Method for detecting intestinal pathogen dientamoeba fragilis

A method and apparatus for producing detectable intestinal parasites. The method includes obtaining an intestinal mucosa sample (e.g. feces) having intestinal parasites, such as Dienamoeba fragilis; and contacting the obtained intestinal mucosa sample with an acridine base compound (e.g. acridine orange and/or acridine yellow, etc.) such that the intestinal parasites become differentially stained and detectable by a human eye when viewed through a fluorescence microscope. The apparatus includes a kit or the like which includes at least one vessel or vial. Preferably, two vials are contained within the kit with one vial having an isotonic salt solution including a salt, such as sodium chloride, potassium phosphate, etc., and the other vial containing an acridine biological staining compound.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention is related to detecting intestinal parasites. More 
particularly, this invention provides a method and apparatus for detecting 
intestinal parasites in feces, especially the protozoa Dientamoebo 
fragilis is in feces, particularly human feces; and samples obtained from 
the mucosa of the intestinal tract, especially the human intestinal tract. 
2. Description of the Prior Art 
A conventional method for the detection of intestinal parasites consists of 
obtaining a fecal sample or intestinal mucosa sample and examining it 
macroscopically and microscopically for the presence of parasites. In the 
case of protozoan parasites,. microscopic examination is necessary for 
identification of trophozoite and cyst stages. Some intestinal protozoa 
can be identified microscopically in fresh, unfixed samples, but 
traditionally, samples are fixed with a fixative, such as formaldehyde, 
polyvinyl alcohol, sodium acetate-acetic acid-formalin, or Schaudinn's 
fixative. Samples may or may not be subjected to concentration techniques 
such as sedimentation and floatation methods. The sample, whether 
concentrated or not or fixed or not, can be examined at this stage for 
protozoa. The sample is commonly subsequently stained using the trichrome 
staining method after fixation of a small amount of sample to a microscope 
slide. 
Dientamoebo fragilis is a pathogenic protozoan parasite of the large 
intestine and cecum of human beings that has no cyst stage. Infection with 
this protozoa has been associated with diarrhea, abdominal pains, pruritus 
and loose stools. It has been suggested that Dientamoeba fragilis causes 
acute and chronic colitis through an invasive ulcerative process. Because 
of the lack of a cyst stage, present laboratory procedures for detection 
of Dientamoeba fragilis in fecal samples are unreliable. A recent study 
has indicated that Dientamoeba fragilis in an infected fecal sample can go 
undetected. 
Therefore, what is needed and what has been invented is a reliable method 
for detecting intestinal parasites, especially Dientamoeba fragilis. 
SUMMARY OF THE INVENTION 
The present invention accomplishes its desired objects by broadly providing 
a method for producing detectable intestinal parasites comprising 
obtaining feces or an intestinal mucosa sample having intestinal 
parasites; and contacting the obtained feces or intestinal mucosa sample 
with an acridine base compound, whereupon the intestinal parasites become 
differentially stained and detectable when viewed by a human eye, 
especially with the employment of a fluorescent microscope. The acridine 
base compound is preferably in a solution such as an aqueous solution. The 
aqueous solution is preferably an isotonic aqueous solution containing a 
compound selected from the group consisting of sodium chloride, potassium 
chloride, potassium phosphate, sodium phosphate, glucose, and mixtures 
thereof. Alternatively, the isotonic aqueous solution may be any salt 
solution, such as commercially available Hank's Balanced Salt Solution 
(HBSS) or Phosphate Buffered Saline (PBS). The isotonic aqueous solution 
may also contain a pH indicator, such as phenol red. More preferably, the 
isotonic aqueous solution has a pH of from about 5.0 to about 8.0 and 
optionally further comprises a fixative, such as formalin. The isotonic 
aqueous solution may optionally further comprise a preservative such as an 
alcohol (e.g. methanol and/or ethanol, etc.). The intestinal parasite 
detected is preferably the intestinal pathogenic protozoan Dientamoeba 
fragilis. 
