MIC456 Microscopy

MICROSCOPY: OBSERVING
MICROORGANISMS
THROUGH THE
MICROSCOPE

LEARNING OUTCOMES

At the end of this topic, students should be able to

▪ Understand the concept of microscopy
▪ Differentiate the different types of microscopes
▪ Describe sample preparation strategies
▪ Explain the process and theory behind staining techniques in

microbiology

MICROSCOPY

▪ Microscopy is a technology that enables
us to visualize things that are too small
to be seen by our naked eyes

▪ There are many types of microscope
available for various functions

▪ The birth of microbiology generally is
due to the invention of the first
microscope by Anton van Leeuwenhoek

▪ Since than, improvement on microscopy
has revolutionized microbiology

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MICROSCOPY

▪ Resolving power → the ability of microscope to identify two objects located closely
as two discrete unit

▪ Resolving power depends on the wavelength of light source used in a microscope
▪ If the wavelength is too long to pass within the space of two closely located objects,

then the object may be seen as one
▪ Thus, it means the shorter the wavelength of the light used the higher the resolution

power of the microscope will be

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SIMPLE MICROSCOPE

▪ Simple microscope make use of only one lens to magnify small things
for examination by the naked eyes.
▪ One magnifying lens and no objective lens

▪ The theory behind it is not much different than that of magnification
glass.

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COMPOUND LIGHT MICROSCOPE

▪ First developed by Zaccharias Janssen, Dutch spectacle maker in 1600 – the
microscope was poor quality

▪ Joseph Jackson Lister (1786-1869) who was the father of Joseph Lister (1827-1912)
commissioned an improved microscope that uses visible light as light source.

▪ Difference between simple and compound microscope is that compound microscope
has two lenses; one ocular lens and one objective lens.

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COMPOUND LIGHT MICROSCOPE

▪ Most compound light microscope https://goo.gl/images/X8j7Mc
nowadays have three or four
objective lenses

▪ 100X (oil immersion lens), 40X
(high-dry), 10X (low power) and
4X (scanning)

▪ The ocular lens is usually 10X, thus
is you see an image using a 10X
objective lens, it means the image
has been magnified 100X it’s original
size

COMPOUND LIGHT MICROSCOPE

▪ 100X oil immersion lens require the use of oil immersion to produce a
clear image.

▪ This is due to the properties of light that are refracted when it travels
through material with different refraction index

▪ The light passes through the microscope glass slide is refracted when
it has to travel to the air before entering the objective lens

▪ Immersion oil has a refractive index of 1.518, which is close to the
refractive index of glass (1.520)

▪ Thus, immersion oil will prevent the light from refracting giving a clear
image.

COMPOUND LIGHT MICROSCOPE

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COMPOUND LIGHT MICROSCOPE

Image without the use of immersion oil Image with the use of immersion oil

Images taken from https://www.microscopeworld.com/t-using_microscope_immersion_oil.aspx

COMPOUND LIGHT MICROSCOPE

▪ Since compound microscope make use of visible white light (550 nm) thus the
resolution power of this kind of microscope is not as high as the electron
microscope.

▪ Compound microscope cannot resolve structures that are less than 220 nm (0.2 µm)
apart

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DARKFIELD MICROSCOPE

▪ Normally used to observe live light-sensitive or
unstained samples

▪ The opaque disc in the darkfield condenser
blocks light that usually enters the objective
lens directly

▪ Only light that is reflected off the sample will
enter the objective lens

▪ Thus the image appears bright with black
background

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Cheek cells bright-field vs dark-field

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PHASE CONTRAST MICROSCOPE

▪ Commonly used to observing live samples
▪ Samples are not fixed or stained
▪ Fixing and staining usually kills the living sample

▪ Allows internal structure to be examined

▪ Special objective lenses together with ring-shaped diaphragm brings
out small differences in refractive indexes of internal structures

▪ Images are often seen as various shades of grey to black resulting
from direct and reflected light rays

Cheek cells HeLa cells

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FLUORESCENCE MICROSCOPE

▪ Make use of ultraviolet light
▪ Depends on samples that are able to absorb UV-light (short

wavelength) and emits light of longer wavelength

▪ Some organisms like Pseudomonas have the natural ability to

fluoresce under UV-light
▪ Other samples may be treated with fluorochrome (ie fluorescent dyes)
▪ Immunofluorescence: a technique that utilizes fluorescent antibodies

to detect specific antigens
▪ Useful in rapid detection of specific pathogens

▪ Images appear as fluorescent objects against dark background

Telophase

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LIGHT MICROSCPE

▪ The microscopes that we have just discussed so far falls under the
category of light microscopes

▪ There are several limitations to light microscopes:

▪ The highest magnification that can be achieved is 2000X
▪ Resolving power up to 0.2 µm

▪ Viruses and most internal structures of the cell cannot be seen
clearly

▪ Thus, new more enhanced microscope was invented → electron
microscope

ELECTRON MICROSCOPE

▪ There are two types of electron microscope:
▪ Transmission electron microscope (TEM)
▪ Scanning electron microscope (SEM)

▪ Electron microscopes were first developed in 1932
▪ It uses beams of electrons which are 100,000X smaller than beams
of light (photon)
▪ Thus it allows scientist to observe structures that were previously
too small to be examined

Scanning Electron Microscope

▪ Provides excellent views of external structures.

