Introduction Flashcards
When focusing a microscope on a new slide/specimen one should always start with the?
a. 10x objective (low power)
b. 40x objective (high dry)
c. 50x objective (oil immersion)
d. 100x objective (oil immersion)
a. 10x objective (low power)
The process and goal of adjusting the microscope condensor to center the light path is called:
a. Aperture diaphragm
b. an annoying and useless bit of trivia
c. Micrometer calibration
d. Kohler illumination
d. Kohler illumination
To increase contrast of the microscopic view one can:
a. lower the condenser
b. open the aperture diaphragm
c. close the aperture diaphragm
d. A and C
d. A and C
When finished using a microscope one should always: Pick the BEST answer.
a. Clean all the objectives with a cotton swab moistened with lens cleaning fluid.
b. Clean the objectives, that had oil used with them, with a cotton swab moistened with lens cleaning fluid. You can use the same swab for more than one lens
c. Do not clean the lenses. Leave that for the next lab
d. Wipe the lenses with a Kimwipe
a. Clean all the objectives with a cotton swab moistened with lens cleaning fluid.
Objects under the microscope can be measured if one has an ocular micrometer scale installed in an eyepiece.
What each line represents in micrometers (um) must be calculated by using a slide (stage micrometer) with established distance markings.
Using the Ocular micrometer Standard Operating Procedure (SOP) reading assignment and the image Fig 1 at the end of the document How many micrometers does one ocular line represent?
a. 0.74 um
b. 7.4 um
c. 74.0 um
d. 1.35 um
e. 13.5 um
f. 135 um
b. 7.4 um
The two lines are superimposed at 27 ocular units on the ocular micrometer and 0.2 mm on the stage micrometer, therefore 27 ocular units = 0,2mm.
1 ocular unit is 0.2mm/27 = 0 ,0074 mm.
There are 1000 um in 1 mm therefore 0.00074 mm = 7.4um.
1 ocular unit = 7.4 um
From the lecture and from https://www.cdc.gov/dpdx/diagnosticprocedures/stool/specimencoll.html Links to an external site.
Stool specimens for microscopic parasite exam should be: (Pick the BEST answer)
a. Read immediately or placed in fridge
b. Read immediately or mixed with preservatives both PVA and 10% formalin
c. Read immediately or mixed with preservatives either PVA or 10% formalin
d. Read immediately or add to preservatives both PVA and 10% formalin. Mixing is not needed
b. Read immediately or mixed with preservatives both PVA and 10% formalin
According to the CDC page, given in the previous question, What protozoan stages tend to be seen the most in different stool specimen consistencies
a. The active motile trophozoite stages tend to be seen more in formed stools While the cysts are more predominant in watery stools.
b. The active motile trophozoite stages tend to be seen more in watery stools While the cysts are more predominant in formed stools.
c. The active motile trophozoite stages and the cysts stages are more predominant in formed stools.
d. The active motile trophozoite stages and the cysts stages are more predominant in watery stools.
b. The active motile trophozoite stages tend to be seen more in watery stools While the cysts are more predominant in formed stools.
It makes sense that motile (swimming) trophozoites would predominate in watery stools.
Also the cyst stage of protozoans are designed to survive in the environment to get to the next host so by the time an infected person is passing a stool (provided it is formed) most of the protozoans have converted to the cyst stage.
NOTE: This is why all stools should be put in the two preservatives PVA and 10% Formalin. Formalin preserves cysts well and also works best for helminth eggs. And while PVA preserves both cysts and trophs of protozoa it is not as good for helminth forms.
According to the CDC link given in the previous question, what things will make a stool sample unsatisfactory for microscopic examination:
a. Barium or bismuth compounds
b. Antimicrobial (anti-parasitic) agents
c. Specimen placed unpreserved in the fridge overnight
d. Stool is a very watery sample
e. all of the above
f. A, B and C
g. None of the above
f. A, B and C
Correct:
While very watery stools can be difficult to obtain enough solid material for a microscope prep they are not rejected
Match the following specimen types with the Methods/reagents used:
a. Formalin preserved specimen: flotation method
b. Formalin preserved specimen: sedimentation method
c. PVA preserved specimen
d. Unpreserved specimen
- Formalin Ethyl Acetate (FEA) concentration
- Trichrome stain
- ZnSO4 or Sheathers sugar concentration
- Immediate microscopic exam only
a. Formalin preserved specimen: flotation method: ZnSO4 or Sheathers sugar concentration
b. Formalin preserved specimen: sedimentation method: Formalin Ethyl Acetate (FEA) concentration
c. PVA preserved specimen: Trichrome stain
d. Unpreserved specimen: Immediate microscopic exam only
TRUE or FALSE. 10% Formalin and other stool preservatives guarantee that all parasitic forms are non-viable
FALSE
Some unhatched Ascarid eggs can survive in formalin for weeks.
Using the Trichrome Staining Procedure https://www.cdc.gov/dpdx/diagnosticprocedures/stool/staining.html Links to an external site.