The present invention further accomplishes its desired objects by also 
broadly providing an apparatus for producing detectable intestinal 
parasites. The apparatus comprises a kit or the like which preferably 
includes at least one vessel or vial, more preferably a pair of vessels or 
vials. One of the vessels or vials contains a solution ranging from a 
hypotonic water to a hypertonic salt solution, preferably an isotonic 
solution exhibiting an osmolality ranging from about 200 milli Osm/liter 
to about 400 milli Osm/liter and containing one or more of the following 
ingredients: NaCl (sodium chloride), KCl (potassium chloride), KH.sub.2 
PO.sub.4 (potassium phosphate), Na.sub.2 HPO.sub.4 (sodium phosphate), and 
C.sub.6 H.sub.12 O.sub.6 (glucose). The solution may also contain a pH 
indicator, such as phenol red, and/or a fixative, such as formalin, and/or 
a preservative, such as an alcohol (e.g. methanol and/or ethanol, etc.). 
The other vessel or vial contains an acridine compound, preferably in a 
solution. More preferably, the other vessel or vial contains an acridine 
staining compound (e.g. acridine orange and/or acridine yellow) in an 
aqueous solution such as, by way of example only, a salt solution 
containing one or more of the following ingredients: NaCl (sodium 
chloride), KCl (potassium chloride), KH.sub.2 PO.sub.4 (potassium 
phosphate), Na.sub.2 HPO.sub.4 (sodium phosphate) and C.sub.6 H.sub.12 
O.sub.6 (glucose). As previously indicated, the aqueous solution may be 
any salt solution, such as HBSS and/or PBS. 
The fluorescent dye (fluorochrome), acridine orange (AO), is a small, 
fluorescent, planar cationic vital dye which, in the unfixed cell, 
complexes with intranuclear nucleic acids as well as other intracellular 
polyanionic biopolymers. Because of its special affinity for the nucleic 
acids, AO has the property of differentiating ribonucleic acid (RNA) from 
deoxyribonucleic acid (DNA). In a solution of optimum acidity, the AO-RNA 
combination fluoresces an orange-pink color and the AO-DNA combination 
fluoresces a yellowish-green color. The intensity of fluorescence in each 
combination depends on the concentrations of respective nucleic acids. 
When a high concentration of RNA is present such as is the case with fast 
growing cells, the cytoplasm and the nucleoli may show a fluorescence of a 
brighter orange-red. 
In a laboratory, it was found that the whole of the RNA-containing 
cytoplasm of the intestinal protozoa Dientamoeba fragilis fluoresces a 
bright orange to red color and that the DNA-containing nucleus or nuclei 
of the amoeba fluoresce(s) yellow to green in a feces or intestinal mucosa 
sample that has been exposed to the AO dye. This occurs while most of the 
remaining objects in the sample, such as bacteria, undigested food, 
mycobacteria, and epithelial cells fluoresce predominantly green, 
providing for easily visible differentiation and subsequent ready 
identification of Dientamoeba fragilis in a sample of fecal or intestinal 
mucosal origin. This bright orange to red fluorescence of Dientamoeba 
fragilis is thought to occur as a function of the high content of RNA in 
the cytoplasm of the protozoa that is rapidly dividing in the cecum and 
colon which are located at the end of the intestinal tract. 
It is therefore an object of the present invention to provide a method for 
producing detectable intestinal parasites, especially the intestinal 
pathogenic protozoan Dientmoeba fragilis. 
It is another object of the present invention to provide an apparatus for 
producing detectable intestinal parasites, especially the pathogenic 
protozoan Dientamoeba fragilis. 
These, together with the various ancillary objects and features which will 
become apparent to those skilled in the art as the following description 
proceeds, are attained by this novel method and apparatus for producing 
detectable intestinal parasites, a preferred embodiment being shown with 
reference to the accompanying drawings, by way of example only, wherein:

DETAILED DESCRIPTION OF THE INVENTION 
Referring in detail now to the drawings wherein similar parts of the 
invention are identified by like reference numerals, there is seen a 
container or kit, generally illustrated as 10 in FIGS. 1 and 9. The 
container or kit 10 may be any suitable container or kit 10 manufactured 
from any material, including pliable material such as pliable plastic, 
nylon, cloth or the like. The container or kit 10 conveniently includes a 
pivotally secured side or door 12 which also may be formed from any 
suitable material including pliable material. The container or kit 10 of 
this invention may be formed or manufactured into any geometric shape, and 
thus, the box-like appearing structure in FIGS. 1 and 9 is not intended to 
limit the scope of the invention. 
