Lab exam 1 Flashcards
Microscope parts and function
MICROSCOPE: Parts Refer Text book Fig 4.4, page 100
Base: supports the microscope
Stage: part where slide with viewing specimen is placed
- has a hole and pair of jaws / clips to immobilize slide
Sub-stage light: located at the base, light passes in the upward direction
Condenser: focuses light on the specimen
- is a lens system located below the stage
- it doesn’t affect magnification
- may include mirrors and prism to deflect light
Iris diaphragm lever: adjust light intensity for best contrast
- is part of the condenser system, below the stage
Coarse and fine adjustment knobs: found on both sides
Coarse knob: allows for gross focusing
Fine focus knob: allows for fine tuning of the focus
Head: supports the objective and ocular lens system
Arm: connects the base and head
Ocular lens: lens in each eyepiece; lens closest to the eyes
- provides additional 10 x magnification when used with objective lens
Objective lens: adjustable lenses on the revolving nosepiece
- 4 types of objective lenses on microscope (scanning, low, high and oil
immersion) 4,10,40,100
Proper Usage of Microscope
PROPER USE AND STORAGE OF MICROSCOPE
- Use both hands when you carry a microscope
DON’T disassemble any part of the microscope without permission from the
instructor.
DON’T apply force on any part of the microscope. - Before and following microscope use, clean all lenses with lens tissue paper.
Don’t use paper towel or Kleenex, as they may scratch the lens. - Plug in the lamp cord into electrical outlet, then turn on the main switch of
microscope - Place slide in the slide holder
- Raise the stage as close to the 4X objective as possible using the coarse adjustment
knob ( on the sides) - Viewing through eyepiece, adjust the stage downward (so that the slide and lens
move apart from each other). - Sharpen focus with the fine focusing adjustment knob.
To maintain sharp focus at all times, use one hand to manipulate the specimen
slide and other hand to turn the fine focusing knob - Before storing microscope back, replace the low objective lens into working
position.
The microscope is always stored with the lowest power objective lens locked in - Place plastic cover to avoid dust on the microscope.
- Always check before changing objective lens to avoid slide breakage.
Concept of resolution
How is resolution affected by magnification?\
How is it corrected?
refraction, refractive index, oil immersion usage.
resolving power, ability of lens to distinguish between 2 points, a specified distance apart. (partial resolution, cmplete/superimposition of image (overlayer), good resolution can tell apart)
resolution is INVERSELY proportional to magnification.
the higher the mag, the lower the ability to resolve/poorer resolution. the lower the mag, the more resolution.
high magnification>low aperture>low resoution
low magnification>high aperture>higher resolution
more light increase resolution!
can be corrected via oil, better lens, more light, decreased magnification.
refraction is bending of light,
refractive index is measure of velocity of light as it passes through medium
oil has same refractive index as glass (thus it prevent the light that couldvebeen refracted in air, and allow light to travel up the microscope more straight) leading t higher resolution, allow light to enter small opening o higher powered lens.
OIL DISPLACE AIR< AVOID REFRACTION\
OIL INCREASE APERTURE THUS IMPROVE RESOLUTION.
real vs virtual image
real image is via objective lens
virtual image via ocular lens (opposite of image)
every time you look via ocular lens, we are looking at virtual image.
should be able to differentiate between eukaryotic and prokaryotic cells (cheek cells staining); use of METHYLENE BLUE (and other basic dye) in cell staining process and concept of contrast
prokaryotic; no nucleus (nucleoid area)
eukaryotic cell: nucleus (DNA is concentrated in one area)
use methylene blue dye b/ it is basic (cationic) cat are good.
the colored portion (chromophore) is positively charged (+) and thus get attracted to negative charge on DNA.
nucleus is dark stained b/c high concentration of negatively charge ion (due to PO4 group) (DNA IS negatively charged!) , cytoplasm is lightly stain bc not as concentrated.
Why do basic dyes adhere to bacterial cells?
Basic dyes “stick” to bacterial cells because the pigmented cations will be attracted to the negatively charged cells and will bind through electrostatic attractions
Why are basic dyes more effective for bacterial staining?
Why are basic dyes more effective for bacterial staining than acidic dyes? Basic stains with a positively charge chromogen are preferred because bacterial nucleic acid and certain cell wall components carry a negative charge that strongly attract and binds to the cationic chromogen.
bacteria cell model
know part and its function
refer to pic:
layers inside to outside:
plasma membrane
cell wall
capsule
chromatophore: pigments are stored here
flagella: long tail
nucleoid area with dna/chromosome
fimbriae/pili
simple staining vs differential staining vs special staining
A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism.
Ex1) (methylane blue in cheek cell exp, stain all cell blue (though we can see certain area stained darker due to higher concetration of negativ charge beign attracted to the stain’s positive charge)
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Differential staining distinguishes organisms based on their interactions with multiple stains.
