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attachments.

9. The Fine Adjustment is just below the coarse ad-
justment. This very gradually raises and lowers the
barrel in order to obtain exact focus.

Illumination

Direct sunlight should be avoided. North light is to be
preferred, as it is the most uniform and steady. The
character and color of artificial light is much improved
by inserting a piece of blue glass between the concave
reflector and the object.

Focusing

Focus the body tube down until the objective touches
the cover glass, then with the eye to the eyepiece focus
up carefully.

Often one acquires the habit of using only the right
or left eye for microscopic work, but it is better to learn
to use both. Both eyes should be open when using the
microscope.

Always examine a specimen first with a low power and
then with a high power objective.

When the oil-immersion objective is used, a drop of
cedar oil is placed between the slide and the end of the



1 8 Clinical Laboratory Technic

objective, and the objective is brought into contact with
this and the observation made through the oil. The oil-
immersion objective is so constructed that when in use
the pencil of light passing through the object to the objec-
tive traverses only media of the same refractive index,
and cedar oil has the same refractive index as glass.

The oil acts as a third lens and increases the magnify-
ing power of the microscope.

A fine hair cemented into one of the oculars serves as
a pointer and is of great aid in singling out some special
object of interest.

Objects are often accidentally present in microscopical
preparations, such as air bubbles. If small, they may look
entirely dark ; if large, they are clear in the middle, with
a broad, dark border, due to refraction of the light.

Linen fibers, well-defined, rounded, slightly twisted.

Cotton fibers, longer, broader, but thinner and more
twisted than linen.

Woolen fibers and hairs have the same structure,
although the wool is finer and is curled.

Dust of the room, showing groups of black particles of
carbon (soot), and shed epithelium-cells derived from
the epidermis.

Molds, with long, branching filaments (hyphae), and
the torula-like particles (spores) from which hyphae may
in some instances be seen sprouting.

Yeast particles or torulse: each torula contains a clear
vacuole and has a well-defined outline, due to a membrane.

Starch granules : fine concentric lines are seen in the
granules, arranged around a minute spot which is placed
near the smaller end of the granule.

Lens paper or fine linen moistened with xylol should



The Microscope 19

be used to wipe the objectives, and this must be applied
carefully, as the lenses are mounted in balsam.

The microscope should be lifted by some one of its solid
parts, and never by any part above the stage.

The glass surfaces should never be touched with the
ringers, and the metal parts should be kept free from
liquids, especially acids and alkalies.



CHAPTER III
URINE

The volume of urine excreted by normal individuals
varies between 1500 and 2000 c.c.

The condition of the kidneys and their capability for
work are determined by accurate chemic and microscopic
examination, and such information is of great importance
in judging the diagnosis and prognosis of disease.

Anuria is the suppression of the secretion in the kidneys
or obstruction in the urinary tract.

Polyuria is the increase in the elimination of urine as
a whole, both fluid and solid.

Hydruria is the increase in the total twenty-four hour
amount out of proportion to the solids.

Oliguria is the diminution in the total excretion of the
urine.

Dysuria is painful or difficult urination.

Consistence

Clear and liquid, frequently turbid and viscid (ropy).
In normal urine this turbidity is caused by precipitated
phosphates or urates ; in abnormal urine it results from
casts, cells, etc. The slight cloud-nubecula which urine
develops on standing is formed by bacteria, desquamated
cells, and traces of mucus.

Odor

Aromatic, due to volatile acids. The odor of ammonia
is important only in fresh urine. If free ammonia is

20



Urine 21

present, moistened red litmus paper will turn blue when
held over the mouth of a tube in which perfectly fresh
urine is boiled. Acetone odor indicates diabetes mellitus ;
putrid odor denotes pus.

Collection for Analysis, Twenty-four Hour Amount

Different voidings may vary greatly in chemical com-
position; therefore quantitative tests are of no value
unless a sample of the mixed 24 hour amount be used.

