Mounting And

Staining PRACTICAL 2 MOUNTING AND

Techniques
STAINING TECHNIQUES

Structure

2.0 Objectives
2.1 Introduction
2.2 Dry Mounts
2.3 Mounting of Living Microorganisms
2.3.1 Preparation of Temporary Wet Mount (TWM)

2.3.2 Hanging Drop Preparation

2.4 General Aspects of Staining Microorganisms

2.4.1 Smears

2.4.2 Simple Stains

2.4.3 Gram’s Staining

2.4.4 Acid Fast Staining

2.5 Practical Aspects of Staining Microorganisms

2.5.1 Preparation of Smears

2.5.2 Simple Staining

2.5.3 Gram’s Staining

2.5.4 Acid Fast Staining

2.6 Let Us Sum Up

2.7 Answers to Check Your Progress

2.0 OBJECTIVES
After completing this unit, you should be able to:

• prepare and observe dry and wet mount slides and hanging-drops slides;
• distinguish different types of microbes in unstained preparation;
• distinguish true motility from Brownian movement;
• make and heat fix a smear;
• explain the basic mechanism of staining;
• perform a simple direct stain;
• list the advantages of showing microorganisms; and
• explain the rationale and procedure for the Gram Staining and Acid Fast
Staining.

30
2.1 INTRODUCTION Mounting And
Staining
Techniques
In Exercise-1 you were familiarised with the use and care of the
compound, monocular and binocular light microscope which is used by
pathologists in the identification of various human and animal pathogenic
microorganisms. You also learnt how to use low and high dry power and oil
immersion objective lens system correctly in exercise 1. In the present
exercise, you will learn the step by step procedure of examining living
microorganisms commonly present in the mouth saliva, urine, stool,
contaminated water etc. You are aware that some of these micro organisms
are responsible for producing diseases. In order to identify specific disease
causing microorganisms you will learn the various methods used for staining
smears such as blood smears, saliva smears etc and micro organisms,
obtained from various sources. Therefore, in this exercise, you will learn
some of the basic methods of staining like simple staining, gram staining and
acid fast staining.

2.2 DRY MOUNTS

Dry mounts are rarely used for microscopic studies of living objects; however
they can be useful for microscopic examination of non-living objects/
materials.

Mounting a hair can be done by obtaining a hair from an individual, placing it

on a glass slide and visualizing it first under low power and t h e n under
high power magnification.

Similarly, you can examine a wool or cotton thread under low power and
then under high power to see its texture.

Exercise 2.1: To prepare and examine a dry mount of hair it under a

light compound microscope.

Aim To prepare a dry mount of hair and examine its structure under the low
power of a light compound microscope.

Material required
Compound, Monocular or binocular light microscope
One strand of your own hair
Glass slide

Procedure

i) Take a clean glass slide and cut and put a piece of your hair on it
ii) Place the slide containing the small piece of hair on the stage of a
compound light microscope and
iii) Observe it under low power (4x or 10x objective) with eye piece/ocular
after adjusting the focus. 31
Mounting And iv) Record your observations. Draw a diagram of the various parts of the
Staining
Techniques hair that you observed under the low power of the microscope.

2.3 MOUNTING OF LIVING MICROORGANISMS

In this exercise you will learn the techniques to prepare wet mounts of
organisms in or from different fluid environments. You will also learn to
examine the prepared wet mounts for assessing the numbers and varieties of
microbes found in nature in the particular liquid environment (blood, sputum
etc.) The microbes studied in wet mounts exhibit either Brownian movement
or true motility or motion. Brownian movement is not true motility but it is
rather the random movement of microscopic particles suspended in a liquid
or gas medium due to the impact of the molecules of the surrounding
medium. In Brownian movement the particles of the medium and the
microorganisms all vibrate at about the same rate and maintain their relative
positions. Actual motility or movement enables microorganisms to move
from one place to another. The motile movement is more directed a n d i s
n o t r a n d o m like the Brownian movement.

Many types of microbes such as protists (protozoa), algae, and bacteria can
be found in pond water and in human organic, fluid matter like urine, stool
and broth culture. Direct examination by the hanging-drop method is very
useful in determining the size, shape, and movement of many types of
protists, algae, and bacteria.

2.3.1 Preparation of Temporary Wet Mount (TWM)

A temporary preparation of a particular microorganism is usually observed as
a wet mount and is prepared by placing a drop of liquid containing a
suspension of a particular microorganism to be studied on a glass slide or if
the material to be studied is dry, by placing it directly on the slide and adding
water or glycerin. The microorganism to be studied may or may not be
stained, depending on its nature and what is required to be studied. The liquid
containing the stained or unstained microorganisms is then covered by a cover
slip. You have to be careful not to trap air bubbles while lowering the cover
slip over the material as it will cause interference in observing the material.
The procedure is shown in Fig. 2.1a & b.

Exercise 2.2: To prepare a temporary, wet mount of a suspension of

nonpathogenic living microorganisms and examine it under a light
compound microscope.

Aim

To Study living nonpathogenic microorganisms paramecium/ bacteria etc

under a light compound microscope in order to study their motility, structure
and shape.

32
Requirement Mounting And
Staining
Techniques
• A hay infusion culture of the protist Paramecium
• A flask with pond water containing nonpathogenic microbes, which is
provided by your counselor.
• Needles
• Blotting paper
• Compound, light microscope
• Microscope glass slides.
• Cover slips.
• Dropping Pipettes.
• Sterile Pasteur pipette
• Sterile water

Procedure

i) Place a drop of sterile water on a clean, glass slide by using a dropper.

ii) With the help of a sterile dropper, gently mix the paramecium culture or
pond water consisting of microorganisms in a glass flask.
iii) Remove a drop (10µL) of the paramecium culture/ or pond water
containing the microbes with the help of a sterile Pasteur pipette, and
transfer it onto the drop of sterile water on the glass slide
iv) Cover the water drop containing the paramecium culture or pond water
placed on the glass slide carefully with a covership in such a way so that
no air bubble gets trapped in the water drop containing the culture/pond
water.

