TOPIC 4: Visualization

‼️ Based on Lecture Slides, DOES NOT INCLUDE VIDEO CONTENT YET

Visible light

(Wavelength of Radiation)

___ ___ is part of a spectrum of electromagnetic radiation

Electrons

(Wavelength of Radiation)

___ are charged particles and when moving, act as waves with wavelengths dependent on the voltage of electron beam

smaller wavelengths

(Wavelength of Radiation)

Radiation of ___ ___ results in enhanced microscopy

Magnification

Apparent increase in size of an object and indicated by number and an “x’ (i.e. 1000x)

Magnification

Results when a beam of radiation refracts as it passes through a lens

1. Brightfield

2. Darkfield

3. Phase Contrast

Types of Light Microscopy

Brightfield

(Type of Light Microscopy)

• Background is illuminated

• Most elementary form of microscope of illumination techniques

• Derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field

Darkfield

(Type of Light Microscopy)

• Specimen is made to appear light against a dark background

Phase Contrast

(Type of Light Microscopy)

• Use the alignment or misalignment of light waves to achieve the desired contrast between a living specimen and its background

Resolution

This is the ability to distinguish between objects that are close together

(1) wavelength

(2) numerical aperture

Resolution distance is dependent on:

1. the (1) ___ of the light or electron beam

2. (2) ___ ___ of the lens

Contrast

Refers to the differences in intensity between two objects or between an object and its background

Stains

What is used to achieve contrast?

Fluorescent Microscopy

Uses invisible UV light to cause specimens to radiate visible light

1. Fluorochroming

2. Immunofluorescence or Fluorescent Antibody Technique (FAT)

Categories of Fluorescent Microscopy

Fluorochroming

(Category of Fluorescent Microscopy)

• Direct chemical interaction occurs between the fluorescent dye or fluorophore and a component of the bacterial cell

Immunofluorescence or Fluorescent Antibody Technique (FAT)

(Category of Fluorescent Microscopy)

• In which an antibody is attached to the dye

Electron Microscopy

Uses a beam of electron instead of light to see the structure of the bacteria

1. Transmission (TEM)

2. Scanning (SEM)

Types of Electron Microscopy

Transmission (TEM)

(Type of Electron Microscopy)

• Can resolve particles with 0.001 micrometer in size

• E.g., x-ray

Scanning Electron Microscopy (SEM)

(Type of Electron Microscopy)

• Scans a focused electron beam over the surface to create an image - can be in a 3D view

1. Direct Wet Mount Preparation

2. Hanging Drop Preparation

3. Intravital Staining

Techniques in Visualization for Unstained, Living State

Direct Wet Mount Preparation

(Techniques in Visualization for Unstained, Living State)

• Used for the examination of motile protozoa and trophozoites

Hanging Drop Preparation

(Techniques in Visualization for Unstained, Living State)

• Using concave slides

Intravital Staining

(Techniques in Visualization for Unstained, Living State)

• Combination of both direct wet mount preparation and hanging drop preparation

• Non-toxic dye is introduced to a microorganism and selectively stains certain cells and tissues

1. Smear Preparation

2. Air Drying

3. Fixation

Techniques in Visualization for Fixed, Stained State

Smear Preparation

(Techniques in Visualization for Fixed, Stained State)

• Use a clean glass slide

Air Drying

(Techniques in Visualization for Fixed, Stained State)

• Used to preserve the morphology of the organism

Fixation

(Techniques in Visualization for Fixed, Stained State)

• Either heat (pass sample 3-5 times under flame) or chemical (immerse in ethanol)

Ethanol

What substance is used for Chemical Fixation?

1. Simple

   1. Positive Staining

   2. Negative Staining

2. Differential

3. Special

Types of Staining

Simple Staining

(Type of Staining)

• Using only one dye

Positive Simple Staining

(Type of Staining)

• Bacteria is stained but not the background

• E.g. Dilute Carbol Fuchsin

Negative/Relief Staining

(Type of Staining)

• Background is stained but not the bacteria

• E.g. India Ink Method

Differential Staining

(Type of Staining)

• Uses 2 dyes

• E.g. gram staining and acid-fast staining

Special Staining

(Type of Staining)

• Used for structures that are difficult to visualize under ordinary stain

• E.g. spores or capsules or metachromatic granules

Gram Staining

(Staining Reaction)

• Differentiates bacteria into two groups

• Staining procedure discovered by Danish scientist Hans Christian Joachim Gram (1884)

Hans Christian Joachim Gram

Discovered Gram Staining

All cocci are gram (+) except the Neisseria Group, Moraxella (formerly Branhamella) catarrhalis (and Veillonella)

What bacteria are Gram (+)?

All bacilli are gram (-) except the ACID FAST ORGANISMS (Mycobacterium, Nocardia), SPOREFORMERS (Bacillus, Clostridium), and Corynebacterium species

What bacteria are Gram (-)?

Hocker’s Method

What is the method of Gram Staining used?

1. Make a smear

2. Flood smear with crystal violet

3. Wash with tap water

4. Cover the slide with gram’s iodine

5. Wash with tap water

6. Now forming a Crystal Violet Iodine (CVI) complex

7. Decolorize with absolute alcohol or mixture of acetone and alcohol

8. Wash with tap water

9. Flood the slide with safranin for 30 secs

10. Wash with tap water

11. Blot dry and examine stain smear under OIO

Steps in Gram Staining (Hocker’s Method)

Crystal Violet

What is the primary stain in Gram Staining?

Mordant

What is the term for the substance that enhances the color of the primary stain?

Gram’s Iodine

What is the substance that acts as the mordant?

Crystal Violet Iodine (CVI) Complex

What is formed upon the addition of Gram’s Iodine?

Absolute alcohol or mixture of acetone & alcohol

What is used to decolorize in Gram staining?

Safranin

What is the counterstain used in Gram Staining?

Oil Immersion Objective

What lens is used to examine the sample in Gram Staining & Acid Fast Staining?

Purple

What color does Gram (+) bacteria ultimately stain?

Pink

What color does Gram (-) bacteria ultimately stain?

Differential Stain

What can a gram stain be called since it differentiates between gram (+) and (-) bacteria?

Magnesium Ribonucleate

What substance is only found in gram (+) cells, which helps (+) bacteria retain the primary dye?

(1) cell wall integrity

(2) old age

(3) autolytic enzymes

Gram (+) cells stain (-) due to the loss of (1) ___ ___ ___ because of (2) ___ ___ or action of (3) ___ ___

(1) 50-90

(2) mycolic

Acid Fast Stain depends on long chain ((1)___-___ C atoms) fatty ((2)____) acids

France Ziehl & Fredrik Neelsen

Who modified the Acid Fast stain by adding phenol or carbolic acid and basic fuchsin?

Ziehl-Neelsen

What is the name of the Acid Fast stain that uses heat?

Cold Kinyoun

What is another variation of acid-fast staining that unlike the Ziehl-Neelsen, it does not require heating so the concentration of carbol fuchsin is increased?

Steps in Acid Fast Staining

Carbol Fuchsin

What is the primary stain in Acid Fast Stain?

Acid Alcohol (i.e., Sulfuric Acid)

What is the decolorizer in the Acid Fast Stain?

Methylene Blue

What is the counterstain in the Acid Fast Stain?

Red

What color does an acid fast bacteria exhibit?

Blue

What color does a nonacid fast bacteria exhibit?