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micro bio lab midterm
Terms in this set (69)
Why is it desirable that the microscope objectives be parafocal?
When switching objectives or changing slides the specimen will still be in focus, which saves times to just use the fine focus knob then having to start from the beginning and searching for the specimen and refocusing it.
Which objective focuses closest to the slide when it is in focus?
Oil immersion lens (1000x objective).
Which controls on the microscope affect the amount of light reaching the ocular lens?
Is your lens corrected for chromatic aberrations?
Yes, it is.
Assume the diameter of field of vision in your microscope is 2mm under low power if one Bacillus cell 2um, how many Bacillus cells could fit end to end across the field? How many 10 um yeast cells could fit across the field?
2mm(1x10^-3m/1mm)(1um/1x10^-6)=2000um <-- diameter of field of vision.
2,000um/2umbacillus cell= 1,000 bacillus cells
2,000um /100 um = 200 yeast cells
Name 2 ways in which you can enhance resolving power?
Shorter wavelength or switching to a higher objective (e.g. oil immersion lens).
What is the advantage of low-power objective over oil immersion objective for viewing fungi or algae?
Eukaryote are larger cells and therefore having a larger field of vision in lower power objective allows us to see the whole organism instead of a small section of the organism as seen in oil immersion lens.
What would occur if water were accidentally used in place of immersion oil?
blurry or fuzzy image. Poor quality due to refracting light water and glass have different refractive index.
Assume you are looking for microorganisms in a tissue sample from a lung biopsy the microbes become apparent when you switch to 1000x what type of microbe is most likely?
How did you distinguish true motility from Brownian movement or motion of the fluid?
long distance movement was true motility in comparison to vibrating in place or particles floating.
What if any, practical values do these techniques have?
Quick and easy to do techniques. Allow us to see organisms in natural habitat and alive.
What is the advantage of the hanging drop procedure over the wet mount?
Hanging drop allows more motility for living organism wet mount movement is more restricted.
Why is petroleum jelly used in the hanging-drop procedure?
Used to seal in the sample, acts like glue to hold cover slip to hanging-drop slide and reduces evaporation of aqueous solution.
Why are microorganisms hard to see in wet preparations?
Lack contrast between organisms and background the microbes and aqueous solution are both clear so they blend into the background.
Can you distinguish prokaryotic organisms from eukartotic organisms? Explain.
Yes because eukaryotes are much larger can be seen in lower objectives and have a nucleus that can be seen inn 1000x objective.
Why isn't oil immersion lens used in hanging-drop procedure?
Hanging-drop slide is thicker than the regular slide so can damage lens of objective if we move to 1000x objective.
Wet mound may be used for quick examination of fecal samples. Is it is most useful for identifying protozoan or bacterial parasites? briefly explain.
Protozoan because they are larger and therefore easier to see.
What is the value of petri plates in microbiology?
Provides larger surface area for examination of colonies. it is a solid cultured media in an easily stored container.
What are bacteria using for nutrients in nutrient agar?
Peptone and beef extract.
What is the purpose of agar?
It is a solidifying agent.
Why is agar preferable to gelatin as a solidifying agent in culture media?
Gel is a protein that is easily degraded by bacteria. Many decompose the gel and uses it as a food source. Few bacteria can degrade agar and it has special properties of liquifying at 100 degrees and solidifying at room temperature.
Did all organisms living in or on the environmental sample grow on your nutrient agar? briefly explain.
No, because some bacteria require different nutrients or environmental conditions for survival. So there is a possibility that some bacteria present were not able to grow on agar plate. Lack of growth could also be because someone has cleaned the environment before sample was taken.
The effectiveness of sanitation procedures in hospitals is determined by sampling surfaces for bacteria. Samples can be taken by rubbing a sterile, moistened cotton swab on a surface and inoculating a nutrient agar plate or with a Rodac environmental sampling plate. The 67-mm plates are designed so that an agar medium can be overfilled, producing a dome-shaped surface for sampling microbes. As the sanitation control officer, you test each method to determine which you will use. what can you conclude from these results?
swab = 5 number of plates with colonies number of plates inoculated = 50
Rodac plate= 6 number of plates with colonies number of plates inoculated = 40
Rodac plate would be more beneficial because it can pick up more bacteria directly from the source without using a swab and less plates are needed with Rodac than with swab plates.
