10

Activity No.!
Kato Thick Smear
(Cellophane Thick s,near)

Objoctlve■ :

Atter complelJng this exercise, the students should be able to:

1 discuss the importance of kato thick technique .
2. perform the Kato thick smear

Discussion:
Kato thick smear technique is simple. time-saving and economical. and is U$elul
in mass stool examination. II ls also good In delectlng all types of helminlh eggs
especially with thlcil shells like Ascaris and Trlchurls but not HoolcwOrm which has a thin
shell.
The technique requires 2 mongo-~ized stools which is placed 011er a glass slide
and is covered with a cut cellophane paper soaked in o miXture of glycerine and
malachite green solution . Tne m~lach1te green 1s used to give oolor to the cellophane to
m1nim1ze the bnghtness ol the m,crosco::,oc lleld. If malachtte green is not available. a
green cellophane maybe used Instead This technique is NOT advisable for diarrhelc
stools and cannot be used for protozoan c.yst and trophozottes.
Material,:
1 Cellophane 1· x1 · soaked in glycenn-malachtte green solution for 24 hours.
2. s•de
3. appllCBtor stick
4. rubber stopper
Procedure:
1. Place approximately Sfl.60 mgs of Slool (the size ol 2 mongo beans) at the
center of a glass Slide and oover with a piece of the pre-heated oellophane.
2. Press the cellophane genuy with a s10yper to spread the stool evenly, taking care
that the specimen does not sprea-:1 beyond the cellophane cover. The
cellophane serves as the cover llip
3 . Leave the prepared Slide a1 room temperature for 10-20 minutes. The
rr,icroscopic field then becomes clear
4 , The Slide ls best examined alter ·,0-20 minutes or within one hour after
preparation.

Preparallon of Glycerin-Malachite Green 1olutlon

Distilled water - 500 ml
Glycerin - 500 ml
3% Malachite Green solutic,n in water - 5 ml
 AcUvlty No. .§
Kato· Katz Technique
(Cellophane fecal thic k ■mea r)

Objectives:
Aller completing this exercise, the students should be able to
1 discuu the lmportonce or Kato- Katt technique
2 . perform tho Kalo-KalZ technique.
D iscussion:
Kato-Katz technique is a modified Kato technique t>eeause the amoum of
feces 10 be eumin.d is measured. This leehnique Is used to quantify the number of
eggs found In a measured fecal sample. EggG of parasfte are counted per gram of fe(;l!S
This technique is Important In determining the intensity of AUBM ,nfectJon II also
determines the egg reduction rate after treatment

Materials and ,.agents

1 . Wooden applicator stickS

2. S181nlesa steel. nylon or piaslic screen: 80-105 mesh.
3 Stainless steel. plastic. or cardboard templates. Templales of dilferent size
have been produced in dllferen1 countnea.
a . A 9 mm d,ameter hole on a t mm thick template can hold 50 mg of
feces:
b . A 6.0 mm diameter hole on a 1 5mm thick template can hold
41 .7 mg; and
c. A 6 5 mm d!ameter hole on a O 5 mm thick template can hold
20 mg
The templates should be standardaed tor the country a nd the $811'18 sl2e of
templates should always be used to e nsure reprocluobdity and comparability ot
prevalence and ,ntensny data
4 Spatul11
5 M, c m ~ slides (75 x 25 mm)
6 Hydroph,ic cellOphano. 40-50 µm thlc:k. stnps 25 x 30 or 25 • 35 mm ,n si.:e
7 Ftat-l>Otlom ,ar With lid
8 Forceps
9 TOllet pa;,er or absorbent t,ssuo
10 Newspaper
11 Glycerol-malachite gree n or gtycer01-me1nylene blue is added to 100 ml or
glycerol and 100 ml ot d1shlled water and ffllXed well This soiuhon 1s poured onto
the cellophane stnps in a ,a, and Jell for at least 24 h pnor to
use ( It gree:i cellophane 1s used, malachde green can be om11teaJ
 1.1

Procedure
a. Place a small mound of fecal material on newspaper or scrap paper and
press the small scseen on lop so lhal some of lhe feces are sJeved through
the ween and accumulole on top.

b. Scrape the flal-!slded spatula across lhe upper surface of lhe screen 10
collect lhe sieved feces.

