BT 2010

Microscopy I

Dr. Sanjukta Patra

Before 17th century, study of microbiology was limited by the lack of
appropriate tools to observe microbes.

Robert Hooke: In 1665 built a compound light microscope and used it to

observe thin slices of cork. Coined the word cell.

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• Anton van Leeuwenhoeck: Father of Microbiology

• In 1673 -first person to observe live microorganisms which he called

“animalcules” (bacteria, protozoa), using microscopes designed by
him.

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Techniques to study - the
microorganisms
Microscope –
1. Bright field, Dark field,
2. Fluorescence, Confocal
3. SEM, TEM etc

Light Microscope Phase Contrast Microscope

SEM TEM
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Microscopy

• Energy source – Light, electrons

• Magnification - Lens
Light
1. Wave nature

2. Particle nature

The nature of light: particle-wave duality

• Absorption
When light passes through an object the intensity is reduced depending upon the
color absorbed. Thus the selective absorption of white light produces colored image.
• Refraction
Direction change of a ray of light passing from one transparent medium to another with
different optical density. A ray from less to more dense medium is bent
perpendicular to the surface, with greater deviation for shorter wavelengths
• Diffraction
Light rays bend around edges - new wave fronts are generated at sharp edges - the
smaller the aperture the lower the diffraction
• Dispersion
Separation of light into its constituent wavelength when entering a transparent medium
the change of refractive index with wavelength, such as the spectrum produced by a
prism or a rainbow
Transmission the light is transmitted from a source on the
opposite side of the specimen from the objective

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Microscope:
Magnification
A microscope consists of two lenses

• Objective-Variable – 10x, 20x, 40x, 100x

• Eyepiece -Fixed

The magnification of a microscope is the product of

the factors of both lenses.

A 40x objective and a 10x eyepiece, provide a 400x

magnification.

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 Resolution
The light wave defines the limit
Resolution is the ability to render two closely adjacent objects distinct.
λ
D=
2 × 𝑛 sin θ
λ=wavelength of energy source
n= refractive index of the media
n=1 for air, n=1.4 for oil
• The value sin θ corresponds to the numerical aperture (NA), the measure of the
light gathering capacity of an objective
• With blue light, the resolution limit is approximately d = 0.2 μm
• With red light, around d = 0.35 μm
• UV objectives attain a resolution just under 0.2 μm. With the naked eye, can not
differentiate structures smaller than 0.2 millimetres
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 θ or μ =half aperture angle

Figure 2- A series of light cones derived from objectives of varying focal length and numerical
aperture.
As the light cones change, the angle μ increases from 7° in Figure 2(a) to 60° in Figure 2(c)
with a resulting increase in the numerical aperture from 0.12 to 0.87, nearing the limit when
air is the imaging medium

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 Empty Magnification: Magnification without resolution

Empty magnification can sometimes be quite useful for making details more easily visible
for the human eye

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Bright-field Images

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Bright-field

Image is a dark sample on a bright background

Sample illumination is transmitted (illuminated from

below and observed from above)

White light and contrast in the sample is caused by

absorbance of some of the transmitted light in dense
areas of the sample

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Ray Diagram

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Light path

• The light path consists of:

• Halogen lamp -transillumination light source
• Condenser lens - focuses light from the light source onto the
sample
• Objective lens - collects light from the sample and
magnifies the image
• Oculars lens - to view the sample image

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 Köhler illumination-
Critical or Köhler illumination to illuminate the sample
minimises internal stray light, allows control of contrast

A=Critical Illumination. Conjugate planes are the B=Köhler Illumination: Conjugate planes are the
illuminating bulb filament and sample plane (O). When illuminating bulb filament and Condenser diaphragm.
adjusted correctly, the image of the filament is seen Second conjugate planes are the Field diaphragm and the
coincident with the sample image. A diffusing glass filter (d) sample plane. When adjusted correctly, the image of the
is used to blur the filament image. FD:Fielddiaphragm field diaphragm and the sample are coincident. The
CD: Condenser diaphragm filament is out of the plane of focus, and thus uniformly
diffuse.
The Microscope Image
• Magnification in the microscope is collectively produced by two
independent optical systems — objective and eyepiece

• The image formed is inverted and reversed, i.e. it is seen upside

down, and left to right is also reversed.

 Maximum Magnification = 1000X

 Resolution = 0.2μ
 Calculate Maximum possible resolution by a light microscope?

