Unit Six

Parasitological Techniques
6.1. Stool Specimen
• Specimen submitted to the laboratory for examination of parasites
must be collected & transported to the laboratory without delay.
Collection and Handling of fecal specimen
• Collected in clean dry wide mouthed containers.
• Collected prior to any medications & avoid contamination with
urine.
• Approximately 5-7 gms of feces collected.
• Safety should be considered (gown, gloves, etc)
Number of specimens
• At least three specimens should be submitted for routine
examination of intestinal parasite before reporting <No ova of
parasite seen>
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Fresh versus preserved specimens
• Liquid stool should be examined within 15 minutes of
passage or be preserved.
• Semi-formed specimens should be examined within 1
hour passage or preserved.
• The three most common preservatives are:
– formalin,
– PVA (polyvinyl alcohol), and
– sodium acetate formalin

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• Formalin: is an effective for long term preservation
of protozoan cysts, helminthes eggs and larvae.
– The proportion is 3 parts of formalin in 1 part of stool
– Formalin does not preserve trophozoites.

• PVA: Proportion 3 parts of PVA in 1 parts of stool.

• Sodium acetate formalin (SAF):used for protozoan cysts &

trophozoites, helminthes eggs & larva, and intestinal coccidian
(such as cryptosporidium parvum).

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 Processing of specimens
– Macroscopic observations
– Microscopic examination
– Serological diagnosis
1) Macroscopic Observations
Consistence Color Elements Stage Parasite contain

1 Hard Black Pulp & fiber nearly pure All stage except trophozoite

2 Formed Brown Conspicuously fibrous All stage except trophozoite

3 Soft Yellow Feces with scanty mucus All stage except trophozoite

4 loose Green Feces with much mucus All stage

5 Diarrheic Clay Mucus with much feces All stage except Cyst

others Others (blood, barium, etc

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 2) Microscopic examination
• Divided in to three steps:
– Direct wet mount
– Concentration technique
– Permanent stain smear
Direct wet mount preparations
• The primary purpose to detect protozoan trophozoites & their
characteristics motility.
• Prepared by mixing a small amount of specimen (approx.
2mg) in a drop of 0.85% saline (physiological saline) on clean,
dry microscope slide.
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 Direct wet Mount
– Saline method (Eosin Method)
– Iodine method

Concentration
• Need for fecal concentration technique:
– When the number of parasite in the sample is too few to be detected by
direct microscopy.
– To check whether treatment has been successful.
– To quantify (enumerate) the parasite.
• Concentration techniques can be:
– Sedimentation technique
– Floatation Technique
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a) Sedimentation Technique(formol-ether)
– is one of the most widely used techniques.
b) Floatation Technique(Zinc_sulphate)

Permanent Stains
• Used for the identification of some of the smaller protozoans by
their detailed cytological morphology.
• Three permanent stains widely used routinely:
– Iron hexatoxyline

– Wheatlev’s trichrome stain

– Modified acid fast stain

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 Preparation of Blood Smears for Parasite examination
• Blood is the specimen of choice for plasmodium species,
Babesia species, trypanosome species, Leishmania species &
Microfilaria (except O. volvulus).
• Blood sample may be obtained from the finger tips, earlobe, or
vein-puncture.
• it may be anticoagulated or non-anticoagulated.
• Two kinds of smears may be made.
– Thin &
– Thick.

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Types of Blood Film
• Thin blood smears are suggested for the observation of
morphologic detail & species identification.
• The thin blood film is made in the same manner as blood smear
used for a differential count in hematology.
• Thick smears are typically used for screening purpose,
particularly in cases where malaria or babesiasis is suspected.
Staining of Blood Film
• Generally, there are two permanent stains that are commonly
used in the clinical laboratory to stain parasite.
1. Wright’s &
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2. Giemsa stains. tafeseb.tufa 9
  
Unit-Seven
Basic Bacteriological Techniques

• General precaution of specimen collection:-

– Correct types of specimen should be collected base up on the
types of pathogen to be isolated.
– Should be collected at correct time for better isolation.
– Must be collected from an active lesion.
– Should be collected under aseptic condition.