At 8 A.M. the bladder is emptied and the urine thrown
away; then save all urine voided until 8 A.M. the next
day, voiding it exactly at 8 and adding to the amount
saved. Collect this 24 hour amount in a perfectly clean,
two-liter bottle, and keep well corked in a cool place. 1

Urine begins to decompose a few hours after it is
voided; therefore add 5 c.c. of toluol, one of the most
satisfactory preservatives, which apparently interferes
with none of the tests. Dust, feces, and expectoration
interfere with analysis, and care should be taken that
these do not enter the bottle.

A single specimen may be obtained for qualitative
examination, and as there is a marked variation in the
urine at different times a day, a specimen should be taken
several hours after a meal, when it contains the greatest
amount of abnormal elements.
Color

Normal urine varies from straw to amber color, and
this color is derived from urochrome and other pigments.

1 Chloral and chloroform reduce Fehling's solution and should not
be used if the urine is to be tested for glucose.

Formaldehyde coagulates albumin and reduces Fehling's.

Thymol is used for saccharine urine, as it has no reducing
action (t gr. to I fl. oz. of urine). Boric acid may be used, 5 grs.
to 4 fl. oz. of urine.



22



Clinical Laboratory Technic



Halliburton's Table



Color

Nearly color-
less.



Dark yellow to

brown-red.

i

Milky.



Orange.

Red or
reddish.



Brown to
brown-black.



Greenish yel-
low, brown
approaching
black.

Dirty green or
blue.



Brown-yellow
to red-brown,
becoming
blood-red
upon adding
alkalies.



Cause of Color
Dilution or diminution
of normal pigment.



Increase of normal or
occurrence of patho-
logic pigment.

Fat globules.
Pus corpuscles.

Excreted drugs, e.g.,

Unchanged hemoglobin.

Pigment in food (log-
wood, madder, bilber-
ries, fuchsin).

Hematin.
Methemoglobin.
Melanin.

Hydrochinone and cate-
chol.

Bile pigment.



A dark blue scum on
surface, with a blue de-
posit, due to an excess
of indigo-forming sub-
stances.

Substances contained in
senna, rhubarb, and
chelidonium, which are
introduced into the
system.



Pathologic Conditions

Various nervous con-
ditions, hydruria,
diabetes insipidus,
granular kidney.

Acute febrile diseases.



Chyluria.

Purulent disease in
urinary tract.

Santonin, chryso-
phanic acid.

Hemorrhage or
hemoglobinuria.



Small hemorrhages.
Methemoglobinuria.
Melanotic sarcoma.
Carbolic acid poison-
ing.

Jaundice.



Cholera, typhus; seen
especially when the
urine is putrefying.



Urine 23



Urine may darken on exposure to the air, owing to the
presence of alkapton, a decomposition product of the pro-
teins found as a crystalline body in the urine.

Reaction

In the majority of cases the mixed twenty- four hour
amount is acid to litmus. The reaction may undergo
marked changes in both physiological and pathological
conditions.

Animal food produces acid urine, and a vegetable diet
may give a neutral or alkaline urine.

The reaction varies according to the time of day the
urine is voided. It may be neutral for some time after a
meal, or alkaline to litmus, as at that time the secretion
of hydrochloric acid into the stomach during the process
of digestion reduces the store of acids in the body. This
change is known as the "alkaline" tide.

Urine may become alkaline owing to the conversion of
urea into ammonium carbonate by micro-organisms.

Microscopically such a urine shows amorphous phos-
phates, ammonium magnesium crystals, and ammonium
urate crystals.

If a urine is alkaline immediately after voiding, it may
be due to a fixed alkali or ammonia, and ammoniacal
fermentation is ordinarily due to cystitis.

Acid fermentation occurs in urine having a normal
acidity when voided, which upon standing becomes
strongly acid. The sediment of such urine shows acid
urates, uric acid, calcium oxalate crystals, and fungi.

Blue litmus paper is changed red by acid urine. Red
litmus paper is changed blue by alkaline urine.

