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6.1. Stool Specimen 6.1. Stool Specimen: 9/18/21 Tafeseb - Tufa 1
6.1. Stool Specimen 6.1. Stool Specimen: 9/18/21 Tafeseb - Tufa 1
6.1. Stool Specimen 6.1. Stool Specimen: 9/18/21 Tafeseb - Tufa 1
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.
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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
3 Soft Yellow Feces with scanty mucus All stage except trophozoite
5 Diarrheic Clay Mucus with much feces All stage except Cyst
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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
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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
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– Collect specimen before instituting antibiotic therapy.
– Used sterile collection devices and containers.
– Place in suitable container, labels appropriately.
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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
• 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.
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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
– Carbon fuchisin
– Crystal Violet
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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?
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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
– 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.
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Differential
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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
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Enrichment
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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.
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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.
• Good for:-
– Chemical screen
– Microscopic examination
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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.
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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
a) Volume
• Usually indicative of the balance b/n fluid ingestion and extra renal loss of water
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• Normal value is depend on the age:
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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
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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.
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• Excretion of constantly small amount of urine (<400ml) called
Oliguria.
• Oliguria may occur due to:-
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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.
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Abnormal colors
• Pale colored urine:- which reflect dilute urine.
– Result from a large volume with corresponding low concentration.
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f) PH
– 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.
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Elevated pH (alkaline)
– 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)
– 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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