Step 2 Step 3
Step 1

Step 4

Fig 2.1: Procedure of wet mount preparation:

Step 1: Place a drop of sterile water on slide

Step 2: Place the specimen/specimens on water drop by using a loop
33
Mounting And Step 3: Put the coverslip over the glass slide on the water drop containing the
Staining specimen/specimens including the mount
Techniques
Step 4: Observe the slide under microscope
v) In order to put the cover slip Fig. 2.2 take a clean glass coverslip and
place one edge of it at an angle, place against the glass slide, making
contact with outer edge of the suspension liquid or water drop near one
edge of the and then gently lower the coverslip with the help of the
needle so that it covers the drop of suspension and prevents the
formation of air bubbles.

With the help of a tissue paper/filter paper, wipe off the excess infusion from
the edges of the

Fig 2.2: The techniques for lowering the cover slip over the glass side

vi) Place the glass slide covered with the cover slip, on the microscope stage
and focus on it with low power 10x eyepiece and 4X objective, under
low light.
vii) Scan and observe the entire slide under low power and record your
observations with help of Fig 2.3. Adjust the iris diaphragm so that only
a small amount of light is admitted. Concentrate your observations on the
larger, more rapidly moving organisms. At this magnification, bacteria
are barely discernible as tiny dots.
viii) After this examine the slide with dry lens objective under high
magnification(10x eyepiece and 40X objective)
ix) In order to observe and scan the wet mount with dry lens objective under
high magnification with 10 x eyepiece and 40 x objective, increase the
light and focus carefully. You can see finer details in the microorganisms
present in the wet mount as they are highly magnified(Refer to Fig
2.2.again)
x) After recording your observations, examine the slide under oil
immersion. Some microorganisms are motile while others exhibit
Brownian movement.
xi) If you want to observe the motile organisms further, place a drop of
alcohol or Gram’s iodine at the edge of the cover slip and allow it to run
under and mix with the infusion. The organisms can now be observed
34 more carefully.
xii) Record your observations, noting the relative size and shape of the Mounting And
Staining
organisms. Techniques
xiii) You can prepare wet mounts from other suspensions/infusions that
contain bacteria, protists and also eggs of parasitic helminthes and
observe them under the microscope using the low and high dry
objectives.
xiv) Record your observations, noting the relative motility, size and shape of
the organisms
xv) Clean all the slides and return them to the slide box as given in exercise
1. Discard the used cover slips in the disinfectant jar.

Precautions

• Avoid using an excess amount of water.

• Handle the coverslip gently. Coverslips crack quickly so manage them
carefully.
• Practice proper staining procedure.
• Don’t press the cover slip over the mount too much.

Fig 2.3: Some microorganisms that can be seen moving in the pond water
35
Mounting And Exercise 2.3: To study a temporary, wet mount/smear of cheek/buccal
Staining
Techniques epithelial cells stained with methylene blue

Aim

To prepare a temporary, wet mount/smear of cheek/buccal epithelial

cells (Fig 2.4), and stain it with methylene blue stain in order to observe
the structure of the buccal epithelial cells.

Materials Required

1) Glass cover slips, glass slides,

2) Slide labels
3) Sterilized disposable spatula or tooth pick
4) 0. 9% NaCl also called physiological saline or normal mammalian saline
5) Methylene blue stain
6) Filter paper
7) Dropper

Procedure

i) Rinse your mouth well with water.

ii) Gently scrape the inside of your cheek with the broad end of a clean
tooth pick or a sterilised disposable spatula.
iii) Spread the cells of buccal cavity on a clean slide to form a wet smear and
then add a drop of 0.9% NaCl. Now place a drop of methylene blue with
the help of a dropper over the wet smear.
iv) Cover the methylene stained smear with a cover slip and gently press it
to flatten the cells. Alternately you can introduce the stain by irrigation
method (see Fig. 2.4)
v) Put the slide to be viewed on the stage. View the slide under low power
with the light, compound microscope. Make sure that your counsellor
sees the slide.
vi) Scan and observe the entire slide under low power (10x eyepiece and
10x objective).
vii) Adjust the iris diaphragm so that only a small amount of light is
admitted. At this magnification, buccal cells are barely seen.
viii) After this examine the slide with dry lens under dry, high magnification
(10x eyepiece and 40x objective= 400x magnification). In order to
observe and scan the wet mount under 400x increase the light and focus
carefully. You can see greater details in the buccal epithelial cells at
higher magnification.
ix) ) Now remove the objective lens without raising or lowering it. Put a small
drop of immersion oil on the coverslip of the glass slide to be viewed
36
 under the microscope and carefully move the immersion oil objective Mounting And
Staining
into place. Focus on the stained buccal smear with the help of the fine Techniques
adjustment knobs of the microscope and observe it under high, oil
immersion magnification (eye piece 10x and objective 100x) of 1000x
after adjusting the iris diaphragm so that the light is neither too low nor
too bright in order to maximize the visibility of the cells After the smear
the slide is in proper focus on the buccal epithelial cells are clearly
visible. Record your observations.

Fig 2.4: Technique of irrigation of liquid suspension/water drop which is covered by a

cover slip

x) Observations:
a) Locate a single cell under dry, high power. Many of the cells will be
crumpled and the cell membrane will appear irregular in outline
because the cell membrane is extremely thin and delicate.
b) The nucleus will be stained dark blue in the centre of each cell.(2.5).
c) If you don't let in too much light through the microscope you will be
able to observe the cell structures better. Compare what you see with
Fig. 2.5. In epithelial cells obtained from females a distinct, darkly
staining body attached to the nuclear membrane can be seen. This is
known as the Barr body.