What other methods can be used to determine motility?
Hanging-drop, flagella staining, semi solid tubes/ deep media tube.
What is the primary use of slants?
provides solid growth surface, it is easier to store and transfer than petri plates
What is the primary use of deeps?
Used to test the oxygen requirements and determine motility.
What is the primary use of broths?
Allow more growth of bacteria.
What is the purpose of flaming the loop before use? After use?
Incinerate any organisms on the loop. It is an aseptic technique used to minimize contamination of sample and environment (lab).
Why must the loop be cooled before you touch the culture? should you let the loop down to cool it? how do you determine when the loop is cool.
It will kill bacteria if the loop is hot, we cannot set the loop down because it will get contaminated with table microbes, we test if loop is cool by touching the side bacteria along the edge of the plate.
Why is aseptic technique important?
To prevent contamination of sample, surrounding air and surrounding surfaces that can put people at risk of exposure to pathogens.
What was the arrangement of lactococcus in the broth culture? was it different from the slant culture.
The broth allows more freedom of movement and ability to grow in any direction. The agar can restrict movement causing the chains to be shorter on slant and longer chains in broth.
For a bacterium what is the evolutionary advantage to forming a pellicle in a liquid medium?
Strict aerobic bacteria can form a pellicle to get more access to air on the top of the tube.
How can you tell that the media provided for this exercise were sterile?
No growth, transparent/ clear media.
A white pellicle has formed on the leftover spaghetti you put into the refrigerator a week ago. what is the pellicle?
Clump of bacteria
Your microbiology lab maintains reference bacterial cultures which are regularly transferred to new media nutrient agar slants. After incubation, the slants were observed and the results indicate that an improper technique had been used. What led to this conclusion?
Appearance of mold and one type of bacteria growth on the slant.
Which bacterium is a rod?
Which bacterium is larger?
Of what value is a simple stain.
Allows easy observation of morphology and arrangement of bacteria by staining bacteria providing contrast between bacterium and the background.
What is the purpose of heat fixing the smear?
Kills bacteria by cell lyses and bacteria stick to the slide. Prevents microbe from moving after staining or from moving after from being washed away.
Another method of fixing smears is to use methanol, instead of heat. How does the alcohol chemical fix the bacteria?
Alcohol denatures proteins and makes bacteria adhere to the glass.
In heat fixing what would happen if too much heat were applied?
Damage cell, stain will wash out bacteria, undergoes plastmolysis.
Methylene blue can be prepared as a basic stain or acidic stain. How would the pH of the stain affect the staining of bacteria?
Basic stain will stain the cells (direct stain), acidic stain stains the background.
Bacteria have an overall - charge so in order for the stain to adhere to the bacterial cell the pH of the stain has to be above 7 (Basic) alkalynic dye with an overall + charge will be attracted to bacteria and stain cell wall. If pH is low or below 7 it will be acidic dye with a - charge chromatophore which will be repelled by the bacteria cell and only stain the background.
Can dyes other than methylene blue be used for direct staining?
Yes, any basic stain with + chromatophore e.g. crystal violet and safranin can be used to directly stain a bacteria cell.
Bacteria can be seen without staining Why then, was Koch's recommendation for fixing and staining important for the discovery of the bacterial causes of disease?
It allowed the development of standard technique that allows researchers to gain better contrast between the bacteria and the background its helpful for identifying bacteria by morphology, arrangement and cell wall composition (gram staining) these differences distinguish different bacterias from each other and this information can be shared by different researchers aiding in identifying a bacteria and linking it to a specific pathogen. The same results can be obtained by different researchers when performing the basic universal staining procedures on the same bacteria.
If quality control staff in a sterilization unit hospital used a simple stain to determine whether bacteria were present in sterilized materials. A simple stain of sterile saline used for respiratory therapy revealed the presence of bacteria. Is the saline contaminated?