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Performance
• Bright field microscopy has low contrast with most biological samples –
absorbtion is less

• Staining increases contrast, but prevents use on live cells

• Bright field illumination is useful for samples which have an intrinsic colour
– chloroplasts

• Magnification is limited by the resolving power possible with the

wavelength of visible light

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Sample preparation
• Smear
• Fixation – heat fixation, glutaraldehyde, formaldehyde
• Stain
• Visualisation

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Staining enhances contrast in the microscopic image

Simple staining – one stain

Simple stains -Methylene blue, Safranin, Crystal violet

Differential staining – multiple stains

Differential stains – Gram staining, Negative stains, flagellar stains,
endospore stains

Use of a colored (usually blue) or polarizing filter on the light source to

highlight features not visible under white light

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• Malachite green - blue-green counterstain to safranin in the Gimenez
staining technique for bacteria. It also can be used to directly stain spores
• Methyl green - used commonly with bright-field microscopes to dye the
chromatin of cells
• Methylene blue - used to stain animal cells, such as human cheek cells, to
make their nuclei more observable
• Neutral red - or toluylene red stains Nissl substance red, is usually used as
a counterstain in combination with other dyes.
• Nile blue - stains nuclei blue, may be used with living cells.
• Nile red - also known as Nile blue oxazone, is formed by boiling Nile blue
with sulfuric acid Nile red is a lipophilic stain, it will accumulate in lipid
globules inside cells, staining them red.
• Osmium tetroxide - is used in optical microscopy to stain lipids, fats, and
is reduced by organic materials to elemental osmium, an easily visible
black substance
• Rhodamine - is a protein specific fluorescent stain commonly used in
fluorescence microscopy
• Safranin - Safranin O is a nuclear stain. It produces red nuclei, and is used
primarily as staining processes which use more than one chemical stain.

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Gram staining
• determine gram status to classify bacteria broadly
• It is based on the composition of their cell wall
• Gram staining uses crystal violet to stain cell walls,
iodine as a mordant, and a fuchsin or safranin
counterstain

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Negative staining

• Smear the sample on to the slide

• Nigrosin (a black synthetic dye) or Indian ink (an aqueous
suspension of carbon particles)
• After drying, the microorganisms may be viewed in bright field
microscopy as lighter inclusions well-contrasted against the dark
environment surrounding them

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• Acid-fast staining is a differential staining used to
differentiate acid fast and non acid fast bacteria.

• It is used to identify acid-fast organisms such as members ACID –FAST STAINING

of the genus Mycobacterium .

• It is also known as Ziehl-Neelsen method

• What are acid fast bacteria?

Acid-fast bacteria are gram-positive, but in addition to

peptidoglycan, the outer membrane or envelope of the acid-
fast cell wall contains large amounts of glycolipids, especially
mycolic acids which makes up approximately 60% of the acid-
fast cell wall. They also contain large amounts of fatty acids,
waxes, and complex lipids and are highly resistant to
disinfectants and dry conditions.

• One common example of acid-fast bacteria is

Mycobacterium tuberculosis.
Structure of an Acid-Fast Cell Wall. In addition to peptidoglycan,
• Presence of large amounts of mycolic acids cause the the acid-fast cell wall of Mycobacterium contains a large amount
bacteria to resist staining by ordinary methods such as a of glycolipids, especially mycolic acids.

Gram stain.
 Principle

• Because the cell wall containing mycolic acid is so

ACID –FAST STAINING
waxy and resistant to most compounds, acid-fast
organisms require a special staining technique.

• The primary stain used in acid-fast staining, carbol

fuchsin, is lipid-soluble and contains phenol, which
helps the stain to penetrate the cell wall. This is
further assisted by the addition of heat.

• The smear is then rinsed with a very strong

decolorizer (acid-alcohol), which strips the stain
from all non-acid-fast cells but does not permeate
the cell wall of acid-fast organisms.

• The decolorized non-acid-fast cells then take up

the counterstain methylene blue.

• Acid fast bacteria will be red while nonacid fast

bacteria will stain blue
PROCEDURE OF ACID-FAST STAINING OF Mycobacterium tuberculosis

Observation under Microscope

Acid fast: Red, straight or slightly curved

rods, occurring singly or in small groups,
may appear beaded.

