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Explain why the body tube of the microscope should not be lowered while you are looking through the ocular lens.
The body tube of the microscope should not be lowered while looking through the ocular lens because you do not want to lower the body tube too much and have it crack the slide.
Why would you adjust the iris diaphragm
in order to change the amount of light entering the lens system
Why would you adjust the coarse-adjustment knob
to move the body tube towards the slide and to adjust the focus in order to see the specimen
Why would you adjust the fine-adjustment knob
to move the body tube in order to focus on the specimen.
Why would you adjust the condenser
adjust so that it is close to the stage in order to achieve optimal focus
Why would you adjust the mechanical stage control
adjust in order to move the slide around the stage to see the different sections of the slide.
How would you correct the following problems with the oil-immersion lens? Inability to bring the specimen into sharp focus
The fine-adjustment knob may need to be adjusted. Also, ensure that the oil is completely enclosing the space between the slide and the oil-immersion lens.
How would you correct the following problems with the oil-immersion lens? Insufficient light while viewing the specimen
Make sure that the iris diaphragm is opened completely and that the condenser is close to the stage.
How would you correct the following problems with the oil-immersion lens? Artifacts in the microscopic field
Remove the slide, and clean all lens systems with the appropriate lens tissue or xylol if the oil-immersion lens is gummy or tacky.
a fixed platform with an opening in the center allowing for the passage of light from an illuminating source below to the lens system above the stage
can be moved vertically or horizontally by means of adjustment controls
directly under the stage and has two sets of lenses that collect and concentrate light as it passes upward from the light source into the lens system, has an iris diaphragm
a shutter controlled by a lever that is used to regulate the amount of light entering the lens system
-Ocular or eyepiece lens- upper end
-Nosepiece with the objective lenses- rotating the nosepiece positions objectives above the stage opening
-Coarse-adjustment and fine-adjustment knobs raise or lower the body tube
enlargement, is the function of the ocular lens and the objective lens. The objective lens produces the real image that is projected up into the focal plane and then magnified by the ocular lens to produce the final image
Resolving power or resolution
-how far apart two adjacent objects must be before a given lens shows them as discrete entities, limits the magnifying power of the lenses.
-dependent on numerical aperature, and the refractive index
a characteristic of each lens- defined as the function of the diameter of the objective lens in relation to its focal length
Resolving power formula
(wavelength of light)/(2x numerical aperture)
-the shorter the wavelength, the greater the resolving power
the bending power of light passing through air from the glass slide to the objective lens
Why are living, unstained bacterial preparations more difficult to observe microscopically than stained preparations?
Bacteria are so small and their refractive index is close to water so there is not much contrast between the two leaving a clear image. Staining adds contrast and makes them easier to see. Also, some bacteria can move around fast making it hard to see them under the microscope.
What is the major advantage of using living cell preparations rather than stained preparations.
With living cell preparations, the motility of the bacteria can be seen, as well as the original shape and size of the bacteria, and binary fission. Staining preparations slightly alter the bacteria .
How do you distinguish between true motility and Brownian movement?
Brownian movement is a small vibrational movement due to the bombardment of water molecules. True movement is self-directed and more than simple vibrations back and forth.
During the microscopic observation of a drop of stagnant pond water, what criteria would you see to distinguish viable organisms from nonviable suspended debris?
Viable organisms will move around and most have intracellular organelles.
Why use hanging drop preparations and wet ounts?
They make movement easier to see because they slow down the movement of water molecules
Organisms used in Negative Staining lab
Micrococcus luteus, Bacillus cereus, Aquaspirillum itersonii
Negative staining procedure
place a drop of nigrosin at one end, place a loopful of the inoculum into the drop of stain and mix with the loop, place a slide against the drop at a 45o and allow the drop to spread along the edge of the slide, push the slide away from the drop forming a thin smear, air dry and examine
regent used for negative stain
Nigrosin (needs to be negatively charged)
using an acidic stain that is negatively charged, so will not penetrate the cells, leaving the unstained cells easy to see against the colored background
Practical applications (2) of negative staining
-Natural size and shape can be seen b/c no heat fixation and no harsh chemicals
-Possible to observe bacteria that are difficult to stain
Why can't methylene blue be used in place of nigrosin for negative staining? Explain
Methylene blue is a basic stain that is positively charged, when an acidic stain that is negatively charged such as nigrosin must be used for negative staining
Why doesn't nigrosin penetrate bacterial cells?
The nigrosin is negatively charged, just like the cell membrane of the bacteria, which means there is a repulsion between the two, it is unable to penetrate.
