DIAGNOSTIC PARASITOLOGY Flashcards
Liquefy 5 g of phenol crystals with a small amount of distilled water, using a warm water bath at 95 celcius. Dissolve 1 g of basic fuchsin in the liquefied phenol. Add 10 ml of 95% ethanol and mix. Add 100 ml of distilled water. Filter and store in a dark flask, well labelled. The solution is ready for use.
Common Reagents and Preparations
Carbol Fuchsin
50 mL formaldehyde + 950 mL distilled water or saline (recommended for all-purpose use and for preservation of protozoan cyst).
Common Reagents and Preparations
Formalin 5%
100 mL formaldehyde + 950 mL distilled water or saline (recommended for helminth eggs and larvae)
Common Reagents and Preparations
Formalin 10%
2 g potassium iodide (KI) + 1.5 g powdered iodine crystals (add after KI dissolves) + 100 mL distilled water. Store in a brown, glass-stoppered bottle at room temperature and in the dark; the expiration date is 1 year. The solution is ready to use. For routine use, put 20 mL in a brown dropper bottle for 10-14 days.
Common Reagents and Preparations
Lugol’s solution
Sodium acetate 1.5 g + acetic acid, glacial 2.0 mL + formalin 4 mL + distilled water 92.0 mL
Common Reagents and Preparations
SAF (sodium acetate-acetic acid-formalin fixative):
simplest and easiest technique to facilitate detection
of intestinal parasites that infected subjects pass in
their feces
DIRECT FECAL SMEAR
In Direct Fecal Smear, presence of _____________ or _________ can be observed directly with a light microscope
intestinal protozoa (trophozoites or cysts)
or helminth eggs
In Direct Fecal Smear, small amount of fresh feces is mixed with either ___________ or __________.
- saline
- lugol/iodine solution
small amount of fresh feces is mixed with _________ (to detect the protozoa motility)
saline
small amount of fresh feces is mixed with _________ (to reveal the parasite structure).
lugol/iodine solution
What can you see in a Iodine Preparation
cysts of protozoa
What can you see in a Saline Preparation
- motile trophozoites and larvae
- red blood cells
- Leukocytes
- Charcot–Leyden crystals
Types of preparation
- Saline Preparation
- Iodine Preparation
When examining diarrheic or liquid feces containing mucus, both preparations should be
applied to the _________ of the stools.
mucous part
- Place 1 drop of saline on the ______ of the slide and 1drop of Lugol’s iodine solution on the _______ of the
slide
PROCEDURE FOR DFS
- left side
- right side
- Take about ____ of faecal specimen (the amount picked up on the end of an applicator stick) and thoroughly emulsify the stool in the _________
PROCEDURE FOR DFS
- 2 mg
- drop of saline
- Place a coverslip on each suspension touching the edge of the drop, then gently lower the coverslip onto the slide so that no _________ are produced
PROCEDURE FOR DFS
air bubbles
- Examine with a ___________.
PROCEDURE FOR DFS
microscope
Recommended for monitoring large-scale treatment
programmes implemented for the control of soiltransmitted helminth infections because of its simple format and ease of use in the field.
Kato-Katz Technique
Manner of Reporting in Kato-Katz Technique
Eggs per gram stool (epg)
Quantify eggs and establishes burden of intestinal infection
Kato-Katz Technique
Kato-Katz Technique Materials and reagents
- Wooden applicator sticks
- screen
- Template
- Spatula
- Microscope slide
- Hydrophillic cellophane
- Flat-bottom jar with lid, forceps and toilet paper
- Newspaper
- Glycerol-malachite green
A hole of 9 mm on a 1 mm thick template will deliver about how many mg of faeces?
Kato-Katz Technique
50 mg
A hole of 6 mm on a 1.5 mm thick template will deliver about how many mg of faeces?
Kato-Katz Technique
41.7 mg
A hole of 6.5 mm on a 0.5 mm thick template will deliver about how many mg of faeces?
Kato-Katz Technique
20 mg
Size of the hydrophilic cellopane for kato-katz
Kato-Katz Technique
40-50 um thick
25 x 30 or 35 mm
In preparation of Glycerol-malachite green, 1 mL of ______________ is added to 100 ml of __________ and 100 mL of ___________ and mix well.
Kato-Katz Technique
- 3% aqueous malachite green
- glycerol
- distilled water
- Prepare all the necessary materials:
Kato-Katz Technique Step by Step Procedure
- template
- nylon screen
- slide
- wooden stick
- Place a small amount of the fecal sample on a ____________ and press a piece of nylon screen on top. Using a _________, scrape the sieved fecal material from the screen.
Kato-Katz Technique Step by Step Procedure
- newspaper
- spatula
- Label a glass slide with the ________ and place a template with hole on the center of a microscope slide. Fill the hole in the template with the sired fecal material, avoiding air bubbles and levelling the feces off to remove any excess material.
Kato-Katz Technique Step by Step Procedure
sample number
- Carefully lift off the template and place it in a bucket of water mixed with _____________ and ____________ so that it can be reused.