37
Mounting And
Staining
Techniques

(a)

(b)

Fig. 2.5: An epithelial cheek cell from a wet mount as viewed by a light compound
microscope under oil magnification power (1000 x) after staining with methylene blue
stain: a) a drawing of the cheek cell and b) a photo of the cheek cell

2.3.2 Hanging Drop Procedure

In this method, a drop of culture is placed on a coverslip that is encircled with
petroleum jelly (or any other sticky material). The coverslip and drop are then
inverted over the well of a depression slide. The drop hangs from the
coverslip, and the petroleum jelly forms a seal that prevents evaporation. This
preparation gives good views of microbial motility.

Exercise 2.4: To study the motility of bacteria by Hanging Drop Method

Aim

To prepare a hanging drop preparation (Fig 2.6), which is a special type of

wet mount (in which a drop of medium containing the bacteria is placed on a
microscope slide), in order to observe the motility of bacteria

Materials Required

i) Cavity microscope glass slides (glass slide with depression) or normal

glass slide with an adhesive or paraffin ring
ii) Paraffin wax
iii) Innoculating culture loop
iv) Coverslip
v) Compound light Microscope (monocular or binocular)
38 vi) Bunsen burner
vii) Young Culture not older than 24hrs froth culture Broth of Mounting And
Staining
Bacillus/Rhizopus sp./Penicillium,/Aspergillus,/pirogyra/Chlamydomonas, Techniques
Volvox, etc.

Procedure

1) Take a clean cavity glass slide and make a ring of paraffin/vaseline/

petroleum jelly around the cavity on the glass slide. If a cavity glass slide
is not available you can take a plain slide and make a small “well” with
the use of plasticine (Fig. 2.6).
2) Hold a clean coverslip by its edges and use a tooth pick to carefully dab
vaseline on the corners of the coverslip.
3) Place the prepared cover slip on a paper towel, with the petroleum jelly
side up.
4) Sterilise an inoculation culture loop over a bunsen burner flame and let it
cool. A loopful of bacteria suspension is taken from the 24-hour old
broth culture aseptically.
5) With the help of the sterile inoculation culture loop, place the loopful of
the fresh broth culture (not older than24-hour old broth culture) to be
observed in the center of the prepared coverslip. Use a light inoculum
(inoculation liquid) so that it is not visibly turbid. Invert the cavity slide
upside down (concavity down) over the drop on the coverslip so that the
vaseline seals the coverslip to the slide around the concavity.
6) Turn the slide over so the coverslip is on top. The slide and cover slip are
pressed together gently, so that the cavity is sealed. Care should be taken
to see that no part of the cavity touches the drop.
7) Allow organisms to “settle” for a minute. The drop can be observed
hanging from the coverslip over the cavity glass slide (Fig2.7).
8) Place the slide on the stage of the microscope and clip it in position (Fig. 2.6).

1. Preparation of the cover slip by

dabbing petroleum jelly on the
corners

2. Place loopful of microbial

suspension into the center of cover
slip

3. Turn the concave depression glass

slide upside down over the cover slip

4. Invert the concave depression glass

slide to produce “ handing drop” of
the specimen

Fig 2.6: Procedure of hanging drop method 39

Mounting And 9) Examine the hanging drop by the ocular/eye piece under low power (10x
Staining
Techniques eye piece and 4x objective) magnification of 40x by locating the edge of
the drop and moving the slide so the edge of the cover slip crosses the
center of the field.
10) Reduce the light of the iris diaphragm and focus on the drop. Observe the
different sizes, shapes, and types of movement of the bacteria.
11) Without raising or lowering the objective tube, switch to dry, high
magnification of 400(10x eye piece and 4x objective) and focus on the
hanging drop with the help of fine adjustment knobs of the microscope.
Since stains are not used in this practical so in order to observe the
bacteria in the hanging drop it is essential to adjust the light of the iris
diaphragm so that the light is neither too low nor too bright in order to
maximize the visibility of the cells. After the slide is in proper focus and
the bacteria are visible then record your observations (Fig 2.7).
12) The cells under dry, high magnification objectives will look either like
dark or slightly greenish, very small rods or spheres. Remember the high
dry objective magnifies a little less than the oil immersion objective.
13) Observe the cells noting their morphology and grouping and determine
whether true motility can be observed.
14) You will observe Brownian movement (vibrational movement) in the
hanging drop at all magnification. However you need to ignore it and
should focus on specific directional movement. If you see only Brownian
movement that means the bacteria in the hanging drop are non motile.
15) Note the following observations of the bacteria (Under 400x
magnification) and write them in your exercise note book:
1) Motility: Motile or non-motile
2) Shape of bacteria -
spherical (cocci) bacteria
rod-shaped (bacilli) bacteria
Comma-like (vibrio)
Spiral (spirochetes)
3) Arrangement of bacteria:
Pairs (diplobacillus/diplococcus)
In fours (tetrads)
In chains (streptococcus/streptobacillus)
Grape-like clusters (staphylococcus)
Cuboidal (sarcinae or octet).
4) Size of bacteria:
By eye observation and estimation, make drawing of the field under
40 oil-immersion objective.
16) When finished, clean your slide, and, discard the used cover slip. Mounting And
Staining
17) Wipe clean the objective lens with lens paper and return your microscope Techniques
to its proper location. Clean your slides well and return them.

Fig 2.7: A hanging drop can be observed, in which the arrows indicate the various
microorganisms present. In a hanging drop we can observe live, unstained, and often
motile bacterial cells under 400x magnification. Some bacterial cells display individual
movement amongst cells. This means their movement is unique and directional. The
hanging drop method is used to distinguish the unique and directional movement of
bacteria from brownian movement which is just a random jiggling motion.

Precautions

1) The broth culture from which the hanging drop preparation is to be made
should not be more than 24 hours old, because bacteria may lose their
motility, as they grow older.
2) Place only a very small quantity of culture on the coverslip so that the
bacteria can be observed clearly.

