(other) Labs Flashcards
Identify the type of lens used in a compound microscope
Objective lenses
Total magnification formula
Total Magnification = Objective Lens Power x Ocular Lens Power
Parfocal
lens or set of lenses where all focal points are aligned in the same plane
Condenser
focus and concentrate the light beam onto the specimen, ensuring even illumination and enhancing image quality by controlling the amount and angle of light that reaches the objective lens
describe one way that aerosols are created in microbiology lab
through the use of a hot loop, specifically when it comes into contact with a broth culture. When a hot loop, not fully cooled, is introduced to a liquid broth, it can cause the broth to boil briefly, creating tiny droplets of bacteria-containing liquid that become airborne as an aerosol
List steps required to perform the gram stain
- Smear bacteria emulsion onto slide.
- Heat fix or chemical fix bacteria with methanol. If chemical fixing bacteria, make sure it’s air-dried before continuing to next step.
- Apply crystal violet dye to slide for about a minute. Rinse off excess dye with water.
- Apply iodine to slide for about a minute. Rinse excess iodine with water.
- Decolorize cells with ethanol until runoff is clear (~20 seconds). Rinse excess ethanol with water.
- Apply safranin counterstain for about 30 sec. Rinse excess with water.
- Gently blot the slide dry.
- Observe the cells under a microscope.
Explain what happens during each step of gram stain
1.Crystal Violet:
The smear is stained with crystal violet, a purple dye, which stains both Gram-positive and Gram-negative bacteria.
- Gram’s Iodine:
The bacteria are treated with Gram’s iodine, a mordant that forms a large crystal violet-iodine complex with the dye, making it more insoluble and preventing it from being washed out. - Decolorization:
A decolorizer, typically alcohol or acetone, is applied. This step differentiates the bacteria. Gram-positive bacteria retain the crystal violet-iodine complex due to their thicker peptidoglycan layer, while Gram-negative bacteria lose the stain as the thinner peptidoglycan layer and outer membrane are degraded by the decolorizer. - Safranin:
A counterstain, safranin, is applied. Gram-positive bacteria, which already retain the crystal violet-iodine complex, remain purple. Gram-negative bacteria, having lost the crystal violet-iodine complex, become stained pink or red by the safranin
Simple stain
-Purpose:
-To visualize the basic morphology of cells, including their shape, size, and arrangement.
-Staining:
-Uses a single stain (e.g., methylene blue, safranin, crystal violet) that colors all cells the same color.
-Result:
-Provides a general overview of cell characteristics without differentiating between different types of bacteria.
Gram stain
-Purpose:
-To differentiate bacteria based on their cell wall composition into Gram-positive and Gram-negative categories.
-Staining:
-Uses a series of multiple stains, including a primary stain (crystal violet), a mordant (iodine), a decolorizer (alcohol), and a counterstain (safranin).
-Result:
-Gram-positive bacteria appear purple/blue, while Gram-negative bacteria appear pink/red, says the Biology LibreTexts.
-Mechanism:
-The difference in cell wall structure, specifically the peptidoglycan layer, determines whether a cell retains the primary stain or not, leading to the observed color difference.
List the steps required to perform an acid fast stain
- Prepare the smear:
Create a thin smear of the sample on a clean glass slide and allow it to air dry. - Heat-fix:
Heat-fix the smear by gently passing the slide through a flame a few times to adhere the bacteria to the slide, according to ATSU. - Stain with carbolfuchsin:
Flood the smear with carbolfuchsin stain and heat gently until it steams. Allow it to sit for 5 minutes, then rinse with water. - Decolorize with acid-alcohol:
Apply acid-alcohol decolorizer to the smear. Rinse with water. - Counterstain with methylene blue:
Apply methylene blue counterstain to the smear for 30 seconds. Rinse with water. - View under microscope:
Blot the slide dry and view under a microscope using oil immersion. Acid-fast bacteria will appear red or pink, while non-acid-fast bacteria will appear blue.
Acid-fast stain
-Purpose:
-To identify bacteria with mycolic acid in their cell walls (acid-fast bacteria).
-Principle:
-Uses carbol fuchsin as the primary stain, followed by decolorization with acid-alcohol, and then counterstaining with methylene blue or malachite green.
-Results:
-Acid-fast bacteria (like Mycobacterium) retain the carbol fuchsin stain, appearing red or pink.
Non-acid-fast bacteria are counterstained with methylene blue or malachite green, appearing blue or green.
-Significance:
-Important for diagnosing infections caused by acid-fast bacteria, such as tuberculosis.
Motility
true motility refers to the directional movement of a cell using structures like flagella
Bowman’s movement
not true motility but rather movement caused by the molecules in the liquid striking an object and causing the object to bounce; the particles and microorganisms all vibrate at about the same rate and maintain their relative position
Compare and contrast the size and movement of prokaryotes and eukaryotes
-Size:
-Prokaryotes: Smaller, typically 0.1-5 micrometers in diameter.
-Eukaryotes: Larger, typically 10-100 micrometers in diameter.
-Movement:
-Prokaryotes:
-Use surface appendages like flagella (which spin) and pili (which pull).
-May exhibit swimming, swarming, gliding, twitching, or floating.
-Eukaryotes:
-Can use flagella and cilia (which beat to create waves) for movement.
-Some eukaryotic cells migrate using actin-dependent processes.
-May exhibit flagellar-dependent swimming and cell migratio
Advantages of wet mounts
-they can be used to view living organisms
-motility can be viewed
-characteristic arrangement, or grouping, of organisms is not interrupted because a heat-fix smear not required
Disadvantages of wet mounts
-quickly dry out, limiting opportunity for viewing
-oil immersion objective cannot be used
-colorless- difficult to see