c. Place template with hole on the cenler or a microscope slide and add feces
from the spa!ula so that the hole is completely filled. Using lhe side of the
spatula. pass over lhe template to remove excess feces from the edge of the
hole (the spatula and screen may be dlscaided or, If carefully washed. may
be reused).

d . Remove the template carefully so thal tne cylinder of feces 1s left oo the slide

e. Cover the fecal material w"h the pre-soaked cellophane strip. The strip must
be very wet if the feces are dry and less so ~ tl)e feces are soft (if excess
glycerol solution 1s present on upper surface of cellophane. wipe with toilet
paper). In dry climates. excess glycerol will retard but not prevent drying

f. lnver1 the microscope slide and firmly press the !ecol sample against the
hydrophilic cellophane strip on an"1her microscope slide or on a smooth hard
surface such as piece ol tile or a flat stone. The fecal material will be spread
evenly between the microscope slide and lhe cellophane strip. 11 should be
possible to read newspaper prlnl through the smear after clarificatk)n

g Carefully the shde by '.,enlly sliding tt sideways to avoid separating the

cellophane strip or lifting it off. Pl11ce the slide on the bench w!lh 1rie
cellophane upwards. Water evaporates while glycerol clears lhe feces.

'i. For al helminth eggs except hookwonn. keep slide for one hour or more al
ambient temperature 10 clear the fecal malerial prior to examinatK>n under lhe
mir'.1oscope To speed up c1eanng and examinalJOn. the slide can be placed
ln a 400C lncubalor or kepi In dir6ct sunlighl for several mlnures

A.scan~ and Tnchuns eggs wlll remain vrsible and recognizat>le for many
monrhs In these prepara11Gns . Hookwonn eggs clear rapidly and w,11 no
longer be vi•lbie after 30 to eo minute•

J. The amear should be examined In o systemallC manner and the number of

eggs d each 1peae1 reponed

Multiply the appropri1te number to give the numbel' ol 1g91 per gram ol
reces :a.by 20 ,f using a 50 mg template: b.by 50 for a 20 mg template; and
c.by 2-4 '°' a 41 .7 mg template
 J~

AcUvlty No. I
Fonnalln•ElherlEthyl AcetalO Concentration Tec:ltnlque

Objective:
After discussing the exen:ise. !he students sltould be able lo:
1. determine lhe proper tecltnique of Foonalln-Ellter or Ethyl Acetate a>ncen1rall0n
lechniquo.
2. explain how to preser,e the sedlmenl posi!lve for paraslles
Dlscuulon:
Then! are two pnnciples Involved ,n recovering parasllos m cases ol li{jht
Infections. One Is sedimentation and the othe< f/olallon.
Formalin Elher Concentration Technique is a c,oncentra!lon technique based on
sedimentation. useful ror both helminlh eggs and proeozoan cys1s. Elher ,s flammable
and expensive. Ethyl eceiale is found as allemative whlclt Is also elfic:ient ,n recoveri119
cestode eggs blld Glarr.ia cysts.

Mai.rlall:
Vial Centrifuge 15ml
Gaiae Applicator slicks
10% formalin Rubber stopper or a>rlt
Ether/Ethyl Acel8te

Procedure:
1 Commlnute a tlalf size thumb S100I with 10 parts of water.
2. Slnlln the fecal emulsion 1h1ough 2 layers ol gauze and COll4!ct In 15 ml
canlnfuge tube
3 Cenlnfuge and throw away the supemalanl. Repeal lhe proceu several limes
using lap water to wash the fecal sediment arid cantnfuge unlll the supemaian1

fluid is clear.
4 . Afte, the last washing. lhe sediment ,s lhen mixed with to ml o! 10% formalin
Let Sland for 10-30 minutes ror fi.1(8tion 10 take place.
S. To tne formaliled sediment 3 mL ether (ethyl acelate) Is - d 10 the tube.
stoppered and shaken vogo,ously Then centriluoe at low spNd for 2 minutes
6 3 layers are found. ether on tap. superlicial debris and formalin are a>mpjelely
:!ecan1ed using an applicator stick 1~ free lhe superfioal debns from lhe

canlnluge lube
7. The se<limenl is lhoroughly mixed w ith lhe fluid Iha! remains from the lube. By
using • p/pelte. transfer to o glass slide and put a cover Slip. Examine at LPO
then HPO. A small drop of 2% Iodine solution maybe used to staln protozoan

cyst
 15

Activity No. !
Stoll Egg Counting Technique
(By Bellzarlo & Solon,}
Objectives:

After ccmpIet.mg th·s

1
exercise , the students should be able to:
1. determine the method of counting e~g in feces.
2. discuss the importance of the technique.

