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

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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

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Light microscopes
Image absorbs light and appears dark to the
observer
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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
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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

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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.
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Bacterial Growth curve
Limited nutrients etc
stationary
log
decline
lag
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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

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Isolation streak on agar in a petri dish
Bacterial Culture and Isolation
Microcolony on growth medium
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Test tube culture

• Agar slants
• Broth

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Selective and differential medium
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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

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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)

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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

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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
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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
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Membrane Filtration
Liquid sample passed through porous membrane
which is then placed on agar Used for
concentrated or dilute samples
Sample
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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
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Dilutions

• .1ml from tubes onto plates
• Incubate plates
• Count the dilution that yields between 30-300
  colonies
• Take average of three plates

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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

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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
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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

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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
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Agglutination
Can be performed on glass slide or in plastic
microtitre (microwell) plates
Y
Y
Y
Y
Y
Y
Y
positive
negative
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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
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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
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Y
Y
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ELISA

• Enzyme Linked Immunosorbent Assay
• Usually performed in microwell plate
• Can be used to detect specific antibody or antigen

Y
Y
Y
Y
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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
  ()

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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

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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