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Dept. of Pharmacy
Chapter 2

Prof A. Akhtar Ahmed

Department of Microbiology
 Introduction
 Ranges of vision
 Type of Microscope
 Preparations of microscopic examination
 Fixed and stained smear
 Microbiological stains
 Simple and differential staining methods
 Wet mount and hanging drop technique
 Microscope
A microscope is an optical instrument
used to see very small objects that are
too small for the naked eye such as
mineral samples or bacteria, fungi,
protozoa, animal or plant cells, typically
magnified several hundred times.

Microscopy is the technical field of using

microscopes to view objects and areas of
objects that cannot be seen with the naked
eye.
 Types of
microscopes:
1. Light -simple or
compound
 Bright field
 Dark field
 Phase contrast
 Fluorescent
2. Electron
 Scanning
 Transmission
 other
 A simple
microscope has Figure 1.2b
 1. Compound Light Microscopy
 Have several lenses:
1. Light originates from an illuminator and passes
through condenser lenses, which direct light
onto the specimen.
2. Light then enters the, which magnify the image.
These are the closest lenses to the specimen:
 Scanning objective lens: 4 X
 Low power objective lens: 10 X
 High power objective lens: 40-45 X
 Oil immersion lens: 95-100 X
3. The image of the specimen is magnified once
again by the ocular lens or eyepiece (10 X).
LIGHT MICROSCOPE
Carrying a Microscope

B
A
1. Compound Light Microscope
 Total magnification: Obtained by multiplying objective lens power by
ocular lens power. (Condenser lenses do not magnify image).

Lens Magnification Ocular Mag. Total

Mag.
Scanning 4X 10 X = 40 X
Low power 10 X 10 X = 100 X
High power 45 X 10 X = 450 X
Oil immersion 100X 10 X = 1000
X
Highest possible magnification with CL microscope is about
2000 X.
 2.
Dark field Microscope
Use:

o examine live or unstained specimens.

o study light sensitive organisms

o study motility of Treponema pallidum and Vibrio cholerae

 Dark field condenser with opaque disc blocks light that would enter
objective lens directly:
 Light reflects off specimen at an angle.
 Only light reflected by specimen enters objective lens.
 No direct background light.
 Image: Light specimen against dark background.
 3
. Phase Contrast Microscope
1. Use
To examine live specimens
2. Advantage
Doesn’t require fixing or staining
 Permits detailed examination of internal
structures.
3. Operation
 Special objective lenses and condenser with ring
shaped diaphragm accentuate small differences in
refractive indexes of internal structures.
 Image: Direct rays and reflected light rays come
together, forming an image with many shades of gray
to black.
4. Fluorescence Microscopy
Fluorescence: Ability of substances to absorb short
wavelengths of light (ultraviolet light) and emit them at
a longer wavelength.
Type
 Natural Fluorescence: Some microorganisms fluoresce naturally
under UV light (Pseudomonas).
 Fluorochrome: Fluorescent dye.
 Acridine orange: Binds to nucleic acids, colors cells orange, green, or

yellow depending on light source.

 Immunofluorescence: Fluorescent antibodies can be used to detect
specific antigens. Very useful for the rapid diagnosis of specific diseases
(e.g.: syphilis).

Image: Luminescent bright object against a dark background.

Limitations of light
microscopy:
 Magnification: Up to 2000 X.
 Resolving Power: Up to 0.2 um.

Because of the limits of

magnification and resolving power,
viruses and most internal
structures of cells cannot be
seen with a light microscope.
5. Electron Microscope
 Electron microscopes were first developed
in 1932, and became widely available in
1940s.
 Use a beam of electrons instead of a
beam of light.
 Wavelength of electron beam is about 100,000
times smaller than visible light.
Use: To examine structures too small to be
resolved with a light microscope.
 Two types of electron microscope:
A. A. Transmission Electron Microscope
(TEM)
B. B. Scanning Electron Microscope (SEM)
5. Electron Microscopy
A. Transmission Electron Microscope (TEM)
 Gives excellent view of internal structures.
 Magnification: 100,000 X or more.
 Resolving power: 2.5 nm or better.
 Two dimensional image.
 Drawbacks of TEM:
 Due to limited penetrating power of electrons, can
only view very thin slices (70-90 nm) of specimen.
 Must slice, fix, dehydrate, and view specimen under a
vacuum. Staining may be used to enhance image
contrast.
 Treatments kill specimen and may cause shrinkage
and distortion of cells (artifacts).
5. Electron Microscope
B. Scanning Electron Microscope (SEM)
 Gives excellent view of external surface.
 Magnification: 10,000 X or more.

 Resolving power: 20 nm or better.

 Three dimensional image.

 More recent invention than TEM. Used mainly to observe

the surfaces of cells and viruses.
 Specimens are covered with a layer of heavy metal (gold

or palladium).
 A narrow beam of electrons (primary electron beam) is

swept across specimen surface.