When urine changes blue litmus paper red and red



24 Clinical Laboratory Technic

9

litmus paper blue, it has an amphoteric reaction, due to
the simultaneous presence in the urine of acid and alkali
salts.

Specific Gravity

The specific gravity of a body is its weight, as compared
with the weight of an equal bulk of a standard body taken
as a unity.

The specific gravity of normal urine varies between
1,015 and 1,025. This means that, taking distilled water
at 77 F. as i, each cubic centimeter of urine weighs
1,015 or 1,025 grams. The urine should be at the same
temperature. If it is not, to every 7 above normal add
one unit of the last order to the last reading; to every
7 below normal subtract one unit of the last order.

EXAMPLE. The urinometer is graduated for 77 F.
and reads 1,018 at 63 F. ; then the specific gravity at
77 F. would be 1,018 0.002, or 1,016. 77 63 =
14, and one unit is subtracted to every 7 below normal;
14 divided by 7 = 2, therefore 2 X o.ooi (one unit
of the last order) = 0.002. 1,018 0.002 = 1,016.

The relative proportion of solid matter in the urine is
shown by the specific gravity, and by knowing the total
amount in twenty- four hours an approximate idea of the
absolute solids is obtained by multiplying the last two
figures of the specific gravity by Long's coefficient, *. e.,
2.6. The solid content in 1,000 c.c. is obtained by multi-
plying the last two figures of the specific gravity observed
at 25 C. by 2.6.

EXAMPLE. Sp. gr. = 1,020. 20 X 2.6 = 52 grams,
the amount of solids in 1,000 c.c. of urine. The total



Urine 25

amount in twenty-four hours is 1,500 c.c. ; therefore it
will contain 1,000 : 1,500 : :$2 :x, or 78 grams,

Meat, drugs (potassium acetate), albumin, and diabetes
mellitus raise the specific gravity.

There is a decrease in chronic interstitial nephritis and
diabetes insipidus.

METHOD. The urinometer glass is filled three-fourths
full of urine ; then introduce the urinometer and wait until
it finds the correct level before reading the scale. The
last mark seen below the surface (the meniscus) is the
correct reading of the scale, and this should be read
through the fluid from below upward.

When the amount of urine is so small .that it is neces-
sary to dilute to get sufficient to take the specific gravity,
add four times as much water as urine, making five
volumes.

EXAMPLE. To one volume of urine add four volumes
of water, making five volumes. If the specific gravity of
this mixed fluid reads 1,005, then that of the urine will
be 1,000 plus (5 X 5), or 1,025.

Albumin

Albuminuria is a condition in which serum albumin or
serum globulin appears in the urine, and these two protein
substances are of the greatest pathologic import.

In the accidental albuminuria the albumin is not ex-
creted by the kidneys, but arises from the blood, lymph,
or some albumin containing exudate coming in contact
with the urine at some point below the kidneys.

The renal type is the more serious, as the albumin is
secreted by the kidneys.



26 Clinical Laboratory Technic

Nitric Acid Test. (Heller's)

Put 5 c.c. of filtered urine in a test tube and introduce
5 c.c. of concentrated nitric acid very carefully with a
pipette to the bottom, forming an underlayer.

Various colored zones, due to the presence of indican,
bile pigment, or to the oxidation of other organic urinary
elements, may form, but these should never be mistaken
for the white zone which alone indicates serum albumin,
serum globulin, albuminose, and peptone.
Magnesium Mixture for Turbid Urine

Filter 10 c.c. of urine; add sufficient magnesium mix-
ture to make the urine alkaline. After testing with litmus
paper, add a few drops of acetic acid. Filter, and test for
albumin with nitric acid.
Albumose Test for "Bence- Jones" Bodies

To 10 c.c. of urine add 5 drops of acetic acid ; boil, then
filter. On the surface of 5 c.c. of nitric acid in a test tube
gently pour some of the cooled filtrate. A white ring at
the junction of the fluids indicates the presence of albu-
mose, and this will disappear on warming, to reappear
again on cooling.
Coagulation or Boiling Test

Fill the test tube half full of filtered urine and gently
heat the upper half to boiling, being careful that this half
of the fluid does not mix with the lower half. A turbidity
indicates serum albumin, serum globulin, or phosphates.
Acidify the urine with 5 drops of acetic acid; the turbid-
ity, if due to phosphates, will disappear. If albumose is
present, a white cloud appears on heating, disappears
on boiling, reappears on cooling.