Step one of the procedure (placing slide in 70% ethanol plus iodine) is required for which of the following preservatives:
a. formalin
b. PVA with Zinc (Zn)
c. PVA with mercuric chloride (Hg)
d. PVA with Copper (Cu)
e. B, C, and D
c. PVA with mercuric chloride (Hg)
Correct.
PVA is the preservative used for stool trichrome stains.
Only the PVA with mercury salts (Hg) need the iodine step.
This is because the iodine is needed to remove the many mercuric chloride crystals that would obscure the microscopic view.
What is the purpose for the Ethyl-Acetate reagent in the Formalin Ethyl Acetate (FEA) concentration method?
a. To remove fatty stool materials from the sample by making them more dense. The fatty debris will end up at the bottom of the tube (the first of 4 layers)
b. To remove fatty stool materials from the sample by making them less dense. The fatty debris will end up above the formalin solution. (the 3rd of 4 layers)
c. To dissolve unwanted bacteria
d. To make the procedure more dangerous, and therefore more exciting.
b. To remove fatty stool materials from the sample by making them less dense. The fatty debris will end up above the formalin solution. (the 3rd of 4 layers)
Using the Formalin Ethyl Acetate Sedimentation Concentration procedure at https://www.cdc.gov/dpdx/diagnosticprocedures/stool/specimenproc. Links to an external site.html
What is the correct order for the steps in concentrating a stool specimen by this method?
a. Add 0.85% saline or 10% formalin to bring the volume in the centrifuge tube to 15mL; then centrifuge at 500g for 10 minutes
b. mix specimen well
c. Add 4 mL of ethyl acetate stopper the tube and shake vigorously for 30 seconds; then centrifuge at 500g for 10 minutes
d. Free the plug of debris from the top of the tube by ringing the sides with an applicator stick. Decant the top layers of supernatant
e. Strain 5mL of the fecal suspension through wetted cheesecloth (This removes larger particles not needed in the microscopic exam)
f. Add several drops of 10% formalin to resuspend the concentrated specimen. Proceed with applicable testing
g. Decant supernatant. Add 10mL of 10% formalin to the sediment and mix throughly
- mix specimen well
- Strain 5mL of the fecal suspension through wetted cheesecloth (This removes larger particles not needed in the microscopic exam)
- Add 0.85% saline or 10% formalin to bring the volume in the centrifuge tube to 15mL; then centrifuge at 500g for 10 minutes
- Decant supernatant. Add 10mL of 10% formalin to the sediment and mix throughly
- Add 4 mL of ethyl acetate stopper the tube and shake vigorously for 30 seconds; then centrifuge at 500g for 10 minutes
- Free the plug of debris from the top of the tube by ringing the sides with an applicator stick. Decant the top layers of supernatant
- Add several drops of 10% formalin to resuspend the concentrated specimen. Proceed with applicable testing
Using the Trichrome Staining Procedure for PVA preserved stool sample slides found at https://www.cdc.gov/dpdx/diagnosticprocedures/stool/staining.html Links to an external site.
Place the following procedural steps in correct order:
a. Destain in 90% ethanol plus acetic acid for 1-3 seconds
b. Place slide in 70% ethanol for 5 minutes
c. Place in trichome stain for 10 minutes
d. Place the PVA slide in 70% ethanol plus iodine for 10 minutes (This step can be omitted if the PVA does NOT mercuric chloride)
e. Examine the smear microscopically utilizing the 100x objective. Examine at least 200-300 oil immersion fields. Ideally one should also spend a few minutes on low power as larger helminth eggs can be detected by this method
f. Place in two changes of 100% ethanol for 3 minutes each
g. Place in second 70% ethanol for 3 minutes
h. Mount slide with coverslip using mounting medium
i. Place in two changes of xylene or xylene substitute for 10 minutes
j. Rinse several times in 100% ethanol
- Place the PVA slide in 70% ethanol plus iodine for 10 minutes (This step can be omitted if the PVA does NOT mercuric chloride)
- Place slide in 70% ethanol for 5 minutes
- Place in second 70% ethanol for 3 minutes
- Place in trichome stain for 10 minutes
- Destain in 90% ethanol plus acetic acid for 1-3 seconds
- Rinse several times in 100% ethanol
- Place in two changes of 100% ethanol for 3 minutes each
- Place in two changes of xylene or xylene substitute for 10 minutes
- Mount slide with coverslip using mounting medium
- Examine the smear microscopically utilizing the 100x objective. Examine at least 200-300 oil immersion fields. Ideally one should also spend a few minutes on low power as larger helminth eggs can be detected by this method
When staining stool samples preserved in PVA fixative: What is/are the advantage(s) of the trichrome stain over the iron hematoxylin stain?
a. Easier to perform
b. The stain is poly-chromatic
c. Simpler procedure with less variability of results
d. All of the above
d. All of the above
Correct:
Due to these advantages most Parasitology labs will use Trichrome stain for PVA preserved stool samples
(By definition "Trichrome" stain is polychromatic. Iron Hematoxylin is monochromatic)
