BLOOD SAMPLES

Safety
Follow universal precautions for the prevention of bloodborne pathogens when working with human
serum and other body fluids. These include:

Wear personal protective equipment such as safety glasses, gloves, laboratory coats.

If you have cuts or abrasions on the skin of your hands, cover them with adhesive dressing.

Use needles and lancets only once, and dispose of them in a "sharps" container for
decontamination.

Remove gloves and wash your hands after completing any task involving the handling of
biological material.

For more information on safety, visit the website of the Centers for Disease Control and
Prevention to view biosafety guidelines or the website of the Occupational Safety and Health
Administration (OSHA) .

Specimen Collection
Timing:
Whenever possible, specimens should be collected before treatment is initiated. When malaria and
babesiosis are suspected, blood smears should be obtained and examined without delay. Since the
parasitemia may fluctuate, multiple smears might be needed. These can be taken at 8 to 12 hour
intervals for 2 to 3 days.
Microfilariae exhibit a marked periodicity depending on the species involved, therefore the time of
specimen collection is critical. If a filarial infection is suspected, the optimal collection time for
demonstrating microfilariae is:



Loa loa—midday (10 AM to 2 PM)
Brugia or Wuchereria—at night, after 8 PM
Mansonella—any time
Onchocerca—any time

Type of Sample:
Venous blood samples provide sufficient material for performing a variety of diagnostic tests, including
concentration procedures (filariasis, trypanosomiasis). However, in some parasitic diseases (e.g., for
diagnosis of malaria in particular), anticoagulants in the venous blood specimen can interfere with
parasite morphology and staining characteristics; this problem can be further compounded by
excessive delays prior to making the smears. In such cases, capillary blood samples are preferable. If

PCR is required, please refer to the molecular diagnosis section for appropriate blood collection
procedures.
Capillary blood obtained by fingerstick:
1.
2.
3.

Label pre-cleaned slides (preferably frosted-end) with the patient's name (or other identifier)
and date and time of collection.
Clean the site well with alcohol; allow to dry.
Prick the side of the pulp of the 3rd or 4th finger (alternate sites include ear lobe, or in infants
large toe or heel).

4.

Wipe away the first drop of blood with clean gauze.

5.

Prepare at least 2 thick smears and 2 thin smears.

Venous blood obtained by venipuncture:
1.
2.
3.
4.

Label collection tubes and pre-cleaned slides (preferably frosted-end) with the patient's name
(or other identifier) and date and time of collection.
Clean the site well with alcohol; allow to dry.
Collect the venous blood in a vacuum tube containing anticoagulant (preferably EDTA);
alternatively, collect the blood in a syringe and transfer it to a tube with anticoagulant; mix well.
Prepare at least 2 thick smears and 2 thin smears as soon as possible after collection.

Specimen Processing
Preparing Blood Smears
If you are using venous blood, blood smears should be prepared as soon as possible after collection
(delay can result in changes in parasite morphology and staining characteristics).

Thick smears
Thick smears consist of a thick layer of dehemoglobinized (lysed) red blood cells (RBCs). The blood
elements (including parasites, if any) are more concentrated (app. 30×) than in an equal area of a
thin smear. Thus, thick smears allow a more efficient detection of parasites (increased sensitivity).
However, they do not permit an optimal review of parasite morphology. For example, they are often
not adequate for species identification of malaria parasites: if the thick smear is positive for malaria
parasites, the thin smear should be used for species identification.
Prepare at least 2 smears per patient!
1.

Place a small drop of blood in the center of the pre-cleaned, labeled slide.

2.

Using the corner of another slide or an applicator stick, spread the drop in a circular pattern
until it is the size of a dime (1.5 cm2).

3.

A thick smear of proper density is one which, if placed (wet) over newsprint, allows you to
barely read the words.

4.

Lay the slides flat and allow the smears to dry thoroughly (protect from dust and insects!).
Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during
staining. The risk is increased in smears made with anticoagulated blood. At room temperature,
drying can take several hours; 30 minutes is the minimum; in the latter case, handle the smear
very delicately during staining. You can accelerate the drying by using a fan or hair dryer (use cool
setting). Protect thick smears from hot environments to prevent heat-fixing the smear.

5.

Do not fix thick smears with methanol or heat. If there will be a delay in staining smears, dip
the thick smear briefly in water to hemolyse the RBCs.

Scratch Method for Thick smears
The scratch method is an alternate method for making thick films that allows for improved adherence
and faster turnaround times. The process is similar to making a normal thick film, but instead of using
a stick to spread the blood, the edge of a glass microscope slide is used, while applying firm pressure
to create small scratches in the underlying slide. The scratches allow for improved adherence of the
blood film to the slide without affecting the smear morphology. The smear can then be stained as soon
as it is dry, generally within 20-30 minutes of smear preparation.
Reference: Norgan AP, Arguello HE, Sloan LM, Fernholz EC, Pritt BS. Malaria Journal 2013; 12: 231.

Thin smears
Thin smears consist of blood spread in a layer such that the thickness decreases progressively toward
the feathered edge. In the feathered edge, the cells should be in a monolayer, not touching one
another.
Prepare at least 2 smears per patient!
A thin smear being prepared.

1.
2.
3.

Place a small drop of blood on the pre-cleaned, labeled slide, near its frosted end.
Bring another slide at a 30-45° angle up to the drop, allowing the drop to spread along the
contact line of the 2 slides.
Quickly push the upper (spreader) slide toward the unfrosted end of the lower slide.

4.

Make sure that the smears have a good feathered edge. This is achieved by using the correct
amount of blood and spreading technique.

5.

Allow the thin smears to dry. (They dry much faster than the thick smears, and are less
subject to detachment because they will be fixed.)

6.

Fix the smears by dipping them in absolute methanol.

Note: Under field conditions, where slides are scarce, national malaria programs (and CDC staff)
prepare both a thick and a thin smear on the same slide. This works adequately if one makes sure
that of the two smears, only the thin smear is fixed.

Special Procedures for Detecting Microfilariae
Blood microfilariae:
A.

Capillary (fingerstick) blood
Since microfilariae concentrate in the peripheral capillaries, thick and thin smears prepared from
fingerstick blood are recommended.

B.

Anticoagulated (EDTA) venous blood (1 ml) should be concentrated by one of the following
methods:
1.

Centrifugation (Knott's technique)
a.
b.

c.
d.
2.

Prepare 2% formaldehyde (2 ml of 37% formaldehyde + 98 ml H2O).
Mix 9 ml of this 2% formaldehyde with 1 ml of patient's venous blood.
Centrifuge at 500 × g for 10 minutes; discard supernatant. Sediment is composed of
WBCs and microfilariae (if present).
Examine as temporary wet mounts.
Prepare thick and thin smears; allow to dry; dip in absolute methanol before
Giemsa staining to enhance staining of microfilariae.
Filtration

a.

Place Millipore® or Nucleopore® membrane filter (5 µm pore) in filter holder
with syringe attachment.

b.

Mix 1 ml of venous blood (in EDTA) with 10 ml of 10% Teepol® 610 (Shell
Co.); allow to stand for several minutes to allow lysis; transfer to a 10 ml Luer-Loc®
syringe; attach the filter apparatus.

c.

Force the solution through the 5 µm pore filter, followed by several syringes of
water to wash out the remaining blood, then 1 or 2 syringes full of air to clear excess fluid.

d.

Prepare a temporary wet mount by removing the filter and placing it on a glass
slide, adding a drop of stain or dye and a coverslip.

e.

For permanent preparations, pass 2 to 3 ml of methanol through the filter
while it is still in the holder; remove filter and dry it on a glass slide; then stain it with
Giemsa stain, horizontally (so that the filter does not wash off the slide); coverslip filter
before examining.

Shipping blood smears for microscopic examination
1.

Place labeled dried blood smears (stained and unstained) in a slide box, with grooves to
separate the slides.

2.

Pack the slide box inside another box, cushioned so that the slides are protected from
breakage.

3.

Include information:
a.

Submitter's name, address and phone number

b.

Physician's name, address and phone number

c.

Patient's name, travel history (places and dates) and treatment information

d.

Specimen collection date

e.

What organism is suspected

4.

Ship via mail or package carrier.

Shipping whole blood for isolation of parasites (culture or animal inoculation),
molecular diagnosis or drug level
1.

Place labeled tube of anticoagulated (EDTA) blood in enough absorbent material to contain any
leakage, and place in a sealed plastic bag or 50 ml screw cap centrifuge tube.

2.

Pack this bag or container in a box, cushioned so that the blood tube is protected from
breakage.

3.

4.

Include information:
a.

Submitter's name, address and phone number

b.

Physician's name, address and phone number

c.

Patient's name, travel history (places and dates) and treatment information

d.

Specimen collection date

e.

What tests are requested and what organisms are suspected

Ship via courier or overnight delivery to permit optimum recovery of parasites; refrigeration
during shipment may be necessary and should be discussed beforehand with the receiving
laboratory.

Staining
Staining Blood Smears
Stain only one set of smears, and leave the duplicates unstained. The latter will prove useful if a
problem occurs during the staining and/or if you wish later to send the smears to a reference
laboratory.

Wright (Wright-Giemsa) stain
Used in hematology, this stain is not optimal for blood parasites. It can be used if rapid results are
needed, but should be followed up when possible with a confirmatory Giemsa stain, so that Schüffner's
dots can be demonstrated.

Giemsa stain
Recommended for detection and identification of blood parasites.
1.

Stock 100× Giemsa Buffer - 0.67 M

2 µm pore). Stable at room temperature for one month. pH 7. Sterile buffer is stable at room temperature for one year. swirling to mix. 5.0.0 mm Absolute methanol. Triton X-100 5% Deionized water (warmed to 56°C) Triton X-100 6. Check pH before use. in the order listed. Put into a 500 ml brown bottle the glass beads and the other ingredients. Working Giemsa Buffer .Na2HPO4 NaH2PO4H2O Deionized water 2. . Should be 7. acetone-free Giemsa stain powder (certified) Glycerol a. Prewarm the deionized water and slowly add the Triton X-100. . Use glassware that is clean and dry. but the following formulation gives more constant results and does not expire) Glass beads. Screw cap tightly. 3. Stock Giemsa stain (Giemsa stain is available commercially. Autoclave or filter-sterilize (0.2 Stock Giemsa Buffer Deionized water 4.2.0067M. 7. 3.