Urine 27

Quantitative Test for Albumin. (Esbach's Method)
This test is made by means of a standard graduated
glass tube or albuminometer.

Add 10 drops of a 10% solution of FeCl 3 to the urine
before introducing the Esbach's reagent ; warm the tube.
Fill the tube with urine to the letter U, then add Esbach's
reagent to R; close the tube with a stopper and invert
several times. Let the tube stand 2 hours, then read off
the number of grams of albumin to the liter, as will be
indicated by the number on the side of the tube on a
level where the albumin settles. If the urine is alkaline,
add a few drops of acetic acid. Urine heavy with
albumin should be diluted with i or 2 volumes of water.
EXAMPLE. The protein precipitate is level with the
figure 2 of the graduated scale, therefore the urine con-
tains 2 grams of the protein to the liter, or .2% (the
amount of protein in per cent).

Test for Lead

Evaporate 2 liters of urine to a tenth of its volume.
Add an equal volume of 20% hydrochloric acid, and
3 grams of potassium chlorate. Heat the mixture on
the water bath to 60 C. Add 3 rrams of potassium
chlorate as soon as the evolution of chlorine has ceased.
Repeat this until the fluid no longer gives off the fumes
of chlorine on the further addition of the chlorate. Water
may be added if the liquid becomes too concentrated.
Cool, dilute with water, then filter. Examine the filtrate
for lead with hydrogen sulphide, sulphuric acid, and
potassium bichromate. If lead is present there will be
precipitates of black lead sulphide, white lead sulphate,
and yellow lead chromate.



28 Clinical Laboratory Techmc

Albumin. (Goodman and Steam's Method)

To 5 c.c. of phosphotungstic acid solution add 2 c.c. of
filtered urine with a pipette graduated in tenths. Shake
after addition of each tenth; add urine until a whitish
cloud appears. The number of tenths is read off and
expressed in terms of 100 c.c. EXAMPLE. If it takes i c.c.
of diluted urine (i-io) there is o.oooi gram albumin,
or i gram in 100 c.c. ; if 0.7 c.c. of diluted urine, then
0.07 c.c. of undiluted urine equals o.oooi gram albumin.
7 c.c. = .01 gram albumin, 700 c.c. = i gram. 700:1.0
::ioo:x, or .142%.

Sugar

Very small amounts of glucose probably occur in every
normal urine. In diabetes mellitus there is a permanent
or persistent glucose excretion. Glucosuria is a transient
type and accompanies various disorders, particularly
cerebral digestive affection, certain forms of poisoning,
morphine, carbon monoxid, chloral hydrate, oil of turpen-
tine, corrosive sublimate, amyl nitrite, and prolonged
hunger. Sometimes perfectly healthy people excrete
glucose in urine after too abundant ingestion of sugar.

Urine containing much glucose presents a light or pale
color and has a high specific gravity.

The most common sugar occurring in urine is glucose.
Levulose is sometimes present with glucose. Lactose is
occasionally found in the urine of nursing women ; laiose,
maltose, and pentose are rare. If albumin is present, it
should be removed before testing for sugar. This is
done by boiling and adding a few drops of acetic acid,
and filtering.



Urine 29

Benedict's Test responds to all the carbohydrates,
and every urine should be tested for sugar by this re-
duction test.

Fermentation Test responds only to glucose, levulose,
and maltose.

Phenylhydrazin Test responds to glucose, levulose,
maltose, lactose, and pentose. This is a very delicate
and reliable test.

o

Polarimetric Test: glucose, lactose, and maltose, and
to a slight extent pentose, rotate the ray of polarized light
to the right; while levulose, /3-oxybutyric acid, and gly-
curonates rotate polarized light to the left. Normal urine
is often slightly dextro-rotary.

