Spore Stain
My Unknown: diploid cocci, negative gram stain (pink) Neisseria
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.)- 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.
- 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.
- 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
- What are some of the unique safety rules of the Microbiology lab? Give the rationale for each.
- Differentiate between resolution and magnification, and describe the microscope parts and techniques which increase each.
- Why are basic dyes used bacteriological stains?
- Why is heat-fixing a bacterial smear necessary?
- List all of the steps (in order) to the preparation of a bacterial stain from a slant culture.
- 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.
- 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)
- 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
- 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.
- 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
- 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
- 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.
- 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****
**** 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.)- 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.
- What is a bacterial colony, and why are colony numbers used to extrapolate numbers of bacteria in an area sampled?
- 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.
- What are some of the unique safety rules of the Microbiology lab? Give the rationale for each.
- Differentiate between resolution and magnification, and describe the microscope parts and techniques which increase each.
- Why are basic dyes used bacteriological stains?
- Why is heat-fixing a bacterial smear necessary?
- 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
- 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.
- 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?
- Why is heat necessary for the spore stain and the acid-fast stain, but not for the gram stain?
- Why are negative stained slides not washed, but placed in a tumbler of disinfectant?
- Why is the capsule stain referred to as a combination negative-positive stain?
- If a positive acid-fast stain is made from bacteria found in a skin lesion, what disease would you suspect? Why?
- Why do you never heat-fix a smear to be used for a capsule stain?
- 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?
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.
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.)
(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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Heat-fixing would fry or boil away the mucoid capsule, and therefore you would not be able to see the capsule on a stain.
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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