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Notes For Micro Chapter 36

MICROBIOLOGY NOTES

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72 views6 pages

Notes For Micro Chapter 36

MICROBIOLOGY NOTES

Uploaded by

dakalonemand
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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14/08/2024

LECTURE 4

CHAPTER 36:CLINICAL MICROBIOLOGY AND IMMUNOLOGY

36.1 The Clinical Microbiology Laboratory Detects Infectious Agents and


Protects Its Workers

What is a Clinical microbiologist?


Isolate & identify pathogenic microorganisms from clinical
specimens
Clinical specimen: portion or quantity of human material that is
tested, examined, or studied to determine the presence or absence
of specific microbes (blood, sputa, urine, cerebrospinal fluid)

What is the purpose of the clinical laboratory?

It also supports public health infrastructure, acting as a sentinel in


the rapid detection of naturally occurring outbreaks of infection and
bioterrorism.
The major goals of the clinical microbiologist are:
o rapid and accurate identification of pathogens from clinical
specimens, and
o antimicrobial susceptibility testing of these organisms

Overview of clinical microbiology laboratory

Clinical microbiology is interdisciplinary, and the clinical


microbiologist must have working knowledge of microbial
morphology, metabolism, growth, reproduction, biochemistry, and
physiology.
Additionally, the clinical microbiologist needs to understand the
principles of microscopy, immunology, molecular biology, genomics,
aseptic technique, sterilization, disinfection, and the dynamics of
host-parasite relationships
Laboratory information system (LIS) which tracks the testing process
& manages the test results

Working with Specimens

Safety concerns – Standard Microbiological Practices have been


established by the Centers for Disease Control and Prevention (CDC)
Standard Microbiological Practices

Are minimum guidelines that should be supplemented with other


precautions based on exposure risks and lab biosafety level
regulations
Goal is to protect workers from contact with agents by their taking
precautions and by working in a safe laboratory environment

Specimen Collection

Numerous methods used; choice of method depends on specimen


o Specimen should represent the diseased area.
o Sufficient quantity for tests.
o Avoid contamination from environment.
o Proper container and promptly sent to laboratory.
o Obtain specimen before antimicrobial treatment.
o Accompanied by a putative diagnosis.
Swab: rayon, calcium alginate or Dacron tipped polystyrene
applicator
Transport medium: preserve/ prevent multiplication of microbes
Needle aspiration: cerebrospinal fluid, pus & blood
Heparin, sodium citrate: prevent clotting & entrapping microbes
Intubation: insertion of tube into body canal or hollow organ
Nasotracheal intubation, specimen from the stomach
Catheter: tubular instrument used for withdrawing/ introducing
fluids from or into a body cavity
Urine specimens
French catheter: soft tube for single specimen
Folely catheter: multiple specimens
“Clean catch method”: collection of midstream urine as it is not
contaminated with transient microbes in the lower urethra
Sputum: mucous secretion (lungs, bronchi & trachea)
Proper labelling & handling of specimen
Speed in transporting specimen to laboratory, medium must
preserve the microbes
Anaerobic specimens:<10 min with an indicator such as resazurin to
show that vial is anoxic
Stool specimens: special buffered preservatives (buffered formalin)
Urine must be analysed <1 hours/refrigerated immediately.
Preservatives: boric acid or polyvinyl alcohol
CSF for meningitis identification:<15 min
Specimens for the isolation of viruses: iced before transport & 4 for
72 h, longer storage at -72
Culture-Based Methods
Media depends on the source of the specimen.
immunomagnetic bead (IMB) technology.
IMBs are small particles coated
with antibody targeting a particular microbe.
A magnet is then used to pull the beads and their attached microbes
out of solution.

Identification of Microorganisms from Specimens

direct identification methods


o Growth/ culture and biochemical characteristics
o microscopy
o molecular methods
o bacteriophage typing
o Gas chromatography
o immunologic tests
indirect identification methods
o Serology
o Immunofluorescence

Bacteria

Most bacteria:
o culturing involves use of numerous kinds of growth media
 can provide preliminary information about biochemical
nature of bacterium
o additional biochemical tests used following isolation
Some bacteria are not routinely cultured –
o rickettsias, chlamydiae, and mycoplasmas
o identified with special stains, immunologic tests, or molecular
methods such as PCR

Identification of microorganisms from specimens

Microscopy

Bacteria

Wet-mount, heat-fixed or chemically fixed specimens


Light microscope, >0.5µm (protozoa, fungi, bacteria)
Stains: Gram stain, not useful for wall-less bacteria
Fluorescence microscope used to identify certain acid-fast microbes (M.
tuberculosis)
Rapid Methods of Identification

Manual biochemical systems


o – e.g., API 20 E system, catalase, oxidase, coagulase, Dnase,
optochin test, bile solubility test, CAMP test, bacitracin test, TSI,
SIM
Automated systems: VersaTREK, MicroScan, VITEK/ VITEK MS, BACTEC
MGIT system, Peptide nucleic acid in situ hybridization (PNA-FISH),
Matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF)
Immunologic systems

Fungi

Presumptive identification: based on hyphae, morphology of


reproductive structures/ spores
Cotton blue (lactophenol aniline) is used to stain fungi
Serology eg. Complement fixation & immunodiffusion (Blastomyces
dermatitidis, Coccidiodes immitis, Histoplasma capsulatum)
Latex agglutination test: C. neoformans in serum & cerebrospinal fluid

Parasites

based on identification of ova, trophozoites & cysts


Staining of blood, body fluids & immunofluorescence
Blood smears for malaria apicomplexan & trypanosome are stained
with Giemsa

Viruses

Fluorescence microscopy: fluorescently labelled antibodies used for


virus detection
Electron microscopy
Cell/ tissue culture, fluorescent antibody, ELISA, radioimmunoassay,
latex agglutination & immunoperoxidase tests, molecular detection
(PCR, nucleic acid probes)
Each type of cell culture specific for growth of different viruses
Replication detected: observing cytopathic effects
Hemadsorption: adherence of red blood cells that were added to cell
culture
Embryonated chicken eggs: virus culture Serology Animals (mice)
Immunofluorescence

Process in which fluorescent dyes are exposed to UV, violet, or blue


light to make them fluoresce
Dyes can be coupled to antibody molecules without changing
antibody’s ability to bind a specific antigen
Can be used as direct fluorescent-antibody (FA) technique or indirect
fluorescent-antibody (IFA) technique assay

Molecular Methods

Analysis of specific proteins and nucleic acids in rapid, accurate, and


sensitive.
Molecular methods widely use:
o Nucleic acid probes
o Real-time polymerase chain reaction (PCR)
o Amplification of DNA
o Digital droplet PCR
o DNA fingerprinting

Ribotyping

Used to identify bacterial genera


Based on high level of 16S rRNA gene conservation among bacteria
rRNA encoding genes or fragments are amplified by PCR
The nucleotide sequence of the amplified DNA is determined and
compared with those in the National Center for Biotechnology (NCBI)

Immune Responses Can Be Measured or Exploited to Detect Infections

Detection of antigens or antibodies in specimens


o especially useful when cultural methods are unavailable or
impractical or antimicrobial therapy has been started
Use of immunological systems has many advantages –
o e.g. easy to use
o e.g. give relatively rapid reaction endpoints
o e.g. are sensitive and specific

Flow cytometry
Forces a suspension of cells through a laser beam, & measure the light
they scatter/ fluorescence emitted
Applications: microbial susceptibility & drug cytotoxicity

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