Just before use. Since good quality control smears are not available commercially. 4.5% Giemsa stain. b. d. Note: As alternates to this 45-60 minutes in 2. Make as many thin smears as possible. One alternate is 10 minutes in 10% Giemsa. Filter a small amount of this stock stain through Whatman #1 filter paper into a test tube. Dry the slides upright in a rack. stock Giemsa stain is stable at room temperature indefinitely (stock stain improves with age). for other size jars.8. anticoagulated with EDTA. 5. that has enough parasites so that at least one is found in every 2 to 3 fields. Choose a patient blood specimen. Staining Procedure: Quality Control To ensure that proper staining results have been achieved. 3. . Add 2 drops of Triton X-100. Remove thin smear slides and rinse by dipping 3-4 times in the Giemsa buffer. according to the directions above. adapt volume but do not change proportions). Pipet from this tube to prepare the working Giemsa stain. the shorter stains yield faster results. Working Giemsa stain (2. Thick smears should be left in buffer for 5 minutes. a positive smear (malaria) should be included with each new batch of working Giemsa stain. Adapt volume to jar size.5%): make fresh for each batch of smears Working Giemsa buffer Giemsa Stain Stock 5% Triton X-100 Staining 1. shake moderately for 30 to 60 minutes daily. 2. Place the bottles at an angle on a shaker. the smears could be stained for shorter times in more concentrated stains. they may be prepared from a patient's blood and stored for future use in the following manner: 1. shake the bottle. for at least 14 days. c. Pour 40 ml of working Giemsa buffer into a second staining jar. Prepare fresh working Giemsa stain in a staining jar. preferably within one hour after the blood was drawn from the patient. but use more stain and might be of less predictable quality. (The 40 ml fills adequately a standing Coplin jar. 2. Place slides into the working Giemsa stain (2.5%) for 45-60 minutes. Kept tightly stoppered and free of moisture.

depending on the clinical context and the availability of laboratory personnel and time. touching front to back. Determination of "No Parasites Found" (NPF): For malaria diagnosis. remove the smear from the box and allow the condensation to evaporate.000 per microliter of blood. in a box without separating grooves. 4. 200. free of stain precipitate. this gives a threshold of sensitivity of 4 parasites per microliter of blood. NCCLS standards recommend examination of at least 300 fields using the 100× oil immersion objective. Fix the smears in absolute (100%) methanol.3. and screening more fields (e. . If you see parasites. Assuming an average WBC count of 8.g. and a rapid screen while the thick smear is still drying. Microscopic Examination Examining thick smears Since the erythrocytes (RBCs) have been lysed and the parasites are more concentrated. date and "Giemsa control slides. screening for parasites if adequate thick smears are not available. 1. 1. 8. or even the whole smear) might be warranted. Store at -70°C (or colder) if the purpose is to make quality control slides. 5. Carefully examine the smear using the 100× oil immersion objective lens. WHO recommends that at least 100 fields. each containing approximately 20 WBCs. 2. Allow the smears to dry quickly. 2." 7. make a tentative species determination on the thick smear and then examine the thin smear to determine the species present. In nonimmune patients. 300. Label the outside of the box with the species. to detect large parasites such as microfilaria. and well-populated with white blood cells (WBCs) (10-20 WBCs/field). First screen the entire smear at a low magnification (10× or 20× objective lens). NCCLS standards recommend examination of at least 300 fields using the 100× oil immersion objective. Just before use. using a fan or blower at room temperature. Examining thin smears Thin smears are useful for species identification of parasites already detected on thick smears. symptomatic malaria can occur at lower parasite densities. 4.. 6. label the slide "+ malaria" and the present date. The smear is now ready for staining since it was previously fixed. Screen at low magnification (10× or 20× objective lens) if this has not been done on the thick smears. the thick smear is useful for screening for parasites and for detecting mixed infections. Select an area that is well-stained. Place them. be screened before calling a thick smear negative. Most often. the thin smear is the appropriate sample for species identification. allow them to dry. 3. Then examine the smear using the 100× oil immersion objective lens.

The method has been applied to malaria parasites (and to a lesser extent. This results in a differential staining of nuclear DNA in green and of cytoplasmic RNA in red. if it is low (e. malaria parasites can be quantified against blood elements such as RBCs or WBCs..000 RBCs (or more). <1%) examine 2.000 WBCs. the tubes are examined using a fluorescence microscope with a stage adapter. an anticoagulant.000 RBCs on the thin smear and express the results as % parasitemia. blood smears on a slide are stained with acridine orange and examined with either a fluorescence microscope or a light microscope adapted with an interference filter system. or otherwise assuming 8. To quantify malaria parasites against WBCs: on the thick smear. count asexual blood stage parasites and gametocytes separately. The QBC method is reported to .g.000 RBCs per microliter blood. Only the former are clinically important and gametocytes of P.g. If this information is needed by the physician. In the Kawamoto technique. African trypanosomes). tally the parasites against WBCs.000. Detection of blood parasites using fluorescent dyes Fluorescent dyes that stain nucleic acids have been used in the detection of blood parasites. Becton Dickinson) method.000> Results in % parasitized RBCs and parasites per microliter blood can be interconverted if the WBC and RBC counts are known. using the WBC count if known. which allows recognition of the parasites. and then are centrifuged in a microhematocrit centrifuge.000 WBCs and 4.000 WBCs per microliter blood. count the parasitized RBCs among 500-2. After centrifugation.Quantifying parasites In some cases (especially malaria) quantification of parasites yields clinically useful information. express the results as parasites per microliter of blood. blood samples are collected in a special tube containing acridine orange. To quantify malaria parasites against RBCs. until you have counted 500 parasites or 1. > 10%) examine 500 RBCs. or a light microscope with a customized fluorescence attachment.. falciparum can persist after elimination of asexual stages by drug treatment. or otherwise (less desirably) by assuming 8. and a float. Malaria parasites concentrate below the granulocyte layer in the tube. whichever comes first. % parasitemia = (parasitized RBCs/total RBCs) × 100 If the parasitemia is high (e. Parasites/microliter blood=(parasites/WBCs) × WBC count per microliter<or 8. In the Quantitative Buffy Coat (QBC®.

Detection and speciation of Plasmodium is done with a two step nested PCR using the primers of Snounou et al 1993.g. The blood smears will be examined first. Alternatively. Species-specific PCR for Diagnosis of Malaria Plasmodium sp. Click to view the DNA extraction protocols recommended for molecular diagnosis of malaria and babesiosis. PCR will be performed only if species determination cannot be made from the blood smears. Blood smears should always accompany the EDTA blood sample. 1 µl of PCR1 amplification product is further amplified using primers specific for each Plasmodium species. and has also been applied (albeit to a lesser extent) to other parasites such as trypanosomes.. microfilaria and Babesia spp. Punching the spots may increase the risk of crosscontamination among specimens. In the first step (PCR1). genomic DNA is extracted from 200 µl whole blood using the QIAamp Blood Kit (Cat. Spot the paper directly from whole blood or finger stick. .com ). Ten microliters of each PCR2 amplified DNA product is electrophoretically resolved on a 2% agarose gel. Prior arrangements should be made to determine the appropriateness of PCR as an adjunct for the diagnosis of malaria and babesiosis. products available through Whatman® http://www.. When species determination cannot be made by microscopic examination. stained for 15 min with ethidium bromide and visualized by UV illumination for analysis of results. Molecular Diagnosis Specimen Collection Microscopic examination of stained blood smears is considered the gold standard for diagnosis ofmalaria and babesiosis. DNA has to be extracted from the blood specimens for PCR detection.whatman. analysis by polymerase chain reaction (PCR) is helpful. Qiagen Inc. Follow allshipment guidelines and requirements. in the second step (PCR2). 1 µl of extracted DNA is amplified using genus specific primers. CA) or a similar product that can yield the comparable concentration of genomic DNA from the same volume of blood. PCR analysis takes approximately one week for completion. Chatsworth. The following procedure describes how a specimen will be accepted for PCR analysis at CDC.have a good sensitivity for detection of malaria parasites. 29106. At this time. No. blood can be collected on filter papers (e. Collect a 1-5 ml blood sample in Vacutainer® EDTA tubes prior to anti-parasitic therapy and ship at 4°C to a reference laboratory.

Use preferably the QIAamp Blood Kit (Cat. 1 µl of extracted DNA was amplified using B. T6050G. d. Pipet 200 µl whole blood. No. 2. CA. Add 200 µl of 96-100% ethanol and mix by vortexing.Species-specific PCR for Diagnosis of Babesia Babesia sp. Before you start. Bab2 and Bab3.g. Valencia. Buffer AW2. 51106. Rochester. 4.5 ml low binding microcentrifuge tube (e. Heat one water bath or heat block to 56°C. Note: This protocol is a summary of the procedure included in the QIAamp Blood kit manual provided by the manufacturer. Preparation of buffy coat is recommended if a higher yield is required. Extraction of DNA from Blood Specimens Whole blood This method is used for whole blood collected in Vacutainer® EDTA tubes. Incubate at 56°C for 10 minutes. microti specific primers. All centrifugation steps should be carried out at room temperature. http://www.com ). e.. Please refer to the manual for detailed product information and protocols. Ensure that buffer AW1. genomic DNA is extracted in the same way as Plasmodium sp. Ten microliters of PCR2 amplified DNA product was electrophoretically resolved on a 2% agarose gel. Use carrier DNA if the sample contains less than 10. in the second step (PCR2).. No. Cat.qiagen. 1 µl of PCR1 amplification product was further amplified using internal primers. be sure to do the following: a. Mix by vortexing. and QIAGEN protease have been prepared according to the instructions. Marsh Biomedical Products. NY). In the first step (PCR1). Bab1 and Bab4. and 200 µl Buffer AL into a 1. Equilibrate samples to room temperature. Procedure 1. 200 µl of the whole blood yields 3-12 µg of DNA. DNA (see above). Detection of Babesia microti is done with a two step nested PCR using the primers of Persing et al. Spin down briefly to remove drops from the inside of the tube. Similar products that can yield the comparable concentration of genomic DNA from the same volume of blood can be used if QIAamp is not available. f. Qiagen Inc. stained for 15 minutes with ethidium bromide and visualized by UV illumination for analysis of results. b. . c. 20 µl QIAGEN Protease.000 genome equivalents. 3.

For PCR amplification. Centrifuge 1 minute at full speed (15. the IsoCode® Stix was not dry and should be discarded. 2.5. Lid locks can be used for this purpose. Do not add the IsoCode® Stix to the tubes yet! Label one additional tube for water to be used in step 7. Centrifuge the tubes again for 5 seconds. Refer to the IsoCode® Stix brochure provided by the manufacturer for detailed product information. Label one 1. Discard the tube containing the filtrate. Spot papers directly from whole blood or finger stick. Vortex the tubes containing the triangles at full power 3× for at least 5 seconds to wash. centrifuge the tubes for few seconds and remove the water with a sterile pipette. 10495015.5 ml low binding microcentrifuge tube. Add 50 µl of deionized sterile water that was exposed to UV (from the additional tube prepared in steps 1 and 2). Do not use blood collected in EDTA to prepare the IsoCode® Stix. At the end of this incubation. Place each tube in a 95°C heat block for 30 minutes. Centrifuge 1 minute at full speed. Use 1µl of extracted DNA for a 50µl PCR.000 × g). 8. 8a. Add enough deionized sterile water in the additional tube to provide 50 µl per sample to be extracted. Place the tubes under ultraviolet light (UV) for 20 minutes. Carefully open the QIAamp spin column and add 500 µl Buffer AW2. Place QIAamp spin column in a clean 2 ml collection tube. 1. place the QIAamp spin column in a new collection tube and spin for 1 minute to remove residual buffer AW2. Schleicher & Schuell. Carefully apply the mixture from step above to a QIAamp spin column. hold the stick with triangle of dried blood over the respective tube containing water that was exposed to UV. now Whatman ). using a new tip for each addition. Alternatively. After vortexing. and pipette off the residual water. Do not expose the IsoCode® Stix to UV light! 3. 10. Open the QIAamp spin column and add 500 µl Buffer AW1 without hitting the rim. 9. Caution should be taken to prevent the lids from opening during incubation. Remove the supernatant to a clean siliconized 1. The DNA will be bound to the filters in the spin columns. NH. Close the cap and centrifuge 1 minute at full speed. use 5 µl of the extracted DNA per 50 µl volume PCR mix. pull the scored end of the stick so that the triangle detaches and falls directly into the tube. Completely immerse the triangle by a brief centrifugation. Extraction from filter containing blood Use preferably the Schleicher and Schuell IsoCode® Stix (Cat.5 ml microcentrifuge for each IsoCode® Stix to be extracted. No. Add 500 µl of deionized sterile water to each tube that will receive the IsoCode® Stix. If the water turns even faintly pink. 2 µl of the extracted DNA per 20 µl of PCR mix can be used. Keene. 4. 6. After incubation. Place column in a clean 2 ml collection tube. Close the cap and centrifuge the QIAamp column for 3 minutes at maximum speed. Add 200 µl Buffer AE or distilled water to the spin column. gently tap tube several times (around 20 times) as most of the fluid is on the tube sides and lid. Incubate at room temperature for 5 minutes to elute the DNA. 6. 7. to each tube. Place the spin column in a clean 1. 9. Spin down tubes for a few seconds to prevent leaking when opening. While closing the cover. 5. Discard the 2 ml collection tube containing the filtrate. 8. 7. . Store at 4°C.5 ml microcentrifuge tube.