Benedict's Qualitative Test for Glucose

Heat to boiling 5 c.c. of Benedict's Solution in a test
tube (put a glass bead in the tube to prevent bumping).
Add 8 drops of urine and boil 2 minutes. If more than
.2 to .3% of sugar is present, the solution will be filled
with a greenish, yellow, or reddish precipitate. With
smaller amounts of sugar the precipitate appears only
on cooling.

The reagent is made as follows:
86 grams sodium citrate
50 grams anhydrous sodic carbonate
400 c.c. distilled water

Stir until entirely dissolved, then add 8.6 grams copper
sulphate that has been dissolved in 50 c.c. of hot water.
Pour the copper solution slowly, and with stirring, into
the alkaline citrate solution. Make up to one liter.



Clinical Laboratory Technic




Benedict's Method for the Quantitative Estimation of Sugar.-
From GradwohFs " Blood and Urine Chemistry"



Urine 31

Benedict's Quantitative Estimation o Glucose

. Dilute 10 c.c. of urine with distilled water up to 100 c.c.
Mix, then transfer to a burette.

Pipette 25 c.c. of the volumetric solution into a Jena
flask (150 c.c. capacity). Add 5 to 10 grams of sodium
carbonate and i gram of powdered pumice.

Heat the mixture to boiling. Run urine in rapidly
from the burette until a white precipitate begins to form,
then more slowly with continuous boiling, until the blue
color entirely disappears. This is the end point.

EXAMPLE. If 8 c.c. of diluted urine were used in
reducing 25 c.c. of Benedict's Volumetric Solution, 1 then
0.8 c.c. of undiluted urine was required, and this amount
contained 0.050 gram glucose. The percentage in the
sample would be calculated thus :

0.8 : 0.050: : 100: x, which equals 6.25%
If 1,400 c.c. of urine were voided in twenty-four hours,
then 0.8: 0.050: : 1,400: x or 87.50 grams glucose.

Benedict's Volumetric Solution
18.0 grams copper sulphate.
100 grams anhydrous or double the quantity of

crystallized sodium carbonate.
200 grams of sodium or potassium citrate.
125 grams of potassium sulphocyanate.
5 c.c. of a $% solution of potassium ferrocyanide.
Make up to I liter with distilled water.
Dissolve the copper sulphate separately in 150 c.c. of
distilled water and then add slowly with constant stir-
ring to a filtered solution (about 800 c.c.) of the other
ingredients. Make up to I liter.

iNote. The 25 c.c. of copper solution are reduced by exactly
50 mg. of glucose.



32 Clinical Laboratory Technic

Fermentation Test

This test is important because the fermentable sugars-
are the pathological ones. Carbon dioxide and alcohol
are formed in the fermentation of sugar, and the produc-
tion of alcohol and the disappearance of sugar lower
the specific gravity.

The urine should be sterilized by boiling, and if not
already acid, acidified with hydrochloric or tartaric acid.
Cool, and determine the specific gravity. Add a small
piece of yeast about the size of a bean. Place 200 c.c. of
this urine in a flask and stopper sufficiently close to pre-
vent the escape of alcohol. 1 Let the flask of urine stand
in a warm room 24 hours, then test with Fehling's solu-
tion. If sugar is still present, let it stand tor another
24 hours. If the reducing substance is still present, it is
not a fermentable sugar. Record the specific gravity.

It has been found that a decrease of o.ooi (a fall of
one point) in the specific gravity corresponds to 0.23%
of sugar in the urine tested.

EXAMPLE. The specific gravity of the urine before
fermentation is 1,030. After the fermentation is com-
pleted, the specific gravity is 1,008, a decrease of 22
points ; and this multiplied by 0.23 gives the amount,
5.06%, of sugar in the urine tested.

Phenylhydrazin Test

Sugars form ozazones when treated with phenyl-
hydrazin.