In the embodiment of the invention and of the container 10 in FIG. 1, the 
container 10 comprises a pair of vessels or vials 14 and 16 having 
removable tops 18 and 20, respectively. Each of the vessels 14 and 16 may 
possess any suitable volume, preferably from about 0.5 ml to about 50.0 
ml, vessel 14 more preferably from about 5.0 ml to about 25.0 ml, and 
vessel 16 more preferably from about 1.0 ml to about 10 ml. Vessel 14 is 
preferably formed with a funnel or cone-shaped bottom 22, and contains or 
includes an aqueous solution 13 ranging from hypotonic water to a 
hypertonic salt solution. Preferably, the aqueous solution 13 in vessel 14 
is an isotonic solution exhibiting an osmolality from about 200 
milliOsm/liter to about 400 milliOsm/liter, more preferably from about 250 
milliOsm/liter to about 350 milliOsm/liter, most preferably about 300 
milliOsm/liter, and containing one or more of the following ingredients: 
NaCl (Sodium Chloride), KCl (Potassium Chloride), KH.sub.2 PO.sub.4 
(Potassium Phosphate), Na.sub.2 HPO.sub.4 (Sodium Phosphate), and C.sub.6 
H.sub.12 O.sub.6 (Glucose). The aqueous solution 13 in vessel 14 may 
optionally include a pH indicator, such as phenol red. The vessel 16 
preferably contains a solution or mixture 15 comprising the aqueous 
solution 13 of or from the vessel 14 (as immediately described in the 
foregoing sentences) and a biological staining agent comprising an 
acridine base compound having the general formula: C.sub.13 H.sub.9 N 
(tricyclic) with an average molecular weight of about 179.2. Acridine 
typically has the properties of being small colorless needles that are 
soluble in alcohol, ether or carbon disulfide, and sparingly soluble in 
hot water. Acridine has the further properties of subliming at about 
100.degree. C., a melting point of about 111.degree. C. and a boiling 
point of about 360.degree. C. As is well known to the artisans skilled in 
the art, acridine is produced by or derived by extraction with dilute 
sulfuric acid from the anthracene fraction from coal tar and adding 
potassium dichromate. The acridine chromate precipitated is 
recrystallized, treated with ammonia and recrystallized. Preferably, the 
acridine base compound is selected from the group consisting of acridine 
hydrochloride [C.sub.13 H.sub.9 N HCl (tricyclic) with an average 
molecular weight of about 215.7]; acridine -9- carboxylic acid [C.sub.14 
H.sub.9 NO.sub.2 (tricyclic) with an average molecular weight of about 
223.2]; acridine mustard; acridine mutagens; acridine yellow [C.sub.15 
H.sub.15 N.sub.3 .HCl (tricyclic) having an average molecular weight of 
about 273.8]; acridine orange base [C.sub.17 H.sub.19 N.sub.3 (tricyclic) 
having an average molecular weight of about 265.4]; and acridine orange 
[3,6-bis [Dimethylamino] acridine]; and mixtures thereof. More preferably, 
the acridine base compound is selected from the group consisting of 
acridine orange, acridine orange base, acridine yellow, and mixtures 
thereof. Most preferably the acridine base compound is acridine orange, 
more particularly acridine hemi (zinc chloride) salt [C.sub.17 H.sub.20 
ClN.sub.3 1/2 ZnCl.sub.2 (tricyclic) having an average molecular weight of 
about 370]. The concentration of the acridine base compound (especially 
the acridine orange) in the solution 15 or mixture 15 ranges from about 
0.04 micrograms/milliliter to about 5 milligrams/milliliter, more 
preferably from about 0.4 micrograms/milliliter to about 100 
micrograms/milliliter, and most preferably from about 5 
micrograms/milliliter to about 40 micrograms/milliliter. 
