Tuesday, July 3, 2012

Lab 1 knowledge Test study notes

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Spore Stain

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My Unknown: diploid cocci, negative gram stain (pink) Neisseria
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Dr. Maxwell and Mr. Bundy in morning lab, July 3, 2012 

*** my answers***ran out of time, its hard study on the 4th of July

Bacteriological Stain Comparisons


Stain
1o dye
+/-
charge
decolorizer
2o dye
counterstain
+/-
charge
Extra steps? Used for?
Simple stain
Carbol fuchsin; crystal violet; safranin; methylene blue




 +
n/a
n/a

n/a
Gram stain
Crystal violet

+
95% alcohol
safranin
+
Iodine: fix first stain
Acid-Fast stain
Carbol Fuchsin
+
Acid alcohol
Methylene blue
+
Heat first stain for 5 minutes: allows dye to penetrate waxy membrane
Endospore stain
Malachite green
+
water
safranin
+
Heat first stain for 5 minutes: allows dye to penetrate spore
Negative stain
nigrosin
-
n/a
n/a

Take second slide at 45 degree angle and drag dye/bacteria towards opposite side
Capsule stain
Nigrosin;

any acidic dye
-
water

Basic dye
+
Must be careful NOT to rinse away background dye



LAB TEST 1
REVIEW SHEET
(You should make sure you understand all of the answers to the questions in the lab manual in addition to using this review sheet as you study for the test.)
  1. Respond to the statement, "Handwashing is useless in disease control, since it does not kill or remove all bacteria from the hands." Do you agree or disagree? Justify your opinion.
Disagree because handwashing kills the extrenous bacteria that live on the surface of your skin, which are most likely the pathogenic ones. It allows the natural flora to live because they typically live in the deeper tissues and are brought to the surface by handwashing. The native flora do not cause disease and are helpful in destroy dead skin tissue and preventing colonization from pathogenic bacteria by removing nutrients that those pathogens would need to live. 
  1. What is a bacterial colony, and why are colony numbers used to extrapolate numbers of bacteria in an area sampled?
Because it is believed that each bacterial colony was formed by one original bacteria from the sampled area. 
  1. Differentiate between extraneous and normal flora organisms, and tell which are the most likely to be found on the "after" section of a handwashing plate.
See answer in question one. Extraneous organisms come from other sources not typically found in the body and most often cause disease. Normal flora




  1. What are some of the unique safety rules of the Microbiology lab? Give the rationale for each.




  1. Differentiate between resolution and magnification, and describe the microscope parts and techniques which increase each.








  1. Why are basic dyes used bacteriological stains?



  1. Why is heat-fixing a bacterial smear necessary?


  1. List all of the steps (in order) to the preparation of a bacterial stain from a slant culture.




  1. Draw the cell wall of a gram negative bacterial cell immediately after the decolorization step of the gram stain. Draw the cell wall of a gram positive cell at the same point. Be able to explain the reason for the differences.




  1. What are the three different decolorizing agents used in bacteriological stains? For which stain is each used? How is their action different based on the purpose of the stain?
95% alcohol (decolorize gram negative but not gram positive cells)
Acid alcohol (decolorize all cells without waxy …. Acid in membrane)
Water (decolorize all of cell except for spore)  

  1. Why is heat necessary for the spore stain and the acid-fast stain, but not for the gram stain?
To force the dye into waxy membrane of acid fast positive bacteria and inner membrane of the spore



  1. Why are negative stained slides not washed, but placed in a tumbler of disinfectant?
They still have live bacteria on them which can be a health hazard.



  1. Why is the capsule stain referred to as a combination negative-positive stain?
Because you use a negative stain to obtain the outline of the cell and a positive stain to stain the cell itself so that you can see the capsule as clear space between the cell and the negative stain



  1. If a positive acid-fast stain is made from bacteria found in a skin lesion, what disease would you suspect? Why?
Mycopasmia leprae, leprosy because they do not have cell walls and have mycelia acid in their cell membrane



  1. Why do you never heat-fix a smear to be used for a capsule stain?
Heat fixing would evaporate the capsule because it is mostly made of water and you would not be able to see the capsule under the microscope.



