(P) Lec 2: Specimen Collection, Procedure and Fixatives Flashcards
Rupert-based
A laboratory section where we test urine and other bodily fluids are processed under the microscope
Clinical Microscopy section
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refers to the eggs stage of the parasites
Ova
This encompasses the other morphologic forms
Parasites
Microscopic examination
A. Collection
B. Transport
C. Fixatives
D. ALL
D
typical stool collection protocol consists:
A. 3 collected in 10 days
B. 5 collected in 14 days
A
Certain medications and substances may interfere with the detection of parasites, except:
A. antidiarrheal medicines
B. antacids
c. mineral oil
d. barium
nota
Stool is collected:
A. 3 and a half days after the completion of therapy
B. a week after antimalarial medications
C. a week after therapy
C
Specimen container:
A. has 4g of stool
B. made of waxed cardboard or plastic
both
Specimen container, except:
A. Travel history
B. Collector’s name
C. Address
D. Clinical findings
C
TOF. Urine contamination is not allowed as it may destroy some parasites.
T
recommended time frame for liquid stool within?
30 minutes
to preserve the stage of amoeba
stage of amoeba
invasive to the skin tissue and fast to die
trophozoites
TOF. Cysts live in watery environment to move and attack tissues.
F (tropho)
Incorrect holding time
A. Formed: 24 hrs
B. Semi-formed: 120 mins
C: liquid stool = 30 mins
B: should be within 1 hr
substances that preserve the morphology of protozoa and prevent further development/stages of certain helminth eggs and larvae
fixatives
ratio of fixative to stool specimens
3:1
hindi tatlong tae
Recovery
Formalin
PROTOZOA and HELMINTHS
protozoa—amoeba, flagelates, ciliated
helminths—nematodes, trematodes, and cestodes (worms)
incorrect concentrations of formalin are commonly used.
A. 5% ideally preserves protozoan cysts
B. 5% ideally preserves larvae
C. 10% concentration preserves helminths eggs
B
10% concentration preserves helminths eggs and larvae
preservative
not for permanent smears:
A. formalin
B. pva
C. schaudinn
A
Advantages of Formalin, except
A. Easy to prep
B. Long shelf life
C. For smear or staining
C
Disadvantages of Formalin
A. Trophozoites cannot be recovered
B. Morphologic details of cysts and eggs may fade
C. Can cause eye irritation or teary eye
All
PVA
A. act as an adhesive
B. useful for the fixation of intestinal protozoans, helminth eggs and larvae
A
PVA
A. preparation of a permanent stained smear
B. added to Schaudinn’s solution
both
A disadvantage of PVA because the solution contains mercuric chloride.
Schaudinn
PVA
A. Long shelf live when stored at cold temperature
B. Antigen test is positive
NOTA
A. warm temperature
B. negative
Methiolate AKA
timerosal
TOF. MIF is a 2 in 1 that acts as a staining and preservative, similar to formalin.
F (formalin is only a preservative)
MIF
A. easy to prepare
B. yields unsatisfactory results
C. easy identification of protozoans
B
useful for the fixation of intestinal protozoans, helminth eggs and larvae.
A. MIF
B. Formalin
C. PVA
A
Sodium Acetate Formalin
A. Toxic-free
B. Multiple vials
NOTA
Can be used for performing concentration techniques and permanent stained smears.
A. MIF
B. SAF
C. PVA
D. Formalin
B
SAF Advantages
A. protozoa is clear
B. addition of albumin to the microscope slide
C. long shelf-life
D. A & C
C
SAF Disadvantages
A. addition of albumin to the microscope slide
B. protozoa is not clear
Both
Alternative Single-Vial System
A. free of formalin
B. usef for concentration techniques
both
Alternative Single-Vial System
A. used for intestinal protozoans
B. free of mercury
C. used for permanent smears
D. B & C
E. A & C
D
Applicable Lab Procedures: Positive Concentration
Choose all that applies
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
All
Applicable Lab Procedures: Positive or negative Concentration
Choose all that applies
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
C & D
Applicable Lab Procedures:
Does not use permanent stain. Choose all that applies:
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
A
Applicable Lab Procedures:
Uses trichrome as a permenent stain. Choose all that applies:
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
C, D, E
Applicable Lab Procedures:
Only uses iron hematoxylin as a permanent stain. Choose all that applies:
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
B
Applicable Lab Procedures: Antigen test is negative
Choose all that applies:
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
C, D & E
Applicable Lab Procedures: Positive antigen tests.