In another preferred embodiment of the present invention and of the 
container 10 in FIG. 1, vessel 14, in addition to containing the aqueous 
solution 13 as more specifically described above and optionally a pH 
indicator, the aqueous solution 13 in vessel 14 comprises from about 1% by 
volume to about 70% by volume, more preferably from about 5% by volume to 
about 15% by volume, of a fixative, such as formalin (i.e. sodium 
acetateacetic acid-formalin) and/or polyvinyl alcohol, and/or Schaudinn's 
fixative and/or a preservative such as an alcohol, e.g., methanol and/or 
ethanol, etc. If a fixative is used in conjunction with a preservative, 
the quantity of each is to be in any suitable percentage by volume, such 
as, by way of example only, the fixative and preservative mixture may 
comprise from about 1% to 99% by volume of the final solution. 
In the preferred embodiment of the present invention and of the container 
10 in FIG. 9, the container 10 contains a vessel or vial 24 having a 
removable top 28 and a funnel or cone-shaped bottom 30. The vessel or vial 
24 may be identical to the vessel 14 and possess any suitable volume, such 
as from about 0.5 ml to about 50.0 ml., more preferably from about 5.0 to 
about 25.0 ml. The vessel or vial 24 contains or includes an aqueous 
solution 32 ranging from hypotonic water to a hypertonic salt solution. 
Preferably, the aqueous solution 32 in vessel or vial 24 is an isotonic 
solution exhibiting an osmolality from about 200 milliOsm/liter to about 
400 milliOsm/liter, more preferably from about 250 milliOsm/liter to about 
350 milliOsm/liter, most preferably about 300 milliOsm/liter, and 
containing one or more of the following ingredients: NaCl (Sodium 
Chloride), KCl (Potassium Chloride), KH.sub.2 PO.sub.4 (Potassium 
Phosphate), Na.sub.2 HPO.sub.4 (Sodium Phosphate), or C.sub.6 H.sub.12 
O.sub.6 (Glucose). The aqueous solution 32 in vessel 24 also comprises the 
biological staining agent comprising the acridine base compound as 
generally and more specifically described above. More particularly and as 
previously indicated, the preferred and more preferred and most preferred 
species of the acridine base compound are the same as previously described 
above. The concentration of the acridine base compound (especially the 
acridine orange) in the aqueous solution 32 in vessel 24 preferably ranges 
from about 0.04 micrograms/milliliter to about 5 milligrams/milliliter, 
more preferably from about 0.4 micrograms/milliliter to about 100 
micrograms/milliliter, and most preferably from about 5 
micrograms/milliliter to about 40 micrograms/milliliter. 
In another preferred embodiment of the present invention and of the 
container 10 in FIG. 9, vessel 24, in addition to containing the aqueous 
solution 32 as more specifically described above and optionally a pH 
indicator, the aqueous solution 32 in vessel 24 comprises from about 1% by 
volume to about 70% by volume, more preferably from about 5% by volume to 
about 15% by volume, of a fixative, such as formalin (i.e. sodium 
acetateacetic acid-formalin) and/or polyvinyl alcohol, and/or Schaudinn's 
fixative and/or a preservative such as an alcohol, e.g., methanol and/or 
ethanol, etc. If a fixative is used in conjunction with a preservative, 
the quantity of each is to be in any suitable percentage by volume, such 
as, by way of example only, the fixative and preservative mixture may 
comprise from about 1% to 99% by volume of the final solution. 
In order to obtain an intestinal mucosa sample if feces is not available, a 
rectal swab technique is employed. The rectal swab technique optionally 
consists of the insertion of a lubricated anuscope (not shown in the 
drawings) into the anal canal of a patient, and the insertion of, and 
subsequent swabbing of the mucosa with one or more sterile swabs, 
generally illustrated as 40. More specifically, each swab 40 includes a 
stick 42 having a cotton tip 44 at one of the distal ends (see FIGS. 2 and 
10). The cotton tip 44 absorbs and/or gathers intestinal mucosa and feces 
when swabbed against and/or within the anal canal of a patient. If the 
intestinal mucosa is to be analyzed for intestinal parasites, especially 
for Dientamoeba fragilis, the rectal swab technique is the more preferred 
procedure for obtaining an intestinal mucosa sample rather than by the 
reception of feces from a patient. 
After the cotton tip 44 of a sterile swab 40 has obtained an intestinal 
mucosa sample and for the embodiment of the invention depicted in FIGS. 