– Sufficient specimen should be collected.

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 – Collect specimen before instituting antibiotic therapy.
– Used sterile collection devices and containers.
– Place in suitable container, labels appropriately.

– All clinical specimens should be considered as potential

biohazards

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Types of specimen
– Cerebrospinal fluid (CSF)
– Feces
– Sputum
– Swabs of Various Fluids
– Blood
– Urine
– Skin smear
– Skin snip
– Skin scraping, etc

NB:- Order the correct test from correct sample, at correct

time, and from correct patient.
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• The major steps bacteria staining for microscopic examination
are:-
Labeling –> smearing –> drying –> heat fixing –> staining

• Labeling of slides:- with wax pencil or glass writing diamond.

• Making smears:- This can be prepared using wire loops, clean

match stick, applicator stick or cotton swabs.

• Drying the smears: - Allow the smears to air dry before fixing.
Do not dry the smears using a flame while the smear is wet.

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• Fixing the smears:- pass the slides over a flame of Bunsen
burners three or four time or alcohol.

• Staining of smears: - This step involves the application of

dyes as desired.
• After staining wipe the back of the slides clean and place in a
drying rack for the smear to dry.
• Examine the smear then with the oil immersion objective.

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 Commonly used staining methods
• In bacteriology, there are three categories
1. Simple stains
2. Differential stains

3. Special stains

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1. Simple staining

• This involves the use of a single dye solution to stain for a specific
period of time.

• All the cells stain uniformly which is useful to show the size, shape
and arrangement of bacterial cells

• The common dyes used for simple staining are:

– Methylene blue

– Carbon fuchisin

– Crystal Violet

N.B. Simple staining is not used for routine laboratory investigation

because it has no differential diagnostic value.
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 2. Differential Staining
• This method involves the application of more than one dye.
e.g. Gram staining & Acid fast staining (AFB)
Gram`s Staining
• It divides bacteria in to two groups:- the gram positive and
gram negative bacteria.
– What is the Principle of gram staining?

– What is the Procedure for gram staining?

– How do you Report gram stain result?

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Theory behind Gram stain

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 Reagents for Gram Stain
• Crystal Violet (purple).
• Primary stain; positive stain
• Stains cell wall purple
• Iodine
• Mordant
• Combines with primary stain to form an insoluble complex that gets
trapped in thicker peptidoglycan layers
• Ethanol
• Decolorizer
• CV-I complex washed out of Gram negative organisms because it
cannot be trapped by peptidoglycan layer.
• Safranin (pink)
• Counterstain
• Simple positive stain that provides contrasting dye for decolorized cells
• Stains all cells, but only the negative ones actually appear pink.

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Procedures and events

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 Ziehel Nielsen staining (Acid fast staining)
 It enables to differentiate b/n AFB such as TB and M. leprosy
from non-AFB.
 Because of their resistance for decolonization with acid and
termed as acid-fast.
 It is due to the presence of high lipid in their cell wall (mycolic
acid).
– What is the Principle of acid fast staining?
– What is the Procedure for acid fast staining?

– How do you Report acid fast staining result?

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Number of AFB found Report
– More than 10 AFB/F (at least 20 fields) ..…………… 3+
– 1-10 AFB /F (at least 100 fields)………………………2+
– 10-100 AFB /100 fields …………………...………….1+
– 1- 9 AFB/100 fields ………………exact number seen (scant)

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 Culture

Depending on uses Culture media can be classified in to:

– Basic
– Selective
– Differential (Indicator)
– Enriched
– Enrichment
– Transport

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Basic
• It can be:
– Nutrient agar
– Nutrient broth

• Use:
 Preparation of enriched media
 Maintain stock cultures of control strains of bacteria.

 Sub-culturing pathogens from differential or selective media

prior to performing biochemical and serological identification
tests.
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 Selective
• Solid media
• Contain substances (e.g. bile salts, dyes, antibiotics) which
inhibit the growth of one organism to allow the growth of
another.
• From a site having a normal microbial flora to prevent
unwanted contaminants overgrowing a pathogen.