11. as confirmed by microscopy. The reported specificities of these tests are high (>90%). Therefore. These tests generate results within 15 minutes and do not require skilled microscopists. Parasite lactate dehydrogenase (pLDH) is produced by asexual and sexual stages (gametocytes) of malaria parasites. ovale. P. aldolase. falciparum malaria. These tests have low sensitivities for detecting infections with low level parasitemias (<100 parasites/µl) and mature gametocytes. Determining the level of mefloquine . intracellular form (amastigote) in stained sections from lesions. falciparum HRP-II only and therefore diagnose onlyP. and P. Slides should be fixed and stained before they are sent unless reagents are not available. They can distinguish P. however. malariae. vivax. but these issues have reportedly been addressed and corrected. Isolation of Organisms The diagnosis of Leishmania spp. and do not provide definitive diagnosis. In contrast. Early tests reported false positives due to cross-reactions with rheumatoid factor. Detection of Parasite Antigens Rapid diagnostic tests for malaria have been developed that employ immunochromatographic methods based on the detection of malarial antigens present in peripheral blood. and parasite lactate dehydrogenase (pLDH). Tests have been developed that detect antigens including the histidine-rich protein II (HRP-II). Most tests use monoclonal antibodies and detect particular malarial antigens in blood specimens. Store extracted DNA at 4°C. The HRP-II antigen is synthesized and released by trophozoite and immature gametocyte stages and persists in peripheral blood. Tests that detect pLDH do not generate persistent positive results following chemotherapy. blood samples are collected and analyzed for the presence of the drug. is made by microscopic identification of the nonmotile. these tests are often not sensitive. particularly for diagnosing cutaneous leishmaniasis. falciparum from the non-falciparum species. but cannot distinguish between P. trained microscopists can diagnose infections with parasitemias as low as 5-10 parasites/µl. When individuals who are presumably on mefloquine prophylaxis exhibit signs of malaria. and by culture of the motile. Serologic tests are also available to detect for anti-leishmanial antibodies. The drug is extracted from the blood and the concentration is determined by high-performance liquid chromatographic methods. Commercially available kits for HRP-II detect P. extracellular form (promastigote) on suitable media. HRP-II tests can remain positive for up to 2 weeks following chemotherapy and parasite clearance. like the HRP-II test. Test kits that are currently available detect pLDH from all four species ofPlasmodium. Special Tests: MQ Testing Mefloquine is recommended by CDC as a prophylactic against malaria.

these specimens are suitable for antigen testing only. Eggs of Ascaris lumbricoides may continue to develop and are infectious even when preserved in formalin. apply cosmetics or manipulate contact lenses in work area.  If you use any sharp instruments.  Do not eat. These risks can be minimized by adopting universal precautions as well as standard microbiological laboratory practices (Biosafety Level 2). Fresh stool should be examined.in the blood helps assess if the individual was adherent with his/her medication. cover them with adhesive dressing. 2. skin penetration by infective larvae.   Decontaminate work surface at least once a day and after any spill of potentially infectious material. smoke. Note: These precautions should be taken even with stool specimens that have been fixed in preservatives because they may still be infectious. For example. These include:  Wear protective safety glasses. Make sure no urine. fixation in formalin takes days or weeks to kill some parasite cysts or oocysts that are protected by a thick shell. Collect the stool in a dry. leakproof container. An exception is specimens kept under refrigeration when preservatives are not available. visit the website of the Centers for Disease Control and Prevention to view biosafety guidelines or the website of the Occupational Safety and Health Administration (OSHA) . or preserved immediately. processed. The image on the right demonstrates the distribution of protozoa in relation to stool consistency and should be taken into consideration when specimens are received. water. soil or other material gets in the container. This procedure is also useful to determine treatment failure due to a mefloquine resistant form of malaria. For more information on safety. Specimen Collection 1.  Remove gloves and wash your hands after completing any task involving the handling of fecal material. and infection by nonparasitic agents found in stool and biologic fluids. . clean.  Use biological safety cabinets as needed. 3. STOOL SPECIMEN Safety Laboratorians working with stool specimens face potential risks including ingestion of eggs or cysts. If you have cuts or abrasions on the skin of your hands. gloves and laboratory coat when processing specimens. dispose of them in a "sharps" container for decontamination. drink.

Add one volume of the stool specimen to three volumes of the preservative. Insure that the specimen containers are sealed well. 5. refer to the molecular diagnosis section to obtain specific information on how to collect. Insure that the specimen is mixed well with the preservative. kaolin. Specimen collection may need to be repeated if the first examination is negative. Certain drugs and compounds will render the stool specimens unsatisfactory for examination. three specimens passed at intervals of 2-3 days should be examined. barium or bismuth (7-10 days needed for clearance of effects). Such substances include: antacids. with the two most commonly used being 10% aqueous formalin and PVA (polyvinyl-alcohol). The specimens should be collected before these substances are administered. 8. If molecular detection (PCR) is required. 10% formalin and PVA (polyvinyl-alcohol). larvae. Insert the container in a plastic bag. and gallbladder dyes (3 weeks). mineral oil and other oily materials. If kits are not available. 7. especially after extended . 6. non-absorbable antidiarrheal preparations. protozoan cysts. using suitable containers. preserve. Preservation of specimens is necessary when stool specimens cannot be examined within the prescribed time interval. Preserve the specimen as soon as possible. follow the kit's instructions. the specimen should be divided and stored in two different preservatives. Various preservatives are available (see table). If possible. antimicrobial agents (2-3 weeks). Reinforce with parafilm or other suitable material. or collection must be delayed until after the effects have passed. Distribution of protozoa in relation to stool consistency Preservative 10% Formalin Advantages  All purpose fixative  Easy to prepare  Long shelf life  Good preservation of morphology of helminth eggs. and ship the specimens. Formed stool needs to be well broken up. If using a commercial collection kit.4. and coccidia Disadvantages  Not suitable for some permanent smears stained trichrome  Inadequate preservation morphology of protozoan trophozoites  Can interfere with PCR.

and chromotrope stains  Compatible with immunoassay kits and UV fluorescence microscopy  Components both fix and stain organisms  Easy to prepare  Long shelf life  Useful for field surveys  Suitable for concentration procedures  LV-PVA (low viscosity polyvinylalcohol)   SAF (sodium acetate-acetic acidformalin)  Good preservation of morphology of protozoan trophozoites and cysts Easy preparation of permanent smears stained with such as trichrome (solution both preserves organisms and makes them adhere to slides) Preserved samples remain stable for several months Suitable for both concentration procedures and preparation of permanent stained smears  Easy to prepare  Long shelf life  Suitable for acid-fast. albumin-glycerin) for adhe of specimens to slides  Permanent stains not as as with PVA or Schaudinn' fixative . safranin.g. coccidia. safranin.Preservative MIF merthiolate-iodineformaldehyde) Advantages  Suitable for concentration procedures and UV fluorescence microscopy  Suitable for acid-fast. and Disadvantages fixation time  Not suitable for some permanent smears stained trichrome  Inadequate preservation morphology of protozoan trophozoites  Iodine interferes with oth stains and fluorescence  Iodine may cause distort protozoa  Inadequate preservation morphology of helminth e and larvae. and microsporidia  Contains mercuric chlorid  Difficult and expensive to dispose of  Difficult to prepare in the laboratory  Not suitable for concentr procedures  Cannot be used with immunoassay kits  Not suitable for acid-fast safranin and chromotrope  Requires additive (e..

Specimen Processing Stool specimens can be examined fresh or preserved. .Preservative Advantages Disadvantages chromotrope stains  Compatible with immunoassay kits   Schaudinn’s Fixative   Modified PVA copper or zinc One-Vial Fixatives (such as Ecofix. and microsporidia  Poor adhesion of liquid o mucoid specimens to slide  Staining not consistent  Organism morphology m poor  Copper-morphology of cy and trophozoites is poor  Zinc-better morphology b not comparable to LV-PVA  Certain one-vial fixatives use certain stains  Color difference of stain  Staining not always cons  Sometimes more expens than formalin and LV-PVA Easy preparation of permanent stained smears    Less suitable for concent procedures Because 10% formalin and PVA have complementary advantages (see table). Commercial two-vials kits are available for this purpose. Parasafe. Preserved specimens can be stored for several months. it is recommended that the specimen be divided and preserved in both types of preservatives (add one volume of stool to three volumes of the preservative). coccidia. STF. Proto-fix. Unifix. and others that may be available) Good preservation of morphology of protozoan trophozoites and cysts Permanent smears can be made and stained with trichrome Zinc is preferred over copper  No mercuric chloride Concentrate and permanent smear can be made out of one vial  Immunoassays can be done on most  No mercuric chloride Contains mercuric chlorid  Inadequate preservation morphology of helminth e and larvae.

and soft specimens (which may contain both trophozoites and cysts) should be examined within one hour of passage. prior to examination. but this must be carried out without delay. If delays cannot be avoided.Examination of fresh specimens permits the observation of motile trophozoites. immunoassay. Specimens preserved in formalin can be tested directly (wet mount. The flow chart on the right shows how specimens preserved in formalin and PVA are processed and tested at CDC. . with overnight refrigeration if needed. the specimen should be preserved to avoid disintegration of the trophozoites. UV fluorescence) or can be concentrated prior to further testing. They are divided into flotation techniques and sedimentation techniques. Liquid (diarrheic) specimens (which are more likely to contain trophozoites) should be examined within 30 minutes of passage (not within 30 minutes of arrival in the laboratory!). chromotrope stain. Concentration procedure separate parasites from fecal debris and increase the chances of detecting parasitic organisms when these are in small numbers. Formed specimens (less likely to contain trophozoites) can be kept for up to one day.