1 Great care should be taken not to plug the fia.sk so tightly as to
cause an explosion. This method is a much more accurate procedure
if the saccharometer of Lohnstein is used.



Urine



33




PHENYLHYDRAZIN TEST FOR SUGARS

In a test tube put nearly ^ in. (i gm.) of phenylhydrazin hydro-
chloric!, an equal quantity of powdered sodium acetate, and
enough of the suspected fluid to half-fill the tube. The acetate
dissolves as the tube is heated. Boil for 2 minutes and examine
after 20 minutes, or, if hurried, examine a drop under the micro-
scope at once without a cover-glass. In 2 or 3 minutes the crystals
form: a, Sheaves and stars of needles glucosazone; b, rosettes
of lance-shaped crystals maltosazone; c t spicules in burr-like
clusters lactosazone.



34 Clinical Laboratory Technic

Benzidin Test for Occult Blood

Add 2 c.c. of a saturated solution of benzidin, in alco-
hol or acetic acid, to 2 c.c. of 3% hydrogen peroxide and
i c.c. of urine. Blood is indicated by the appearance of
a green or blue color.

Gerhardt's Test for Acetone

To 10 c.c. of urine in a test tube add 10 drops of acetic
acid, then add 10 drops of freshly prepared sodium nitro-
prusside solution 1 and mix; carefully overlay with 2 c.c. .
of concentrated ammonia.

If acetone be present, a violent- red ring will develop
at the point of contact. The amount of acetone will be
increased in fever, with starvation, with purely meat diet,
in diabetes mellitus, in certain forms of digestive disturb-
ance, and in some cases of carcinoma.

Acetone is a physiological as well as a pathological
constituent of the urine.

Acetone is said to be excreted under the following
pathological conditions :

Diabetes mellitus

Pneumonia

Nephritis

Deranged digestive function

Fasting

Autointoxication

Anesthesia

Scarlet and typhoid fevers

Phosphorous poisoning

Grave anemias

1 Sodium nitropmsside solution: To 5 grams sodium nitroprusside
add 5 c.c. water; or a crystal of sodium nitroprusside may be added to
the acidified urine.



Urine 35

Normal adults on a mixed diet excrete 3 to 15 mg. of
combined acetone and acetoacetic acid per day; more is
considered pathological.

In severe diabetic acidosis 6 grams or more may be
excreted, Acidosis is due mainly to a disturbance in
the metabolism of fats.

Diacetic Acid Test

To 5 c.c. of urine add an excess of a 10% solution
of ferric chloride. A Bordeaux-red color indicates dia-
cetic acid. If this color is masked by the precipitate
of ferric phosphate, the fluid should be filtered. The red
color, if due to diacetic acid, disappears on heating.
The presence of antipyrin, aspirin, phenacetine, salicylic
acid, and sodium acetate give a similar red color, which
does not disappear en heating.

Test for Indican or Potassium Indoxyl Sulphate

The test for indican is based upon the fact that an excess
of HC1 will liberate the indoxyl, and by the addition of
an oxidizing agent this is converted into indigo blue, and
finally this can be recognized in small amounts by extrac-
tion from the bulk of urine with chloroform. The pres-
ence of more than a trace indicates the existence of un-
desirable decomposition in the intestinal tract.

METHOD. Add 10 c.c. of urine to the same amount of
Chemically Pure concentrated HC1. Mix and add 3 drops
of freshly prepared \% solution of potassium perman-
ganate. If indican is present, a purplish cloud will form.
Then add a few drops of chloroform and then a few more
drops of permanganate solution. Shake vigorously and



36 Clinical Laboratory Technic

the color will change to a deep blue, due to the precipita-
tion of indican by the chloroform.

Test for Melanin

In cases of melanotic sarcoma, the urine treated with
iron chlorid assumes a deep black color.

Urochromogen and Diazo Tests in Tuberculosis

(Journal A. M. A., October 10, 1914)
Place i c.c. of limpid urine diluted with 3 c.c. of water


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