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Differential

• Dyes or other substances are added to differentiate

microorganisms.
• It can differentiate among Microorganisms spps
• E.g.
– Sabouraud’s agar
– Blood agar
– TCBS /Thiosulphate Citrate Baro Sulfate/ can support the growth of V.
cholera.

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Selective -differential
• Mannitol salt agar
– Both selective and differential medium.
– High salt concentration - inhibits most bacteria.
– Selective for Staphylococcus sp.
– Differentiate between Staphylococcus sp. by the sugar mannitol
fermentation .
• MacConkey agar
– Both selective and differential.
– Crystal violate and bile salts inhibit gram positive bacteria.
– Lactose fermenters are red colonies and non lactose fermenters are
light pink colonies.

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Enriched

• Required for the growth of organisms with exacting growth

requirements (Fastidious): H. influenzae, Neisseria species,
Streptococcus species.
• Enriched with whole or lyzed blood, serum, peptones, yeast
extract, vitamins and other growth factors.
• Low number of organisms.

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 Enrichment

• Applied to fluid selective media.

• Contain substances that inhibit the growth of unwanted

organisms.
• Rappaport-Vassiliadis broth used as an enrichment medium for
Salmonella serovars in faeces.

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 Culture & Identification

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Examples: Pathogen Mixed with Normal Flora & a Pure
Culture Obtained by Subculture.

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 Unit Eight - Urinalysis
• Definition: - is a analysis of the physical, chemical and microscopic
properties of the urine.
– Physical properties: - simple observation of volume, odor, color,
transparence of urine.
– Chemical property- test of bilirubin, glucose, protein, ketone, etc
– Microscopic properties:- urinary sediments like:
• Cells – RBC, WBCS, epithelial cell
• Organisms – parasites, bacterial, yeast
• Casts and crystals.
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 8.1. The Composition of urine
Normal urine constituent’s
– Water about 95% of urine
– Urea
– Creatinine
– Uric acid
– Electrolytes
Abnormal urine constituents
– Glucose
– Protein
– Bile pigments
– Blood cells
– Pathogenic bacterial, yeasts, parasites.

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 8.2. The factors affecting the composition of urine
– Diet and nutritional status.

– Condition of body metabolism.

– Ability of kidney function.

– Level of contamination with pathogenic microorganisms (bacteria).

8.3. Collection of urine specimen

– The container used should be dry and clean.

– The collection procedure → label, keep time of collection, volume.

– The condition of storage and preservation.

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8.4. Types of Urine Specimen
A) First morning specimen:- a specimen obtained during the
first urination of the day.
– Most concentrated
– Bladder incubated
• Best for: Nitrite, Protein, Microscopic examination, HCG test.
B) Second- voided specimen: - in this case first morning
specimen is discarded & the second specimen is collected.
• Such type of specimen is good for:-
– Reflection of blood glucose.

– Keeping of formed elements intact.

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C) Random specimen:- a specimen obtain at any time during
examination
– Most convenient
– Most common

• Good for:-
– Chemical screen
– Microscopic examination

D) Postprandial:- a specimen obtained two hours after meal

– Good for glucose

E) 24 hour specimen:- a specimen obtained within 24hrs

– Necessary for quantitative tests, especially for quantitative
determination of protein and volume.

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F) Supera pubic Urine: taken by syringe directly from the
bladder
– Best for the bacteriological analysis
G) Mid stream specimen:- a specimen obtained from the middle
part of the first time.
– It is commonly used for routine urinalysis
– It is also important for bacteriological urine culture.
H) Clean catch urine specimen
– Used for microbial culture and routine urinalysis
– Urine sample obtain after cleaning the genital area by water and soap.
I) Catheterization
– Urine sample directly obtained from the bladder by inserting the
catheter.
– Used for the microbiological analysis.

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Sources of Errors in the collection of urine:
• Bacteriologically or chemically contaminated specimen.
• Wrong type/amount of preservative.