Flotation techniques (most frequently used: zinc sulfate or Sheather's sugar) use solutions which have higher specific gravity than the organisms to be floated so that the organisms rise to the top and the debris sinks to the bottom. The main advantage of this technique is to produce a cleaner material than .This flow chart shows how specimens preserved in formalin and PVA are processed and tested at CDC.

7. Add 4 ml of ethyl acetate. Specimens preserved in PVA are mostly used for permanent staining with trichrome. Mix the specimen well. however. 8.g. Slides may be trichrome stained or kept for several months in a protective slide tray or box for future staining. thus hindering identification. Centrifuge at 500 × g for 10 minutes. MIF or SAF. Free the plug of debris from the top of the tube by ringing the sides with an applicator stick. Insure that the specimen is well mixed. Formalin-Ethyl Acetate Sedimentation Concentration 1. Sedimentation techniques are recommended for general diagnostic laboratories because they are easier to perform and less prone to technical errors. (Conical paper cups with the tips cut off are sufficient). Add several drops of 10% formalin to resuspend the concentrated specimen. The disadvantages of most flotation techniques are that the walls of eggs and cysts will often collapse. 2. 6. 3. Proceed with applicable testing. Prepare a smear using 2 to 3 drops of the specimen depending on density. *Commercial fecal concentration tubes are available that decrease processing time and supplies needed for concentrating specimens (e. 10. Fecal Parasite Concentrator. 5. Centrifuge at 500 × g for 10 minutes. . Use a cotton-tipped applicator to remove debris from sides of the centrifuge tube. Blastocystis hominis may be deformed or destroyed. and shake vigorously in an inverted position for 30 seconds. 2. 9. Evergreen Scientific).the sedimentation technique. Add 10 ml of 10% formalin to the sediment and mix thoroughly with wooden applicator sticks. they are processed as follows: 1. Prior to staining. 4. Also. Carefully remove the stopper. Add 0. and which can be used with specimens preserved in formalin. Heat fix on slide warmer set at 60°C for 5 minutes or air dry completely at room temperature. The sedimentation technique used at CDC is the formalin-ethyl acetate technique. 3.85% saline or 10% formalin through the debris on the gauze to bring the volume in the centrifuge tube to 15 ml. stopper the tube. Decant the top layers of supernatant. Sedimentation techniques use solutions of lower specific gravity than the parasitic organisms. some parasite eggs do not float. Distilled water may be used.. Decant supernatant. a diphasic sedimentation technique that avoids the problems of flammability of ether. thus concentrating the latter in the sediment. Strain 5ml of the fecal suspension (more or less depending on its consistency) through wetted cheesecloth-type gauze placed over a disposable paper funnel into a 15 ml conical centrifuge tube.

In these cases. make sure your package will arrive on a weekday and willnot arrive at CDC on the weekend or a federal holiday. 2.gov/od/ohs/biosfty/shipregs. depending on the volume: Volume not exceeding 50 ml. Packaging Stool specimens must be packaged so as to avoid leakage. conveyer belts. 3. county or state health department laboratory (seehttp://www.htm. 1. vial. The material to be shipped shall be placed in a securely closed.cdc. There are two sets of instructions for packaging. unpreserved stool is requested in order to isolate a known or suspected pathogen (i. The sender must ensure that the specimen remains cold during transport by using available packing materials such as cold-packs. Shipment of Preserved Specimens The rules for the shipment of preserved specimens are the same as those for unpreserved specimens except that they do not need refrigeration. the specimen must be placed in a clean container as quickly as possible and kept under refrigeration until necessary arrangements are made for pick-up and delivery by an overnight courier. watertight container referred to as the secondary container. Shipment of Unpreserved Specimens On some occasions. That facility will refer specimens to CDC if necessary. or similar container that is referred to as the primary container. Click on the following link to see proper labeling and packaging of specimens: (http://www.org ) for processing and examination. CDC follows regulations describing the requirements for proper packaging and shipment of biomedical materials (42 CFR Part 72 . Several primary containers can be placed in one secondary container as long as the combined volume does not exceed 50 ml.Shipment Submit the specimen to the appropriate city. .Interstate Shipment of Etiologic Agents) which can be located at http://www. The package should be able to withstand rough handling and passage through routinely used sorters.aphl. The primary container is then placed in a durable. etc.e. PCR testing). Note: When you ship a specimen to CDC.cdc. watertight tube.htm). culture for microsporidia.gov/od/ohs/biosfty/bmbl4/b4acf1..

two or more primary containers. biopsies) may also be stained. Other types of clinical specimens such as duodenal fluid. There must be enough material to absorb all of the contents of the primary container in case of leakage. which may be difficult to detect with routine stains such as trichrome. Store at room temperature. however. should be placed on all sides around the secondary container before placing in the outer shipping container. 3. and Cyclospora). whose combined volume does not exceed 1000 ml can be placed in a single secondary container. The maximum amount of specimen within one shipping container may not exceed 4000 ml. Staining Procedures Modified Acid-Fast Staining Procedure This technique is useful for the identification of oocysts of the coccidian species (Cryptosporidium. 5. Shock absorbent material. Each set of primary and secondary containers is then placed in an outer shipping container constructed of corrugated fiberboard. or other like material. 6. in volume at least equal to that of the material used between the primary and secondary containers. Single primary containers shall not contain more than 1000 ml. Packaging of these larger volumes of material must comply with all of the requirements of the above but in addition: 1. Absolute Methanol 2. Specimen: Concentrated sediment of fresh or formalin-preserved stool may be used. Reagents: There are four steps to this procedure requiring the following solutions: 1. Cold packs along with absorbent material must be placed around the primary containers.4. Acid Alcohol: 10 ml Sulfuric Acid + 90 ml Absolute ethanol. Kinyoun's Carbol fuchsin: may be purchased commercially. wood. The absorbent material should not be particulate such as sawdust or vermiculite. bile. cardboard. pulmonary samples (induced sputum. . bronchial wash. this stain does not require the heating of reagents for staining.Biomedical Material" label affixed in a prominent location. 2. Volume greater than 50 ml. 3. The shipping container must have a "Etiologic Agents .Cystoisospora. Unlike the Ziehl-Neelsen Modified Acid-Fast Stain.

Do not make the smears too thick! 2. 5. The background should stain uniformly green. 4. 6. Quality Control: A control slide of Cryptosporidium spp. Heat fix on a slide warmer at 60°C until completely dry (5-10 minutes). Destain with acid alcohol for 2 minutes. Rinse briefly with distilled water and drain.4. Examine 200 to 300 fields using 40× or higher objectives. 7. 3. stains a pinkish-red color. Specimen: Prepare a thin smear using approximately 10 µl of 10% formalin fixed stool suspension (unconcentrated) on a glass slide. Stain with Kinyoun's carbol fuchsin for one minute. 3% Malachite green: dissolve 3 g of malachite green in 100 ml of distilled water. from a 10% formalin preserved specimen should be included with each staining run. 2. Procedure: 1. Mount with a coverslip using desired mounting media. Rinse briefly with distilled water and drain. Counterstain with Malachite green for 2 minutes. Fix with absolute methanol for 30 seconds. Absolute methanol Chromotrope Stain: Chromotrope 2R Fast green . Rinse with distilled water and drain. Reagents: There are six steps to this procedure requiring the following solutions: 1. Chromotrope Staining Procedure This staining method was developed at CDC using various components of the trichrome staining method to differentiate microsporidia spores from background fecal elements. Cryptosporidium spp. Prepare a smear with 1 to 2 drops of specimen on the slide and dry on a slide warmer at 60°C until dry. use 100× oil immersion objective. Store at room temperature. Dry on a slide warmer at 60°C for about 5 minutes. To confirm internal morphology. Formalin concentrates may also be used but the number of organisms will be essentially the same as before concentration.

Destain in acid alcohol for only 1 to 3 seconds. higher magnifications are better. A microscope with good optics is recommended for accuracy. Use at least 100× objective oil immersion magnification to detect organisms. Place in two changes of 100% ethanol for 3 minutes each. 95% ethanol 100% ethanol Xylene or xylene substitute Procedure: 1. permount).Phosphotungstic acid Glacial acetic acid 3.. 2. Quick-Hot Gram-Chromotrope Staining Procedure This is an alternative stain to the chromotrope procedure that is a fast. Change all solutions subsequent to chromotrope stain after every 10 slides to obtain proper rinsing and dehydration. Place in chromotrope stain for 90 minutes. it is recommended that a second reader confirm a positive diagnosis. . Rinse in 95% ethanol by dipping. reliable. Examine smear after drying using at least 100× objective oil immersion or higher. Examine at least 200 to 300 oil immersion fields.g. 6. 6. and simple method of staining smears to demonstrate microsporidian spores in fecal and other clinical specimens. Prepare fresh for use every month. Drain slide and mount with coverslip using mounting media (e. 5. Spore walls of microsporidia stain a pinkish-red color and measure about 1 µm. Quality Control: A control slide of microsporidia from a 10% formalin preserved specimen should be included with each staining run. Mix ingredients and allow to stand for 30 minutes. Place in two changes of xylene or xylene substitute for 10 minutes each. Fix smear in absolute methanol for 5 minutes. Acid alcohol: 90% ethanol Glacial acetic acid 5. 7. 7. Because of the difficulty of identification of these small spores. 4. Then add 100 ml of distilled water. 3. 4.

Cool to room temperature. 4. Perform Gram's stain omitting the safranin step as follows: a. and bring the slides to water before performing the Gram's stain. paraffin-embedded tissue sections. Mix dry ingredients then add acetic acid. b. For formalin-fixed. sputum. Reagents: 1. Heat-fix smear (3 times for 1 second each over a low flame or 5 minutes on a slide warmer set at 60°C). For tissue sections. saliva. urine. Procedure: 1. 2. . Let stand for 30 minutes and then add 100 ml distilled water. hydrate in a series of alcohols. and cell culture supernatant) and air dry.Specimen: Prepare a thin smear of the material to be stained (such as feces. a hot plate will suffice. Since only the chromotrope stain needs to be warmed. 2. Acid alcohol: 90% ethanol Glacial acetic acid 5. Gram Stain Kit Chromotrope Stain: Chromotrope 2R Fast green Phosphotungstic acid Glacial acetic acid 3. 95% ethanol 100% ethanol Note: In addition to the listed reagents. a method for heating reagents and maintaining a specific temperature is required. deparaffinize as usual. Flood slides into gentian violet solution and let stand for 30 seconds. Rinse off excess stain gently with water. 6. extend time to 1 minute. Prepare fresh for use every month.