1-8, the cotton tip 44 is inserted into the previously described aqueous 
solution 13 (e.g. Hank's balanced salt solution (HBSS)) within the vial 14 
and swirled such that the intestinal mucosa sample may pass into the 
aqueous solution 13 (see FIG. 2). If feces is available, a quantity (e.g. 
0.1 to 1.0 cubic centimeters) of the feces is emulsified in the aqueous 
solution 13. Subsequently, the vial 14 is centrifuged to produce 
supernatant 50 and sediment 52 (see FIG. 3). The supernatant 50 is slowly 
decanted or otherwise removed from the sediment 52, leaving the sediment 
52 in the bottom of the vial 14, as best shown in FIG. 4. 
The solution or mixture 15 (i.e. the aqueous solution 13 plus the 
biological staining agent (an acridine base compound)) from vessel 16 is 
added to sediment 52 in vial 14, as best shown in FIG. 5. More 
specifically the mixture 15 is added to the sediment 52 in a weight or 
volume ratio varying from about 1:10 to about 10:1, preferably in about a 
1:1 volume ratio. After such addition, the solution or mixture 15 is mixed 
with the sediment 52 to produce an admixture 85 as best shown in FIG. 6. A 
convenient quantity of the admixture 85 (e.g. one drop or approximately 50 
ul) is placed on a microscope slide 70 (see FIG. 7) and a coverslip 72 is 
superimposed on slide 70 to sandwich the quantity 85. The quantity of 
admixture 85 is subsequently examined for parasites using a microscope 76 
equipped with an ultraviolet light source (e.g. a fluorescence 
microscope). When the quantity of the admixture 85 is viewed through the 
fluorescence microscope, the following by way of example only and not by 
limitation may be seen; intestinal parasites 185 (e.g. Dientamoeba 
fragilis), typically having an orange to red cytoplasmic color 86 and 
including nuclei with a green color 87; bacteria 88 and undigested food 
material 89 also both typically having a green color; and a residual field 
or background 200, typically having a black color. 
For the embodiment of the invention depicted in FIGS. 9-14, after the 
cotton tip 44 of a sterile swab 40 has obtained an intestinal mucosal 
sample, the cotton tip 44 is inserted into the previously described 
aqueous solution 32 (e.g. HBSS and one of the previously described 
acridine based compounds) within vial 24 and swirled such that the 
intestinal mucosa sample may pass into and be suspended in the aqueous 
solution 32 (see FIG. 10). If feces is available, it is emulsified and 
from about 0.1 to about 1.0 cubic centimeters of the emulsified feces is 
suspended in the aqueous solutions 32. Subsequently, the vial 24 is 
centrifuged to produce supernatant 92, stained sediment 94 (see FIG. 11). 
The supernatant 92 is slowly decanted or otherwise removed from and/or off 
the stained sediment 94, leaving the stained sediment, as best shown in 
FIG. 12. A convenient quantity of the stained sediment 94 (e.g. one drop 
or approximately 50 .mu.l) is placed on the microscope slide 70 (see FIG. 
13) and the coverslip 72 is placed over the stained sediment 94 and over 
the slide 70. The quantity of stained sediment 94 is subsequently examined 
for parasites using the microscope 76 equipped with an ultraviolet light 
source (e.g. a fluorescence microscope). When the quantity of the 
admixture 94 is viewed through the fluorescence microscope, the following 
by way of example only and not by limitation may be seen; intestinal 
parasites 185 (e.g. Dientamoeba fragilis), typically having an orange to 
red cytoplasmic color 86 and including nuclei with a green color 87; 
bacteria 88 and undigested food material 89 also both typically having a 
green color; and a residual field or background 200, typically having a 
black color. As previously indicated, the biological staining agent (i.e. 
the acridine base compound) differentially stains the parasites such that 
a human eye looking through the microscope 76 can detect the parasites. As 
further previously indicated above, when acridine orange is employed and 
Dientamoeba fragilis is to be detected in the intestinal mucosa sample, a 
sharp contrast results from the Dientamoeba fragilis having an orange to 
bright red color when contrasted against a background, which is a 
different color from an orange to bright red color. 
