Lab Diagnosis - Prac. Microbiology

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Transcript of Lab Diagnosis - Prac. Microbiology

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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.

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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.

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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

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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.

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Specimen Collection

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I) DIRECT METHOD2) Microscopic Examination: usually before further processing of specimens stained /unstained (wet)preparations different types of microscopes

Staphylococci in pus

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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.

 

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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).

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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

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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.

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Skin Test

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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.

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Microscopy

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Light Microscope

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Stained Preparations Unstained preparations

Bacterial morphology Motility

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Bacterial MorphologySize

Shape

Special arrangement Staining affinity

Spore formation

Capsule formation

Motility

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Bacterial Shape

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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

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Spore formation

Morphological characters of bacterial spores:

* Shape. * Position. * Staining.

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Bacterial spores

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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

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Smearing out of the sample

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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

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Differential stainingPrinciples of differential stain * Application of the main stain.

• Decolourization. *Application of the counter-stain.

e.g. Gram stain & Ziehl-Neelsen stain

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1. Gram Stain

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Components

Primary Stain: Methyl violet + Gram’s iodine

Decolourizing agent: 95% ethyl alcohol

Counter stain: dil. Carbol fuchsin

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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 )

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Gram Staining Technique

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Gm+ve cocci & G-ve bacilli

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2. Ziehl-Neelsen

Stain

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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.

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Components

Primary Stain: Conc. Carbol fucshin

Decolourizing agent: 20% H2SO4

OR 3% HCL in

alcohol

Counter stain: methylene blue

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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)

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Ziehl-Neelsen Stain Technique

4 5 6

7

1 2 3

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Questions

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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

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