▪ With magnification of 10,000X or more and resolving power of 20 nm
▪ Image appears as 3D and are called electron micrograph

▪ Mechanism

▪ Samples are coated with heavy metals (gold or palladium)
▪ A narrow beam of electron is applies to the surface of the sample which creating

a secondary electron bean → collected and amplified to produce image

Scanning Electron Microscope

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Transmission Electron Microscope

▪ Allow scientists to observe and study internal structures of samples
▪ Magnification same as SEM but resolving power of 2.5 nm or better
▪ Image is 2D and are called electron micrograph

▪ Disadvantage:
▪ Can only observe very thin samples
▪ Processing of samples are tedious involving slicing, fixing,
dehydrating. Sometimes staining may be used.
▪ Viewing of sample has to be done in vacuum state
▪ Distortion of sample are often seen especially due to sample
processing

Transmission Electron Microscope

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SAMPLE PREPARATION FOR LIGHT MICROSCOPE

1. Smear preparation
▪ Smear is a preparation of a thin film or layer of sample over a solid
surface eg. microscope slide
▪ There are several types of smear –

▪ blood smear - thin blood smear or thick blood smear
▪ bacterial smear
▪ buccal swab/smear

▪ Correct techniques in the preparation of smear is crucial in
obtaining good image in microscopy

Thin blood smear

Images taken from https://www.agric.wa.gov.au/livestock-biosecurity/blood-smear-technique-veterinarians

Thick blood smear

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Wet Mount

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Buccal swab/smear

• Samples are taken using sterile swab from mucosal site – oral, vaginal or anal

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Bacterial smear

▪ Smear can either be made from liquid or solid media

▪ If from solid media, a drop of sterile saline is usually need in order to get an evenly
spread smear.

▪ Often bacterial smear require fixing in order to
▪ kill bacteria
▪ ensure sample adheres to the glass slide
▪ preserve and minimize distortion of cells

▪ Two types of fixing
▪ Heat fixing – running of the slide with air-dried smear on top of a flame
▪ Chemical fixing – applying methanol over air-dried smear for about 1 minute

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SAMPLE PREPARATION AND STAINING METHODS FOR
LIGHT MICROSCOPE

2. Staining
▪ Once smears has been prepared, the next step would be staining
▪ Function of staining

▪ Allow cells to be seen more easily
▪ Differentiate the types of cells
▪ Observe certain structures of the cell
▪ Differentiate different status of cells

▪ Stains or dyes are generally salts of positive or negative ions
called chromophores

▪ Generally there are two types of dyes – acidic dyes or basic dyes

Acidic dyes

▪ The chromophores are negatively charged (anions)
▪ Often used for staining of background (negative staining)
▪ Bacteria are not readily stained by acidic dyes – bacterial cell wall

are slightly negatively charge
▪ In microbiology, can be used to in capsule staining
▪ Examples include:

▪ Eosin
▪ Nigrosin
▪ Indian ink

Capsule staining

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Basic dyes

▪ The chromophores are positively charged (cation)
▪ Often used for staining of bacterial cells in microbiology
▪ Bacteria are readily stained by basic dyes – bacterial cell wall are

slightly negatively charge
▪ Examples include:

▪ Safranin - red
▪ Iodine – yellowish brown
▪ Crystal violet - purple
▪ Methylene blue - blue

TYPES OF STAINING METHODS

1. Simple staining
▪ Used generally to observe the morphology and appearance of
bacteria
▪ Make use of only one dye
▪ Procedure:

▪ Stain is allowed to come in contact with prepared bacterial smear for
a certain amount of time

▪ The stain is then washed off with water and the stained smear is
allowed to air-dry before cells are observed under microscope

▪ Mordant is some times used to intensify the stain color

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TYPES OF STAINING METHODS

2. Differential staining - Gram staining
▪ Was first developed by Hans Christian Gram in 1884 – thus the “g”
in Gram has to be capitalized since it’s a person’s name
▪ Make use of two dyes, one mordant and one decolorizor
▪ Frequently used in microbiology especially medical microbiology
▪ Differentiates between Gram positive and Gram negative bacteria
▪ Drawback:

▪ Cannot stain acid fast bacteria
▪ Works well on young culture (16-18 hr). Old cultures of Gran positive

bacteria results in Gram variable





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TYPES OF STAINING METHODS

3. Differential staining – Acid fast staining
▪ Also known as Ziehl-Neelsen staining

▪ Use to staid acid fast bacteria (Mycobacteria)

▪ Contains large amount of lipids in their cell wall
▪ Normal basic dye cannot be readily absorbed

▪ Make use of carbol fuchsin, acid alcohol and methylene blue

▪ Acid fast bacteria will appear bright red against blue background

Steps in acid fast staining

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TYPES OF STAINING METHODS

1. Special staining – Endopore staining
▪ Endospores are spores produced by certain bacteria such as

Bacillus subtilis especially when nutrient is limited

▪ Endospores are extremely resistant to harsh environment such as
desiccation and heat

▪ Ordinary staining method cannot be used to stain endospore due to
their thick wall

▪ Schaeffer-Fulton method is one of the most commonly used endospore
staining procedure.

▪ Make use of malachite green and safranin

TYPES OF STAINING METHODS

1. Special staining – Endopore staining
▪ After staining, bacterial cell will appear red (due to safranin) while
the endospore will appear green (due to malachite green)

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