Check Your Progress 1

1) From your theory course recall the names of two motile and two non-
motile bacteria.
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
41
Mounting And 2) Motile bacteria possess as organ of locomotion.
Staining
Techniques 3) What biological specimens can be observed in an unstained saline
mount? What bacterial features do you think can be observed in an
unstained mount being examined?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
4) How do you recognize true motility in the bacterial present a hanging
drop examination?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
5) Can you list the organs of locomotion in protozoa?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

2.4 GENERAL ASPECTS OF PREPAING A

BACTERIAL SMEAR AND STAINING IT
FOR IDENTIFICATION OF
MICROORGANISMS
Most cells or microorganisms or organisms or tissues, when observed under
the compound, light microscope in their natural state, lack both color and
contrast. This makes it difficult, if not impossible, to detect important cellular
structures and their distinguishing characteristics. In order to overcome these
limitations we use stains and dyes so that we are able to see the structures
more clearly and are able to differentiate the cellular structures easily. Thus,
staining is the technique that is usually employed to enhance contrast in
samples of organisms, generally at the microscopic level. In this subsection
we will discuss the use of specific techniques of air drying, chemical fixation
and staining etc. for sample preparation of cells or microorganisms or
organisms or tissues for staining. We shall then also proceed to learn the
various clinically important and relevant staining techniques used that
enhances the contrast between cells/ bacteria/other organisms/tissues and the
surrounding material and permits observation of greater detail and resolution
42 than the wet mount procedures.
One method used for the study and identification of cells/microorganisms/ Mounting And
Staining
tissues is preparing of a smear of the cells/ microorganisms/tissues by Techniques
preparing a smear on to a g l a s s microscope slide for staining.

2.4.1 Preparation of bacterial or micro organism Smears

In the present subsection, we will explain in brief the preparation of a
bacterial smear so that you can prepare the bacterial smear in the practical
exercise 2.5 subsection 2.5.1. In order to prepare a bacterial or microbial
smear, a drop of growth or culture media containing the bacteria is
transferred to a glass microscope slides with the help of an inoculating loop.
An inoculating loop is a nichrome (alloy of nickel and chromium) wire held
with an insulated handle. Before using an, the inoculating loop it is sterilized
by heating or flaming over a Bunsen burner (Fig 2.8). When the loop is cool,
a loopful of bacterial culture is transferred by it on a clean glass slide. After
this, the loop is again sterilised by flaming before i t i s s e t down in its
designated area on the work bench.

A smear of the bacteria suspension transferred, by the inoculating loop is

prepared on the glass slide by spreading it and allowing it to air-dry. The air
dried bacterial smear is then heat fixed by passing it through a Bunsen burner
flame several times. Heat fixing, kills the bacteria thus, stopping their
movement and metabolism while preserving the integrity of their cellular
components for observation. In addition to this, heat fixing also enhances the
attaching of the bacterial cells to the microscope slide.

Unstained bacterial cells are nearly transparent when observed by light

microscopy and hence are difficult to see. However, most bacteria can
easily be stained. Staining involves applying a staining reagent on a
bacterial/microbial smear on a glass slide.

2.4.2 Simple Stains

Let us now learn some common terminologies used with reference to
staining. Most stains or dyes used for staining biological material, especially
in microbiology are synthetic chemicals derived from nitrobenzene or aniline
(coaltar derivative). Stains are used commonly in microbiology to increase
the contrast between microorganisms or parts of its and the background, so
that they can be easily visible. The process of giving colour to particular
organism or components of its is known as staining.

Each stain or dye is composed of three components.

i) Benzene ring: the colourless part of a dye and is the basic structural
component of a dye.
ii) chromophore: is the functional group of a dye that give colour to the
stain. The benzene ring and chromophore is collectively known as
chromogen.
iii) Auxochrome: The group that gives ionic property to the stain 43
Mounting And Stains, or dyes, contain salts made up of a positive ion or a negative ion.
Staining
Techniques Depending on the type of dye, the positive or the negative ion may be the
chromophore (the colored ion); the other, uncolored ion is called the
counterion. If the chromophore is the positively charged ion, the stain is
classified as a basic dye. However if the negative ion is the chromophore, the
stain is considered an acidic dye (Fig 2.9 a & b).

Types of stains

Based on the nature of chromogen, there are three types of stain.

1) Acidic stain (Anionic stain)

2) Basic stain (Cationic stain)
3) Neutral stain

Fig 2.8: Sterilization of inoculation loop over a Bunsen burner

1) Acidic stain (Anionic stain)

• Chromogen of acidic stain is negatively charged. so, it is also known as

anionic stain
• Acidic stains are used to stain the positively charged components such as
background staining.
• Histone protein is positively charged so it can be stained by acidic stain.
• Acidic stain cannot stain the bacterial cell due to repulsion of same
charge.
• Examples: Eosin, Nigrosin, India ink

2) Basic stain (Cationic stain)

• Chromogen or coloured part of basic stain is positively charged and so, it

is also known as cationic stain.
• Basic stain are used to stain negatively charged components such as
bacterial cell.
44
Examples: methylene blue, safranin, malachite green, basic fuschin, crystal Mounting And
Staining
violet Techniques

3) Neutral stain

• In neutral stain, both cation and anion are coloured, so that the net charge
is neutral.
• A neutral stain is actually a salt of acidic and basic stain.
• Example: Giemsa stain.

Dyes as must have realized by now are chosen for staining biological
material based on their property and the chemical properties of the dye and
the being observed, which determine how the dye will interact with the
specimen. In most cases, it is preferable to use a positive stain /basic dye.
Positive Stains as you are aware colour negatively charged molecules and
structures, such as nucleic acids and proteins. Thus basic dyes are absorbed
by the cells or organisms being observed, and so as a result colour structures
or organelles of interest which thus stand out against the background. In some
instances however, it is advantageous to use a negative stain, which leaves
the bacteria or organisms under study unstained but stains the background as
it absorbs the stain. Thus negative stains colour the background and not the
specimen. Negative staining produces an outline or silhouette of the
organisms against a colorful background. (Fig. 2.9)

(a) (b)

Fig 2.9: Simple staining: a) Basic staining with Methylene blue b)Negative staining
with India ink, nigrosine

Most bacteria are stained when a basic stain permeates the cell wall and
adheres by weak ionic bonds to the negative charges of the bacterial wall.
Staining procedures that use only one stain are called simple stains. Simple
stains can be used to, and size and arrangement of the bacterial cells. Simple
stains, contains only one reagent simple or single staining is used to
determine cell morphology which also includes observe cell shape (cocci,
bacilli, vibrio, spirilli). It is also used to the determine the arrangement
(single, pair, tetrad, chain, cluster) of bacteria.