Choose all that applies:
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
All except PVA
Applicable Lab Procedures: Positive or negative antigen test
Choose all that applies:
A. 10% formalin
B. SAF
C. PVA
D. Modified PVA
E. Single-vial system
D & E
Elements which may found in stool specimens
A. macrophages for amebic cysts
B. epithelial cells for trophozoites
C. animal hairs may look like trophozoites
B
Correct
A. macrophages for amebic trophozoites
B. epithelial cells for trophozoites
C. animal hairs may look like helminth larvae
- Is used to measure objects observed microscopically accurately.
- Is a disk that is inserted into the eyepiece of the microscope.
- Calibration is necessary
Ocular Micrometer
Ocular Micrometer
diagnostic stages of parasites detected microscopically are measured in units known as
A. nm
B. µm
C. mm
B. microns
Ocular Micrometer
TOF. Obsolete
T
Direct Wet Preparations
A. the presence of cysts is being detected
B. 0.85% saline solution (NSS) is the reagent of choice
B
A. the presence of motile protozoan trophozoites is being detected
suggested size for the glass slide
3x2
size of the square cover slip
22mm
Direct Wet Preparations
Trophozoites can be stained to demonstrate the nuclear morphology using?
Nair’s buffered methylene blue solution (BMB)
Nair’s buffered methylene blue solution (BMB)
A. Entamoeba cytoplasm stains pale blue
B. nucleus stains darker blue
both
TOF. If the specimen is received in fixative, the direct wet preparation can be done from the O&P procedure.
F (eliminated from the procedure)
the concentrate and permanent stain techniques are performed.
Objective
fixed or smear slides
OIO
Objective
fresh slides
LPO or HPO
Objective
pus cells or rbcs
HPO
Objective
epithelial cells or muscle fibers
LPO
Direct Wet Iodine Preparation
A. enhance the detail of the protozoan TROPHOZOITES
B. destroy the cysts stage of protozoan
NOTA
May be made to enhance the detail of the protozoan cysts.
It destroy the TROPHOZOITES stage of protozoan.
Direct Wet Iodine Preparation
Stain
Lugol’s or D’Antoni’s formula
Lugol’s or D’Antoni’s formula
A. cytoplasm will stain orange
B. nucleus will be pale and refractile
C. glycogen will be deep red
B
the cytoplasm will stain golden yellow, the nucleus will be pale and refractile, and the glycogen will be deep brown.
Direct Fecal Smear
A. Adapted from (WHO 1994).
B. Basic microscopic technique
Both
Kato-Thick Smear
stool is placed over a glass slide and covered with cut?
cellophane paper
Kato-Thick Smear
Weight of stool
50 to 60 mg of stool (approximately the size of two mongo beans)
Kato-Thick Smear
The cellophane paper is soaked in
A. glycerine
B. carbon fusin
A
glycerine and malachite green solution
Kato-Thick Smear
a clearing solution
glycerine
Kato-Thick Smear
used to give color to the cellophane in order to give a pale green background to the eggs and to minimize the brightness of the microscopic field
Malachite green
Kato-Thick Smear
can also be used if malachite green is not available
Green cellophane
Kato-Thick Smear
A. Ascaris and Trichuris
B. Hookworm
C. protozoan cyst and trophozoites
A
detecting eggs with thick shells such as in Ascaris and Trichuris
Kato-Thick Smear
TOF. If the preparation is too long before examination, hookworm eggs become too transparent or distorted making identification very difficult.