Perform chromotrope stain as follows: a. d. extend time to 1 minute. extend time 30 seconds. Wash the slide gently with cold water to remove excess decolorizer solution. should stain either dark violet or pinkish-red and are easily differentiated from microsporidia spores. Take extra care during this step to achieve correct staining of spores. 3. Hold the slide at an angle and add the decolorizer solution dropwise until it flows off the slide colorless. b. Place the slide in warmed (50° to 55°C) chromotrope stain for at least 1 minute. following instructions. in 100% ethanol (two separate containers are required for this step). Remove Gram's iodine solution by gently rinsing with decolorizer solution. if present. Including Paraffin-Embedded Tissue Sections. microsporidia spores should appear as dark staining violet ovoid structures against a pale green background. d. Schwartz DA. et al 1997. The modified safranin technique produces a more uniform . In fecal samples. Let dry then mount with Cytoseal60 (Stephens Scientific) or other suitable sealer. c. Occasionally. microsporidia spores should stain deep violet to pink violet and may contain gram-positive granules. Quality Control: A control slide of microsporidia spores from a 10% formalin-preserved specimen should be included for each staining run. Modified Safranin Technique (Hot Method) Staining Procedure For Cyclospora. the oocysts stain variably from nonstaining to full staining leading to possible misidentification. Bornay-Llinares F. 30 seconds each time. Reference: Moura H. Arch Pathol Lab Med. Yeast cells.c. we recommend that the slides be washed briefly in a solution of 50% ethyl alcohol/50% xylene for 15 seconds. A new and Improved "Quick-Hot GramChromotrope" Technique That Differentially Stains Microsporidian Spores in Clinical Samples. Flood slides into Gram's iodine solution and allow to remain on the slide for 30 seconds. and Cystoisospora species: Oocysts of Cyclospora in clinical specimens are routinely demonstrated using modified acid-fast stain (cold). Cryptosporidia. with that technique. However. Take extra care during this step to achieve correct staining of spores. In cytologic preparations. For tissue sections. e. Rinse in 90% acid-alcohol for 1 to 3 seconds. For tissue sections.121:888-893. spores will demonstrate a prominent equatorial belt-like stripe. before mounting. Rinse in 95% ethanol for 30 seconds. Rinse twice. For tissue sections.

Dry the slide and mount with a coverslip using desired mounting media. yeast. Quality Control: A control slide of Cyclospora spp. Safranin stain (Available in Gram stain kits) 3% Malachite Green: dissolve 3 g of malachite green in 100 ml of distilled water. 8. Rinse briefly with distilled water. human cells. 7. Procedure: 1. Rinse with distilled water. 3. 3. Cyclospora spp. from a 10% formalin preserved specimen should be included with each staining run. The stain needs to be heated to boiling using either a hot plate or microwave. 6. The Wheatley Trichrome technique for fecal specimens is a modification of Gomori's original staining procedure for tissue. 5. requiring the following solutions: 1. Store at room temperature. 2. oocysts will stain reddish-orange. Other types of clinical specimens such as duodenal fluid may also be stained. . The background should stain uniformly green. 2. Rinse with distilled water. Small protozoa. Reagents: There are three steps to this procedure. Place in boiling safranin for 1 minute. Trichrome Staining Procedure It is generally recognized that stained fecal films are the single most productive means of stool examination for intestinal protozoa. Counterstain with malachite green for 1 minute. Store at room temperature in a tightly closed container. Specimen: Concentrated sediment of fresh or formalin-preserved stool may be used. It is a rapid. and artifact material. 4. Acid Alcohol (3%HCL/Methanol): slowly add 3 ml of hydrochloric acid to 97 ml of absolute methanol. missed by wet mount examinations (of either unconcentrated or concentrated samples) are often seen on the stained smear.staining of these oocysts. The permanent stained smear facilitates detection and identification of cysts and trophozoites and affords a permanent record of the protozoa encountered. Fix in acid alcohol for 5 minutes. which produces uniformly well-stained smears of the intestinal protozoa. simple procedure. Prepare a thin smear on the slide and dry on slide warmer.

Place in two changes of xylene or xylene substitute for 10 minutes. Examine the smear microscopically utilizing the 100× objective. 70% Ethanol (twice) 3. 10. 9. 5. Place in two changes of 100% ethanol for 3 minutes each. permount). To use. 3. follow the manufacturer's instructions.g. Place in Trichrome stain for 10 minutes. 90% Acid Ethanol 90% ethanol Acetic acid (glacial) 5. 100% ethanol (twice) 7. 2. Examine at least 200 to 300 oil immersion fields.Specimen: The specimens usually consist of fresh stool or stool fixed in polyvinyl alcohol (PVA) smeared on microscope slides and allowed to air dry or dry on a slide warmer at 60°C. Rinse several times in 100% ethanol. For other fixatives. Reagents: There are seven steps to this procedure. Mount with coverslip using mounting medium (e. Place in second 70% Ethanol for 3 minutes 4. 8. dilute the stock with 70% alcohol until a dark reddish brown color or strong tea color is obtained. Omit the iodine step for preservatives that do not contain mercuric chloride.. 70% Ethanol plus iodine: prepare a stock solution by adding iodine crystals to 70% alcohol until you obtain a dark solution. Destain in 90% ethanol plus acetic acid for 1 to 3 seconds. 6. 2. requiring the following solutions: 1. 7. . 95% ethanol 6. Xylene or xylene substitute (twice) Procedure: 1. Place slide in 70% Ethanol for 5 minutes. place the slide in 70% ethanol plus iodine for 10 minutes. Trichrome Stain: may be purchased commercially 4. For PVA smears. Stool preserved in sodium acetate-acetic acid-formalin (SAF) or some of the one-vial fixatives can also be used.

from a PVA preserved specimen should be included with each staining run. 3.01% calcofluor white reagent for 1 minute. Rinse with distilled water and let the smear dry. Fix the smear in methanol for 30 seconds. also known as optical brightening agents. Absolute methanol 2.2 Procedure 1. Fungi-Fluor or Uvitex 2B. and Dirofilaria spp. Reagents There are two steps to this procedure requiring the following solutions: 1. the cytoplasm of protozoan trophozoites will have a blue green color sometimes with a tinge of purple. When the smear is thoroughly fixed and the stain is performed correctly. are rapid and inexpensive screening agents. Acanthamoeba spp. red blood cells. Examine with an UV fluorescence microscope equipped with a blue violet filter cube with a wavelength of 400 nm or less. urine. 6. bacteria) and Charcot-Leyden crystals have a red color sometimes tinged with purple. Specimens may include stool. Pneumocystis jiroveci. pH 7. 4. Prepare a thin smear of fecal. 0. such as Calcofluor. Nuclei and inclusions (chromatoid bodies. This test should be used as a quick screening tool and not for species identification. Mount with a #1 thickness cover slip. Heat fix on a slide warmer at 60°C until completely dry (5-10 minutes). 2.01% Calcofluor white reagent: Prepare a 0. These reagents are sensitive but nonspecific since many objects and organisms other than parasites may fluoresce. The background material usually stains green providing a nice color contrast with the protozoa. culture. culture or other types of samples.1M Tris-buffered saline. 5. or other sample material. Specimen: Prepare a thin smear using approximately 10 µl of fresh or preserved specimens on a glass slide.Quality Control: A control slide of a known protozoan such as Giardia spp. . Stain with 0.01% solution in 0. Cysts tend to be slightly more purple. Calcofluor White Staining Procedure This chemofluorescent technique is useful for the detection of microsporidia. Chemofluorescent agents. Glycogen is dissolved by the solvents and appears as a clear area in the organism..

as well as the number of millimeters on the stage micrometer.01 mm) divisions of the scale. Adjust the stage micrometer so that the "0" line on the ocular micrometer is superimposed with the "0" line on the stage micrometer. This calibration should be done for each of the microscope's objectives.6 mm x 48 ocular micrometer spaces = 0. 0. Quality Control A control slide of microsporidia preserved in 10% formalin prepared from culture or from a stool specimen should be included with each staining run. find a point as distant as possible from the two superimposed "0" lines where two other lines are also exactly superimposed. between the two points of superimposition. Microscopic Examination Calibration of Microscopes Using an Ocular Micrometer: A correctly calibrated microscope is crucial because size is an important characteristic for identification of parasites.7. This section assumes that an ocular micrometer disk has been installed in one of the oculars and that a stage micrometer is available for calibrating the ocular micrometer. Calibration readings should be posted on each microscope and the microscope should be recalibrated after every cleaning or changing of objectives or oculars. examine the smear with a 50× or 100× oil immersion objective. The microsporidia spores will fluoresce a brilliant blue-white color on a black background. For example: Suppose 48 ocular micrometer spaces (units) equal 0.125 mm ocular space × 1000 µm/mm = 12. To screen for microsporidia.0125 mm/ocular micrometer space Since most measurements of microorganisms are given in µm rather than mm.1 mm) and the small (0. Determine the number of ocular micrometer spaces. For example: 0.6 mm. .5 µm. Calculate the number of mm/ocular micrometer space. 1 ocular micrometer space (unit) is the equivalent of 12.5 µm/ocular micrometer space Thus in this case. Place the stage micrometer on the microscope stage and focus on the micrometer scale. the value calculated above must be converted to µm by multiplying it by 1000 µm/mm. until you can distinguish between the large (0. Follow the above steps for each objective. Without changing the stage adjustment.

The objectives and oculars used for the calibration procedure should be used for all measurements on the microscope.Wet Mount Preparation: Figure A Figure B Figure C Before preparing a wet mount slide. . The calibration factors should always be posted on the side of the microscope. the microscope should be calibrated.

digital images) should be available by the workstation to compare morphological details and organisms. For other fixatives. It must be heated to approximately 70°C to both mix and use. If the specimen is unpreserved. . obtain a microscope slide and the stool specimen. add a drop or two of saline to the specimen and mix. Other suitable sealing preparations can be used if desired. of which one can be stained with iodine. a higher magnification may be necessary. CAUTION: Bringing high power objectives too near the edge of the slide will result in the sealant smearing the objective and interfering with the optors. Thickness of the wet mount should be as Figure A on the right illustrates. Normally 3 × 1 slides are used to prepare permanent stained slides. A preparation of petroleum jelly and paraffin in a 1:1 ratio can be applied with a cotton tip swab as illustrated in Figure B on the right.Protozoan trophozoites. After the staining process is complete. Stained Slide Preparation: Permanent stained slides are used for identification of protozoan trophozoites and cysts and for confirmation of species. Examine at least 200 to 300 oil immersion fields. If necessary dilute feces with saline. To prepare a wet mount. Spread a thin layer around the edges. Report protozoa seen as either trophozoites and/or cysts as applicable. If desired the coverslip(s) can be sealed. apply two or three drops of the specimen to the slide and with a rolling motion or an up and down dabbing motion spread the specimen evenly to cover an area roughly the size of a 22 by 22 mm coverslip. If the stool specimen is still somewhat solid. Ideally. photographs. It also permits consultation reference and diagnosis when needed as well as providing a permanent record of organism(s) observed. Sealing the coverslip keeps organisms from moving when using oil immersion objectives and prevents the preparation from drying out. Take a small amount of the specimen and place it on a microscope slide. two smears can be prepared on one slide. Positive microscope slides as well as reference material (plates. If something suspicious is seen. check manufacturers instructions. For PVA fixed specimens. oocysts. systematically examine the smear microscopically utilizing the 100× oil objective. To seal. secure the four corners by placing a drop of hot sealant to anchor the coverslip. prepare a thin even smear of the material by streaking the material back and forth on the slide with an applicator stick. cysts. Systematically scan the entire coverslip area using the 10× objective as illustrated in Figure C on the right. and helminth eggs and larvae may be seen and identified using a wet mount identification technique. Refer to the staining section of stools for additional information regarding which stains to use. The microscope should be calibrated before examination begins.

