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