Module 2 microbiology Flashcards
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.
Compound light microscope (inventor? use? lens system? mag and res? light source? viewing? specimen prep? oil immersion lens? suitability?)
- Inventor: Zacharias Janssen, around 1590.
- Common Use: Widely used in laboratories. used to look at prokaryotic and eukaryotic cells in glass slides
- Lens System: Includes both ocular and objective lenses.
- Magnification and Resolution: Offers up to 1000x magnification but has poor resolution, producing 2D images.
- Light Source: Illuminated from the bottom by a lamp. passes through air
- Viewing: Requires the human eye’s mechanics (retina) to see the image, which appears inverted.
- Specimen Preparation: Specimens are typically transparent and need staining for better visibility. absorbs light
- Oil Immersion Lens: Can be used, but requires the specimen to be dead.
- Suitability: Good for observing some structural aspects of specimens, but not detailed features.
Dissection Microscope (inventor? lens system? mag and res? lens specifics? dual light source? use case? design purpose?)
- Horatio Greenough - 1886: Met with the Zeiss brothers, first mass producers of microscopes.
- Lens System: Features an ocular and a stereo lens system.
- Magnification and Resolution: Offers low magnification and low resolution, capable of producing 3D images.
- Lens Specifications: Ocular lens at 10X and stereo objectives at 1x, 2x, or 3x, varying by microscope model.
- Dual Light Sources: Equipped with light sources from both the top and bottom.
- Use Case: Ideal for observing larger, live specimens like Daphnia magna in a dissection dish. specimens absorb light
- Design Purpose: Configured for low magnification of three-dimensional objects, suitable for larger or thicker specimens than what a compound microscope can handle.
Dark Field (DF) Microscope
(Inventor? lens system? mag and res? light technique? use? DF technique? light source? visual? specimen type? max mag?)
*you can make one with external light by blocking the light at the bottom of a light field microscope with a black piece of paper above the condenser.
- J.J. Lister - 1830: Developed a microscope design.
- Lens System: Includes both ocular and objective lenses.
- Magnification and Resolution: Provides low magnification and low resolution, producing 2D images.
- Lighting Technique: Observes specimens using light scattered by them.
- Ideal Use: Suitable for live, unstained biological samples.
- Dark Field Technique: Uses an opaque disc under the condenser lens to allow only scattered light to reach the eye, creating a dark field effect.
- Alternative Dark Field Method: Light is emitted from an external source onto the specimen without the opaque disc.
- Visual Effect: Background appears black while the specimen is illuminated.
- Specimen Types: Effective for observing yeast, bacteria, small protists, and motility.
- Maximum Magnification: Up to 100X.
Phase Contrast Microscope (inventor? lens system? light source? mag and res? live microorganisms observation? human eye limitations? Phase Plate and Condenser Annulus? contrast enhancing technique? image capture? staining? image types?)
based on interference of light beams
- Frits Zernike - 1934: Developed an advanced microscope design.
- Lens System: Includes ocular and objective lenses, plus a phase plate and condenser annulus.
- Light Source: Located at the bottom of the microscope.
- Magnification and Resolution: Offers good magnification (up to 1000x) and resolution.
- Live Microorganisms Observation: Utilizes a hanging drop slide and cover slip for observing live microorganisms with an oil immersion objective lens.
- Human Eye Limitation: Can detect changes in light amplitude but not phase shifts.
- Phase Plate and Condenser Annulus: Capture light phase shifts, creating high-contrast images.
- Contrast-Enhancing Technique: Ideal for producing high-contrast images of transparent and live specimens.
- Image Capture: Can be done electronically with a camera or viewed directly with the human eye.
- No Staining Required: Specimens can be observed live without staining.
- Image Types: Capable of producing both positive and negative phase plate images, depending on the phase plate used.
if there is no sample on the PCM what will your eyes see?
blackness, nothing
what are the two parts of a PCM that makes it unique?
1)phase ring/ phase plate
2)annular ring/ condenser annulus
which of the two parts for the PCM is found in the condenser?
Annular ring. condenser annulus
P=S+D what do each of these letters represent?
P= particular wave or total beam
S= surrounding wave
D= defracted wave
when s wave and d wave are identical =?
contructive interference “inphase state” (indentical to S and D but bigger)
when s wave and d wave are opposite=?
destructive interference “out of phase state” no wave (straight line) they cancel each other out
when s wave and d wave are slight identical=?
slightly out of phase (they partially line up)
Confocal Microscopy (who?, Laser diode?, Pinhole technique? laser focusing? Using Immunofluorescence? Viewing Cell Layers? Fluorescent Chemical Labeling? Software for 3D Imaging?)
-
Minsky’s 1957 Innovation
- Developed a method to view cell details.
- A breakthrough in cellular imaging.
-
Laser Diode’s Role
- Used for both creating and capturing cell images.
- Key in imaging technology.
-
Pinhole Technique
- Filters blurry signals for clear images.
- Essential for precise imaging.
-
Laser Focusing Method
- Targets specific areas within a cell.
- Allows for detailed observation.
-
Using Immunofluorescence
- Labels parts of the cell for easy identification.
- Enhances cell component visibility.
-
Viewing Cell Layers
- Technology can focus on various cell depths.
- Allows for multi-layered cell analysis.
-
Fluorescent Chemical Labeling
- Highlights specific cell parts like DNA.
- Used for detailed cell component study. (specimen needs to be coated in fluorescent markers
-
Software for 3D Imaging
- Transforms captured signals into 3D images.
- Facilitates advanced cell analysis.
TRANSMISSION ELECTRON
MICROSCOPY(TEM) (Inventor? Electron Beam? Specimen Thickness? Specimen Staining?)
Invention: Developed the electron microscope in 1931.
Maximum Magnification: Capable of magnifying up to 1,000,000 times.
Electron Beam: Uses a static beam of electrons to examine specimens.
Specimen Thickness: Requires extremely thin specimens for analysis. coated in gold
Specimen Staining: Involves staining with heavy metals to enhance contrast and detail in the images.
SCANNING ELECTRON
MICROSCOPY(SEM) (Inventor?, Electron Beam Scanning?, Imaging Focus?, Specimen Prep?)
Development: Created by Manfred von Ardenne in 1937.
Maximum Magnification: Offers a magnification of up to 100,000 times.
Electron Beam Scanning: Utilizes a beam of electrons that scans over the specimen.
Imaging Focus: Primarily images the surface of the specimen.
Specimen Preparation: Requires the specimen to be dried and coated with a metal layer before scanning. placed on collodion or other supporting material on copper grids 3mm across.
Review diagram “Light path of compound microscope”
https://microscopeinternational.com/content/resources/compound-microscope-labeled.jpg
Review diagram “Dissecting microscopy”
https://tse1.mm.bing.net/th?q=dissecting%20microscope%20parts%20and%20functions%20pdf
Review Diagram “Dark field microscopy”
https://assets.coursehero.com/study-guides/lumen/images/microbiology/instruments-of-microscopy/OSC_Microbio_02_03_Darkfield3.jpg
Review Diagram “Phase contrast microscopy”
https://thebiologynotes.com/wp-content/uploads/2022/02/Phase-Contrast-Microscope-1024x537.jpeg
Review Diagram “Confocal microscopy”
https://microbiologynote.com/wp-content/uploads/2020/07/nihms-1063368-f0001.jpg
Review Diagram “SEM microscopy” and “TEM microscopy”
https://microscopewiki.com/wp-content/uploads/2021/12/Difference-between-Light-microscopy-Transmission-electron-microscopy-and-Scanning-electron-Microscopy.jpg