enzyme immunoassay (EIA). antigen detection tests using blood or serum are available forPlasmodium and Wuchereria bancrofti. The primary drawback of these assays is the requirement for fresh. unpreserved stool specimens. Despite the age of the specimen or sample. dispar group are available in the U. Organisms of both the pathogenic E. antigen detection tests have been developed as alternatives using direct fluorescent antibody (DFA). and rapid. If this filter set is not available. andTrichomonas vaginalis. Antigen detection methods can be performed quickly and do not require an experienced and skilled morphologist. duodenal fluid. a less intense green fluorescence can be obtained with blue excitation (450-490 nm). Specimens for antigen detection Fresh or preserved stool samples are the appropriate specimen for antigen detection testing with most kits. Cyclospora oocysts appear as refractile spheres (8-10 µm) with a distinct oocyst wall. Cyclospora oocysts exhibit intense blue color when observed under a fluorescence microscope (UV excitation filter set at 330-365 nm). dipsticklike tests. or small intestine biopsy specimens. it does not provide a permanent stained slide that can be archived.S. These assays use monoclonal antibodies that detect the galactose-inhibitable adherence protein in the pathogenic E. Detection of Parasite Antigens The diagnosis of human intestinal protozoa depends on microscopic detection of the various parasite stages in feces. histolytica/E. histolytica. but refer to the recommended collection procedures included with each specific kit. Entamoeba histolytica. Giardia duodenalis. However. The utilization of both bright-field (DIC) and fluorescence microscopy provides an efficient and reliable approach to diagnosis. In addition. resulting in commercially available reagents for the intestinal parasites Cryptosporidium spp. Much work has been accomplished on the development of antigen detection tests. Under bright-field (differential interference contrast or DIC) microscopy. Amebiasis EIA kits are commercially available for detection of fecal antigens for the diagnosis of intestinal amebiasis.UV Fluorescence Microscopy Procedure: The demonstration of Cyclospora oocysts in wet preparations is greatly enhanced by using UV fluorescence microscopy. Since fecal examination is very labor-intensive and requires a skilled microscopist.. histolytica and the nonpathogenic Entamoeba dispar strains are morphologically identical.. . but only the TechLab kit is specific for E. histolytica. Several EIA kits for antigen detection of the E.

Cryptosporidiosis Immunodetection of antigens on the surface of organisms in stool specimens.. or Wheatley's trichrome stain for Giardia spp. Laboratories which use these EIA kits need to be aware of potential problems with false-positive results and take steps to monitor kit performance. The Meridian Merifluor DFA Kit for Cryptosporidium/Giardia. especially acid-fast staining. The most sensitive (99%) and specific (100%) method is reported to be the DFA test. Giardiasis Detection of antigens on the surface of organisms in stool specimens is the current test of choice for diagnosis of giardiasis and provides increased sensitivity over more common microscopy techniques. histolytica. Concentrated or polyvinyl alcohol-treated (PVA) samples are unsuitable for testing with available antigen detection EIA kits. There are commercial products (DFA. are used at CDC for routine identification of these parasites. EIAs. and show good correlation with the DFA test. A combined DFA test for the simultaneous detection ofCryptosporidium oocysts and Giardia cysts is available. These techniques can be used to confirm suspicious or discrepant diagnostic results. and rapid tests) are available in the United States for the immunodiagnosis of giardiasis. DFA assays may be purchased that employ FITC-labeled monoclonal antibody for detection of Giardia cysts alone or in a combined kit for the simultaneous detection of Giardia cysts and Cryptosporidium oocysts. The kits are reportedly superior to microscopy. histolytica. and test cost must be considered when determining the test of choice for individual laboratories. single versus batch testing. IFA. Giardia. or sodium acetate-acetic acid-formalin (SAF) fixed stool specimens. These offer the advantage of short test time and multiple results in one reaction device. technical skill and time. Initial evaluations indicate comparable sensitivity and specificity to previously available tests. EIA. The sensitivity and specificity of these kits were both . Rapid immunochromatographic assays are available for the combined antigen detection of either Cryptosporidium and Giardia or Cryptosporidium. Giardia. Factors such as ease of use. Kit sensitivities and specificities reportedly range from 93 to 100% when used in a clinical setting. Commercial products (DFA. using monoclonal antibody-based DFA assays. is the current test of choice for diagnosis of cryptosporidiosis and provides increased sensitivity over modified acid-fast staining techniques. Several kits are combined tests for Cryptosporidium. and rapid tests) available in the United States for the diagnosis of cryptosporidiosis. and E. and E. modified acid-fast stain for Cryptosporidium spp. which identifies oocysts in concentrated or unconcentrated fecal samples by using a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody. Some commercial EIA tests are available in the microplate format for the detection of Cryptosporidium antigens in fresh or frozen stool samples and also in stool specimens preserved in formalin.

and E. Giardia. These offer the advantage of short test time and multiple results in one reaction device. Organism Cryptosporidium spp. is a common sexually transmitted disease. Diagnosis is made by detection of trophozoites in vaginal secretions or urethral specimens by wet mount microscopic examination. or SAF fixatives. modified acid-fast stain for Cryptosporidium spp. an infection caused by Trichomonas vaginalis. the manufacturer's evaluation indicated good sensitivity and specificity. DFA staining of specimens. EIA kit sensitivity rates were recently reported as ranging from 94-100% while specificity rates were all 100%. Trichomoniasis Trichomoniasis. Kit name Manufacturer distributora Ty T Crypto CELISA Cellabs EIA PARA-TECT™ Cryptosporidium Antigen 96 Medical Chemical Corporation EIA ProSpecT Rapid Remel EIA ProSpecT Remel EIA Cryptosporidium TechLab EIA Cryptosporidium Wampole EIA . Rapid immunochromatographic assays are available for the combined antigen detection of either Cryptosporidium and Giardia or Cryptosporidium. These techniques can be used to confirm suspicious or discrepant diagnostic results. The Meridian Merifluor DFA Kit for Cryptosporidium/Giardia. They may be used for quantitation of cysts and oocysts. Sensitivity of the assays were reported as 60% for wet mounts and 86% for DFA when compared to cultures.100% compared to those of microscopy.or enzyme-labeled monoclonal antibodies for use in a DFA or EIA procedure is available for detection of whole parasites in fluids. A kit which employs FITC.. and thus may be useful for epidemiologic and control studies. or Wheatley's trichrome stain for Giardia spp. histolytica. or culture. Concentrated or PVA samples are not suitable for testing with EIA kits. Some commercial EIA tests are available in the microplate format for the detection of Giardia antigen in fresh or frozen stool samples and also in stool specimens preserved in formalin. Initial evaluations indicate comparable sensitivity and specificity to previously available tests. MIF. are used at CDC for routine identification of these parasites. A latex agglutination test for antigen detection in vaginal swab specimens is available.

Organism Cryptosporidium spp./Giardia duodenalis Cryptosporidium spp. histolytica II TechLab EIA ProSpecT Remel EIA Giardia CELISA Cellabs EIA PARA-TECT™ Giardia Antigen 96 Medical Chemical Corporation EIA ProSpecT Remel EIA Giardia II TechLab EIA Giardia Wampole EIA GiardiaEIA Antibodies. EIA Giardia CEL Cellabs IFA ProSpecT Remel Rap . dispar Giardia duodenalis Kit name Manufacturer distributora Ty T Crypto CEL Cellabs IFA XPect Crypto Remel Rap PARA-TECT™ Cryptosporidium/Giardia DFA 75 Medical Chemical Corporation DFA Merifluor Meridian DFA ProSpecT Remel EIA Crypto/Giardia CEL Cellabs IFA ColorPAC* Becton Dickinson Rap ImmunoCard STAT!* Meridian Rap XPect Remel Rap Triage BioSite Rap Entamoeba CELISA Cellabs EIA E./Giardia duodenalis/Entamoeba histolytica/dispar Entamoeba histolytica Entamoeba histolytica/E. Inc. histolytica Wampole EIA E.

these specimens must be stored cold or frozen and shipped either refrigerated (4°C) or frozen (shipped with dry ice). Stool specimens in these preservatives can be stored and shipped at room temperature. parvum or C. the specimen must be collected in a preservative that is compatible with molecular detection. . Specimen Collection If PCR is being requested on a stool specimen. the stool specimen can be analyzed using molecular techniques such as polymerase chain reaction (PCR). LVPVA. For specific applications or when commercial fixatives are not an option. modified PVA (Zn. cayetanensis) should accompany the stool specimen when requesting PCR for any of these protozoa. however. dispar) or acid-fast smears (for C. PCR will not be performed. DNA Extraction It is necessary to extract DNA from the stool specimens for PCR detection.5% (1:1 dilution) or in absolute ethanol (1:1 dilution) and shipped refrigerated. the stool can be mixed in potassium dichromate 2. Trichrome stained smears (for E.or Cu-based). histolytica/E. Fixatives/preservatives that are not recommended for molecular detection include formalin. Unifix. Click to view the DNA extraction protocols recommended for molecular diagnosis of intestinal parasites. Fixatives/preservatives with acceptable performance for PCR include TotalFix. All stained smears will be read first and if an identification of the parasite can be made. and Protofix. PCR amplified fragments can be analyzed by using restriction fragment length polymorphisms (RFLP) or DNA sequencing if further characterization is needed. SAF. and Ecofix. Alternatively. stool specimens can be collected in a clean vial and kept unpreserved. If an unequivocal identification of the parasite can not be made.Organism Wuchereria bancrofti Manufacturer distributora Kit name Ty T Simple-Read Giardia Medical Chemical Corporation Rap Filariasis CELISA Cellabs EIA Molecular Diagnosis Microscopic examination is still considered the "gold standard" for the diagnosis of parasitic diseases. Click here for more information about shipping stool specimens to CDC.

Assays using SYBR Green can be easier and less expensive. dispar is performed at CDC by both conventional PCR and real-time PCR. thus avoiding false-positive results. Probe-based assays have the advantages of high specificity and the possibility to detect multiple targets in the same vessel by combining probes labeled with dyes with different fluorescent spectra. Amplified DNA fragments are electrophoretically resolved on an agarose gel for analysis of results. Click on the links above to learn more about the specific tests and to view analysis of PCR results for the respective parasites listed. The fluorescence signal is measured every cycle and is proportional to the amount of accumulated PCR product. call the Division of Parasitic Diseases at (404) 718-4120.PCR Analysis Molecular detection of Cyclospora cayetanensis. For additional information on molecular diagnosis using stool specimens. Entamoeba histolytica. The real-time PCR assays for parasite detection at CDC use either the DNA-binding dye SYBR Green or sequence-specific TaqMan probes as fluorescence detection mechanisms. and E. See illustrations for details about the fluorescence detection mechanisms using SYBR Green and TaqMan probes. The specificity of these assays can be improved by including a dissociation (melting) curve analysis. Conventional PCR: DNA preparations extracted from fecal samples are tested by PCR with diagnostic primers. but caution should be exercised since all double-stranded DNA is detected. including primer-dimers and other PCR artifacts. the DNA amplification is monitored by measuring the fluorescence signal generated in the reaction vessel. Conventional PCR is available for microsporidia. Real-Time PCR In real-time PCR. This helps to distinguish the correct product from possible artifacts. Real-time PCR Illustrations SYBR Green .