The invention will be illustrated by the following set forth examples which 
are given by way of illustration and not by any limitation. All parameters 
such as mixing proportions, time(s), etc. submitted in these examples are 
not to be construed to unduly limit the scope of the invention. 
EXAMPLE I 
Swab a rectal canal of a patient with sterile cotton tip 44 of one or more 
swabs 40 to obtain an intestinal mucosa sample; or obtain sample from 
feces of the patient. Immediately suspend sample in HBSS or PBS (or the 
aqueous solution 13 as previously described) by swirling the cotton tip 44 
in the solution 13; or, in the case of feces, emulsify and suspend 
approximately 0.5 cubic centimeters in the solution 13. Within 24 hours in 
the case of the solution 13 containing no fixative and/or preservative, 
within four days in the case of the solution 13 being refrigerated at 
about 4.degree. C. or containing a preservative such as an alcohol, and 
within 6 months, in the case of solution 13 containing a fixative such as 
formalin, centrifuge sample to produce a supernatant 50 and sediment 52 
and subsequently slowly decant supernatant to obtain a sediment. Add an 
equal volume of 14.4 micrograms/milliliter acridine orange in HBSS or PBS 
(i.e. the solution 15) to the sediment to obtain a sample/stain mixture 
85. Place 1 drop (approximately 50 .mu.l) of sample/stain 85 on a 
microscope slide 70 and place a coverslip 72 on top of sample/stain 85 
drop. Examine for parasites using microscope equipped with an ultraviolet 
light source (e.g. a fluorescence microscope). Parasites are detectable by 
being differentially stained by acridine orange as depicted in FIG. 8. 
EXAMPLE II 
Swab a rectal canal of a patient with sterile cotton tip 44 to obtain an 
intestinal mucosa sample; or obtain sample from feces of the patient. 
Immediately suspend sample in HBSS or PBS (or the aqueous solution 32 as 
previously described) with 14.4 micrograms/milliliter of acridine orange 
stain by swirling the cotton tip 44 in the solution 32 or, in the case of 
feces, emulsify and suspend approximately 0.5 cubic centimeters in the 
solution. Within 24 hours centrifuge sample to produce supernatant 92, and 
sample/stain sediment 94. Slowly decant supernatant 92 and place 1 drop 
(approximately 50 .mu.l) of sample/stain sediment 94 on a microscope slide 
70 and place a coverslip 72 on top of drop. Examine for parasites using 
microscope 76 equipped with an ultraviolet light source (e.g. a 
fluorescence microscope). Parasites are detectable by being 
differentially-stained as best shown in FIG. 14. 
EXAMPLE III 
Swab intestinal mucosa in a rectal canal of a patient with sterile cotton 
tip 44 of one or more swabs 40 to obtain an intestinal mucosa sample; or 
obtain sample from feces of the patient. Immediately suspend sample in 
HBSS or PBS (or the aqueous solution 13 as described above) by swirling 
the cotton tip 44 in the solution; or, in the case of feces, emulsify and 
suspend approximately 0.5 cubic centimeters in the solution 13. Within 24 
hours in the case of the solution 13 containing no fixative and/or 
preservative, within four days in the case of the solution 13 being 
refrigerated at about 4.degree. C. or containing a preservative such as an 
alcohol, and within 6 months, in the case of solution 13 containing a 
fixative such as formalin, centrifuge sample to produce a supernatant 50 
and sediment 52 and subsequently slowly decant supernatant to obtain a 
sediment. Spread a thin layer of emulsified bottom sediment on a 
microscope slide and allow to dry. Stain emulsified dried bottom sediment 
by placing enough acridine orange solution (14.4 micrograms/milliliter in 
HBSS or the solution 15 as described above) to cover the thin layer of 
dried bottom sediment for 2 minutes. Then rinse side of microscope slide 
having dried bottom sediment covered with acridine orange solution with 
water and allow to dry. Place one drop (approx. 50 .mu.l) of PBS or HBSS 
or suitable mounting medium on the rinsed side of the slide 70 and cover 
with cover slip 72. Examine for parasites using microscope equipped with 
an ultraviolet light source (e.g. fluorescence microscope). Parasites are 
detectable by being differentially stained as best shown in FIG. 8. 