 Electrons on the specimen surface are knocked out,

creating a secondary electron beam which is collected

and amplified to produce an image.
Electron Microscope
. Scanning Tunneling Microscope and
Atomic Force Microscope (AFM)

 Developed in the 1980s.

 Used to observe structure and surface of
biological molecules and silicon computer chips.
A. Scanning Tunneling Microscope (STM)
Uses a thin metal probe that scans the surface of a
specimen.
B. Atomic Force Microscopy (AFM)
Uses a diamond and metal probe that scans
surface of specimen.
Advantages of both microscopes:
 Higher resolving power than electron microscopes
 No special specimen preparation required
Preparation of Specimens for Light
Microscopy
1. Smear: Spread a thin film of material
containing microorganisms over slide surface.
Allow to air dry.
2. Fixing: Process that kills microorganisms
and attaches them to a microscope slide. Fixing
preserves and minimizes distortion of cells.
Two main methods of fixation:
 Heat fixation: Pass over Bunsen burner flame
several times.
 Chemical fixation: Cover with methanol for 1
minute.
Preparation of Specimens for Light
Microscopy

3. Staining: Coloring microorganisms with a dye that

emphasizes certain structures. Before staining a sample, it
must be fixed.
Stains are salts composed of a positive ion (cation) and a
negative ion (anion).
The colored ion is called the chromophore.

Two types of dyes:

A. Basic dyes
B. Acidic dyes
Preparation of Specimens for Light Microscopy

A. Basic dyes:
 Chromophor is in positive ions.
 Most commonly used dyes.
 Bacteria are slightly negatively charged at
pH 7, therefore they stain with basic dyes.
 Examples:
Crystal violet
 Methylene blue
 Saffranin
Preparation of Specimens for Light Microscopy

B. Acidic dyes:
 Color is in negative ions.
 Stain the background: negative staining.
 Bacteria do not stain with acidic dyes.
 Used to observe cell shape, size, and capsules.
 Minimal distortion because heat fixing is not necessary an dye is
not taken up by cells.
Examples:
1. Eosin
2. Nigrosin
3. India ink.


Preparation of Specimens for Microscopy

1. Simple Stains
 Aqueous or alcohol solution of a single basic
dye.
 Primary purpose is to stain entire
microorganism to view cell shape and basic
structures.
 Procedure:
 Stain is applied for a certain time, and then washed off.
 Slide is dried and examined.
 Mordant: May be used to increase stain intensity.
Increases affinity of stain for specimen.
 Examples: Safranin, methylene blue, crystal
violet, and carbolfuchsin.
 Preparation of Specimens for
Microscopy
2. Differential Stains
React differently to different types of bacteria.
 Can be used to distinguish among different
groups of bacteria.
 There are two important differential stains
used in microbiology:

A. Gram stain
B. Acid-Fast stain
 ***REQUIREMENT FOR
GRAM’S STAINING

tain – Gentian violet/ Crystal violet/ Methylene blue

ordant – Ligule's iodine

ecolorizer - Ethyl alcohol (75%)/ acetone

ounter stain – Safranin/ carbol fuchsine

ap water

lass slide

noculating wire loop

prit lamp

linical specimen
28
icroscope
2. Differential Stains
A. Gram Stain
Developed in 1884 by Hans Gram, a Danish
microbiologist.
Gra
m staining consists of four components:

rimary stain (Crystal violet, methyl violet or Gentian

violet)

ordant (Gram's Iodine)

ecolourizing (ethyl alcohol, acetone or 1:1 ethanol-

acetone mixture)

ounterstain (Dilute carbol fuchsin, safranin or neutral

red)
Steps of Gram Stain
1. Primary stain: Cover a heat fixed smear
with a basic dye (crystal violet) for 20 sec.
 All cells, gram-positive and gram-negative, are
stained with crystal violet (appear purple).

2. Mordant: After smear is rinsed with

water, an iodine mordant solution is
applied for 30 sec.
 Crystal violet-iodine [CV-I] complex forms
Steps of Gram Stain
3. Decolorizing: Slide is washed with
alcohol, which will remove stain from Gram-
negative cells but not from Gram-positive
cells.
 Gram-negative cells will be decolorized.
 Gram-positive cells will remain purple.
4. Counterstain: Alcohol is rinsed off.
Safranin is applied for 20 sec., which will
stain cells that were decolorized.
 Gram-negative cells are stained pink.
 Gram-positive cells remain purple.
 primary
stain

mordant

decolorization

counterstain
positive
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negative
What accounts for the differential
staining between Gram-positive and
Gram-negative cells?
Gram-positive cells have very thick peptidoglycan cell
walls, whereas gram-negative cells have very thin cell
walls and more lipid content. Crystal violet easily
penetrates both cell types.
Because of its larger size, the crystal violet-iodine
complex [CV-I] is not easily removed from gram-
positive cells, due to their thick cell wall. The CV-I
complex is readily washed out of gram-negative cells
with alcohol.
 Counterstain only colors gram-negative cells.
 Gram Stain
 Gram positive bacteria retain the purple stain
 Examples include species of- Staphylococcus (Cocci), Streptococcus
(Cocci), Clostridium (bacilli), Corynebacterium (bacilli) etc.