While differential stains consist of more than one reagent and are used to 45
Mounting And stain bacteria, in such a way so that different parts of the bacteria react
Staining
Techniques differently to the different reagents present in the differential stain. Gram
stain and acid fast stain are differential stains.

2.4.3 Gram’s Staining

The Gram staining technique gets its name from the Danish bacteriologist
Hans Christian Gram who first introduced this staining method, in 1884. The
Gram Stain is a differential stain and is very useful for identifying and
classifying bacteria. The gram stain helps us to classify bacteria as either
gram-positive or gram-negative (Fig 2.10). The gram staining technique is
as follows:

The first step in gram staining of bacteria is the use of the primary stain,
namely crystal violet (CV) dye which stains all bacteria purple. The next
step, also known as fixing the dye, involves the use of a mordant(a reagent,
such as tannic acid, in the form of iodine that acts to fix dyes to cells, tissues,
or textiles or other materials).The mordant causes the formation of crystal
violet- iodine complex in the bacterial cell that prevents the easy removal of
the dye. Subsequently, a decolourising agent also called a decolorizer, which
is often a solvent such as of ethanol (ethyl-alcohol) or acetone (ethyl-alcohol
acetone) is used to remove the primary dye. The primary stain is washed out
(decolorized) by the decolourising agent in some bacteria while in others it is
retained. The basic principle of gram staining involves the ability of the
bacterial cell wall to either retain or lose the primary crystal violet dye during
solvent decolourising treatment.
The bacteria which remain unaffected by the decolourising agent and retain
the crystal violet dye are called as Gram-positive bacteria. Gram-positive
bacteria are able to retain the crystal violet dye despite treatment with a
decolouriser (solvent) because their cell walls have a thick layer of protein-
sugar complexes called peptidoglycan and also have low lipid content. The
action of the decolorizing agent on the bacterial the cell causes the thick cell
wall of the bacteria to dehydrate and shrink, which closes the pores in the cell
wall and prevents the stain from exiting the cell. Thus the solvent (ethanol or
acetone) cannot remove the crystal violet-iodine complex that is bound to the
thick layer of peptidoglycan of gram positive bacteria and causes the thick
layer of peptidoglycan appear blue or purple in colour. The bacteria which
are unable to retain the primary crystal violet dye during solvent treatment
and become decolourised are referred to as gram negative bacteria. This
decolourisation in gram negative bacteria occurs due to the fact that though
the cell wall of the gram negative bacteria, also takes up the crystal violet dye
-iodine complex initially, however because its cell wall is formed of a thin
layer of peptidoglycan and a thicker outer layer of lipids, the CV-iodine
complex gets washed off because the decolourizer dissolves the lipids in the
cell walls, which allows the crystal violet-iodine complex to leach out of the
cells. Then when the Gram negative bacteria are stained with safranin, after
decolourizationthe peptigly can layer of gram negative bacteria takes the
safronin stain and appears red in color.
46
The final step in gram staining is to use a basic fuchsin stain which gives the Mounting And
Staining
decolorized gram-negative bacteria pink color for easier identification. It is Techniques
also known as a counterstain. Some laboratories use safranin as a
counterstain; however, basic fuchsin stains gram-negative organisms more
intensely than safranin. Similarly, Hemophilus spp., Legionella app, and
some anaerobic bacteria stain poorly with safranin.

Fig 2.10: Principle of Gram’s staining

2.4.4 Acid Fast Staining

Acid Fast Staining is the differential staining method which was first
developed by Ziehl and later modified by Neelsen About which You learnt in
this Unit of Block 1 of this course. This method is also called the Ziehl-
Neelsen staining technique.

Acid fast staining method is used for those microorganisms which cannot be
stained by simple or Gram staining method, particularly members of genus
Mycobacterium, which are resistant to simple and gram staining and can only
be visualized by acid-fast staining. The main aim of this staining is to
differentiate bacteria into acid fast group and non-acid fast grouping.

Ziehl-Neelson procedure is the most widely used acid fast staining technique.
In the Ziehl-Neelson procedure, the smear is flooded with the Stain
carbolfuchsin, which has a high affinity for a particular chemical component
of the bacterial cell. Ziehl introduced the use of carbolfuchsin (containing 5%
phenol) to replace aniline dye in staining. The smear is when stained with
Carbolifuchsin stain is heated in order to facilitate the penetration of the stain
into the bacteria. After this all cells appear stained red. The stained bacterial
smear is washed with an acid-alcohol decolorizing mixture (3% HCL in 95%
alcohol) that easily decolorizes most bacteria except the acid-fast microbes.
The non-acid fast organism are decolorized and so are colourless. After this
the smear is stained with the counterstain, methylene blue which enables the
observation of the non-acid fast bacteria. The acid-fast bacterial cells still
retain the red color. 47
Mounting And
Staining
2.5 PRACTICAL ASPECTS OF PREPAKING A
Techniques BACTERIAL SMEAR AND STAINING
You should keep certain points in mind while doing the practicals in
microbiology because you will be handling live organisms and you want to
b e s a f e a n d a l s o get good results in your practicals.

1) Always use clean slides and cover slips.