EXAMPLE IV 
Swab intestinal mucosa in a rectal canal of a patient with sterile cotton 
tip 44 of one or more swabs 40 to obtain an intestinal mucosa sample; or 
obtain sample from feces of a patient. Immediately suspend sample in HBSS 
or PBS (or the aqueous solution 13 as previously described) by swirling 
the cotton tip 44 in the solution 13 or, in the case of feces, emulsify 
and suspend approximately 0.5 cubic centimeters in the solution 13. Within 
24 hours in the case of the solution 13 containing no fixative and/or 
preservative, within four days in the case of the solution 13 being 
refrigerated at about 4.degree. C. or containing a preservative such as an 
alcohol, and within 6 months, in the case of solution 13 containing a 
fixative such as formalin, centrifuge sample to produce a supernatant 50 
and sediment 52 and subsequently slowly decant supernatant to obtain a 
sediment. Slowly decant supernatant and spread a thin layer of emulsified 
bottom sediment on a microscope slide and allow to dry. Fix dried bottom 
sediment by covering with 30% by volume ethanol/70% by volume HBSS for 2 
minutes, rinse side of microscope slide having dried bottom sediment 
covered with ethanol/HBSS with water and air dry. Stain rinsed side of 
slide by placing enough acridine orange solution (14.4 
micrograms/milliliter in HBSS or the solution 15 as described above) to 
cover the rinsed fixed layer for 2 minutes. Then, rinse side of microscope 
slide having the stained, fixed, dried bottom sediment with water and 
allow to dry. Place one drop (approx. 50 .mu.l) of PBS or HBSS or suitable 
mounting medium on the rinsed side of slide and cover with cover slip 72. 
Examine for parasites using microscope 76 equipped with an ultraviolet 
light source (e.g. fluorescence microscope). Parasites are detectable by 
being differentially stained as best shown in FIG. 8. 
EXAMPLE V 
Swab intestinal mucosa in a rectal canal of a patient with sterile cotton 
tip 44 of one or more swabs 40 to obtain an intestinal mucosa sample; or 
obtain sample from feces of the patient. Immediately suspend sample in 
HBSS or PBS (or the aqueous solution 13 as previously described) by 
swirling the cotton tip 44 in the solution 13; or, in the case of feces, 
emulsify and suspend approximately 0.5 cubic centimeters in the solution 
13. Within 24 hours in the case of the solution 13 containing no fixative 
and/or preservative, within four days in the case of the solution 13 being 
refrigerated at about 4.degree. C. or containing a preservative such as an 
alcohol, and within 6 months, in the case of solution 13 containing a 
fixative such as formalin, centrifuge sample to produce a supernatant 50 
and sediment 52 and subsequently slowly decant supernatant to obtain a 
sediment. Slowly decant supernatant and spread a thin layer of emulsified 
bottom sediment on a microscope slide and allow to dry. Fix dried bottom 
sediment by covering with 30% by volume ethanol/70% by volume HBSS for 2 
minutes, rinse side of microscope slide having dried bottom sediment 
covered with ethanol/HBSS with water and air dry. Stain rinsed side of 
slide by placing enough acridine orange solution 15 (14.4 
micrograms/milliliter in HBSS or the solution 15 as described above) to 
cover rinsed, fixed bottom sediment layer for 2 minutes, then rinse side 
of microscope slide having the stained, rinsed fixed bottom sediment with 
water and allow to dry. Permanently fix sediment on slide by covering the 
rinsed, stained bottom sediment with 10% formalin for 2 minutes, rinse 
side of microscope slide having the permanently fixed, stained bottom 
sediment with water and allow to air dry. Place one drop (approx. 50 
.mu.l) of PBS or HBSS or suitable mounting medium on the rinsed side of 
the slide and cover with cover slip 72. Examine for parasites using 
microscope 76 equipped with an ultraviolet light source (e.g. a 
fluorescence microscope). Parasites are detectable by being differentially 
stained as best shown in FIG. 8. 
While the present invention has been described herein with reference to 
particular embodiments thereof, a latitude of modification, various 
changes and substitutions are intended in the foregoing disclosure, and it 
will be appreciated that in some instances some features of the invention 
will be employed without a corresponding use of other features without 
departing from the scope of the invention as set forth.