Microbiology Session III

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Feb 22, 2014 (3 years and 3 months ago)

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Microbiology Session III

Parasitology



Session Objectives

Parasitology



Introduction



Perform Wet Prep from stool (formalin fixed
concentration) for possible parasitic infection



Examine Positive trichrome stain smears



Examine Giemsa stained blood film slides
for Malaria and other blood parasites






Parasitic Inflection Dx


Microscopic identification of eggs in the stool is
the most common method for diagnosing
intestinal parasites.



The recommended procedure is as follows:


Collect a stool specimen.


Fix the specimen in 10% formalin.


Concentrate using the formalin

ethyl acetate
sedimentation technique.


Examine a wet mount of the sediment.

Specimen Collection


Collect the stool in a dry, clean, leak proof container.


Make sure no urine, water, soil or other material gets in
the container.


Fresh stool should be examined, processed, or
preserved immediately


Preserve the specimen as soon as possible


Specimen collection may need to be repeated if the first
examination is negative.




If possible, three specimens passed at intervals of 2
-
3
days should be examined

Preservation of specimens


When stool specimens cannot be
examined within 30 minutes Preservation
is necessary




Various preservatives are available



The two most commonly used are 10%
formalin and PVA (polyvinyl
-
alcohol).



Examination of fresh
specimens


permits the observation of motile trophozoites,
(must be done without delay)




Liquid

(diarrheic) specimens (which are more
likely to contain trophozoites) should be
examined within 30 minutes of passage



Soft

specimens (which may contain both
trophozoites and cysts) should be examined
within one hour of passage.




If delays cannot be avoided, the specimen
should be preserved to avoid
disintegration of the trophozoites.




Formed

specimens (less likely to contain
trophozoites) can be kept for up to one
day if refrigerated, prior to examination.


Concentration procedure


Separate parasites from fecal debris and
increase the chances of detecting parasitic
organisms when these are in small
numbers.



They are divided into: flotation techniques
and sedimentation techniques.

Flotation techniques


Most frequently used: zinc sulfate


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 the sedimentation technique.




The disadvantages of most flotation techniques are that
the walls of eggs and cysts will often collapse, thus
hindering identification.


Also, some parasite eggs do not float.


Sedimentation techniques


use solutions of
lower specific gravity

than the
parasitic organisms, thus concentrating the
latter in the sediment.




Sedimentation techniques are
recommended

for general diagnostic laboratories because they
are
easier to perform

and less prone to technical
errors.




The sedimentation technique used is the
formalin
-
ethyl acetate technique


Formalin
-
Ethyl Acetate
Sedimentation Concentration



Mix the specimen well.


Strain 5ml of the fecal suspension through
gauze placed over a disposable paper funnel
into a 15 ml conical centrifuge tube.



Add 0.85% saline or 10% formalin through the
debris on the gauze to bring the volume in the
centrifuge tube to 15 ml.



Centrifuge at 500
×

g

for 10 minutes.


Decant supernatant.




Add 10 ml of 10% formalin to the sediment and
mix thoroughly with wooden applicator sticks.


Add 4 ml of ethyl acetate, and shake vigorously
in an inverted position for 30 seconds.




Centrifuge at 500
×

g

for 10 minutes.


Free the plug of debris from the top of the tube
by ringing the sides with an applicator stick.


Decant the top layers of supernatant.


Use a cotton
-
tipped applicator to remove debris
from sides of the centrifuge tube.


Add several drops of 10% formalin to resuspend
the concentrated specimen.




Proceed with testing.


Staining Procedures



Permanently stained smears provide laboratories with a
permanent record

and a specimen that can be
reexamined as needed
.




In addition, when organisms with unusual morphology
are encountered, or when identification is difficult, the
slides can be sent to a reference laboratory
.




For the above reasons, the permanent stained smear is
recommended for use with every stool specimen
submitted for a routine parasite examination.


Trichrome Staining Procedure


Stained fecal films are the single most productive means
of stool examination for intestinal protozoa.




The permanent stained smear facilitates detection and
identification of cysts and trophozoites


Small protozoa, missed by wet mount examinations are
often seen on the stained smear.



The Trichrome technique for fecal specimens is a rapid,
simple procedure, which produces uniformly well
-
stained
smears of the intestinal protozoa


















Trichrome Stain

Specimen


The specimens usually consist of fresh
stool or stool fixed in polyvinyl alcohol
(PVA) smeared on microscope slides and
allowed to air dry

Trichrome stain Procedure



For PVA smears, place the slide in 70% ethanol plus iodine for 10
minutes.


For other fixatives, follow the manufacturer's instructions.


Omit
the iodine step for preservatives that do not contain mercuric chloride.


Place slide in 70% Ethanol for 5 minutes.


Place in second 70% Ethanol for 3 minutes


Place in Trichrome stain for 10 minutes.


Distain in 90% ethanol plus acetic acid for 1 to 3 seconds.


Rinse several times in 100% ethanol.


Place in two changes of 100% ethanol for 3 minutes each.


Place in two changes of xylene for 10 minutes.


Mount with cover slip using mounting medium


Examine the smear microscopically utilizing the 100
×

objective.


Examine
at least 200 to 300 oil immersion fields.


Ascaris lumbricoides



E. histolytica

trophozoites


Entamoeba histolytica

cysts


E. coli

trophozoites
stained with
trichrome


E. coli

cyst in a
concentrated wet
mount stained with
iodine


Hookworm eggs in
unstained wet
mounts, taken at
400
×

magnification.


Eggs of
Diphyllobothrium

sp.
in an unstained wet
mount

Giardia Lambilia
Trophozoites/cyst



Eggs of
Hymenolepis
nana


Adult male of
E.

vermicularis

from a
formalin
-
ethyl acetate
(FEA) concentrated
stool s


The eggs of
Enterobius
vermicularis



Two trophozoites of
T. vaginalis



Eggs of
S. mansoni

in
unstained wet mounts


Eggs of
S.
haematobium
in wet
mounts of urine
concentrates


Free
-
living adult male
S. stercoralis
. Notice
the presence of the
spicule (red arrow).



Adult free
-
living
female
S. stercoralis

alongside a smaller
rhabditoid larva

Blood Parasites


Giemsa stain

-

Recommended for
detection and identification of blood
parasites.

Malaria


Ring
-
form
trophozoites (rings)
of
Plasmodium
falciparum


Blood stage parasites of
Plasmodium falciparum




Ring
-
form
trophozoites of
P.
vivax

usually have a
thick cytoplasm with
a single, large
chromatin dot


T. cruzi

trypomastigote in a
thin blood smear
stained with Giemsa

THANK
YOU








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