2) Use sterile saline for preparing suspensions/infusions.
3) Use separate pipette for each specimen.
4) Always heat-sterilize the bacterial inoculating loop before and after use
5) Cool the heat-sterilized loop before taking a bacterial suspension drop
for examination.
6) All unstained preparations must be freshly made and examined
microscopically under dry objective lens (10x, 40x) with reduced light
by adjusting the condenser.
7) Stained smears for bacteria and parasites should be examined under dry
objective at high magnification and then under oil immersion objective
(100x) with adequate light by adjusting the condenser.
8) Insects should be examined with a hand-lens or under the low power
(10x) of the microscope.
9) Some bacteria/parasites are alive and infectious; hence precautions (such
as wearing gloves and masks) should be taken in order to avoid getting
infected.
10) Discard infected material, slides, pipettes in a disinfectant kept in a glass
jar. Wash your hands after the practical.

2.5.1 Preparation of Smears

Exercise 2.5: To prepare a bacterial smear for staining
Aim I
To prepare a simple bacterial smear for staining with either methylene
blue or crystal violet stain or Nigrosin stain

Materials Required
i) Microscope glass slide
ii) Paraffin wax
iii) Inoculating loop
iv) Bunsen burner
v) Young broth culture/bacterial suspension (not older than 24-hr old) or
bacterial culture in a petri dish
Procedure
48
1) You have to keep in mind the following points while preparing smears. Mounting And
Staining
2) Clean well, with water and dry the glass microscope slides which you Techniques
will be using.
3) Handle the clean glass slides by their ends. Label one edge of the glass
slide appropriately with the name of the bacterial culture sample and date
of preparation of smear.
4) Use a marking pencil to make a circle in the centre of each slide of
approximately 20 mm diameter. The circle should be on the bottom or
lower end of the slide.
5) For preparing a bacterial smear from a solid bacterial culture
contained in a petri dish(Fig 2.11) sterilize the inoculating loop in the
flame and cool it. Use the sterilized inoculating loop by taking one
loopful of distilled water with the inoculating loop and placing it in the
center of the circle drawn by you earlier on the slide.
Scrape a small amount of the bacterial culture kept in the petridish and
place it in the drop of distilled water which is on the slide. Spread the
bacterial suspension in the distilled water drop so that it to fills the
marked circle, forming a thin smear . The smear should look like diluted
skim milk. Flame your loop again before you put it down.
6) For preparing a bacterial smear from a liquid bacterial suspension
you are not required to put a drop of distilled water on the glass slide.
Instead: sterilize the inoculating loop in the flame and cool it. Gently
shake the bottle/ flask containing the liquid bacterial suspension, Flame
its neck or top edges to sterilize it. When it has cooled, pick a loopful of
the liquid bacterial suspension with the sterilized, inoculating loop and
place it in the centre of the circle you drew earlier on the glass slide and
make a smear of it with the help of the inoculating loop, without adding
distilled water. Flame the inoculating loop before you put it down.
7) Allow the smear to air dry completely on the slide. Do not blow and do
not flame the slides.
8) When the slides are dry heat fix the smears by passing the slides
through a Bunsen flame 2 or 3 times. (Fig. 2.12 – step 1)

CAUTION: Certain infectious diseases can be transmitted through

saliva. Avoid any contact with another person's saliva. Do not share a
spatula with anyone else.

2.5.2 Simple Staining

Exercise 2.6: To prepared stain the bacterial smear with methylene blue
or nigrosin stain

49
Mounting And Aim
Staining
Techniques
To stain the prepared a bacterial smear with the simple stain of
methylene blue (Fig 2.11) or nigrosin stain (Fig. 2.12) and study it under
the microscope

Materials Required

i) Microscope glass slide with freshly prepared bacterial smear

ii) Coverslip
iii) Clothespin
iv) Wash bottle of distilled water
v) Inoculating loop
vi) Bunsen burner
vii) Methylene blue stain
viii) Nigrosin or India ink stain
ix) Simple acid stain safranin/eosin
x) Polystyrene mountant such as DePeX or Canada Balsam
xi) Immersion oil
xii) Compound light microscope
Procedures
1) Stain one slide at a time ((Fig 2.11 & 2.12). Use a clothespin to hold the
slide in place or place it on a staining rack.
2) With the help of a dropper cover the freshly prepared smear with
methylene blue (Fig 2.11) and leave for 30 to 60 seconds.
3) Carefully wash off the excess stain with distilled water from a wash
bottle by keeping the slide in a tilted position.
4) Gently blot the smear with an absorbent paper and let it air dry.
5) Covering the smear with a coverslip is optional. Usually cover slips are
not used. However, for keeping the simple, stained smear slides for a
longer duration covering the smear with a coverslip is better.
6) In case of covering the simple, stained smear with a coverslip place a
drop of polystyrene mountant such as DePeX or Canada Balsam on the
stained smear and cover it with a cover slip. Once the stained smear is
coverslipped the slides can be stored indefinitely. In case you do not
cover the coverslip then put the oil directly on the smear while viewing it
with oil immersion under the microscope. However if the stained smear
is coverslipped then put the immersion oil on the surface of the coverslip.
7) Examine the stained smears microscopically using the low, high dry and
oil immersion objective lenses
8) Record your observations and draw well drawings in your practical note
book.
50
 Mounting And
Staining
Techniques

Fig 2.11: Steps of Simple Staining of smear with methylene blue

9) In case you do not cover the stained simple smear with a coverslip then
after viewing the smear under immersion oil by means of the
microscope, remove the slide from the microscope. Place the slide in
xylene for a few minutes to remove the immersion oil and wipe it gently
and store it in a slide box.

Fig. 2.12: Steps of Simple Staining with nigrosin 51

Mounting And 10) In case of the coverslipped simple, stained smear, wipe off the
Staining
Techniques immersion oil on the coverslip after viewing under the microscope, with
a tissue paper dipped in xylene and then with alcohol and (refer to
section 1.6 in unit 1) store it in a slide box.
11) Clean the oil from the objective lens. Remove the oil from the slides by
blotting with an absorbent paper and store it refer to section 1.6 in unit 1

12) Return the microscope to its proper place after taking

all the care it needs.
2.5.3 Gram’s Staining
Exercise 2.7: To stain a freshly prepared bacterial smear from a
bacterial culture with simple gram stain

AIM

To stain a bacterial smear by Gram staining and also to interpret the

observation of Gram’s stained bacteria under the compound light
microscope.

