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

Specimen coated with gold or other metal that will be stimulated by electron beam

electron microscopes: SEM,TEM, STM, AFM

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

lag

log

stationary

decline

Limited nutrients etc…

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

Isolation streak on agar in a petri dishMicrocolony on growth medium

Bacterial Culture and Isolation

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

SampleSample

Liquid sample passed through porous membrane which is then placed on agar

Used for concentrated or dilute samples

Dilutions

Sample with unknown density of bacteria

test tubes, each with 9ml of sterile buffered water

Transfer 1ml from sample to first tube, then 1ml from first to second etc…

10X 10X 10X 10X

Final dilution factor?

Dilution factor for each step

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

YYY

Known antibodies mixed with unknown antigen

Y YY

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

YY

Y

Y

Y

YY

Y

YYY

Precipitated (out of solution) antibody-antigen complex, will appear cloudy while rest of tube is clear

Agglutination

YY

Y

Y

YY

Y

positive negative

Can be performed on glass slide or in plastic microtitre (microwell) plates

Coomb’s 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

YY

Y

YY

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

YY

Y

YYY

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

Ψ Ψ Ŧ § § Ŧ §

YY

Y Y

ELISA

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

YY Y

Y

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

Gel made of translucent, porous matrix through which molecules can move when exposed to an electric field

Samples added to wells in matrix

-

+

Western Blot

Antigens separated by gel electrophoresis

Paper placed on gel

Proteins diffuse from gel to paper

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

Y

+

-