Kato-Katz Technique Step by Step Procedure
- concentrated detergent
- disinfectant
- Place one piece of cellophane, which has been soaked overnight in __________, over the fecal sample
Kato-Katz Technique Step by Step Procedure
glycerol solution
- Invert the microscope slide and firmly press the sample against the __________ on another microscope slide or on a smooth hard surface to spread the feces in a circle
Kato-Katz Technique Step by Step Procedure
cellophane strip
- Carefully pick up the slide again by gently sliding it sideways to avoid separating the cellophane strip or lifting it off. Place the slide on the bench with the cellophane ________. Water evaporates while ________ clears the feces. When clarified it should be possible to read newspaper print through the stool smear
Kato-Katz Technique Step by Step Procedure
- upwards
- glycerol
For all except hookworm eggs, keep the slide for____________at _________ to clear the fecal material prior to examination under the microscope.
Kato-Katz Technique
- one or more hours
- room temperature
To speed up clearing and examination, the slide can be
placed in a __________ or kept in direct sunlight for
several minutes.
Kato-Katz Technique
40 °C incubator
_________ and __________ will remain visible
and recognizable for many months.
Kato-Katz Technique
- A. lumbricoides
- T. trichiura eggs
Hookworm eggs clear rapidly and will no longer be
visible after ____________
Kato-Katz Technique
30–60 minutes.
___________ may be recognizable for up to several months but it is preferable to examine the slide preparations within 24 hours.
Kato-Katz Technique
Schistosome eggs
The smear should be examined ____________.
Kato-Katz Technique
systematically
Then, multiply by the appropriate FACTOR to give the number of eggs per gram of feces
____ if using a 50 mg template
____ if using a 41.7 mg template
____ if using a 20 mg template
Kato-Katz Technique
- x 20
- x 24
- x 50
Light intensity infections of Ascaris lumbricoides
Classification of Intensity of Infection
1- 4,999 epg
Moderate intensity infections of Ascaris lumbricoides
Classification of Intensity of Infection
5,000 - 49,999 epg
Heavy intensity infections of Ascaris lumbricoides
Classification of Intensity of Infection
> 50,000 epg
Light intesity infections of Trichuris trichiura
Classification of Intensity of Infection
1 - 999 epg
Moderate intensity infections of Trichuris trichiura
Classification of Intensity of Infection
1,000 - 9,999 epg
Heavy intensity infections of Trichuris trichiura
Classification of Intensity of Infection
> 10,000 epg
Light intensity infections of Hookworms
Classification of Intensity of Infection
1 - 1,999 epg
Moderate intensity infections of Hookworms
Classification of Intensity of Infection
2,000 - 3,999 epg
Heavy intensity infections of Hookworms
Classification of Intensity of Infection
> 4,000 epg
Light intensity infections of Schistosoma mansoni
Classification of Intensity of Infection
1 - 99 epg
Moderate intensity infections of Schistosoma mansoni
Classification of Intensity of Infection
100 - 399 epg
Heavy intensity infections of Schistosoma mansoni
Classification of Intensity of Infection
> 400 epg
2 types of blood film for malaria parasites
- Thick blood smear
- Thin blood smear
Blood smear used to determine if parasite is present
Thick
Blood smear used to confirm the Plasmodium species present
Thin
Lysed RBCs
Thick or Thin Blood Smear
Thick Film
larger volume
Thick or Thin Blood Smear
Thick Film
0.25 ul blood/100 fields
Thick or Thin Blood Smear
Thick Film
blood elements more concentrated
Thick or Thin Blood Smear
Thick Film
good screening test
Thick or Thin Blood Smear
Thick Film
positive or negative
Thick or Thin Blood Smear
Thick Film
parasite density
Thick or Thin Blood Smear
Thick Film
more difficult to diagnose species
Thick or Thin Blood Smear
Thick Film
fixed RBCs, single layer
Thick or Thin Blood Smear
Thin Film
smaller volume
Thick or Thin Blood Smear
Thin Film
0.005 ul blood/100 fields
Thick or Thin Blood Smear
Thin Film
good species differentiation
Thick or Thin Blood Smear
Thin Film
requires more time to read
Thick or Thin Blood Smear
Thin Film
low density infections can be missed
Thick or Thin Blood Smear
Thin Film
To quantify malaria parasites against RBCs, count the parasitized RBCs among ____________ on the thin smear and express the results as % parasitemia.
500-2,000 RBCs
% parasitemia =
= (parasitized RBCs/total RBCs) × 100
If the parasitemia is high [e.g., > 10%], examine _______
500 RBCs
If it is parasitemia is low [e.g., <1%] ,examine ___________
2,000 RBCs (or more)
In Quantifying Malarial Parasites, Count ___________ and _________ separately.
- asexual blood stage parasites
- gametocytes
Only the asexual blood stage parasites are clinically important and gametocytes of ____________ can persist after elimination of asexual stages by drug treatment.
P. falciparum
In Quantifying Malarial Parasites againsts WBC, what microscope lens is used?
