Module 2: Principles of Microscopy

Microorganism measuring system

Metric system

Units in the metric system/Microbiology

• Micrometre (μm) = 0.000001 m (10-6 m)

• Nanometre (nm) =  0.000000001 m (10-9 m)

E. coli length and diameter

about 2 μm in length and 0.5μm in diameter.

Microscopy

the use of light or electrons to magnify small objects.

Light microscopy

the use of a microscope that employs visible light to view a specimen.

Magnification

the process of enlarging an object in appearance only.

A simple compound light microscope will have:

will have 3 objective lenses, each with a different magnifying power.

• eg 10x (lower power), 40x (high power), 100x (oil immersion)

The ocular lens always has a magnifying power of

10x

How is the total magnification calculated?

by multiplying the magnification of the specific objective lens being used with that of the ocular lens.

What is resolving power?

the ability of the lenses to distinguish two points as distinct and separate.

In general resolving power increases with:

decreasing wavelength of light used to visualize the specimen.

Resolving power of the white light used in a compound light microscope

0.2μm

What does it mean to have a resolving power of 0.2μm

This means that points that are < 0.2μm apart will not be seen as distinct and separate.

Why do we use oil immersion?

• Because bacteria are so small, we need to use the 100x objective lens to see them.

• But, the 100x lens is small, and so most light from the illuminator is lost due to refraction when the light passes from the glass slide to air

• To ensure that as much light as possible enters the objective lens, we add oil in between the glass slide and the 100x lens. 

• The oil minimizes refraction of the light rays since it has a similar refractive index as the glass slide

What the most common type of light microscope?

Brightfield microscopy

What needs to be done to be able to see a specimen on the brightfield microscopy?

• The specimen, it has to be naturally pigmented (coloured), or stained prior to microscopy.

• Staining increases the contrast between the specimen and surrounding medium.

What is phase-contrast

a type of light microscopy that utilizes a compound light microscope with a special condenser.

What is a fluorescence microscopy

another form of light microscopy that uses ultra-violet (UV) light to illuminate the specimen.

What diagnostic technique is fluorescent microscopy used in

The fluorescent-antibody (FA) technique or immunofluorescence.

How is the fluorescent-antibody (FA) technique or immunofluorescence used

• A clinical sample suspected to contain the suspect pathogen is placed on a microscope slide, and the fluorochrome-conjugated antibody is added to the specimen.

• If the pathogen is present, the antibody will bind to the cells, and the cells will be visible when viewed with a fluorescent microscope

Electron microscopy

uses a beam of electrons instead of light for visualizing microbes.

The two types of electron microscopy

• transmission electron microscopy (TEM)

• scanning electron microscopy (SEM).

Transmission electron microscope (TEM)

an electron beam is passed through an ultrathin section of a specimen

How does a transmission electron microscopy (TEM) work?

• The specimen is strained with a metal strain that absorbs electrons.

• Electrons that are not absorbed by the specimen will pass through the specimen and then through the electromagnetic objective lens and projector lens to a screen.

• The final image contains dark and light areas depending on how many electrons are absorbed by the different parts of the specimen.

TEM magnification and resolution

can achieve very high magnification (10,000 – 10,000,000x) and resolution (0.2 nm).

What can you see on the TEM and not the light microscopy

It can also be used to see viruses, which are too small to be visualized using light microscopy.

What can a TEM and SEM not be used for

It cannot be used to see living microorganisms.

Scanning electron microscopy (SEM)

the specimen is coated with a metal strain, and a primary electron beam is passed over the surface of the specimen.

How does the SEM work?

• the specimen is coated with a metal strain, and a primary electron beam is passed over the surface of the specimen.

• This causes secondary electrons to be emitted from the specimen surface.

• The secondary electrons are  collected, amplified, and used to generate an image of the specimen.

SEM achieves a magnification and resolution of:

achieves high magnification (1,000 – 500,000x) and resolution (0.5 μm), though not as high as TEM.

Steps of preparation of specimens for brightfield microscopy

• Spread culture in thin film over slide

• Dry in air

• Pass slide through flame to heat fix

• Flood slide with stain; rinse and dry

• Place drop of oil on slide; examine with 100x objective lens

Preparing smears for staining:

• Before cells can be stained, they must undergo fixation where they are made to attach to the microscope slide so that they do not get washed off during the staining process.

• First, a smear is prepared by spreading a thin film of material containing the microbes on the surface of the glass slide

• The smear is allowed to air dry, and then the slide is passed over the flame of a Bunsen burner several times

• The heating step fixes the cells to the slide, and it simultaneously kills the cells.

Staining smears:

• Smears are stained by flooding the slide with a staining solution

• Stains are salts made of positive (+) and negative (-) ions, one of which is coloured (called the chromophore).

With basic stains, the chromophobe is the

(+) ion

Basic stains are commonly used to strain

bacterial cells, which have a slightly negative (-) charge.