• Partial loss of specimen or inclusion of two morning specimen in

the 24hr collection.
• Inadequate mixing of specimen before examination.
• Careless measuring of the 24hr volume.

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8.5. Preservation of Urine Specimen
• The maximum time that urinary contents to be maintained in
urine specimen is 1 hr.
• Long standing of urine at room temperature can cause
– Oxidation of bacteria.
– Break down of urea to ammonia by bacteria leading to an increase in
the PH of the urine and this may cause the precipitation of calcium and
phosphate.
– Oxidation of urobilinogen to urobilin.
– Destruction of glucose by bacteria.
– Lysis of RBCs, WBC, and casts.
– Acetone evaporates.
– Acetoacetic acid will be converted in to acetone.
– Bilirubin oxidized to biliverdin.

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 Method of preservation of urine specimen
a) Physical Method
• Used refrigerator (4-80C) for 6-8 hours

b) Chemical method
• Toluene:- is a liquid working by preventing growth of bacteria
• Thymol:- is a crystalline working by preventing growth of
bacteria
• Formalin:- a liquid acting by fixing the formed elements.

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8.6. Examination of urine

8.6.1. Physical examination of urine

• is the first part of routine urinalysis.
• It usually gives hint for the subsequent urinalysis.

a) Volume

• Usually indicative of the balance b/n fluid ingestion and extra renal loss of water

• The test requires good collection and time specimen.

• Volume of urine excreted is related to:

– Individual fluid intake
– Body temperature
– Climate
– Individual’s health status

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• Normal value is depend on the age:

– New born birth- 3 days ………….. 20 - 30ml/24hrs

– New born 5 - 10 days…………….… 100 - 350ml/24hrs
– Children 1 year……………………… 300 - 600ml/24hrs
– Children 10 year……………………..750 - 1500ml/24hrs
– Adults ………………………………..750 - 2000ml/24hrs
Note:- the ratio of night to day excretion is 1:2 or 1:3 for adult.

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 Clinical significance
• When an individual excretes more than 2000 ml of urine 24hr,
consistently (for long period) it is called polyuria.
• It may occur due to:
– Diabetic mellitus

– Certain tumors of brain and spinal cord

– Acromegaly
– Some type of tubular necrosis

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 • Any increased amount of urine volume, even if for short
period, is called dieresis.
• It is usually due to excessive fluid intake.
• Polyuria may result physiologically after consumption of:-
– Intravenous glucose or saline.

– Coffee, alcohol, tea, caffeine.

– Pharmacological agent, such as thiazides and other
diuretics.

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• Excretion of constantly small amount of urine (<400ml) called
Oliguria.
• Oliguria may occur due to:-

– Dehydration or poor blood supply to kidney that may be due to

prolonged vomiting, diarrhea, etc.
– Obstruction of some area of the urinary tract.
– Cardiac insufficiency.
– Various renal diseases such as glomerulonephritis, etc.
– Excessive salt intake, etc.

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• Complete absence of urine excretion is (<100ml) called Anuria.
• Anuria may occur due to:-
– Complete urinary tract obstruction
– Acute renal failure

– Acute glomerulonephritis
– Hemolytic transfusion reaction, etc.

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b) Odor
• Normally fresh urine has faint aromatic odor, normally found in
urine mostly ammonia.
Clinical Significance
• Abnormal urine odor may result from aging of urine, disease and
diet.
– If the urine specimen is old i.e. after collection ammonical
(pungent) odor.
– Cystinuria & homcystinuria have sulfurous odor.
– “Sweet fruity” odor due to ketone bodies.
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 c) Foam
• Normally the urine is slightly foam when it is shaken.
• Normally the foam is white.
• The presence of bile pigments (bilirubin) will cause a vivid
yellow foam.

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d) Color
• Normally color of urine may vary within a day.
• Correlates with concentration of urine.

• Influenced by – Variety of metabolic products.

– Foods, Drugs, Pigments (dyes)
•  Normal urine is varies from straw (light yellow color) to dark
amber (dark yellow)

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Abnormal colors
• Pale colored urine:- which reflect dilute urine.
– Result from a large volume with corresponding low concentration.

