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Bench-level Standard Operating Procedure

PARASITES IN STOOL - DIRECT SMEAR 

MICROSCOPIC EXAMINATION

P– 001

Page 1

Authorised signature:                           

Issuing Date: April 12, 2000                             Next Revision Date: April 12, 2001

Staff able to perform test:     Laboratory Assistant and higher

Principle of the Test Method:

Many parasites cause disease in man.  Some of these parasites are excreted in stool; they are called intestinal parasites.  Intestinal parasites can be identified by examination of fresh stool samples.  In stool samples we can find worms (eg. Ascaris lumbricoides) and segments of worms (e.g. Taenia species) visible to the eye.  By microscopic examination of fresh stool samples, we can find eggs (e.g. Hookworm)  and larvae of worms (e.g. Strongyloides stercoralis). We also find protozoa  trophozoites (e.g. Amoeba)  and cysts (e.g. Cyclospora cayetanensis).  In heavy and moderate infection, a direct smear examination with normal saline and/or iodine to stain cysts, is usually sufficient.  For light infections, a concentration of the stool sample might be required to find helminth (worm) eggs and protozoa by microscopic examination.

 

Clinical Significance of the Test:

 Many pathogenic parasites are excreted in stool.  Often, when a person is infected with intestinal parasites, other symptom such as anaemia, eosinophilia, diarrhoea and malabsorption are also present.  However diagnosis by physical examination is not sufficient to identify intestinal parasitic infection.  Stool examination is essential to identify parasites that cause the disease.

Specimen:

Fresh stool samples.

Equipment Requirements:

-          Glass slides

-          Cover slip (20 x 20 mm)

-          Wooden applicator

-          Grease pencil.

-         Microscope.

Reagents & Stain Requirements:

  • Normal Saline (0.9% Sodium chloride solution)

  • Lugol’s iodine. 

How to prepare 0.9% Sodium chloride solution –Normal Saline

Sodium chloride NaCl …………………… 9 g

Distilled water…………………………1000 ml

How to prepare Lugol’s iodine:

Potassium iodide…………………      2 g

Iodine…………………………….        1 g

Distilled water……………up to    100 ml

Mix the potassium iodide with about 200 ml of water until it is completely dissolved. Add the iodine to the potassium iodide solution, mix until dissolved.  Add water until the 1000 ml mark.  Store in dark brown bottle.  Use a small amount in a brown dropper bottle.  Prepare a new solution if colour fades.

 

PARASITES IN STOOL - DIRECT SMEAR 

MICROSCOPIC EXAMINATION

P– 001

Page 1

 Authorised signature:                                          

Issuing Date: April 12, 2000                                                  Next Revision Date: April 12, 2001

Staff able to perform test:     Laboratory Assistant and higher

Test Procedure Instructions:

·          Always first examine the stool sample macroscopically (with your eyes).  

·          Note the colour, consistency and look for mucus, blood stains and worms.

·          Label a glass slide with the patient name and/or lab number.

·          Put one drop of Normal Saline in the middle of the left half of the slide.

·          Put one drop of Lugol’s iodine in the middle of the right half of the slide. 

·          You can examine the slide using Normal Saline only but you would not been able to see cysts well. Therefore it is advisable to examine each stool sample with Lugol’s iodine and Normal Saline.  

·        Take a small piece of stool with the wooden applicator. (About this size     ).

·          If the stool is formed take the piece from inside and the surface of the sample.

·          If the stool is liquid take a drop.  Any part is o.k.

N.B. If the specimen is very liquid place one or two drops of stool directly onto the slide and cover it with the cover glass, do not add the saline as this would further dilute the specimen.

·          Mix the sample first with the drop of Normal Saline on the left half of the slide.

·          If the stool contains mucus or bloodstained parts, prepare a second slide with a drop of Normal Saline and take the piece from the mucus or blood-stained part.

·          Amoeba tropozoites are often more easily found in mucus. Careful, do not mix the mucus with the Lugol’s iodine.

N.B.  The Iodine preparation is useful to identify cysts but kills all living organisms in the specimen.  0.5 % Eosin solution helps in the search for trophozoites of protozoa.

·          Take a small piece again and mix it with the drop of Lugol’s iodine on the right half of the slide.

·          The iodine preparation is useful to identify protozoa cysts.

·          Place a cover slip over each drop.

·          Put the cover slip slowly letting it move down from the side to avoid air bubbles.

·          Examine the entire coverslip systematically.

·          For the saline preparation use the 10x objective and the 40x objective.