With acidic stains, the chromophore is the

(-) ion

Acidic stains are commonly used to

stain the background surrounding bacterial cells (negative staining).

What is the purpose of the fixation step when preparing a specimen for Brightfield microscopy?

The fixation step is performed to attach (adhere) the cells to the slide. This ensures that the cells do not get washed off the slide during the staining step.

Types of staining techniques

1. Simple stains,

2. Differential stains, and

3. Special stains.

Simple stains:

• Use a single basic dye (eg crystal violet, methylene blue, safranin) to stain microbial cells.

• They are used to visualize the entire microbe so that the cell shape and basic structures can be observed.

Mordant

• A chemical that is sometimes added to simple stains to increase the affinity of the stain for the cell. 

• Mordants can also be used to coat fine cellular structures (eg flagella) to make them thicker and easier to see after staining

Differential stains

use two different coloured dyes that react differently with different types of bacteria, and thus are used to distinguish them.

The 2 most common differential stains that are performed

the Gram Stain and the Acid-Fast Stain.

Gram stain

• One of the most useful staining procedures as it classifies bacteria into two main groups:

  1. Gram-positive

  2. Gram-negative

• Bacteria in these groups have:

  • Different cell wall structures, and

  • Different susceptibility to antibiotics

Simplified steps in gram stain procedure

1. Application of crystal violet (purple dye)

2. Application of iodine (mordant)

3. Alcohol wash (decolorization)

4. Application of safranin (counterstain)

Step 1 in gram stain procedure

Add crystal violet (primary stain) and leave for 1 min, then rinse with water.

• Primary stain: imparts colour to all cells

Step 2 in gram stain procedure

Add Gram's Iodine (mordant), leave for 1 minute, then rinse with water.

• Mordant helps to increase the affinity of the stain for the cell. Iodine functions as a mordant by forming a complex with crystal violet (I-CV) that is too large to escape through the cell wall.

Step 3 in gram stain procedure

Decolourizing step: Add alcohol (decolonizing agent), leave for 10 seconds, then rinse. Repeat step.

Step 4 in gram stain procedure

Add counterstain (safranin O), leave for 1 min, then rinse.

• Counterstain: provides contrasting colour to the primary stain

Gram-negative cells appear

Pink

Gram-positive cells are

Purple

Examples of gram-positive bacteria

• Staphylococcus aureus

• Streptococcus pyogenes

• Clostridiodes difficile

• Mycobacterium tuberculosis

Examples of gram-negative bacteria

• Escherichia coli

• Klebsiella pneumoniae

• Vibrio cholerae

• Neisseria gonorrhoeae

Acid-fast stain classifies bacteria as either

• Acid-fast or

• Non-acid fast

Mycobacterium tuberculosis

Causes tuberculosis

Mycobacterium leprae

Causes leprosy

Mycolic acid

Waxy lipids in the cell walls of mycobacteria

Steps in acid-fast stain procedure

1. Carbolfuchsin (primary stain) is applied to a heat-fixed smear. The slide is heated gently for 5 min to enhance penetration and retention of dye. The slide is cooled and rinsed with water.

2. Next, acid-alcohol (decolourizing agent) is applied to the smear. This removes the red dye from cells that are not acid fast, while acid-fast cells remain red.

3. Finally, the smear is treated with methylene blue (counterstain). After 1 min, the slide is washed with water, blotted dry, and examined using a microscope

What colour are acid-fast bacteria at the end of the procedure

Red

What are special stains used for

used to visualize specific structures of microbial cells, such as endospores, flagella, capsules.

Negative staining for capsules

• The capsule is a polysaccharide covering that is present on some microbes. It plays an important role in the virulence of some pathogens.

• To visualize the capsule, a negative staining procedure is used.  

• First, nigrosin (an acidic stain) is applied to stain the surrounding background. The cells remain unstrained.

• Then, safranin (basic stain) is used to stain the cells. The capsules appear as unstained halos surrounding the stained cells

What are endospores

highly resistant, dormant structures that are produced within certain bacterial cells.

• eg Bacillus spp., Clostridium spp.

Why can’t endospores be stained by normal staining methods?

This is because the stains cannot penetrate the wall of the endospore.

Steps in an endospore staining procedure

1. Apply malachite green (primary stain) to a heat-fixed smear. Heat the slide to steaming for 5 min. This enables the stain to penetrate the spore wall. 

2.  Wash the slide in water for 30 sec to remove the stain from all cell parts except the endospores. 

3. Apply safranin (counterstain) to the smear for 1 min, then rinse and blot dry.The endospores will appear green within red or pink cells

Bacterial flagella

are structures that are used for cellular motility. They are too small to be seen with a light microscope without special staining

Flagella staining

• The flagella staining procedure uses a mordant to increase the diametre of the flagella.

• The flagella are then stained with carbolfuchsin to visualize them with a microscope