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Microbiological Methods

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Selective/differential media (utilization of specific nutrients, resistance to chemicals) ... Facultative-means growth will occur under certain conditions if necessary ... – PowerPoint PPT presentation

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Title: Microbiological Methods


1
Microbiological Methods
  • Scientific Research and Medical Diagnostics

2
Overview Of Microbiological Methods
  • Phenotypic tests
  • Microscopy
  • Culture techniques
  • Immunological techniques
  • Genotypic tests

3
Methods for Studying Microbes
  • By phenotype (expressions of genes, physiology)
  • Cell morphology, colony morphology, behavior
  • Growth conditions (aerobic, anaerobic)
  • Selective/differential media (utilization of
    specific nutrients, resistance to chemicals)
  • Test for various enzymes e.g. oxidase, catalase
  • Serology, Antigen/antibody binding
  • By genotype (genetic sequence or structure)
  • Based DNA profiles based of RE digestion
  • Based on sequence of DNA or PCR

4
Compound Microscope
  • A variety of lenses can be used to magnify small
    objects from 40X to 1000X
  • Only used for thin, transparent objects lt1mm
    thickness

5
Light microscopes
Image absorbs light and appears dark to the
observer
6
Fluorescence Microscopy
  • Dye or labeled antibody binds to object and
    fluoresces under UV light
  • May provide greater resolution for small bacteria
  • Dye may indicated a specific physiological state
    of the bacterium

7
Electron Microscopy
electron microscopes SEM,TEM, STM, AFM
Specimen coated with gold or other metal that
will be stimulated by electron beam
8
Preparations for light microscopy
  • Used to identify phenotypic characteristics of
    certain taxa
  • Usually performed on microscope slides using
    sample of pure culture
  • Some tissue samples may be examined directly
    (CSF) or after staining
  • Common staining procedures for bacteria include
    Gram stain, acid fast stain, endospore stain,
    capsule stain
  • Does not usually identify to the species level
    but is useful in combination with other methods
    of identification

9
Binary Fission
  • Most common method of bacterial reproduction
  • Allows for vary fast population growth

8 cells after 3 generations 64 cells after 6
generations 512 after 9 generations.
10
Bacterial Growth curve
Limited nutrients etc
stationary
log
decline
lag
11
Culture Media
  • Culture- maintenance of a lineage, usually
    implies in vitro
  • Culture medium- the substrate on/in which the
    culture is maintained
  • Natural or Synthetic
  • Selective and/or Differential
  • Enrichment
  • Solid or Liquid

12
Isolation streak on agar in a petri dish
Bacterial Culture and Isolation
Microcolony on growth medium
13
Test tube culture
  • Agar slants
  • Broth

14
Selective and differential medium
15
Culturability
  • Most microorganisms are difficult to culture or
    not culturable
  • Culturable does not mean ALWAYS culturable
  • VBNC or VNC (viable but non-culturable)
  • Some organisms such as (E. coli) are very easy to
    culture
  • Metabolic factors are proximate factors that
    influence an organisms culturability.
  • Remote factors include environmental conditions,
    chemicals, pH etc

16
Variation in growth conditions
  • Aerobic-utilizing oxygen
  • Anaerobic-not using oxygen
  • Facultative-means growth will occur under certain
    conditions if necessary
  • Obligate- strictly limited to specified
    conditions
  • Fastidious organisms are difficult to grow in lab
  • Organisms with highly specialized lifestyle
    utilizing unusual compounds for growth and
    survival
  • Obligate Intracellular symbionts require growth
    in living growth medium (e.g. lab animal or their
    tissues, HeLa cells)

17
Methods of Enumeration
  • Yields information about growth, risks etc..
  • Only an estimate of actual population density
  • Several methods
  • Direct microscopic counts
  • Spread plate
  • Membrane filtration
  • Most probable number
  • Spectrophotometry
  • Flow cytometry

