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Microbiology
The Study of Microorganisms
Definition of a Microorganism
• Derived from the Greek: Mikros, «small» and
Organismos, “organism”
– Microscopic organism which is single celled
(unicellular) or a mass of identical
(undifferentiated)
– Includes bacteria, fungi, algae, viruses, and
protozoans
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Microorganisms in the Lab
Growth Media
Goals
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Growth under controlled conditions
Maintenance
Isolation of pure cultures
Metabolic testing
Types
• Liquid (Broths)
– Allows growth in suspension
– Uniform distribution of nutrients, environmental
parameters and others
– Allows growth of large volumes
• Solid media
– Same as liquid media + solidification agent
• Agar: Polysaccharide derived from an algae
Growth in Broths
Non inoculated
clear
Turbid + sediment
Turbid
Clear + sediment
Growth on Agar
• Growth on solid
surface
• Isolated growth
• Allows isolation of
single colonies
• Allows isolation of
pure cultures
Single colony
Solid Media (Cont’d)
• Slants
– Growth on surface and in depth
– Different availabilities of oxygen
– Long term storage
• Stab
– Semi-solid medium
– Long term storage
– Low availability of oxygen
Microscopy
Staining
Simple Staining
• Positive staining
– Stains specimen
– Staining independent of the species
• Negative staining
– Staining of background
– Staining independent of the species
Methods
• Simple stain:
– One stain
– Allows to determine size, shape, and aggregation
of bacteria
Cell Shapes
• Coccus:
– Spheres
– Division along 1,2 or 3 axes
– Division along different axes gives rise to different
aggregations
– Types of aggregations are typical of different
bacterial genera
Cocci (Coccus)
Axes of division
Arrangements
Diplococcus
Streptococcus
(4-20)
Tetrad
Staphylococcus
Hint: if name of genus ends in coccus, then the shape of the
bacteria are cocci
Cell Shapes (Cont’d)
• Rods:
– Division along one axis only
– Types of aggregations are typical of different
bacterial genera
The Rods
Axes of division
Arrangements
Diplobacillus
Streptobacillus
Hint: if name of bacteria genus is Bacillus, then the shape of the
bacteria are rods
If it doesn’t end in cocci, it’s probably a rod.
Counting Microorganisms
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Turbidity measurements: Optical density
Viable counts
MPN
Direct counts
Turbidity measurements
• Measures the amount of light that can go
through a sample
• The less the amount of light which goes
through the sample the denser the population
• Mesures optical density or percent
transmission
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Turbidity measurements
• Spectrophotometer (A600):
– Measuring optical density
Light
Detector….reading
600nm
Different reading
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O.D. 600nm
2.0
1.0
0
% Transmission
0
Inverse relationship
Cellular density
50
100
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Viable Counts
• Serial dilutions of sample
• Spread dilutions on an appropriate medium
• Each single colony originates from a colony forming
unit (CFU)
• The number of colonies represents an approximation
of the number of live bacteria in the sample
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Bacterial
culture
CFU
CFU
CFU
• 63 CFU/0.1ml of 10-5
• 630 CFU/1.0ml of 10-5
• 630 CFU/ml X 105 = 6.3 x 107/ml in original sample
What if there were 100 ml in the flask?
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Viable Counts
• Advantages:
– Gives a count of live microorganisms
– Can differentiate between different microorganisms
• Limits:
– No universal media
• Can’t ask how many bacteria in a lake
• Can ask how many E. coli in a lake
– Requires growth
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– CFU
one bacteria
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=
=
• Ex. One CFU of Streptococcus  one of E.coli