If you have a problem in

remembering the staining
reagents used in Gram’s
staining procedure and
their order of use you can
remember this sentence
“Come in and stain” i.e.,
the order is
- Crystal Violet
- Iodine
Alcohol/Acetone and final
one is Safranin.

Fig. 2.13: Steps of gram staining here- from above

Materials required

i) Gram staining reagents: Crystal violet, Gram’s iodine, Ethyl alcohol or

Acetone, safranin.
ii) Wash bottle of distilled water
52
iii) Glass Slides Mounting And
Staining
iv) Innoculating loop Techniques

v) Young cultures of Staphylococcus (Staph), Streptococcus (Strept),

Escherichia coli (Esch. coli) and Bacillus subtilis (Bacillus).
vi) A few permanent prepared stained demonstration glass slides of any of
the following species of bacteria:
Gram Positive spherical (cocci) — Staphylococcus, Streptococcus,
Micrococcus.
Gram negative spherical (cocci)— Neisseria, Veillonella
Gram Positive rod-shaped (bacilli) bacteria — Bacillus sp,
Corynebacterium,
Clostridium
Gram Negative rod-shaped (bacilli) bacteria — Salmonella sp, Shigella
sp, Escherichia
coli, Pseudomonas sp, Bacteroides sp.

Steps of Gram’s staining (Fig2.8)

1) Prepare a bacterial smear one at a time as described in subsection 2.5.1,

on the glass slides, from each of the bacterial culture provided
(Staphylococcus, Streptococcus, Escherichia coli and Bacillus subtilis)
2) Fix the prepared smear by gently heating over a flam the lower end of
the glass slide containing the smear.
3) Stain the smear with crystal violet stain solution for 1 minute—wash the
slide carefully with distilled water.
4) Apply Gram’s iodine for 1 minute to the smear stained with crystal violet
stain solution.
5) Decolourise for one second, the smear that has been stained with crystal
violet stain solution and treated and fixed with mordant Gram’s iodine
with acetone or ethyl alcohol.
6) Wash the decolourised smear with tap water. You should not over-
decolourise.
7) Counterstain the decolourised smear with safranin for half a minute,
wash and dry it with absorbent paper.
8) Covering the smear with a coverslip is optional. Usually cover slips are
not used. However, for keeping the simple stained smear slides for a
longer duration covering the smear with a coverslip is better.
9) In case of covering the Gram’s stained smear with a coverslip place a
drop of polystyrene mountant such as DePeX or Canada Balsam on the
stained smear and cover it with a cover slip. Once coverslipped the slides
can be stored indefinitely.
53
Mounting And 10) In case you do not cover the coverslip then put the oil directly on the
Staining
Techniques smear which viewing it under the high magnification of a compound
light microscope. However if the stained smear is coverslipped then put
the immersion oil on the surface of the coverslip while viewed it under a
light, compound magnification under high power magnification.
11) Examine the stained smear microscopically using the low, high dry and
oil immersion objective lenses (Fig 2.14 a & b).
12) Examine the stained slide under low, high-dry and oil immersion lens
(100x) and record your findings.
13) Repeat the staining procedures with other smears of various cultures.
14) Observe the demonstration slides.

Interpretation

The smears stained using the Gram Stain protocol, when viewed with a
compound, light microscope under high magnification by using oil
immersion reveal the shape, size and gram reaction of the bacterial cells
present in the stained smear. The arrangement of the bacterial cell can also be
viewed under the microscope. As the photo in figure 2.14 a ,shows the
stained smear is of Gram positive bacteria which are coloured blue/purple
and are rod shaped, while the photo in fig 2.14 b shows the stained smear is
of Gram negative bacteria which are coloured pink/red and are rod shaped.

a b

Fig 2.14 Gram’s stained bacterial slides viewed with immersion oil under a compound
light microscope: a) Gram positive bacteria and b)Gram negative bacteria(Source:
https://www.atsu.edu/faculty/chamberlain/mosdoh/GramStainingRules.htm

Procedure
(1) Record the following features of the gram positive and gram negative
bacteria present in the permanent, prepared stained glass slides provided
by your counselor.
Exercise 2.7
To study, prepared, stained, permanent demonstration glass slides of a few
selected bacterial species.
Aim; To study and identify the various gram negative and gram positive,
54
selected bacterial species on basis of stain clour, shape size and arrangement. Mounting And
Staining
15) Record the following features under the oil- immersion objective: Techniques

1) Stain of bacteria:
• Gram Positive spherical (cocci) — Staphylococcus, Streptococcus,
Micrococcus.
• Gram negative spherical (cocci) — Neisseria, Veillonella
• Gram Positive rod-shaped (bacilli) bacteria — Bacillus sp,
Corynebacterium,
• Clostridium
• Gram Negative rod-shaped (bacilli) bacteria — Salmonella sp,
Shigella sp,
• Escherichia coli, Pseudomonas sp, Bacteroides sp
2) Shape of the bacteria -
• spherical (cocci) bacteria
• rod-shaped (bacilli) bacteria
• Comma-like (vibrio)
• Spiral (spirochetes)
3) Arrangement of bacteria:
• Pairs (diplobacillus/diplococcus)
• In fours (tetrads)
• In chains (streptococcus/streptobacillus)
• Grape-like clusters (staphylococcus)
• Cuboidal (sarcinae or octet).
4) Size of bacteria:
Make a drawing of the field containing bacteria which is viewed
under oil- immersion objective.
16) Record your observations.
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55
Mounting And Exercise 2.8: To stain a freshly prepared bacterial smear from
Staining
Techniques a bacterial culture with simple gram stain.

2.5.4 Acid Fast Staining

Exercise 2.9: To stain a freshly prepared bacterial smear with

acid fast stain
AIM

To stain a bacterial smear with acid fast stain since it does not stain with
Gram’s stain and also to make a note of observation of the acid fast
stained bacteria under the compound light microscope.