  1. You are gram staining alone on a Saturday night, and realize you've run out of crystal violet. Making do with what you have, which stain would be the best one to use as the primary stain - carbol fuchsin or methylene blue? Why?
Methylene blue to give contrast to the sarfanin stain used as the secondary dye. Carbol fuchsin is pink and it would be hard to tell the difference between it and the sarfanin dye.

**** teacher answers****


LAB TEST 1

REVIEW SHEET
(You should make sure you understand all of the answers to the questions in the lab manual in addition to using this review sheet as you study for the test.)

  1. Respond to the statement, "Handwashing is useless in disease control, since it does not kill or remove all bacteria from the hands." Do you agree or disagree? Justify your opinion.

  2. The population of bacteria which are most likely to be removed by handwashing are the extraneous organisms, which you pick up from other people or inanimate objects which you touched recently. These are much more likely to be pathogenic than your normal flora, which remain after washing. For this reason, disease control IS accomplished by thorough handwashing.
  3. What is a bacterial colony, and why are colony numbers used to extrapolate numbers of bacteria in an area sampled?

  4. A bacterial colony is a visible mound of bacterial cells produced by the cloning of an original bacterial cell placed on the agar's surface. Since each organism placed on the surface should produce a colony, the number of colonies should represent the number of individual bacteria present on the surface of the agar originally. (Note: This is true only for the organisms which can grow with the nutrients, temperature, and oxygen levels provided.)
  5. Differentiate between extraneous and normal flora organisms, and tell which are the most likely to be found on the "after" section of a handwashing plate.

  6. (See question 1 above) There will be both extraneous and normal flora organisms in the "before" section of a handwashing plate, but only normal flora in the "after"section of the plate, since the extraneous are surface only, while normal flora live down in the creases of the hands and are flushed to the surface during the washing process.
  7. What are some of the unique safety rules of the Microbiology lab? Give the rationale for each.

  8. No eating, drinking, or putting of ANY object in the mouth - contamination risk
    Keep jackets, purses, and bookbags off benchtops - contamination of objects, potential fire hazard, or staining of objects.
    Put all unwanted bacterial cultures in biohazard bag or basket - sterilization of all culture materials reduces chance of bacteria in waste disposal of city.
    Use broom and dustpan to clean up glass breakages rather than fingers - glass slivers could act to inoculate bacteria into the body.
    Disinfect benchtop before and after each lab exercise - prevent contamination of cultures from previous workers, as well as to protect the next worker from any invisible spills you may have had during your work.

  9. Differentiate between resolution and magnification, and describe the microscope parts and techniques which increase each.

  10. Resolution is the clarity of the image. This is enhanced by the use of high quality lenses, as well as the use of immersion oil. Since immersion oil has the same refractive index as glass, the use of the oil prevents scattering (diffraction) of light as the light passes from the specimen into the optical system of the objective lens. Maintaining the substage condenser at the correct height (close to the stage) also maximizes resolution by focusing the light on the specimen.
    Magnification is simply the enlargement of the image formed by a lens system. This is accomplished by the use of ocular and objective lenses which enlage the image.

  11. Why are basic dyes used bacteriological stains?

  12. Basic dyes have a net positive charge, which causes them to be attracted to and adhere to the negatively charged cell surface of most bacterial cells.
  13. Why is heat-fixing a bacterial smear necessary?

  14. This serves both to kill the bacteria, making the smear safe to work with, and to affix the smear to the surface of the slide, so that the bacteria do not wash off the slide and down the drain during the staining and rinsing process.
  15. List all of the steps (in order) to the preparation of a bacterial stain from a slant culture.

    • wash slide thoroughly with abrasive powder, rinse, and dry
    • flame slide thoroughly on both sides to remove oil, lint, and bacteria
    • use the loop to put a drop of water on the slide
    • holding the needle in your dominant hand like a pencil, and with the slant resting lightly against the fingers of your other hand, loosen the cap of the slant tube
    • flame needle until red hot, cool slightly
    • remove the cap from the tube with the pinky of the dominant hand
    • flame tube opening
    • gently touch the needle tip to surface of slant
    • flame tube and recap immediately after removing needle from tube. Set slant down in rack or cup.
    • smear bacteria in drop of water in a circular motion over the surface of the slide to make a thin, slightly cloudy smear
    • flame loop
    • allow smear to air-dry
    • heat-fix smear by passing the BOTTOM ONLY of the slide through the flame twice slowly.
    • Your smear is ready to stain or hold for staining at a later time

  16. Draw the cell wall of a gram negative bacterial cell immediately after the decolorization step of the gram stain. Draw the cell wall of a gram positive cell at the same point. Be able to explain the reason for the differences.