Materials:

o.1 N Sodium Hydroxide prepared as follows :

NaOH pellets 4.0 gram
Distilled Water_ _ _ _ _ _ _ 100 ml
Stoll flask
Glass Beads
4 gram fecal specimen
Stool pipette
Rubber stopper

Procedure:
1. Fill the special Stoll flask with 0.1N NaOH up to 56 ml ~ark. .
2. Using 2 applicator sticks add sufficient feces to the fluid, to raise the level to the
60ml mark.
3. Add 6-8 glass beads and firmly insert a rubbr stopper
4 . Shake vigorously to secure a homogencus suspension.
5. Routinely, it is best to run the sample to this point in the aftemoonand set it aside
overnight, then reshake the contents and proceed.
6. The ova and debris will begin to settle down immediately after shaking stops. Using
a special pipette calibrated to 0.15 ml, quickly withdraw such amount from the
center of the flask. This is to decrease the error due to settling.
7. Expel the entire contents on the slide and count all the ova in the preparation ,
starting at low power.
8. Multiply the number by 100,this will yield the total ova per cc of formed feces. It is
best to convert all counts to a formed stool basis using the following factors:
Normally formed x 1
Mushy x2
Diarrheic x4

By: Markell, Voge & John

1. Save the entire 24 hour stool specimen and determine weight in grams.
2. Weigh out accurately 4 grams of feces.
3. Place feces in calibrated bottle er large test tube, add sufficient N/1O NaOH to
bring volume to 60ml.
4. Add a few glass beads and shake vigorously to make a uniform suspension. If
the specimen is hard , the mixture maybe placed in a refrigerator overnight
before shaking , to aid in its comminution.
5. With a pipette, remove immediately 0.15ml suspension and drain onto a slide.
6. Do not use a cover slip; place slide on mechanical stage and count all the eggs
on the slide.
7. Multiply egg count by 100 to obtain the number of eggs per gram of feces, and
by weight of specimen to get the total number of eggs per 24-hour specimen.
 17

Activity No. I
Zinc Sulfata C.ntrlfugal Flotation Technique

ObJectivn:
After completing this exercise. the students should be able to;
1. perform the correct procedure or Zinc Sulfate Cenlrdugal F101a1,on Technique
2 . recover paraskes lrom previously reportl!d negative stools.
3. differentiate 1he sedimentation lrom notation technique.
Principle:
Prolozoan cysts ond helmlnthic ova will float to the surface In solutions that have
higher specillc gravky lhan that of the egg or cyst. Zinc sullate with a specific gravny 01
1. 180 Is used to float the cy51s or eggs which can then be colleded lrom the surface

Materials/Reagent
1. Cenlriluge 5. Applicator sticks
2. Test lubes 15 ml 6. 33% ol ZnSO, (Sp.gr. 1. 11l-1.20)
3 . Funnel 7. Fecal specimen
4. Gauze/cheese cloth
Procedu,.:
1. Place abo'Jt i gram (size ol a pea) of leces In a test tube and commlnute wrth 10
!Imes its volume of water.
2. Strain through double layer gauze and ~pir. about 5 ml of the filtrate at 2.500
rpm ror 5 minu1es .
3. Decant and add about 1 ml of water and shake a break of the sediment. fill up
with water to the original volume and spin at the same speed of 11me
4. Repeal the procedure 3 times or unm the supernatant is clear.
5. After the last centrifugation. decant completely: then add about t ml of ZnSO,.
and break off sediment. add more ZnSO, up to about ½ inch from the rim or the

tub4:! and spin at 2,500 rpm lor 1 minute

6. Place preparation ,n a test lube rack and ollow standing lor 2 m1nu1es without

disturbing.
7. Obtain malenal lrom surface film by means of wire loop and transfer 10 a clean

gla&s llide
8. Add one drop Iodine solulion and mount preparabOn wnr. cover slip.
9. Examine under the m1Ctoscope,
Modification• -After &tep 5 you may proceod •• follows
6. Place preparation with more ZnSO, but do not allow overflowing
7. Carefully place cover slip on top and allow standing lor 2 minutes.
8. Pull up carefully the cover slip and add a drop of Iodine.
9. Invert on microscope slide and do the microscopy.