• Pale foamy:- large amount of protein.

• Yellow- Brown or “Beer brown” : Indicates bilirubin.

• Green color:-is due to formation of biliverdin.
• Clear- red – caused by RBCS ( intact RBCS)

• Cloudy red:- caused by RBCs (intact RBCs)

– Hematuria can be differentiated from hemoglobinuria by microscopic
examination.

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f) PH

• A test that determine acidity, alkalinity or neutrality of a solution.

– PH =7 indicates neutrality

– PH <7 indicate acidity

– PH>7 indicate alkalinity

• Normally, freshly voided urine PH range from 5-6 in healthy.

Procedure of the test

– Litmus paper

– Nitrazine paper

– Dipstick

– Glass electrode
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Clinical Significance
• Kidney regulate PH of blood at 7.4 +/- 0.05.
• Persistent alkaline urine (PH>6) may be caused by:
– UTI
– Renal failure
– Vomiting
– Anorexia nervosa
– Alkalosis (metabolic or respiratory e.g. due to accumulation CO 2 in our
body.
– Alkalizing drugs i.e. during in take of drugs such as streptomycin,
kanamycn, etc. e.g. for UTI
– certain vegetables, fruits, and milk products also may cause alkaline urine,
which is not pathological.

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• Persistent acid urine (PH<6) may be caused by:
– Diarrhea

– Malabsorption syndromes
– Diabetic Katoacidosis

– Dehydration
– Fever

– Starvation

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8.6.2. Chemical Analysis of Urine
• Urine contains normal chemical compositions.
• But in abnormal (pathological) conditions its composition varies
in kind and quantities.
• So, the chemical changes of urine can indicate disease at early
stage.
Urine reagent strip test
• Test for the rapid determination of Leukocytes, RBCs, Glucose,
micro albumin, bilirubin, pH, specific Gravity, ketones, protein,
nitrite, creatinine and calcium ion in urine.
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 Chemical Reaction Chart

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 Chemical Exam
• When the test strip is dipped in
urine the reagents are activated
and a chemical reaction occurs.

• The chemical reaction results in a

specific color change.

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 Elevated pH (alkaline)

(Normal for comparison)

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Storage and handling
– Store in a cool, dry place at temperatures between 40C-300C.

– Do not store the strips in a refrigerator or freezer.

– Store away from moisture and light.

– is stable up to the expiry date printed on the container.

Visual test procedure

– Dip the strip into the urine up to the test area for once only.

– Turn the strip on its side up.

– Excessive urine on the strip may cause the interaction of chemicals.

– compare the test results carefully with the color chart on the vial label
under good light.
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8.6.3. Microscopic Examination of Urine

Introduction
• Microscopic examination of urine is one of the routine tests of
urinalysis.
• It can be considered as renal biopsy because it reveals more
about the function of the kidneys.
• Urine sediments can be grossly be categorized in to organized
and non-organized sediments based on the substances they are
composed of.

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 Urinary sediments
a) Organized Elements (Formed from Living Materials)
Sediments
– RBCs/HPF
– WBCs/HPF
– Epithelial cells/LPF
– Casts/LPF
– Parasites/LPF
– Bacteria/HF
– Yeast cell/LPF
– Mucus trade/LPF
– Spermatozoa

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b) Non-organized Elements (Non-living Materials)

i) Acid urine crystals

a) Normal crystal
– Amorphous urates
– Uric acid crystals

– Calcium phosphate
– Cholesterol
– Ammonium buirates

– Calcium sulfate(urates)
– Calcium carbonates
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 b) Abnormal crystal
– Bilirubin
– Cystine crystal
– Tyrosine
– Leucine
ii) Acidic, neutral or slightly alkaline urine crystals
– Calcium oxalate crystals
iii) Alkaline, Neutral, or Slightly acidic urine
– Triple phosphates
iv) Alkaline urine crystals
– Amorphous phosphate
 

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 Thank you!
End of the class

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 Question, comments &suggestion!!

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