  17. gram negative: I can't do this on the web, sorry! It would be colorless, though! The LPS layer is dissolved and removed, as well as dehydration of protein portion of thin peptidoglycan layer leading to porous remaining wall losing primary (purple) dye.
    gram positive: Purple cell, since without any lipid (no LPS) to dissolve, and having a thick peptidoglycan layer without significant alteration after dehydration of proteins, the primary dye remains trapped in the cell wall layers.

  18. What are the three different decolorizing agents used in bacteriological stains? For which stain is each used? How is their action different based on the purpose of the stain?

  19. 95% alcohol - gram stain (dissolves LPS to remove dye from Gr-)
    water - spore stain (primary stain is only slightly basic and does not stick to cells well, so simple water washing removes dye from all but the re-solidified spore coat)
    acid-alcohol - acid fast stain (harsh acid decolorizes even gram positives with thick peptidoglycan, leaving only the waxy-walled (mycolic acid) Mycobacterium colored.


  20. Why is heat necessary for the spore stain and the acid-fast stain, but not for the gram stain?

  21. Both the spore and the acid-fast cell normally resist dyes, due to the thickness (spores) or waxy nature (acid-fast cell wall). In order to force dye into the cells, heat softens and loosens the surface to allow entry of the dye into these hardy structures.
  22. Why are negative stained slides not washed, but placed in a tumbler of disinfectant?

  23. Since a negative stain is not heat-fixed, there are still live bacteria in a negatively stained slide. You should not touch this with your hands to wash it, since you risk contaminating yourself with live, potentially pathogenic bacteria.
     
  24. Why is the capsule stain referred to as a combination negative-positive stain?

  25. A negative staining procedure is used first, to stain the background and leave the capsule (with the cell inside) unstained. In order to see the morphology and arrangement of the cell producing the capsule, a basic dye is added to color the cell, leaving the capsule a colorless "halo" on a black background with a colored cell.
  26. If a positive acid-fast stain is made from bacteria found in a skin lesion, what disease would you suspect? Why?

  27. Organisms belonging to the genus Mycobacterium are the only ones which stain acid-fast. I would suspect M. leprae in a skin scraping, since this organisms is the causative agent of leprosy.
  28. Why do you never heat-fix a smear to be used for a capsule stain?

  29. Heat-fixing would fry or boil away the mucoid capsule, and therefore you would not be able to see the capsule on a stain.
  30. You are gram staining alone on a Saturday night, and realize you've run out of crystal violet. Making do with what you have, which stain would be the best one to use as the primary stain - carbol fuchsin or methylene blue? Why?

  31. Probably the better stain for you to use would be the methylene blue, since the counterstain in the gram stain is safranin (pink), the pink dye carbol fuchsin would not provide sufficient contrast with the counterstain to offer an easy distinction between gram positives and gram negative.
 

Acid fast stain


Capsule stain



endospore



A. gram + cocci
B. gram - bacilli



Negative stain bacilli


MICROBIOLOGY LAB TEST 1
Sec. 02
Spring 2012                                                                                         “I have abided by the academic                                                                                                                     integrity policy on this test.”

                                                                                                                                                                       
                                                                                                                        (signature) + printed name

Multiple Multiple Choice:  Circle the letter(s) of ALL OF the correct answer(s) to each question below.  NOTE: There may be multiple choices to circle for complete credit, and there may be NO correct choices listed.  @30pts.