Non-acid fast: Blue color, in addition,

background material stain blue
• Mycobacteria can be stained using a variety of
techniques, including the Ziehl–Neelsen (ZN)
stain, auramine-rhodamine stain, and carbol
fuchsin stain:
• Ziehl–Neelsen stain: A two-step process that
stains all cells pink in the first step with basic
fuchsin, then decolorizes all cells except acid-
fast cells in the second step with acid
alcohol. Acid-fast cells retain the color and
appear red. The ZN stain is used to identify
acid-fast organisms, mainly Mycobacteria.
• Carbol fuchsin stain: Mycobacteria stained with
carbol fuchsin typically appear as red rods that
are 1–10 μm long and 0.2–0.6 μm wide. They
can also appear beaded, banded, coccoid, or
filamentous.
• Auramine-rhodamine stain: Another acid-fast
stain used to visualize mycobacteria.
• Mycobacteria can also be identified by Gram
staining, which reports them as Gram-neutral or
Gram-ghost
Questions:
1. What is Magnification?
2. What is Resolution?
3. What are the factors on which resolution depends? Explain their significance?
4. What is Empty Magnification? Give one utility of empty magnification?
5. Explain with ray diagram the working of Bright field microscope.
Dark field microscopy
Transmitted light microscopy

• Both diffracted (interacting rays with specimen) and non diffracted

(undeviated rays through specimen) are collected by objective and
contribute to image formation

• The component of non diffracted background light is very large

• This results in bright low contrast images with poor details

• Removal of non diffracted waves can be a solution

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Dark field

Image is bright on a dark background

Image is composed solely of diffracted wave component

Unscattered (non diffracted) beam from the image is excluded

As non diffracted light is excluded, the field around the specimen (where there is no
specimen to scatter the beam) is generally dark

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Principle
In dark field, an opaque disc is placed underneath the condenser lens

Only light that is scattered by objects on the slide can reach the eye

The light is reflected by particles on the slide

Bright image against a dark background

Pigmented objects are often seen in false colors - the reflected light is of a color
different than the color of the object

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May be false colour visualisation

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Light path
• Light enters the microscope for illumination of the sample
• The patch stop blocks some light from the light source, leaving an outer ring
of illumination
• The condenser lens focuses the light towards the sample
• The light enters the sample, some is scattered from the sample
• The scattered light enters the objective lens, while the directly transmitted
light misses the lens
• Only the scattered light produces the image, while the directly transmitted
light is omitted

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When to use dark field
• Dark field illumination is most readily set up at low magnifications, can be
used with any dry objective lens

• In a liquid sample, dark field is preferred

• Dark field is especially useful for finding cells in suspension

• For initial examination of suspensions of cells such as yeast, bacteria, small

protists, or cell and tissue fractions including cheek epithelial cells,
chloroplasts, mitochondria, even blood cells

• Initial survey and observation at low powers of pond water samples, hay or
soil infusions, protist as Paramecium or metazoan cultures

• Examination of unstained/lightly stained prepared slides

• Initial location of any specimen of very small size for later viewing at higher
power

• Determination of motility in cultures

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Advantages

• Live sampling
• Recovery of samples
• Technique well suited for live and unstained biological samples
• No sample preparation

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Disadvantages
• The main limitation is the low light levels seen in the final image

• This requires the sample to be very strongly illuminated, which damage the
sample

• Not only the specimen, but dust and other particles scatter the light and are
easily observed

• Sample materials need to be spread thinly, too much material on the slide
creates overlapping layers leading to difficult interpretation

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 Hanging drop:

It is used to observe the motility germination or fission of microorganisms

a cavity slide, which has a circular concavity in the centre, is used

The periphery of the concavity on the cavity slide is smeared with Vaseline

A drop of liquid microbial culture is placed in the centre of the cover glass

If the culture is solid, it is mixed with a drop of distilled water before placing on the cover
glass

The cover glass is inverted over the concavity so that the drop hangs freely and the edge of
cover glass adheres tightly to the Vaseline coated periphery of the concavity
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The microorganisms present in the hanging drop are now observed under the microscope
Wet mount method:

• A drop of liquid containing microorganism to be examined is put on a glass

slide
• A cover slip made of thin glass is placed on it
• The fluid spreads out in a thin layer between cover slip and slide
• The mount is now examined under the microscope

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Questions:
1. Why is staining required in bright field microscopy?
2. What is the difference between bright field, dark field and negative staining
images?
3. What modifications do you need to make in bright field microscope to convert it to
dark field?
4. What is hanging drop and wet mount technique.