 Gram negative bacteria appear pink after the counter stain

 Examples include species of Neisseria, Haemophilus, Salmonella, Shigella,
Vibrio, Klebsiella, Brucella, Yersinia, Coli forms. Only Neisseria is Cocci,
and the rest are bacilli

34

Gram - Gram +
 RESULT OF MICROSCOPIC EXAMINATION OF
GRAM’S STAINED FILM

umber of bacteria

ram reaction of bacteria

orphology of bacteria

resence & number of pus cells

resence of yeast cell & epithelial cells

 Requirements for Ziehl
Neelsen Staining
icroscope

tain – Carbol fuchsine

ecolorizer – H2SO4 solution & alcohol

ounter stain – Methylene blue

ap water

lass slide

prit lamp

noculating wire loop

linical specimen
36
Principle & procedure
(Acid-fast staining )
•Mycobacteria do not stain well by the Gram
technique or by simple stains such as methylene
blue

•This is due to high lipid content, presence of

mycolic acid and cell wall integrity of
Mycobacterium
•They can however be stained with strong dye
e.g. fuming Carbol fuchsin combined with phenol

•Then an acid or acid-ethanol decolorizing solution is applied

37
 Principles of Ziehl-Neelsen Stain

 Acid-fast staining
 waxy chemical in the cell wall allows
the organisms to retain the stain

38
PROCEDURE OF ACID-FAST
STAINING
Flood the slide with
strong Carbol fuchsin & until steam rises. Allow the stain to act for 5-7
sec. with intermittent heating. Never too much to produce boiling or
charring. The stain must not be allowed to evaporate.

ash with water

ecolourize with 20% H2SO4 - 5-7 sec.

ash in running tap water

ounter stain with Methylene blue - 1-2 sec.

ash in running tap water

lot dry, observe under oil immersion lens

bservation:
Acid-fast bacilli --------------------- Pink
Tissue &other organisms -------- Blue
39
esult: Smear positive for Acid fast bacilli
Application of Oil

40
 SYSTEMATIC
EXAMINATION OF
SMEARS

41
MICROSCOPIC EXAMINATION OF WET MOUNT

Wet
mount is the simplest and easiest technique
for the examination of urine and faeces

Typ
es of wet mount preperation:
Sali
ne
Iod
ine
Buf
fered methylene blue.
 THE SALINE WET MOUNT

Used to
observe:

orm's eggs

orm's larvae

rotozoan trophozoites, and cysts.

ed blood cells

hite blood cells.

43
 THE IODINE WET MOUNT

odine is used mainly to stain

glycogen and the nuclei of cysts

The buffered methylene blue (BMB)

wet mount is used to observe amoebic
trophozoites in a saline wet mount.

44
 Procedure of Wet Mount
Preparation
1.
Write the patient’s name/number and the date at
the left-hand end of the slide with a wax pencil
2.
Place a drop of saline in the center of the left half
of the slide and place a drop of iodine solution in
the center of the right half of the slide.
Note: If the presence of amoebic trophozoites is
suspected, warm saline (370C) should be used.
3.
With an applicator stick (match or tooth pick),
pick up a small portion of the specimen (size of a
match head) and mix the drop of saline.

45
How to make a wet-mount
slide
[Link] a clean slide and coverslip
[Link] ONE drop of water in the middle of the slide.
Don’t use too much or the water will run off the edge and
make a mess!
4. Slowly lower the cover slip on top of the drop.
[Link] the edge of the cover slip on one side of the
water drop.
Cover Lower
Slip slowly

5. Place the slide on the stage and view it first with the
red-banded objective. Once you see the image, you can
rotate the nosepiece to view the slide with the different
objectives.
You do not need to use the
stage clips when viewing wet-
 HANGING DROP PREPARATION

Preparation of samples for live observation (ex.

Hanging drop)
 EXAMINATION
1.
Put the slide on the microscope stage and
focus with the x10 or low-power objective.
2.
Regulate the light in the microscope field
with the sub stage diaphragm. Too much or
too little light is not good.
3.
Examine the entire coverslip area with the
x10 objective; focus the objective on the
top left-hand corner and move the slide
systematically backwards and forwards, or
up and down.

49
 CONT.

4
. Switch the x10 or low-power to the high-
dry objective, when organisms or
suspicious material are seen, and
increase the light by opening the sub
stage diaphragm.
-
This is a systematic examination. If
mounts are examined in this way, any
parasites present will usually be found. If
the mount is not examined systematically,
parasites may be missed. Examine each
microscope field carefully, focusing up
and down, before moving to the next field.
50
Thank You