18
Direct microscopic counts
  • Known volume of sample added to microscope slide
  • Slide is marked with special grid to aid with
    counting the number of observed cells per unit
    area
  • Does not usually allow for inferring that cells
    are viable however some chemicals can be added
    that indicate viable cells only

Glass slide
grid
19
Spread Plating
Sample spread evenly over surface of medium Only
works for samples with density of 300 CFU/ml or
greater
Colonies appear after incubation
20
Membrane Filtration
Liquid sample passed through porous membrane
which is then placed on agar Used for
concentrated or dilute samples
Sample
21
Dilutions
Transfer 1ml from sample to first tube, then 1ml
from first to second etc
Dilution factor for each step
10X
10X
10X
10X
Final dilution factor?
Sample with unknown density of bacteria
test tubes, each with 9ml of sterile buffered
water
22
Dilutions
  • .1ml from tubes onto plates
  • Incubate plates
  • Count the dilution that yields between 30-300
    colonies
  • Take average of three plates

23
Identifying organisms by the presence of certain
biochemical reactions
  • Many tests revolve around the observation or
    measurement of bacterial enzymes, which are
    phenotypic characteristics
  • Enzymes can be detected by adding a substrate
    either in vitro or in vivo to a bacterial sample
    and observe reactions (e.g. catalase test)
  • Growth media may allow biochemical reactions to
    be tested
  • Commercially available kits allow for multiple
    tests to be performed simultaneously

24
Immunological (serological) tests
  • Can be used to detect specific antibodies or
    specific antigens
  • Either the antibody or antigen will be
    hypothesized the other will be known

Antibodies from patients serum mixed with known
antigen sample
Known antibodies mixed with unknown antigen
Y
Y
Y
Y
Y
Y
25
Detection
  • The binding of antibodies and antigens may be
    detected or visualized in several ways
  • Precipitation
  • Agglutination
  • Fluorescence
  • RBC lysis (Complement fixation)
  • Enzymatic color reaction
  • Electrophoresis and staining

26
Precipitin Tests
Y
Y
Precipitated (out of solution) antibody-antigen
complex, will appear cloudy while rest of tube is
clear
Y
Y
Y
Y
Y
Y
Y
Y
Y
27
Agglutination
Can be performed on glass slide or in plastic
microtitre (microwell) plates
Y
Y
Y
Y
Y
Y
Y
positive
negative
28
Coombs antiglobulin test
  • Antibodies may be formed against other antibodies
  • The resulting complex allows for amplification
    of agglutination
  • First antibody X made in animal A then injected
    into animal B
  • Animal B produces antibodies Z to first antibody

Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
29
Immunofluorescence
  • Antibody with fluorescent label binds to specific
    antigen from sample
  • Can be done on slide and viewed through
    microscope or in
  • Can be done in microwell plates

Y
Y
? ? T T
30
Y
Y
31
ELISA
  • Enzyme Linked Immunosorbent Assay
  • Usually performed in microwell plate
  • Can be used to detect specific antibody or antigen

Y
Y
Y
Y
32
Complement fixation test
  • Serum sample taken which is hypothesize to
    contain antibodies to specific antigen
  • Known antigen added to serum
  • Complement added to serum/antigen mixture
  • If serum contains antibodies compliment will be
    fixed at this point otherwise, it will remain
    free in the serum/antigen mixture and will be
    fixed at next step
  • RBCs bound to antibodies added to mixture and
  • If lysed then serum did not contain suspected
    antibodies(-)
  • If not lysed, then serum did contain antibodies
    ()

33
Gel Electrophoresis
Samples added to wells in matrix
-
Gel made of translucent, porous matrix through
which molecules can move when exposed to an
electric field

34
Western Blot
  • Antigens separated by gel electrophoresis

-
Labeled antibodies applied to paper and cause
color change where specific binding occurs

Paper placed on gel
Y
Proteins diffuse from gel to paper
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