Materials required

i) Acid-fast staining reagents—Ziehl’s carbolfuchsin, Acid-alcohol(3%

HCL in 95% alcohol), Methylene Blue
ii) Absorbent paper
iii) Boiling water bath
iv) Wash bottle with distilled water
v) Clean glass Slides and coverslips
vi) Cultures of Mycobacterium phlei and Escherichia coli
vii) Demonstration slides : Acid-fast sputum slides.
viii) Compound light microscope
ix) Turgitol in case a water bath is not available.

Procedure of Acid fast staining

1) Prepare and heat fix a smear of one culture at one time as per procedure
given in subsection 2.4.1.
2) Cover the smear lightly with a small piece of absorbent paper.
3) Add a copious amount of Ziehl’s carbol fuchsin stain (Fig 2.15).
4) Heat the slide by setting it on a boiling water bath. Steam for 5 minutes,
adding more stain if needed. Discard the absorbent paper
5) In case you do not have a water bath then put both carbol fuchsine stain
and turgitol on the smear present on the slide for 3 to 5 minutes
6) Wash the stained, smear well, with distilled water; then decolorize it for
10 to 15 seconds with acid-alcohol. Wash with distilled water.
7) Counter stain for about 30 seconds with methylene blue.
8) Wash with distilled water and blot dry.
9) Prepare acid-fast stained smear of the other cultures by the same
procedure.
56 10) Observe microscopically the acid-fast stained slides under 1000X
 magnification i.e. under immersion oil and record your observations. Mounting And
Staining
11) Observe the bacterial smear prepared and stained by you and compare Techniques
them with the demonstration slides provided by your counsellor.

Fig. 2.15: Steps of acid-fast staining

Interpretation of acid fast staining (Fig 2.16 a & b)

Acid fast stained bacteria under immersion oil with the light compound
microscope will appear bright red to intensive purple while non-acid fast
bacteria will appear blue color. In addition, the background of material of
non-acid fast bacteria should stain blue.

a b

Fig 2.16 Acid-fast staining photographs at 1000x (oil immersion) magnification (eye
piece 10X and objective 100X): a)of positive stained acid fast bacteria Mycobacteria
and b) Negative stained acid fast bacteria Escherichia coli .
57
Mounting And Check Your Progress 2
Staining
Techniques
1) Name 2 important Gram positive and 2 negative bacilli and cocci
bacteria .
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2) How are staphylococci different from streptococci?
.....................................................................................................................
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3) Give the name of one acid-fast stained bacteria and one negative stained
acid fast bacteria
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4) How is Corynebacterium diphtheriae (C. diphtheriae) recognized in a
smear?
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5) Draw labelled diagrams of ova/cysts of pathogenic parasites after
observing a prepared, permanent demonstration slide of a human stool
provided by your counsellor.
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58
2.6 LET US SUM UP Mounting And
Staining
Techniques
Observing live microbes and staining them is exciting but at the same time
dangerous as a large number of them are pathogenic and should be handled
with extreme care. You should always wear protective clothings like gloves,
protective glasses and a lab coat when handling them.

You have seen various shapes and sizes of microbes in your practical work.
Many kinds of microbes can be found in pond water and in infusions of
organic matter. Direct examination by the hanging-drop method is very
useful in determining size, shape and movement of microorganisms. The
procedures of air drying, chemical fixation and staining allows you to be
more accurate in your observations and preservation of slides. Staining
bacteria enhances the contrast between bacteria and the surrounding material
and permits observation of greater detail. Microorganisms are prepared for
staining by smearing them onto a microscope slide. Staining techniques
involve either a simple stain or a differential stain.

Self Activity

List all the stains that are used in your hospital for the study of microbes.
Classify them according to their use/utility.

2.7 ANSWERS TO CHECK YOUR PROGRESS

Check Your Progress 1

1) 2 Motile bacteria — Escherichia coli, Salmonella typhi

2 Nonmotile bacteria — Shigella dysenteriae, Klebsiella pneumonia
2) Flagellae
3) In order to observe bacteria, ova and cysts of parasites, pus cells, RBC
etc. Also to observe the following features in bacteria:
1) Motility: Motile or non-motile
2) Shape of bacteria:
Spherical (coccus)
Rod-shaped (bacilli)
Comma-like (vibrio)
Spiral (spirochetes)
3) Arrangement of bacteria:
Pairs (diplobacillus/diplococcus)
In fours (tetrads)
In chains (streptococcus/streptobacillus)
Grape-like clusters (staphylococcus)
Cuboidal (sarcinae or octet). 59
Mounting And Size of bacteria
Staining
Techniques 4) 1) Truly motile organisms move actively in a directed direction.
5) Pseudopodia, flagella, cilia.

Check Your Progress 2

Give the name of any 2 of the following types of bacteria

1) i) Gram Positive Bacilli — Bacillus sp, Corynebacterium,

Clostridium
ii) Gram Negative Bacilli — Salmonella, Shigella, Esch. coli,
Pseudomonas, Bacteroides.
iii) Gram Positive cocci — Staphylococcus, Streptococcus,
Micrococcus.
iv) Gram negative cocci — Neisseria, Veillonella
2) Name of Bacteria Staphylococcus Streptococcus
i) Morphology in bunches in chains
ii) Catalase positive negative
3) Positive stained acid fast bacteria- Mycobacteria and Negative stained
acid fast bacteria Escherichia coli
4) Corynebacterium diphtheriae (C. diphtheriae) is recognized because it is
a Gram positive bacilli which occurs in pairs in the form of a Chinese
letter arrangement Metachromatic granules can be seen in its smear
when stained with Albert’s stain.
5) You may observe some of the following structures in the slide
I) Cysts: i) Giardia sp, ii) E. coli, iii) E. histolytica
II) Ova: ii) hook worm, ii) round worm, iii) tape worm.

60