100x oil immersion lens
In Quantifying Malarial Parasites againsts WBC, select area with _______
10-20 WBCs per field
In Quantifying Malarial Parasites againsts WBC, count the number of _________ and ______________ in the same fields on thick smear.
- asexual parasites
- white blood cells
In Quantifying Malarial Parasites againsts WBC, you need to count how many WBCs
> 200 WBCs
In Quantifying Malarial Parasites againsts WBC, assume WBC is ____________
8000/ul
parasites per ul =
Parasites counted/WBC counted x WBC count/ul
Cellophane swab is used for the diagnosis of:
- Enterobiasis
- Taeniasis
Enterobiasis caused by a nematode called?
Enterobius vermicularis
Taeniasis caused by tapeworm called?
Taenia solium or Taenia saginata
Cellophane Swab is Specimen of choice for the detection of _____________
Enterobius
vermicularis (pinworm) eggs
Adult female pinworms may also be seen in __________
Cellophane Swab
At night, when the body is at rest, gravid (pregnant) adult female worms exit the host, typically a child, through the rectum and lay numerous eggs in the
________________.
perianal region
Time of Collection for Cellophane swab
Early in the morning before the
patient washes or defecates.
In cellophane swab, standard protocol for specimens collected daily for the number of negative tests that should be performed to rule out a pinworm infection is ______
5
Each worm lay about how many eggs?
10,000 - 11,000 eggs
The eggs of Enterobius vermicularis can spread everywhere and become infective in ________
4 - 6 hours
In cellophane swab, ____________ may need to explain the procedure to patients, their families, and/or other health care professionals.
Laboratory technicians
In cellophane swab, when instructing others, it is also critical to emphasize the importance of exercising ____________ and ___________ during specimen collection to avoid spreading infectious eggs into the environment
- proper hygiene
- preventive measures
In Adhesive tape test, what side of the tape to you press on the slide?
Sticky side
These procedures allow for the detection of parasitic elements that may be missed when examining only a direct wet smear.
Concentration (sedimentation and flotation)
This procedure leads to recovery of ll protozoan cyst and oocysts, helminth eggs and larvae present in the stool specimen
Formalin - ethyl acetate sedimentation concentration
It is recommended as being the easiest to perform and the least subject to technical error, allowing recovery of the broadest range of parasitic elements.
Formalin - ethyl acetate sedimentation concentration
The specimen can be fresh or fixed stool
Formalin - ethyl acetate sedimentation concentration and concentration by flotation
This preparation will often contain more debris than that obtained with the flotation and other procedures
Formalin - ethyl acetate sedimentation concentration
Formalin - ethyl acetate sedimentation concentration is not reccomended for ______________________ and ____________________.
- eggs of Fasciola spp.
- larvae of Strongyloides stercoralis
In concentration by sedimentation, mix about ____ of faeces with _____ of fixative (SAF or fomalin 5-10%), and leave for at least _______.
- 1 g
- 10 ml
- 30 minutes
In concentration by sedimentation, strain the suspension into a ____________ through a sieve or double layer of gauze allocated into a small funnel and centrifuge at _______ for __________
- 15 ml conical tube
- 500 g
- 10 minutes
In concentration by sedimentation, the contenst in the tube will separate into four layers:
- sediment (contain parsitic elements)
- saline
- plug of faecal debris
- Top layer of ethyl acetate (or ether or gasoline)
This technique allows separation of parasitic elements fromo the coarsest organic debris, using a high specific density flotation solution.
Concentration by flotation
Eggs, cysts and oocysts, with a specific density lower than the flotation solution, will rise to the top of the suspension
Concentration by flotation
The most widely used flotation solution are:
- zinc sulfate solution
- sodium chloride
Heavy eggs such as those of _____________ or _____________ not efficiently concentrated with Concentration by flotation
- Fasciola
- infertile Ascaris eggs
In Concentration by flotation, eggs and cyst tend to lose their typical shape after ________
40 - 60 minutes
In Concentration by flotation, how many grams of stool do you need to use?
3 g
In concentration by flotation, what fixative can you use?
Formalin 5-10%
How many g and how long is the first centrifuge of concentration by sedimentation?
- 500 g
- 10 mins
How much saline will you add to the sediment after the first centrifuge in concentration by sedimentation?
7 mL
How much ethyl acetate will you add to the sediment after adding saline and mixing in concentration by sedimentation?
3 mL
In concentration by sedimentation, after adding ethyl acetate, how long will you shake it vigorously?
30 seconds
How many g and how long will you centrifuge the second time in concentration by sedimentation?
- 500 g
- 3 mins
After the second centrifugation, what are the layers you need to pour off?
concentration by sedimentation
Top 3 layers
What g and how long is the first centrifuge in concentration by flotation?
- 1500 g
- 3 mins
after the second centrifugation how much flotation solution will you use to resuspend the sediment?
concentration by flotation
10 mL
How many g and how long is the third centrifugation
concentration by flotation
- 800-1000 g
- 5 mins
Where will you harvest the parasites in concentration by flotation?
upper part of the meniscus
What will you use to harvest the upper part of the meniscus in concentration by flotation?
cover slip