a culture containing a single unadulterated species of cells
a solution containing nutrients- soluble low-molecular-weight substances that are frequently derived from the enzymatic degradation of complex nutrients
a liquid medium lacking a solidifying agent, can supplement with agar (solidifying agent) to get a solid or semisolid medium
-extract of seaweed, a complex carbohydrate composed mainly of galactose, acts as solidifying agent
-liquefies at 100 and solidifes at 40
What temp does agar melt and solidify
liquefies at 100C solidifies at 40C
requires an agar concentration of about 1.5-1.8%, gives a hardened surface on which microorganisms can be grown
forms with less than 1% agar
allowed to cool and harden in a slanted position, used for maintaining pure cultures
Agar deep tubes
tubes allowed to harden in the upright position- used primarily for the study of the gaseous requirements of microorganisms
poured into petri dishes and allowed to solidify- provides large surface areas
the process of rendering a medium or material free of all forms of life
transferring microorganisms from one vessel to another
-an incubator used to maintain optimum temperature
-uses dry heat so must maintain a moist environment by placing a beaker of water in the incubator during growth)
Shaking waterbath (advantage and disadvantage)
-Advantage- provides a rapid and uniform transfer of heat to the culture vessel, and its agitation provides increased aeration, resulting in acceleration of growth
-Disadvantage- can be used only for cultivation of organisms in a broth medium
What organism used in Lab 1- culture transfer techniques
Explain why the following steps are essential during subculturing
-Flaming the inoculating instrument prior to and after each inoculation
to maintain sterility
Explain why the following steps are essential during subculturing: Cooling the inoculating instrument prior to obtaining the inoculum
excess heat can fixate the samples or kill the organisms
Explain why the following steps are essential during subculturing: Holding the test tube caps in the hand to form a V
There are bacteria and contamination on our hands, and we don't want to contaminate the caps or tubes
Explain why the following steps are essential during subculturing: Flaming the neck of the tubes immediately after uncapping and before recapping
If there are microbes on the neck of the tube, you don't want to accidently bring those into the nutrient base, also you don't want to spread the microbe you were incubating by spreading it through the neck
Purposes of the subculturing procedure
To separate microbes in order to do tests on them. Also, it can be used to prepare and maintain stock cultures. The procedure is done aseptically in order to reduce the risk of contamination
Explain why a straight inoculating needle is used to inoculate an agar deep tube.
the inoculum needs to be drawn out from the bottom of the tube in a straight line, along the line of insertion
There is a lack of orange-red pigmentation in some of the growth on your agar slant labeled S. marcescens. Does this necessarily indicate the presence of a contaminant? Explain.
No it doesn't mean that there is contamination. It could just meant that you failed to inoculate a bacteria. Also, it could mean that the environment was wrong for the bacteria to grow.
Upon observation of the nutrient agar slant culture, you strongly suspect that the culture is contaminated. Outline the method you would follow to ascertain whether your suspicion is justified.
You could grow the culture more to see if cultures of unknown stuff grows.
What organisms used in lab 2- Techniques for isolation of pure cultures
Serratia marcescens, micrococcus luteus, escherichia coli
essentially diluting- involves spreading a loopful of culture over the surface of an agar plate (streak and turn, streak and turn...)
requires a previously diluted mixture of microorganisms. During inoculation the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent glass rod while the Petri dish is spun on a "lazy Susan" turntable
requires a serial dilution of the mixed culture by means of a loop or pipette. The diluted inoculum is then added to a molten agar medium in a Petri dish, mixed, and allowed to solidify
Can a pure culture be prepared from a mixed-broth or a mixed-agar-slant culture? Explain.
Yes, the components from the mixed culture just need to be separate first. This can be done by doing a streak plate or diluting the mixture and using a spread-plate technique. Then when you have individual colonies you can grow them as a pure culture.
Observation of a streak-plate culture shows more growth in Quadrant 4 than in Quadrant 3. Account for this observation.
This could be due to human error, no properly sterilizing the inoculating loop or spreading some of quadrant 1 into quadrant 4. This can also be due to the fact that a wider streak is used, so if the microbe is not diluted enough, it can grow more
Why is a needle used to isolate individual colonies from a spread plate or streak plate.
With a needle less microbes are picked up. This limits to possibility of getting two different microorganisms by accident.
How can you determine if the colony that you chose to isolate is a pure culture?
The cultures that grow should all look the same, they should have the same cultural characteristics.
What microorganisms use in lab 3- Cultural Characteristics of microorganisms
Pseudomonas aeruginosa, Bacillus cereus, Micrococcus luteus, Escherichia coli, Mycobacterium smegmatis
differences in the macroscopic appearance of the growth of microorganisms on different media. Used to separate microorganisms into taxonomic groups
Nutrient agar slants- how to evaluate
-abundance of growth
Form- agar slants
-Arborescent- (treelike growth)
-Rhizoid- (rootlike growth)mooth edges)
-Beaded- (nonconfluent to semiconfluent colonies)
-Echinulate- (continuous, threadlike growth with irregular edges)
-Effuse- (thin, spreading growth)
-Filiform- (continuous, threadlike growth with smooth edges)
Consistency- agar slants
Dry, buttery (moist and shiny), mucoid (slimy and glistening)
Nutrient agar plates- how to evaluate
size, pigmentation, form, margin, elevation
Form- agar plates
-Circular- (unbroken, peripheral edge)
-Irregular- (indented, peripheral edge)
-Rhizoid- (rootlike, spreading growth)
Margin- agar plates
-Entire- (sharply defined, even)
-Lobate- (marked indentations)
-Undulate- (wavy indentations)
-Serrate- (toothlike appearance)
-Filamentous- (threadlike, spreading edge)
Elevation- agar plates
-Raised- (slightly elevated)
-Convex- (dome-shaped elevation)
-Umbonate- (raised, with elevated convex central region)
Nutrient broth cultures- how to evaluate
-Uniform fine turbidity- (finely dispersed growth throughout)
-Flocculent- (flaky aggregates dispersed throughout)
-Pellicle- (thick, padlike growth on surface)
-Sediment- (concentration of growth at the bottom of broth culture may be granular, flaky, or flocculant)
Nutrient gelatin- how to evaluate
-Crateriform- (liquefied surface area is saucer-shaped)
-Napiform- (bulbous-shaped liquefaction at surface)
-Saccate- (elongated, tubular)
-Stratiform- (complete liquefaction of the upper half of the medium)
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