Experiment No. 4 - ROUTINE STOOL EXAMINATION: MICROSCOPIC Flashcards
The microscopist should be familiar with the different structures found in the feces such as
trophozoites and cysts of Amoeba , helminth eggs and larvae macrophages, WBC, fungi, plant cells (pollen grains and spo , RBC, res), epithelial cells, crystals like Calcium oxalate and triple phosphate, bacteria, plant fibers, root hairs and animal cells are similar to helminth ova.
Most common errors in the identification of parasites may be due to the following:
(1) Lack of familiarity with the parasites
(2) Cursory examination of the slide
(3) Confusion of parasite with artifacts.
This is the simplest and moderately efficient procedure for examinations of feces.
Direct Fecal Smear (DFS) or Direct Wet Mount Technique
The (?) of the intestinal tract usually results in an even distribution of parasites in stool.
mixing action
DFS preparations from (?) specimens should be mandatory.
soft, loose and watery
It is highly recommended for the detection of (?) in liquid, diarrheic and bloody mucoid specimens, however, if the number of organisms are few, this method may be of low sensitivity and insufficient to reveal their presence.
motile trophozoites
Direct Fecal Smear (DFS) or Direct Wet Mount Technique
- Comminute approximately (?) of feces with a drop of NSS on a clean slide using an applicator stick until a uniform suspension without fibers or gritty materials obtained.
- Place the cover slip above the preparation and immediately examine under the LPO of the microscope in a (?) (figure A). Confirm identification under HPO.
- Wet mounts may be preserved for several hours by ringing the cover glass with (?).
2 mgsy
stematic method
paraffin or nail polish
The amount of feces used for the direct mount is important. The (?) recommended approximates what would form a low cone at the end of a wooded applicator stick.
2 mg
When less than 2 mg of feces is used, the suspension will be (?) and may have (?), whereas the use of more than 2 mg results in a suspension that is and parasites may be (?).
too thin
blank spaces
too thick
hidden under fecal debris
(?) may be used with wet mount preparations to aid in location and identification of protozoa but are not necessary for eggs and larvae.
Temporary stains
(?) are the most commonly used temporary stains and are most useful for recognition of cyst stage; however, they kill and distort the trophozoites.
Dilute iodine solutions
In cysts, the visibility of (?) is enhanced so that their number and morphological features are more clearly seen.
nuclei
The use of (?) is not recommended since they do not stain organisms well.
weak iodine solutions
Likewise, the use of an (?) will stain the organisms so darkly that morphological features cannot be seen.
iodine solution that is too strong
Dilute iodine solutions that are recommended are (?).
D’ Antoni’s, Dobell and O’Connor’s and Lugol’s
This technique is based on the fact that when a layer of stool is in contact with glycerin-soaked cellophane for a period of time, it becomes clarified and helminth eggs become visible.
Kato - Thick Smear Preparation
Kato - Thick Smear Preparation
- Place a pea-size stool sample at the center of glass slide and cover with a square piece of (?).
- With the aid of a (?), press the cellophane gently to spread the stool, taking care that the specimen does not spread beyond the cellophane cover, the cellophane also serves as a cover slip.
- Leave the prepared slide at room temperature for (?). During this time the microscopic field becomes clear due to the action of glycerin on the stool constituents.
- The slide should be examined (?). Allowing the slide to stand for long period of time will cause drying and shells of hookworm ova will become be difficult to see.
pre-treated cellophane
rubber stopper
10-20 minutes
after 10-20 minutes or within one hour after preparation
virtually ensures the detection of even small numbers of organisms that would go undetected if only direct smears of permanent-stained slides were examined.
Formalin - Ether Concentration Technique (FECT)
Concentration procedures generally fall into two categories - (?).
flotation and sedimentation
(?) can be achieved by simple gravity or centrifugation.
Sedimentation
Since the sediment will generally contain all the parasites occurring in the stool sample, this procedure has greater diagnostic sensitivity.
Sedimentation
An added advantage is that the technique can be readily applied to both fresh and preserved feces.
Sedimentation
Formalin - Ether Concentration Technique (FECT)
- Thoroughly comminute approximately [?] of fresh feces in [?] in a suitable container. Let it stand for [?] or longer to achieve adequate fixation. For very loose or watery fecal sample, use [?] of material.
- Strain the suspension through two layers of wet gauze and pour into a [?] conical centrifuge tube.
- Fill tube with NSS; centrifuge at [?].
- Discard the supernatant if [?]; the sediment should be resuspended then centrifuged again using NSS. Proceed to the next step only after the supernatant is clear.
- Resuspend the sediment in 10% formalin to a volume of [?]; add [?] of ether (or ethyl acetate) then shake vigorously for [?]. If ether is used, hold tube with stopper directed away from face.
- Centrifuge at [?]. .When tube is removed, it will be seen to consist of four layers
- Decant tube, discard [?], mix small amount of fluid left with the remaining sediment and pipette out.
- Prepare [?] for examination
1.0 to 1.5 g
10 ml 10% formalin
30 minutes
5-6 ml
15 ml
400 - 500 g for 1 – 2 minutes
cloudy
10 ml
3 ml
30 seconds
400 - 500 g for 2 -3 minutes
iodine and unstained wet mounts
iodine and unstained wet mounts
iodine and unstained wet mounts
top 3 layers
When tube is removed, it will be seen to consist of four layers:
a. a top layer of ether
b. a plug of debris adhering to the wall
c. a layer of formalin
d. sediment for examination
Recently, concern over storage and use of ether, a potentially flammable and explosive material, has led to the use of (?) as a substitute.
ethyl acetate
(?) appears to be somewhat more efficient than ether when specimens contain Taenia or Hymenolepis nana eggs or Giardia cysts since there are fewer tendencies for these eggs or cysts to become trapped in the plug of debris.
Ethyl acetate
However, one disadvantage is that ethyl acetate, when used as a solvent for fresh feces, is not as efficient as ether in (?) which may interfere with the microscopic examination of the sample
extraction of fatty or mucoid materials
A. Direct Fecal Smear (DFS) or Direct Wet Mount Technique
Simplified:
● [?] feces to a glass slide
● Add [?] then mix w/ applicator slip then cover w/ a coverslip
● View under [?]; [?] for parasite
● Count by [?]. Take a picture of each field
2 mg
NSS
LPO; HPO
battlement method
B. Kato - Thick Smear Preparation
Simplified:
●[?] feces to a glass slide
● Use.a [?]
● examine after [?]
2 mg
single glycerin strip
10-20 mins
C. Formalin - Ether Concentration Technique (FECT)
Simplified:
● [?] feces to a test tube w/ cap; [?] for watery stool
● add [?] to preserve
● stand for [?]
● strain suspension in [?] of wet gauze
● put in a [?]
● fill tube w/ NSS until capacity of the tube (?)
● centrifuge for [?]
● discard supernatant (liquid NSS) if [?] by decanting unit NSS is clear
● resuspend sediments in [?]
● add [?] of ether
● shake for [?] vigorously
● centrifuge for [?]
● examine [?] (ask to sign)
● decant the tube, discard [?], leave fecal material
● prepare [?] for examination (1 drop of iodine and NSS)
1-1.5 grams; 5-6 teaspoon
10 mL % formalin
30 mins
2 layers
centrifuge tube w/ cap
15 mL
1-2 mins
cloudy
10% formalin
3 ml
30 secs
4 layers
3 layers
iodine and unstained wet mount
