Microbiological Methods
Scientific Research and Medical
Diagnostics
Overview Of
Microbiological Methods
• Phenotypic tests
– Microscopy
– Culture techniques
– Immunological techniques
• Genotypic tests
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
Compound Microscope
• A variety of lenses can be used
to magnify small objects from
40X to 1000X
• Only used for thin, transparent
objects <1mm thickness
Light microscopes
Image absorbs light and appears dark to the observer
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
Electron Microscopy
electron microscopes:
SEM,TEM, STM, AFM
Specimen coated with gold
or other metal that will be
stimulated by electron beam
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
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….
Bacterial Growth curve
Limited nutrients etc…
stationary
log
lag
decline
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
Bacterial Culture and Isolation
Isolation streak on agar in a petri dish
Microcolony on growth medium
Test tube culture
• Agar slants
• Broth
Selective and differential medium
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
organism’s culturability.
• Remote factors include environmental conditions,
chemicals, pH etc…
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)
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
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
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
Membrane Filtration
Sample
Liquid sample passed through
porous membrane which is then
placed on agar
Used for concentrated or dilute
samples
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
Dilutions
• .1ml from tubes
onto plates
• Incubate plates
• Count the dilution
that yields between
30-300 colonies
• Take average of
three plates
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
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 patient’s
serum mixed with known
antigen sample
Known antibodies mixed
with unknown antigen
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
Precipitin Tests
Y
Y
Precipitated (out of solution) antibodyantigen complex, will appear cloudy
while rest of tube is clear
Agglutination
Can be performed on glass
slide or in plastic microtitre
(microwell) plates
Y
Y
Y Y Y
Y
Y
positive
negative
Coomb’s antiglobulin test
Y
Y
Y
Y
Y
Y
Y
• 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
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
Ŧ
Y
ΨΨ
§§
Ŧ
§
Y
Y
ELISA
Y Y
Y Y
• Enzyme Linked Immunosorbent Assay
• Usually performed in microwell plate
• Can be used to detect specific antibody or antigen
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 (+)
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
+
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