The principle of SYBR Green detection in real-time PCR is outlined in the figure above. since the denaturation step contains predominantly single-stranded DNA (6). 1 The fluorescent dye SYBR Green is added to the PCR mixture (1). The signal is only detectable during annealing and extension. As the reaction proceeds and PCR product accumulates. SYBR Green is a DNA binding dye that fluoresces strongly when bound to double-stranded DNA. and so the fluorescent signal detected by the thermocycler is low (3). very little double stranded DNA is present. the amount of double-stranded DNA increases and with it the fluorescence signal (4-5). At the start of the reaction. TaqMan .

and 3) (1). As long as the probe is intact and the reporter and the quencher dyes are in close proximity. 1The TaqMan probe is designed to be 5 complementary to a specific sequence spanned by the PCR primers. After the annealing of the TaqMan probe (2) and the primers (3).The principle of TaqMan real-time PCR is depicted in the schematic above. the primers are extended by the DNA polymerase. it uses its exonuclease activity to remove the probe one nucleotide at the time (4). 2. As the polymerase reaches the TaqMan probe. . The TaqMan probe has a reporter dye at its 5 end and a quencher dye at its 53 end. no fluorescence signal is emitted due to the quenching effect (black arrow in 1. This releases the reporter from the proximity of the quencher and allows for the release of a fluorescence signal from the reporter (5).

laboratory coats. and dispose of them in a “sharps” container for decontamination. Specimen Submission To submit a specimen for antibody testing for parasitic diseases: 1.References SERUM/PLASMA SPECIMENS Safety Universal precautions for the prevention of bloodborne pathogens must be followed when working with human serum and other body fluids.1 ml neat fluid (no washings). The specimens will be referred to CDC if necessary.5 ml. county or state health department laboratory for processing and examination (see http://www. Acceptable only for cysticercosis and baylisascariasis testing. submit the specimen to the appropriate city. acute and convalescent specimens are not necessary.org ). Acceptable only for toxocariasis. For routine requests. gloves. visit the website of the Centers for Disease Control and Preventionto view biosafety guidelines or the website of the Occupational Safety and Health Administration (OSHA) Specimen Requirements Serum/plasma is required for all parasitic disease immunodiagnostic tests. CSF and eye fluids (vitreous or aqueous) are acceptable for selected diseases (see below) and MUST be accompanied by a serum specimen.  Remove gloves and wash your hands after completing any task involving the handling of biological material. Eye fluids: 0.  Use needles and lancets only once. Serum for all tests: 0. cover them with adhesive dressing. CSF: 0. A single sample is usually sufficient.5 ml serum/plasma separated from RBC's before shipping. These include:  Wear personal protective equipment such as safety glasses. For more information on safety.aphl. .  If you have cuts or abrasions on the skin of your hands.

5. 4. 3. Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.phmsa. (International Air Transport Association) http://www. Part 72. Label the shipping container "Clinical Specimen" on the outside of the package.gov/od/ohs/biosfty/shipregs. 2. patient's name.dot. Ship at room temperature by overnight/2 day carrier. the shipping container).2. address. Shipping Requirements Clinical specimens such as serum.htm Antibody Detection Tests Offered at CDC Disease Organism Test Acceptable Specimens Amebiasis Entamoeba histolytica Enzyme immunoassay (EIA) Serum Babesiosis Babesia microti Babesia sp.e. fax number.org/ps/publications/dgr/Pages/index. and e-mail address. and a durable outer package for shipment as required by international regulations for biological agents of human disease.gov/hazmat/regs Title 42 Code of Federal Regulations. Parts 100-185. and test(s) requested.htm): 1. 4.iata. Include the following information: submitter's name. 3. Place the secondary package in the outer packaging (i. and travel history. Wrap the tube in absorbent packing material. Figure 1shows correct labeling and packaging of specimens (http://www. (World Health Organization ) The IATA Dangerous Goods Regulations. Interstate shipment of etiologic agents (Department of Health and Human Services)http://www. 2.cdc. call the Immunodiagnostic Laboratory of the Division of Parasitic Diseases at (770) 488-4431 to make arrangements. specimen collection date. CSF. Blot Serum Filariasis Wuchereria bancrofti and Brugia malayi EIA Serum . age. Label the specimen tube with the patient ID and seal with waterproof tape.. brief clinical history. Additional information about shipping regulations may be found at: 1. phone number. and eye fluids must be triple packed in a primary receptacle.cdc. water tight secondary packaging. Place the specimen tube in secondary packaging.gov/od/ohs/biosfty/bmbl4/b4acf1. For an emergency. WA1 Immunofluorescence (IFA) Serum Baylisascariasis Baylisascaris procyonis Immunoblot Serum or CSF Chagas disease Trypanosoma cruzi IFA Serum Cysticercosis Larval Taenia solium Immunoblot (Blot) Serum. CSF Echinococcosis Echinococcus granulosus EIA.aspx Title 49 Code of Federal Regulations. Hazardous Materials regulations (Department of Transportation)http://www.

vitreous fluid EIA Serum Trichinellosis(Trichinosis) Trichinella spiralis OTHER SPECIMENS Shipment Note: When you ship a specimen to CDC. county or state health department laboratory (see http://www. please refer to shipping regulations and guidelines at the following addresses: a. donovani L. Interstate shipment of etiologic agents (Department of Health and Human Services)http://www. haematobium S.aspx Title 49 Code of Federal Regulations.gov/hazmat/regs Title 42 Code of Federal Regulations. japonicum L.cdc. (World Health Organization ) The IATA Dangerous Goods Regulations. b.gov/od/ohs/biosfty/shipregs. 2. 1. tropica FAST-ELISA Serum Blot Strongyloidiasis Strongyloides stercoralis EIA Serum Toxocariasis Toxocara canis EIA Serum. Parts 100-185.gov/od/ohs/biosfty/bmbl4/b4acf1. d. Figure 1 shows correct labeling and packaging of specimens (http://www.cdc. For routine requests. 4. e.cdc. vivax IFA Serum Paragonimiasis Paragonimus westermani Blot Serum Schistosomiasis Schistosoma sp.aphl.htm Title 42 Code of Federal Regulations. Part 72.gov/od/ohs/biosfty/bmbl/bmbl-1. If shipping a specimen. malariae P. That facility will refer specimens to CDC if necessary.selectagents. Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens. submit the specimen to the appropriate city. f.dot. mansoni S. Additional requirements for facilities transferring or receiving select agents (Department of Health and Human Services)http://www.iata. (International Air Transport Association)http://www. call the Epidemiology Branch of the Division of Parasitic Diseases at (770) 488-7760 to make special arrangements.org/ps/publications/dgr/Pages/index.htm Tissue Specimens . 3.Disease Organism Test Acceptable Specimens Leishmania braziliensis Leishmaniasis IFA Serum Malaria Plasmodium falciparum P.gov/ Biosafety in Microbiological and Biological and Biomedical Laboratories (CDC/NIH)http://www. make sure your package will arrive on a weekday and willnot arrive at CDC on the weekend or a federal holiday. Hazardous Materials regulations (Department of Transportation) http://www. ovale P. S.phmsa. Part 72. In emergencies.org ) for processing and examination.6.htm). c.

Slides should be fixed with methanol and Giemsa stained before they are sent unless reagents are not available. Balamuthia. and biopsy smears can be performed at CDC.  unstained slides (for indirect Immunofluorescence.  unfixed brain tissue or CSF for PCR. these tests are often not sensitive. including sectioned and stained slides. an accurate identification of the parasite can be established in tissue sections. Isolation of Leishmania Organisms The diagnosis of leishmaniasis is made by microscopic identification of the nonmotile. including biopsies. Tissue Specimens for Free-living Amebae (FLAs) Tissue specimens. with prior notification of the appropriate state health laboratory. For PCR. Occasionally. particularly for diagnosing cutaneous leishmaniasis. Specimens may be submitted in several forms. Unfixed specimens for culture should be sent at ambient temperature by overnight priority mail. collected for possible detection of parasites. as they can be damaged in shipment if not packed in a crush proof container. The desired specimens include:  tissue slides stained with hematoxylin and eosin (H&E). CDC can provide culture medium (typically Novy-MacNeal-Nicolle (NNN) medium). Specimen submission forms for free-living amebae testing can be requested by emailing dpdx@cdc. Ship samples according to shipping guidelines and requirements (see shipping guidelines). In most instances. sterile unfixed specimens or specimens in 70-90% ethanol should be sent by overnight priority mail on ice packs. additional sections are required to further the diagnosis. however. intracellular form (amastigote) in stained sections from lesions. and by culture of the motile. as they can be damaged in shipment if not packed in a crush-proof container.  unfixed corneal scrapings (for Acanthamoeba). and many times. surgical or necropsy specimens.  paraffin-embedded tissue block. Leishmanial culture is also available on needle aspirates or punch biopsy lesions. surgical or necropsy specimens. should be submitted to the hospital pathologist. Specimens may be forwarded to the Division of Parasitic Diseases at CDC if further identification is necessary. and do not provide a definitive diagnosis. Care should be taken to pack glass slides securely. or preserved tissues from cases that have a high suspicion of containing parasite material. or IIF).gov. Care should be taken to pack glass slides securely. Serologic tests are also available to detect for antileishmanial antibodies. may be collected for the detection of free-living amebae (Naegleria.Tissue samples. tissue blocks. impression smears. routinely stained hematoxylin and eosin sections are suitable for examination for parasitic agents. Keep this refrigerated until it is used . including biopsy. and Acanthamoeba). Examination of scrapings. Ship samples according to shipping guidelines and requirements (see shipping guidelines). extracellular form (promastigote) on suitable media.

