OBSERVING
MICROORGANISMS
THROUGH
MICROSCOPE
LEARNING OUTCOMES
At the end of this topic, students should be able to
Explain and use the metric system
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
UNIT OF MEASUREMENT
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UNIT OF MEASUREMENT
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UNIT OF MEASUREMENT
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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
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
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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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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 ren (due to safranin) while
the endospore will appear green (due to malachite green)
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TYPES OF STAINING METHODS
2. Special staining – Capsule staining
Capsule is a semi-rigid polysaccharide layer produced by some
bacteria as protection against
Phagocytosis – virulence factor
Dryness
Chemicals – such as detergent
Viruses
Since capsule does not readily retain dyes, negative stainig methos is often used
whereby the cells are stained with safranin or crystal violet (basic dye) prior to
flooding the smear with Indian ink to stain the background
Capsules appear as a halo ring around the red-stained dye/ purple-stained dye
Steps in Capsule staining
1. Place a drop of Indian ink on one end of your slide.
2. Then add your bacteria to the Indian ink and make a smear (like a blood smear)
3. Air-dry the smear BUT DO NOT HEAT FIX
4. Flood the smear with safranin for 30 seconds to 1 minute – be gentle because the
smear has not been heat-fixed
5. Rinse the stained smear and allow the slide to air-dry.
6. Observe under a microscope.
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TYPES OF STAINING METHODS
3. Special staining – Flagella staining
Flagella are long filamentous appendages that some bacteria possess to
facilitate motility
They are generally too thin to be seen under the microscope
Staining procedures are often difficult and usually require mordant and coating
of the flagella surface with metal or dyes
In medical microbiology, the number and arrangement of flagella can help in
making diagnosis
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LEARNING OUTCOMES
At the end of this topic, students should be able to
Explain and use the metric system
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