EX1: gram stain help distinguish between gram pos and gram neg. gram pos stain purple becase their thick peptidoglycan retain primary stain. gra neg stain red because their thin peptidoglycan loses primary stain and takes up the red counter stain (safarin)
EX2: (spore stain (spore is green, mother cell is red),
EX3: acid fast stain (for cel with waxy mycolic acid cell wal over their thick peptidoglycan cell wall preventing gram stain to stick), in a mix smear the acid fast positive is stained red while acid fast neg is stained blue via counter stain)
EX4) capsule stain/negative stain
using acid dye neg charge) to identify bacteria cell capsule (which are also neg charge)
leading them to repel each other causing a “halo effect’
acidic and basic dye, concept and usage.
acidic dye have negative charge (anion, anna is bad)
basic dye have positive charge (cation, cat are good)
basic dye is attracted to po4/dna, etc in cells and is useful to stain those organelles since they are positively charged (opposite attracts).
acidic dye is useful because it is negatively charge thus, it can be used to “repel” other negative things.
example: bacteria capsule can be revealed via acidic dye (negative staining). the acidic dye (-) will repel against the bacteria capsule (-) creating a “halo effect” so we can identify the capsule.
Capsule stain/ advantage of negative staining
Capsule stain/negative stain
using acid dye neg charge) to identify bacteria cell capsule (which are also neg charge). (casule is a bunch of negative charged surrounding the cell/bacteria)
leading them to repel each other causing a “halo effect’
Gram staining (differential) and PRINCIPLE
Today we use Gram’s staining techniques to aid in the identification of bacteria, beginning with a preliminary classification into one of two groups: Gram positive or Gram negative.
The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process.
gram positive have thick peptidiglycan which can retain primary stain(thus stain purple), gram negative have thin peptidoglycan leading them to lose primary stain and instead take on secondary stain (safarin) and stain red instead.
Gram staining involves four steps.
1) primary stain: First cells are stained with crystal violet,
2) mordant: f the addition of a setting agent for the stain (iodine). create I-CV complex
3) Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. (beacause gram positive have thick peptisoglycan, gram neg have thin peptidoglycan that cant retain primary stain.
4) secondary stain, safranin, is added, which counterstains the decolorized cells pink (gram neg)
Acid fast staining (differential)
principle.
should we identify between acid fast pos and acid fas neg?
cord factor?
Some bacteria produce the waxy substance mycolic acid when they construct their cell walls. This waxy barrier prevents stains from penetrating the cell, which is why the Gram stain does not work. Thus acid fast is the alternative.
(gram positive cell are the same as acid fast positive, they just cant be stained via gram staining due to their waxy coat (mycolic acid) and can only be stain via acid fast method.
yes we can distinguish them apart (acid fast pos and neg) acid fast pos stains pink. acid fast neg stains blue.
Pathogenic acid fast bacteria; MYCOBACTERIUM TUBERCULOSIS
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CORD FACTOR: virulence factor, enhance disease causing ability.
secreted protein, stick cells together to give advantage to bacteria. help bacteria void and escape host defense cell. prevent it from being eliminated.
MYCOBACTERIUM TUBERCULOSIS (id this on slide!) refer to folder of slides
(clustered together, in reality they are just A TON bacilli (rods)
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1) heat-fixed smear (mix of acid fast positive and acid fast negative, in order to later produce contrast) is flooded with the primary stain carbol fuchsin (pink)
2) mordant: The heat; “melts” the waxy cell wall and permits the absorption of the dye by the cells. drives it in.
3) solution of acid and alcohol is added as a decolorizer. Cells that are “acid-fast” because of the mycolic acid in their cell wall resist decolorization and retain the primary stain. All other cell types will be decolorized.
4) counter stain: Methylene blue is then used as a counterstain. In the end, acid-fast bacteria (AFB) will be stained a bright pink color (from primary stain), and all other cell types will appear blue (acid fast negative).
this create contrast to easily see the acid fast positive in a background of blue (acid fast neg).
spore staining (differential)
principle and role of mordant and counterstain
Because the endospore coat is highly resistant to staining, a special method was developed to make them easier to see This method, called the endospore stain,
1) primary stain: ( malachite green). stain all (both spore and vegatative cel/mother cell)
2) mordant: heat/stream: uses either heat or long exposure time to entice the endospores to take up the primary stain. soften the keratinsised spore coat.
3) decolorization step, which removes the dye from the vegetative cells in the smear,
4) the counterstain: safranin is applied to provide color and contrast. (since mother cell/vegative cell lost green priamry dye, it takes up the counter stain(red)
When stained by this method, the endospores are green, and the vegetative cells stain pink/red.
advantage and disadvantage of gram stain
advantage:
rapid, useful, provide clue to identify.
valueable info to initiate antimicrobial tx
disadvantage:
- often, disease causing bacteria do not have distinct stain chaacteristic
- stain is NOT specific enough to diagnose the cause of infection
- some bacterial cell stain poorly or not at all, freshly grown bacteria is preferred.
endospore, spores
oval shape, glass bead apperance.
no metabolic activity, spore itself is dry dehydrated structure made by bacteria itself under unfavorable condition.
Trepnema pallidum (Syphilis)
identify on slide
one of the 3 spirochete.
AKA Syphilis bacteria.
fyi:
A bacterial infection usually spread by sexual contact that starts as a painless sore.
Syphilis develops in stages, and symptoms vary with each stage.
The first stage involves a painless sore on the genitals, rectum, or mouth. After the initial sore heals, the second stage is characterized by a rash. Then, there are no symptoms until the final stage which may occur years later. This final stage can result in damage to the brain, nerves, eyes, or heart.
Syphilis is treated with penicillin. Sexual partners should also be treated.