After the aspirate is obtained. Ascaris lumbricoides larvae. then centrifuge. The needle aspirate should be obtained by first drawing approximately 0.0 to 3. Sputum should be obtained from the lower respiratory passages rather than a sample consisting mainly of saliva. . isoenzyme studies will be performed for species identification. hookworm larvae.0ml syringe. Use 1 specimen for culture and the other for impression smears. For biopsy specimens.Strongyloides stercoralis larvae. Aspirates Duodenal aspirates may be useful in demonstrating Giardia duodenalis or Strongyloides stercoralis larvae.  The specimen may be preserved in 10% formalin and a formalin-ethyl acetate concentration procedure may be completed and the sediment examined using either a wet mount or a stained preparation. An unfixed specimen can be examined immediately or if the specimen cannot be examined within 1 to 2 hours after collection. it should be preserved in 10% formalin. place the punch biopsy into the culture medium. call 404-718-4195 or 404-718-4110. and rarelyEntamoeba histolytica. Material collected following intubation through the nose and stomach into the upper small intestine may be submitted to the laboratory. Move the needle back and forth repeatedly under the skin simultaneously rotating the syringe and applying gentle suction until pink-tinged tissue juice is noted in the hub of the syringe.1 ml of preservative-free sterile 0.(stable for 2-4 weeks) and bring it to room temperature right before inoculation. Centrifuge the specimen at 500 × g for 2 to 3 minutes and examine the wet mount. and examine the sediment.9% NaCl into a 1. The cultures will be kept 4 weeks at CDC and if the culture becomes positive and grows. If culture medium is needed. For culture. Insert the needle (23-27 gauge) through intact skin into the dermis of the active border. The culture tubes should be kept at room temperature prior to and during shipment. keep the culture at room temperature and mail as soon as possible by overnight mail. Once inoculated. Sputum specimens should be collected first thing in the morning. A sputum sample can be examined in several ways:  The unfixed specimen may be centrifuged and then the sediment examined as a direct wet mount. obtain 1 or 2 full thickness punch specimens at the active border of the lesion. an equal volume of 3% sodium hydroxide may be added. Sputum Specimens Microscopic examination of sputum is used in identifying Paragonimus westermani eggs.  If the sputum is too viscous.  The specimen may also be preserved in PVA if protozoa are suspected and stained with trichrome stain. discharge into the leishmanial culture medium.

jiroveci. histolytica at the species level. 4. Material from the mucosal surface or from visible lesions should be aspirated. A real-time PCR test is available for confirmation of amebiasis that detects E. To examine these specimens for cysts of P. the material can be stained using trichrome stain and examined for trophozoites of Entamoeba histolytica.Sigmoidoscopy material and abscesses of the liver and lung may demonstrate amebic trophozoites. Preparation of the specimen Refer to safety section prior to handling the specimens (link) 1. centrifuge once more following conditions described above and discard the supernatant.. material obtained by needle aspiration from bone marrow or spleen can be used to demonstrate amastigote stages. If molecular diagnosis is necessary. specimens should be unpreserved and kept refrigerated or frozen. Skin ulcers may demonstrate the amastigote stages in cutaneous and mucocutaneous leishmaniasis. 3. Centrifuge at 500 × g for 5 minutes. saponin*). If this is the case. Sputum.g.. induced sputum. Smears can then be prepared by fixing in methanol and staining with Giemsa stain. The latter will prove useful if a problem occurs during the staining and/or if you wish later to send the smears to a reference laboratory. and spleen may be examined for the presence of motile trophozoites of Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense.85% saline wet mount preparation (or part of this material could be placed in formalin) or can be fixed in PVA. Treat specimens containing mucus (sputum mainly) with a mucolytic agent (e. 2. bone marrow. induced sputum and bronchoalveolar lavage (BAL) for Pneumocystis jirovecii Sputum. Treat sediment containing blood with red cell lytic agent (e. prepare smears of the sediment and examine after staining with the recommended procedure (Giemsa or methenamine silver stain). and bronchoalveolar lavage (BAL) material are commonly used for diagnosing Pneumocystis jirovecii (previously classified as Pneumocystis carinii) infections. Lymph node material. This material can be examined immediately in a 0. . Once fixed in PVA. Sputalysin) by mixing the agent in a 1:1 ratio with the specimen.g. Discard supernatant. Staining Procedures Stain only one set of smears and leave the duplicates unstained. Permanent stained smears made with these specimens by fixing in methanol and staining with Giemsa stain. ForLeishmania donovani infections.

Working Giemsa Buffer . 8. Resuspend the remaining sediment. Spread the drop on the slide with a pipette to evenly distribute on the slide.0067M.5. 5. separate an aliquot that has not been subjected to lyses and prepare smears from that as well. Place drops of the sediment on the center of a microscopic slide.2 µm pore).0 mm .0. 7. Sterile buffer is stable at room temperature for one year.2 Stock Giemsa Buffer Deionized water 4. Stable at room temperature for one month. Triton X-100 5% Deionized water (warmed to 56°C) Triton X-100 6. Autoclave or filter-sterilize (0. Air dry and stain following the required procedure. *For specimens containing blood. Should be 7. Stock Giemsa stain Giemsa stain is available commercially. pH 7. 3.* 7. swirling to mix.67 M Na2HPO4 NaH2PO4H2O Deionized water 2. Stock 100× Giemsa Buffer . but the following formulation gives more constant results and does not expire Glass beads. Check pH before use. 6. Prewarm the deionized water and slowly add the Triton X-100. 3.2. Giemsa Stain 1.0.

Remove thin smear slides and rinse by dipping 3-4 times in the Giemsa buffer. Pour 40 ml of working Giemsa buffer into a second staining jar. Add 2 drops of Triton X-100. c. Prepare fresh working Giemsa stain in a staining jar.5%) for 45-60 minutes. Working Giemsa stain (2.Absolute methanol. adapt volume but do not change proportions). Place slides into the working Giemsa stain (2. Kept tightly stoppered and free of moisture. Adapt volume to jar size. . 4. shake moderately for 30 to 60 minutes daily. Filter a small amount of this stock stain through Whatman #1 filter paper into a test tube. (The 40 ml fills adequately a standing Coplin jar. Pipet from this tube to prepare the working Giemsa stain. shake the bottle. Put into a 500 ml brown bottle the glass beads and the other ingredients. for other size jars. 5.5% Giemsa stain. but use more stain and might be of less predictable quality. b. Screw cap tightly. 3. Thick smears should be left in buffer for 5 minutes. the shorter stains yield faster results. d. for at least 14 days. 2. one alternate is 10 minutes in 10% Giemsa. Use glassware that is clean and dry. in the order listed. according to the directions above. the smears could be stained for shorter times in more concentrated stains. Dry the slides upright in a rack. stock Giemsa stain is stable at room temperature indefinitely (stock stain improves with age). Just before use. a positive smear (malaria) should be included with each new batch of working Giemsa stain. Note: As alternates to this 45-60 minutes in 2.5%): make fresh for each batch of smears Working Giemsa buffer Giemsa Stain Stock 5% Triton X-100 Staining 1. Staining Procedure: Quality Control To ensure that proper staining results have been achieved. a. acetone-free Giemsa stain powder (certified) Glycerol 8. Place the bottles at an angle on a shaker.

F. CrO3 10% solution (stable for 1 year) CrO3 Distilled H2O 2. (yellow) (CI no. AgNO3 Distilled deionized H2O 4.Methenamine silver stain (Gomori's silver methenamine) Store reagents at room temperature (25°C) except the silver nitrate. 5 H2O Distilled deionized H2O 8. Sodium borate (Borax) 5 % solution (stable for 1 year) Na2B4O7 . Stock Light green (stable for 1 year) Light green. 1. Silver Nitrate 5% solution (stable for 1 month). S. Methenamine 3% solution (stable for 6 months) (CH2)6N4 (Hexamethylenetetramine) Distilled H2O 3. which must be stored at 4°C. 10 H2O Distilled deionized H2O 5. Gold Chloride 1 % solution (stable for 1 year) Gold chloride Distilled deionized H2O 7. 42095) . Sodium Thiosulfate 5% solution (stable for 1 year) Na2S2O3 . Sodium bisulfate 1% solution (stable for 1 year) Na2HSO3 Distilled deionized H2O 6.

Wash slide with distilled H2O. 5. 8. Leave the slides in the hot methanine solution for 1-3 minutes. cover with 5% sodium thiosulfate solution and incubate for 1 minute at room temperature.5 ml of 5% sodium borate. Microwave on 600 Watts for 35 seconds. 10. Dip the slides several times in the in the 1% gold chloride solution. 12. Alcohols Ethanol 100% and 95% Methanol 100% 11. Cover slides with 1% sodium bisulfate. 1 ml of 5% silver nitrate. Working Light Green (stable for 1 month) Stock Light Green Distilled deionized H2O 10. 7. If a microwave oven is not available. Wash slides with distilled H2O. Drain the excess of water and air dry. prior to adding the slides. 4. Prepare working methanine solution by mixing 20 ml of 3% methanine. 13. Gently rotate the jar and microwave again for 35 seconds. Microwave it at 50% power for 35 seconds. 3. Prewarm the solution by putting the Coplin jar in the bath in the bath for 6 minutes. use a water bath heated at 80°C. 9.Distilled deionized H2O CH3COOH 9. Incubate for 10 min at room temperature. . Wash the slides with distilled H2O. Place slides in a Coplin jar containing the working methanine solution. 1. Incubate for 1 min at room temperature. 14. 6. Examine the slides. Add chromic acid 10% solution to cover the smears. Add the slides and incubate in the water bath for additional 5 minutes. 2. Wash the slides with distilled and then with deinonized H2O. Place the slides on a rack. and 17 ml of distilled deionized H2O. Wash the slides with distilled H2O. Xylene or Xylene substitute PROCEDURE: 1. Cover with a cap and place in a microwave oven. 11. Cover the slides with the working light green solution (counterstain) and incubate for 1 minute at room temperature.

Specimens should be collected on three consecutive mornings prior to bathing. An increased number of eggs is shed in the urine . vaginalishave been reported and may be increasing. but currently it is considered a fungus. There are no standardized guidelines for the identification and treatment of these organisms. This test is performed at the Division of Parasitic Diseases on isolates that are received from across the country. 1756 Sulfur Spring Rd. However. cases of resistant T. Vaginal Swabs for Detection and Susceptibility of Trichomonas Demonstration of Trichomonas vaginalis trophozoites is usually done by preparing wet mounts made from vaginal swabs or scrapings. It can be used to determine the extent of resistance of a trichomonad isolate to the 5 nitroimidazoles. If the specimen cannot be examined immediately. vaginalis is usually highly susceptible to metronidazole. LA = latex agglutination Cellulose Tape or Swube Tube Procedure for Demonstration of Pinworm Eggs The most reliable and widely used technique for demonstrating pinworm eggs (Enterobius vermicularis) is the cellulose tape or swube tube procedure. Test results are forwarded to the CDC/NCHSTP/DSTDP and are then communicated to the submitting physician. haematobium eggs in urine.G. 28835 Single Oak Dr. Susceptibility Testing T. it should be preserved in PVA and stained smears examined later. Commercially available tests for detection of Trichomonas a. Temecula. Baltimore. MD 21227 DFA = direct fluorescent antibody. The adhesive part of the swube tube or tape is applied to the perianal area first thing in the morning. an in vitro drug susceptibility test was developed by J. PanBio InDx. Urine Specimens The definitive diagnosis of urinary schistosomiasis (Schistosoma haematobium) is established by demonstration of S. b. If an infection is present.. CA 92590. eggs and sometimes adult worms of Enterobius vermicularis will be present on the tape and can be seen under the microscope. In 1978. Chemicon. jiroveci was previously classified as a protozoa.P.. Meingassner.

The specimen should be immediately centrifuged at 400 × g and the sediment examined by wet mount. especially in infected male patients. so an optimum urine specimen for diagnosis should be collected at noon. Trichomonas vaginalis motile trophozoites may also be found in the urine. are also helpful. the urine specimen should be centrifuged at 400 × g. To look for the presence of trophozoites. .around midday. the sediment mixed with a drop or two of saline. such as methylene blue or malachite green. and examined by wet mount. Temporary stains.