion oil) only need to be cleaned by lens
paper or tissue paper.
12) Wash your hands after performing the exercise.

1.6 SAFETY INSTRUCTIONS FOR WORKING

IN THE LABORATORY
From the beginning you should develop the habit of handling all cultures in a
correct and safe manner. Regard all cultures as potentially infective.

1) A white coat or gown must be worn while conducting praticals in the lab
in order to prevent contamination of your clothes.
2) Keep the working space on the lab bench, free of unnecessary books and
papers.
3) Lunch packets should not be kept on the lab bench and eating any food
material while at the lab bench is not permitted.
4) Do not moisten labels, pencils etc. with your tongue.
5) Keep your fingers and hands away from your eyes, nose and mouth
while you are working in the laboratory.
6) Report all cuts, pricks and abrasions, however slight or minor to the
instructor. If you spill or splash any infective material report immediately
to one of the Laboratory staff and also to the instructor.
7) Any personal article that falls on the floor or on the contaminated surface
should be disinfected thoroughly or discarded.
8) Do not remove cultures from the laboratory. Culture tubes must be kept
in containers or kept upright in the racks and are not to be placed on the
bench. Cotton wool plugs should be held in hand without touching the
part that goes inside the tube and should not be laid on the bench.
9) Tubes and bottles must be stoppered except when actually in use. The
Tube rim or bottle rim should be flamed immediately before and after
use.
10) The inoculating loop must be thoroughly sterilized before and after use
26 by heating the loop until the wire is red hot. Never put down the
 inoculating loop before sterilization. Microscope Study and
Identification of
11) Used slides, coverslips and pipettes should be placed in the jar Pathogenic Microbes

containing disinfectant liquid. Filter papers, cotton etc. must be put in the
disposal bucket provided and not in sinks.
12) Take good care of the microscope. Clean all lenses with the lens paper or
a clean silk or lint cloth. The oil immersion lens should be carefully
cleaned with a xylol swab, then with alcohol swab and dried by lens
cleaning paper. The microscope including the stage and other metal parts
should be wiped off after use and the microscope should be placed in its
cabinet or under a dust cover.
13) If you spill or splash any infective material report immediately to
one of the Laboratory staff.
14) Wash your hands thoroughly with soap and water before leaving the
laboratory.

Check Your Progress

1) Explain the following terms:

a) Condenser
..............................................................................................................
..............................................................................................................
..............................................................................................................
..............................................................................................................
b) Iris diaphragm
..............................................................................................................
..............................................................................................................
..............................................................................................................
..............................................................................................................
c) Parafocal high power lens
..............................................................................................................
..............................................................................................................
..............................................................................................................
..............................................................................................................
d) Numerical aperture ie. the numerical aperture of a microscope
objective
..............................................................................................................
..............................................................................................................
..............................................................................................................
..............................................................................................................
27
Mounting and 1) Why are two types of surfaces of the mirror provided in a compound,
Staining
Techniques light microscope which uses an external light source?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
2) What is the power of an oil immersion objective lens of a light,
compound microscope?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
3) What is the advantage offered by an oil immersion lens system?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
4) What is the range of magnification that may be obtained using
microscopes?
.....................................................................................................................
.....................................................................................................................

1.7 LET US SUM UP

• The light, compound microscope is an instrument that is always required
in a microbiology laboratory. The magnification it provides enables us to
see the microorganisms and their structures which are invisible to the
naked eyes. The magnifications of the light, compound microscope
ranges from 40x-1000x.
• The light, compound microscope comprises of the following parts:
• Base, microscope body/ tube, arm, objective lens system/objective,
nosepiece, eyepiece/ occular/lens, coarse and fine adjustment knobs,
stage assembly, condenser lens, iris diaphragm, mirror/iIluminator.
• A compound light microscope contains multiple lenses to enlarge the
image of a specimen/object or its parts.
• A compound light microscope is typically, used for viewing image of a
specimen/object or its parts at high magnification (40 - 1000x), which is
achieved by the combined effect of two sets of lenses: the ocular
lens (typically with 10x magnification within the eyepiece and
the objective with lenses that typically have magnification of 4x or 10x
or 40x or 100x) . The objective is located above and close to the
specimen/object).
28
• The total magnification of a compound light microscope is calculated by Microscope Study and
Identification of
multiplying the magnification of the ocular/eyepiece lens by the Pathogenic Microbes
magnification of the objective lens.
• In addition to the light, compound microscope, several different types of
microscope are available but they are not discussed in this practical
course. The slide preparation methods for viewing specimens/objects and
their parts differ with different types of microscopes.
• The light, compound microscope is used in medical microbiology for
studying and identifying various human pathogenic microorganisms such
as bacteria, fungi animal-like protists etc. This is done by viewing the
prepared, unstained or stained microscope slides of these human
pathogenic microorganisms through the eyepiece of the light, compound
microscope and recording the observations.

1.8 ANSWER TO CHECK YOUR PROGRESS

1) a) The Condenser is a part of the microscope located just below the
specimen stage. It focuses the beam of light on the specimen from
any illuminated source. The condenser lens is useful in getting sharp
images of magnification 400x and above.
b) Iris diaphragm is a device placed below the stage of the microscope
and is used to control the amount of light entering the objective lens.
c) Parafocal high power lens means that the high power lens is co-
focused with the low power lens. Once the image viewed by the
occular lens/eyepiece under the low power objective lens is focused,
then the shift to high power objective lens will also keep the image
almost in focus.
d) The numerical aperture of a microscope objective is a measure of
its ability to gather light and resolve fine specimen detail at a
fixed object distance Numerical aperture is the measure of the
ability of the objective to collect light rays diffracted by passage
through the specimen.
2) The plane mirror surface reflects light rays from an artificial source of
light, such as an external lamp onto the objective, and the concave mirror
surface collects the scattered rays from a natural and focuses them on to
the objective lens.
3) Oil immersion lenses attain higher numerical aperture (upto 1.4) by
virtue of the fact that the RI of immersion oil is the same as that of glass
and forms a homogeneous medium for the light rays to pass from
specimen to the lens. Due to higher NA, more light is able to pass
through the specimen and clear observation is possible.
4) 40 to 100 X.

29