T
10-20 minutes is the preparation to best examined the smear
These techniques use differences in specific gravity and centrifugation to separate the parasites from fecal debris and increase their recovery.
Concentration Methods
Concentration Methods
A. might be detected using wet preparations
B. used for low concentration of parasites
C. be performed on fresh or preserved stool specimens.
D. trophozoites do not survive
All of the Above
Two types of Concentration methods
Sedimentation and floatation
Parasites are concentrated in the sediment of the tube following centrifugation; the parasite settles at the bottom
Sedimentation
the parasites are less dense than the solutions used and, during centrifugation, they float to the surface
Floatation
FEAS Procedure
A. Methyl acetate is added to a saline-washed formalin-fixed sample
B. Based on specific gravity
B
Formalin- Ethyl Acetate Sedimentation Procedure
Ethyl acetate is added to a saline-washed formalin-fixed sample and the tube is then centrifuged.
Formalin- Ethyl Acetate Sedimentation Procedure
Disadvantage:
A. Difficult to perform
B. Does not provide good recovery of most parasites
C. contains more fecal debris than a flotation technique
C
Acid Ether Concentration Technique (AECT)
A. Reagents are 20% HCl and Ether
B. also for animal stool
C. does not recover Trichuris, Capillaria, and trematode eggs especially Schistosoma
D. most widely used sedimentation technique.
B
AECT
A. possible destruction of protozoan cysts
B. loss of parasite to the plug of debris
Both
Formalin Ether Concentration (FECT)
A. recovery of both helminth eggs and protozoan cysts
B. uses 10% formalin
Both
FECT
A. Only uses formalin
B. More parasites can be recovered from formalin-preserved samples
B
Can also be done with formalin-preserved and PVA- preserved stools.
FECT
TOF. Parasite morphology is also better preserved in formalin than in PVA.
T
Formalin Ether Concentration (FECT)
creates 4 layers which are?
- ether and dissolved fats,
- fecal debrie,
- formol water
- and sediment
FECT
At what layer are the parasites present?
Sediment
Flotation Technique
based on differences in specific gravity between the sample debris, which in this case is heavy and sinks to the bottom of the test tube, and potential parasites, which are lighter and float toward the top of the tube
Zinc Sulfate Flotation Technique
Formol-Ether Sedimentation Technique was adapted from?
Cheesbrough 2009 (modified), WHO 1994
Zinc Sulfate Flotation Technique
Zinc sulfate with a specific gravity of
A. 1.130 - 1.200
B. 1.180 - 1.200
C. 1.200 - 1.480
B
Zinc Sulfate Flotation Technique
Main reagent
33% ZnSO4 solution is the main reagent. in 1000ml of water
Sheather’s Sugar Flotation
A. BEST for the recovery of trophozoites
B. use of a phase microscope
C. Boiled sugar solution preserved with formalin
B
With this procedure, visualization of oocysts can be better appreciated through the use of a phase microscope.
Sheather’s Sugar Flotation
Is considered as the BEST for the recovery of coccidian oocysts, mainly (3)
Cryptosporidium, Cyclospora and Isospora
Sheather’s Sugar Flotation
Boiled sugar solution preserved with?
phenol
Brine Flotation
A. saturated table salt solution
B. specimen directly mixed with brine solution
C. not useful for operculated eggs
All
Brine Flotation
A. Centrifugation is needed
B. hookworm and Schistosoma become badly shrunken
C. Useful for Clonorchis, and heterophyids
B
- Centrifugation is not needed since helminth eggs rise to the surface of the solution.
- Not useful for operculated eggs like Clonorchis, and heterophyids because these do not float in brine solution
final procedure in the O&P examination
A. Direct Wet Preparation
B. Concentration methods
C. Permanent Stains
C
as a microscope slide that contains a fixed sample that has been allowed to dry and subsequently stained
A. Smear
B. Permanent Stain
C. Direct Wet Iodine Preparation
B