1.    Extraneous bacteria:
a.    Are typically non-pathogenic
b.    Are present in higher numbers on unwashed hands than washed hands
c.    Are present in the creases and crevices of hands

2.    Heat-fixing slides
a.    Involves flaming front of slides
b.    Involves flaming back of slides
c.    Kills bacteria
d.    Sticks bacteria to slides
e.    Is necessary for the penetration of some dyes

3.    95% alcohol
a.    Dissolves lipids
b.    Decolorizes gram + cells
c.    Decolorizes gram – cells
d.    Decolorizes acid-fast cells

4.    Acid alcohol
a.    Dissolves lipids
b.    Decolorizes gram + cells
c.    Decolorizes gram – cells
d.    Decolorizes acid-fast cells

5.    The spore stain can be used to identify the pathogens which cause:
a.    Tuberculosis
b.    Tetanus
c.    Pneumonia
d.    Anthrax

6.    Heating during the primary stain step is done
a.    In the spore stain
b.    in the negative stain
c.    In the gram stain
d.    to help dye penetrate
e.    to kill cells
f.     to affix cells to the slide

7.    All differential stains MUST involve
a.    Application of primary dye
b.    Application of secondary dye
c.    Decolorization
d.    Application of mordant
e.    Heating during stain application
f.     At least 2 possible outcomes
g.    Rinsing with water between all steps
h.    Blotting dry between all steps

8.    Thorough handwashing with antibacterial soap will
a.    Reduce the overall number of bacteria found on skin
b.    Reduce the number of normal flora found on skin
c.    Reduce the number of extraneous bacteria found on skin
d.    Increase the overall number of bacteria found on skin
e.    Increase the number of normal flora found on skin
f.     Increase the number of extraneous bacteria found on skin
g.    Help prevent the spread of disease
h.    Increase likelihood of bacterial resistance to antiseptics

9.    Use of waterless hand sanitizer
a.    Removes some normal flora
b.    Removes some extraneous bacteria
c.    Kills some normal flora
d.    Kills some extraneous bacteria
e.    Increases the overall number of bacteria found on the skin
f.     Reduces the overall number of bacteria found on the skin

10. Safranin is
a.    Used as the primary stain in the gram stain
b.    Used as the secondary stain in the spore stain
c.    Used as the primary stain in the acid-fast stain
d.    Used as the secondary stain in the negative stain
e.    An acidic dye
f.     a positively charged dye
g.    repelled by bacterial cells

11. Acid-fast cells:
a.    Are gram -, due to lipid content of walls
b.    Contain waxy mycolic acids in their wall
c.    Retain the primary dye when decolorized with 95% alcohol
d.    Retain the primary dye when decolorized with acid alcohol
e.    Is diagnostic because the organisms do not grow quickly in culture

12. In making a smear from a broth culture
a.    The needle is used for obtaining culture
b.    Water is placed on the slide prior to adding culture
c. the loop is used for obtaining culture


13. Failing to flame the loop after making a smear would
a.    Contaminate your culture
b.    Contaminate your desktop


SHORT ANSWER:  Briefly explain the following.
14. Describe the two reagents used in the capsule stain (name, charge), explain what each reagent stains, and describe the procedure.  Be sure to include the steps that are MISSING (when compared to the gram stain, for example), and why those steps must be omitted in order to see the spores.   @6pts.










15. Differentiate between magnification and resolution, and list the microscope parts and procedures that are responsible for increasing each.  @5pts.











16. You have an organism with large, oval, subterminal spores suspected based on your gram stain.  You were supposed to perform the spore stain, but you realize belatedly that you actually did the acid-fast stain on your organism instead. Will this work to see spores better?  Why or why not?  Draw below what you would actually see (colors, locations) when you view this slide. @6pts.














PRACTICAL QUESTIONS:  View the materials at each station to answer the following:

17. Name the microscope part at the pointer, and give its function.  @2pts.


18. Give the total magnification in use, showing calculations. @2pts.



19. Record the microbiological term for the shape of this organism. Is the dye used here basic or acidic?  @2pts.



20. Give the gram reaction, morphology, and arrangement of the organism shown.  @3pts.



21. Give the gram reaction, morphology, and arrangement of the organism shown.  @3pts.



22. What unusual substance is found in the walls of the pink bacilli, seen here stained with the acid-fast stain?  (Hint: responsible for its staining properties with this stain procedure.)  @2pts.



23. Record the characteristics of this particular organism's spores. (hint: 3 of them) @3pts.





24.  Explain why this plate has so many colonies present on it.  @2pts.

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