Module 2 microbiology Flashcards
(44 cards)
Conversions
Mega (10^6), Kilo(10^3), Hecto(10^2), Deca(10^1), base, Deci(10^-1), Centi (10^-2), Milli(10^-3), Micro(10^-6), Nano(10^-9), Angstorm(10^-10)
What are the two major methods used in microbiology used for examining microorganisms?
Direct examination technique and staining technique
List two advantages of examining microorganisms using the wet mount technique?
It’s a fast method and living microorganisms can be examined
What is the one disadvantage of examining a microorganism using the wet mount technique?
This form of examination is not suitable for long periods of time.
List the three classifications of stains.
Basic, acidic, neutral
There are two factors that control what stain a microbial cell will absorb, what are these
factors?
The chemical makeup of the microbial cell and the pH of the surrounding environment
If a cell contains mostly acidic components, of the three classifications of stains, which will the cell most likely attract?
The Basic Stains
Basic Stains have _____________ charged ions
Basic stains have positively charged ions.
Acidic stains have ___________ charged ions.
Acidic stains have negatively charged ions.
Provide four examples of basic stains
Methylene blue
Basic fuchsin
Iodine
Crystal violet
Safranin
Acid stains behave differently than basic stains, explain the behavioral difference?
Acid stains are generally repelled by bacteria, dye does not color the cell but the background
Provide four examples of acidic stains
Nigrosin
Malachite green
Acid fuchsin
India ink
List three examples of neutral stains
Giemsa
Wright
Leishman
Why is it so important to understand the staining process in microbiology?
Because the staining properties of bacteria aid in the identification process
List five steps taken when preparing a bacterial smear?
- Two loops of water on slide
- Add a loop of the bacteria
- Spread and swirl it out on the slide
- Allow to air dry
- Fix bacteria to slide by passing through flame
List the five main types of staining that take place in microbiology?
- Simple staining
- Differential staining
- Staining of microbial structures
- Negative staining
- Staining with fluorochrome dyes for fluorescence microscopy
Place the letter of the staining process employed in front of the clue provided.
Staining Process
a) Simple Staining
b) Differential Staining
c) Staining of microbial structures
d) Negative Staining
e) Fluorochrome Staining
Clue Staining Process
B 1. Uses a combination of multiple stains
A 2. Used to look at general morphology of the
cell
D 3. Used to look at the capsule of a cell. structures
D 4. Because cells are generally negatively
charged or in a neutral environment, this
stain works best.
A 5. Uses only one dye
B 6. Uses a counterstain
B 7. Used on cells with high lipid components
A 8. All cells are of the preparation are one color
E 9. Requires UV light
C 10. Stain process requires both basic fuchsin
and a mordant.
B 11. The acid fast stain is an example
E 12. requires a counterstain of potassium
permanganate
C 13. Sometimes requires heat, e.g. Spore stain
D 14. Nigrosin or India Ink
E 15. Rapid screening is possible with this
staining process
Gram Stain Method
Crystal Violet (1 min)
Stains all of the cells a violet color, ability to penetrate gram +ve cell wall.
Iodine (1 min)
Forms a crystal violet-iodine complex which fixes the color into the gram +ve cell
Alcohol (15 – 30 seconds)
Does not affect the C-I complex. Color of all of the cells remain purple.
Safranin (15 seconds)
A counterstain does nothing to the gram +ve cell, unless the slide was dipped into the alcohol
too many times, it will then stain the cells red, although they are gram +ve cells.
2 Lens system of microscope and how image is created using our eye and lenses
Objective lens system (closest to the specimen)
ocular lens system (closest to the eye)
In a microscope, the objective lens first magnifies the object to create a real, internal image. Then, the eyepiece (ocular lens) further magnifies this real image, creating a much larger virtual image that appears as if it’s floating. This is the detailed image you see when you look through the microscope.
review illumination system
To adjust a microscope for Köhler Illumination in a simplified way, follow these steps
Center the Light Source: Make sure the microscope’s light source is centered using 10 x objective
Adjust the Condenser Height: Move the condenser up or down until it is close to the slide.
Open the Field Diaphragm: This controls the amount of light. Open it fully.
Focus the Microscope: Use a slide and focus on the specimen with the objective lens.
Adjust the Field Diaphragm: Close it until its edges are visible in the field of view.
Center the Field Diaphragm: Use the condenser controls to center the light in the field of view.
Adjust the Condenser’s Aperture Diaphragm: This controls the contrast. Adjust it for the best image quality.
Reopen the Field Diaphragm: Open it just beyond the field of view.
Wavelength and Resolution, their “relationship” (0.2
um or 200 nm)
- Resolution: The ability to clearly distinguish fine details in an image.
- Wavelength of Light: Key factor affecting resolution; shorter wavelengths improve resolution.
- Visible Light Range: Extends from about 400 to 700 nanometers, from blue (shorter wavelength) to red (longer wavelength).
- Resolution Limit: Best compound microscopes can’t resolve details closer than about 200 nanometers.
- Inverse Relationship: There is an inverse relationship between wavelength and resolution; as wavelength decreases (toward blue light), resolution increases.
- Blue Light for Microscopy: Utilizing blue light enhances resolution because of its shorter wavelength compared to red light.
Numerical Aperture – define – how it relates to
resolving power
Definition: Measures how much light a lens can gather from the specimen.
Higher NA = Better Detail: Lenses with a higher NA can see finer details (better resolution).
Example: A lens with NA 1.52 sees finer details than one with NA 0.25.
Use of Immersion Oil: Adding oil between the lens and specimen increases NA, improving the ability to see fine details
limitations of the compound microscope
A compound microscope has limitations such as being most effective for dark or light-bending objects, a resolution cap of about 0.2 micrometers due to light bending, difficulty in studying live cells with low natural contrast, and the necessity to kill and fix cells for better visibility using stains.
