Lab Diagnosis - Prac. Microbiology
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Transcript of Lab Diagnosis - Prac. Microbiology
Infectious diseasesThey are clinically evident diseases with the
potential of transmission from one person or species to another.
They result from the presence of pathogenic microorganisms (very small organisms that are invisible to the naked eye) that are able to cause disease in human beings.
Pathogenic micro-organisms include bacteria, viruses, fungi, protozoa, and multicellular parasites.
Practical microbiology sessions focus on the diagnosis of these infectious agents.
Laboratory diagnosis of infectious diseases
Direct method Indirect (serologic) method
Detection of: microorganisms, their structural components their products
in specimens collected from the patient (e.g. urine, blood, sputum, CSF……etc).
Detection of antibodies against the microorganism in the patient’s serum.
I) DIRECT METHOD1)Specimen CollectionA ’good quality’ clinical specimen.
2) Microscopic Examination:usually before further processing of specimens
3) Microbial Detection:a) Culture technique: b) Non-culture technique
I) DIRECT METHOD1)Specimen Collection
A ’good quality’ clinical specimen
Collecting specimens before the start of antibiotics.
Choosing the appropriate specimen (representing the infection site).
Using sterile containers and avoiding contaminating the specimen.
Transporting the specimen properly to the lab. as early as possible.
Specimen Collection
I) DIRECT METHOD2) Microscopic Examination: usually before further processing of specimens stained /unstained (wet)preparations different types of microscopes
Staphylococci in pus
I) DIRECT METHOD3) Microbial Detection: a) Culture Technique: Isolation of the organism in pure culture →inoculating the specimen onto appropriate artificial culture media
followed by Identification of the isolate by e.g. :
microscopic examination biochemical reactions reaction with specific antibody (serologic identification of the organism) DNA probes
Which of these approaches is used and in what sequence depends upon the type of specimen and organism.
Antibiotic sensitivity After growing the organism in pure culture.
I) DIRECT METHOD3) Microbial Detection: b) Non-Culture Technique:
I. Identification of a specific microbial antigen such as :
a structural component (e.g. cell wall antigen, capsular polysaccharide…etc) or a product (e.g. an exotoxin) by reacting with specific antibody
OR:
II. Identification of a specific gene sequence (i.e. nucleic acid of the organism)
by the application of different molecular methods (e.g. PCR, DNA probe).
I) DIRECT METHOD
yield more rapid results (minutes or hours) (do not depend on growth and multiplication of the organism) However, antimicrobial susceptibility cannot be determined (although the presence of resistance genes can be determined by
molecular methods).
Non-culture techniques are mainly applied if:
a rapid diagnosis is needed The microorganism cannot be cultured on artificial
media A slowly growing micro-organism
II) INDIRECT (SEROLOGIC) METHOD
Serologic diagnosis of infectious diseases involves the use of known microbial antigens to detect antibodies against the microorganism in the patient’s serum.
current (active) infection is diagnosed by the
detection of one of the following
specific IgM antibodies rising titre of specific IgG antibodies (4-fold or greater rise) a single high titre of IgG antibodies in certain diseases
N.B. Skin tests based on cell-mediated hypersensitivity may also help in the diagnosis of certain diseases.
Skin Test
Diagnosis of infectious diseases
Diagnosis of infectious diseases
DIRECT METHOD DIRECT METHOD
INDIRECT METHOD
(SEROLOGICAL)
INDIRECT METHOD
(SEROLOGICAL)Specimen Specimen
Culture technique Culture
techniqueNon-culture technique
Non-culture technique
Isolation on culture media
Isolation on culture media
Identificatione.g.
• .microscopical.ex•bioch. Reactions•DNA probe•serology
Identification e.g.
•microscopical. ex.•bioch. Reactions•DNA probe•serology
Antibiotic sensitivityAntibiotic sensitivity
• detection of specific antigen (serology)• detection of specific gene sequence (mol. tech.)
• detection of specific antigen (serology)• detection of specific gene sequence (mol. tech.)
Detection of antibodies in serum
•IgM•rising titre of IgG
Detection of antibodies in serum
•IgM•rising titre of IgG
Microscopical ex.Microscopical ex.
Microscopy
Light Microscope
Stained Preparations Unstained preparations
Bacterial morphology Motility
Bacterial MorphologySize
Shape
Special arrangement Staining affinity
Spore formation
Capsule formation
Motility
Bacterial Shape
Bacterial arrangement
Chains.
Pairs (diploids).
Clusters (group).
No special arrangement.
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Bacterial arrangement
Cocci
Irregular ClustersChains Pairs
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Bacterial arrangement
Bacilli
chains No special arrangement Pairs
Spore formation
Morphological characters of bacterial spores:
* Shape. * Position. * Staining.
Bacterial spores
Bacterial capsule
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Staining of BacteriaStaining of Bacteria
Bacteria cells Bacteria cells are are almost almost colorlesscolorless and and
transparenttransparent
A staining technique is often applied to the A staining technique is often applied to the
cells to cells to color themcolor them → →
Their Their shapeshape and and size size can be easily can be easily determined under the microscope. determined under the microscope.
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Smear preparationSmear preparation
S Fixation
Smearing out of the sample
Types of Stains1- simple stain: Single basic dye e.g. Methylene blue All bacteria take the color of the dye
2- Differential stain: Two dyesseparated by a decolorizing agent e.g. Gram stain & Ziehl-Neelsen stain 3- Special stain: e.g. Fontana stain
Differential stainingPrinciples of differential stain * Application of the main stain.
• Decolourization. *Application of the counter-stain.
e.g. Gram stain & Ziehl-Neelsen stain
1. Gram Stain
Components
Primary Stain: Methyl violet + Gram’s iodine
Decolourizing agent: 95% ethyl alcohol
Counter stain: dil. Carbol fuchsin
Principle
Primary Stain Methyl violet + Gram’s iodine → All Violet
Decolourizing agent: 95% ethyl alcohol
Not decolourized ( violet ) decolourized ( colourless )
( Gram + ve) (Gram –ve )
Counter stain: dil. Carbol fuchsin→ colourless→pink(Gram -ve )
Gram Staining Technique
Gm+ve cocci & G-ve bacilli
2. Ziehl-Neelsen
Stain
Mycobacterium A 3rd type of cell envelope (high lipids
content of cell wall)
Not readily stainable with ordinary stains.A strong stain e.g., concentrated carbol
fucsin + heat.
Resist decolorization by strong mineral acids or acid-alcohol →
Acid-fast.
Components
Primary Stain: Conc. Carbol fucshin
Decolourizing agent: 20% H2SO4
OR 3% HCL in
alcohol
Counter stain: methylene blue
Principle
Primary Stain Conc. Carbol fucshin → All Red
Decolourizing agent: 20% H2SO4
OR 3% HCL in alcohol
Not decolourized ( red ) decolourized
( colourless ) ( acid-fast) (non
acid-fast)
Counter stain: methylene blue → Colourless →blue (non acid-fast)
Ziehl-Neelsen Stain Technique
4 5 6
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1 2 3
Questions
5.Acid fast bacilli stained by Z-N stain appear:a) Violetb) Bluec) Redd) Colourlesse) brown
6.The steps of Gram’s stain is as follows:a) methyl violet / ethyl alcohol / dil. carbol fuchsin / iodineb) methyl violet / ethyl alcohol / iodine / dil. carbol fuchsinc) dil. carbol fuchsin / methyl violet / ethyl alcohol / iodined)dil. carbol fuchsin / iodine / methyl violet / ethyl alcohole)methyl violet / iodine /alcohol / dil. carbol fuchsin