Yes, because there was a presence of bacteria.
What microscopic technique gives a similar in appearance to that seen in the negative stain?
carbolfuchsin can be used as direct stain and as a negative stain. As direct stain the pH is___.
Unlike nigrosin, acidic congo red does not contain suspended particles, but it can give the appearance of a negative stain. What is the basis for this stain?
Dye and bacteria are both negatively charged so they repel each other, so the dye stains the background and leaves the capsules surrounding the bacteria clear.
Treponema denticola is easily missed in a direct stain of a tooth or gum scraping because the cells are distorted by the staining procedure. Why can this bacterium be seen with negative staining?
Because in negative staining the cells do not intake the dye becoming shrunken and distorted instead the negative stain is repelled by the bacteria and the background is stained instead providing contrast between the background and the bacteria. Making bacteria appear larger and not distorted or dehydrated by the dye.
Why will gram + cells more than 24 hours old stain gram -?
Old cells lose cell wall integrity, which results in not being able to hold the dye.
Can Iodine be added before the primary stain in a Gram stain?
No, because it must complex with the primary stain.
List the steps of Gram staining procedure in order (omit washings), and fill in the color of gram+ cells and gram - cells after each step.
step chemical appearance Gram + and Gram -
step chemical appearance Gram + and Gram -
1 crystal violet purple purple
2 iodine purple purple
3 ethanol (alcohol) purple colorless
4 safranin purple pink
Which step can you omit without affecting determination of the Gram reaction?
Suppose you are viewing a Gram stained field of red rods and purple cocci through the microscope. What do you conclude?
Bacteria Gram+ culture is old some bacteria cells lost ability to retain primary dye.
Why can't human cells be gram stained?
because human cells do not have cell walls made of peptidoglycan which is what is dyed in Gram staining technique.
Considering you can't identify bacteria from a Gram Stain, why might a physician perform a Gram Stain on a sample before prescribing an antibiotic?
To determine antibiotic susceptibility.
How would an endospore stain of Mycobacterium appear?
What type of culture medium would increase the size of a bacterial capsule?
Polyssacharide (sugar) rich media.
Describe the microscopic appearance of encapsulated Sreptococcus if stained with safranin and nigrosin.
Cells would appear red surrounded by a clear capsule against a black background.
In the Dorner stain a smear covered with Carbolfuchsin is steamed, then decolorized with acid-alcohol and counterstained with nigrosin. Describe the microscopic appearance after this procedure.
Edospores red in colorless cell against a black background.
Endospores appear as colorless areas in a simple stain and Gram stain. Why is it necessary to perform an endospore stain to identify Clostridium difficile in health care settings?
Colorless areas could be something beside endospores. The endospore stain will confirm it as an endospore.
The flagella stain uses crystal violet, as does the Gram stain. Why do all cells stain purple in the flagella stain but not in the Gram stain?
the gram stain procedure uses a decolorizer to differntiate between Gram + and Gram- cells.
Of what advantage to Clostridium is an endospore?
when Strict anaerobe are in unfavorable conditions, the formation of an endospore allows the bacteria to remain in a dormant state until conditions become favorable. This helps them to survive the unfavorable conditions.
How might flagella contribute to pathogenicity?
Flagella make bacteria motile and allow them to move away.
How might a capsule contribute to pathogenicity?
What are the Gram reactions of Clostricium and Bacillus?
both are gram +
Of what morphology are most bacteria possessing flagella?
rods and spirals
What prevents the cell from appearing green in the finished endospore stain?
cell decolorized with water and safranin
How did the appearance of the 24-hour and 72 hour- Bacillus cultures differ? How do you account for this difference?
72 hour B. subtilis had more endospores and were not in the cell. there was no cell wall visible in 72- hour culture but there was at 24- hours
What are the types of sterility indicators
dissemination of coccidio and blasto is similar but which one spreads to meninges?
what does micrococcus luteus look like?
What are limitations of UV radiation?
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