·          For the Lugol’s iodine preparations use the 40x objective, as there you might find cysts.

N.B. When searching for Cryposporidium  and Cyclospora cayetanensis it is better to prepare a modified Ziehl Neelsen stained smear.


 

PARASITES IN STOOL - DIRECT SMEAR

MICROSCOPIC EXAMINATION

E – 001

Page 3

 Authorised signature:                                                              

Issuing Date:                          April 12, 2000                                                                                                  Next Revision Date: April 12, 2001

Staff able to perform test:     Laboratory Assistant and higher

Reporting and Interpretation of Results:

 

Macroscopic examination:

/         Always report the colour of the stool sample: The colour of the stool changes with dietary intake.  Normal stool in adults is brown or light brown.  In infants it is yellow or curd-like.

/         Always report the consistency of the stool sample: Formed, semi-formed, soft or watery.

/         Always report the visible presence of blood, mucus or parasites.  Look for adult worms of Ascaris lumbricoides or Trichuris trichuria, and segments of Taenia species.

 

Microscopic examination:

/         Report the name of the parasite found and the quantity (few, some, many).

/         Also report the quantity of white blood cells and red blood cells if found.

 

The most common intestinal parasites found in Nepal are:

Eggs of Helminths:                                                                            Larvae of Helminths:

Ascaris lumbricoides (Round worm)                              Stongyloides stercoralis (Thread worm)

Hookworm                                                                                                       Hookworm larvae, especially if stool is not fresh!

Trichuris trichura (Whip worm)

Hymenolepis nana ( Dwarf Tape worm)

Taenia species (Tape worm)

Enterobius vermicularis (Pin worm)

Cysts or trophozoites of Protozoa:

Giardia lamblia

Entamoeba histolytica

Cryposporidium  and Cyclospora cayetanensis (usually found in watery stool. Better seen in Modified Ziehl Neelsen stain. See separate sheet.)

 

Notes:

/         Other eggs are less common, so always refer to a chart or picture atlas, when in doubt or preserve the specimen in 10% Formalin and send it for examination to the National Public Health Laboratory!

/         Schistosoma mansoni has recently been found in Nepal.

/         Eggs of Enterobius vermicularis are seldom found in the stool sample it is better to collect an anal swab for diagnosis. See separate sheet.

 


 

PARASITES IN STOOL - DIRECT SMEAR

MICROSCOPIC EXAMINATION

E – 001

Page 4

 Authorised signature:                                                              

Issuing Date:                          April 12, 2000                                                                                                  Next Revision Date: April 12, 2001

Staff able to perform test:     Laboratory Assistant and higher

Internal Quality Control Procedures and Sources of Error:

/         Follow proper collection procedures to ensure accurate diagnosis, e.g. Amobic trophozoites begin to degenerate within 1-2 hours after collection.

/         Cystes, flagelates and eggs also undergo changes especially if the stool is left at high temperatures.

/         Properly label the specimen with patient name and lab number to avoid confusion.

/         Only accept fresh specimens and refuse specimens contaminated with dirt or urine.

/         If you can not examine specimens immediately, leave them in a cool place and not exposed to sun.

/         Always examine watery and blood-stained specimens first.

/         Store Lugol’s iodine in brown bottles.  Prepare Lugol’s iodine fresh every two weeks.

/         Never use tincture iodine as it contains alcohol and it would destroy amoeba trophozoites 

/         When preparing the smears, select portions of the stool that are coated with blood or mucus.

/         Keep prepared slides in a wet chamber to prevent them from drying up.

/         Do not touch the stool or the smear with your bare fingers.  Stool may contain infectious material, health hazard!

/         Refer to pictures and charts if you are in doubt about structures that resemble eggs, cysts or trophozoites.

/         If in doubt preserve the stool in 10% formalin for examination by a visiting export or for referral of the specimen.

/         Always examine the slide systematically.

 

Reference:

-          WHO (1991) Basic Laboratory Methods in Medical Parasitology. WHO, Geneva

-         Chessbrough, Monica (1998). District Laboratory Practice in Tropical Countries. Tropical Health Technology, Cambridgeshire.

-          Sherchand, Jeevan B. et.al. (1998) A cross Sectional Study on Intestinal Parasitic Infections in Primary School Children of Godar VDC Nepal. 1-1-98, p.61-64. JONAMELS, Kathmandu.

-         Mallapaty, Gabriele (1984) Practical Manual for a Rural Laboratory, GLRA, Germany.

Copyright ©2002 Gabriele Mallapaty

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The Public Health Care Laboratory - 2001 © Gabriele Mallapaty