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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) cells
– Includes bacteria, fungi, algae, viruses, and
protozoans
2
Microorganisms in the Lab
Growth Media
Goals
•
•
•
•
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
Counting Microorganisms
Methods
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•
•
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Turbidity measurements: Optical density
Direct counts
Viable counts
MPN
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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Turbidity measurements
O.D. 600nm
2.0
1.0
0
% Transmission
0
Inverse relationship
Cellular density
50
100
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Direct Counts
• The sample to be counted is applied onto a
hemacytometer slide that holds a fixed
volume in a counting chamber
• The number of cells is counted in several
independent squares on the slide’s grid
• The number of cells in the given volume is
then calculated
Direct Counts
• Advantages:
– Quick
– Growth is not required
– No information about organism required
• Limits:
– Does not discriminate between live and dead
– May be difficult to distinguish bacteria from
detritus
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Using a hemacytometer
Using a hemacytometer (Cont’d)
Hemacytometer
• This slide has 2 independent counting
chambers
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Using a hemacytometer (Cont’d)
Determining the Direct Count
• Count the number of cells in three independent
squares
– 8, 8 and 5
• Determine the mean
– (8 + 8 + 5)/3 =7
– Therefore 7 cells/square
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Determining the Direct Count (Cont’d)
1mm
Depth: 0.1mm
1mm
• Calculate the volume of a square:
= 0.1cm X 0.1cm X 0.01cm= 1 X 10-4cm3 or ml
• Divide the average number of cells by the the
volume of a square
– Therefore 7/ 1 X 10-4 ml = 7 X 104 cells/ml
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Problem
• A sample is applied to a hemacytometer slide
with the following dimensions: 0.1mm X
0.1mm X 0.02mm. Counts of 6, 4 and 2 cells
were obtained from three independent
squares. What was the number of cells per
milliliter in the original sample if the counting
chamber possesses 100 squares?
Viable Counts
• A viable cell: a cell which is able to divide and
form a population (or colony)
1. A viable cell count is done by diluting the original
sample
2. Plating aliquots of the dilutions onto an appropriate
culture medium
3. Incubating the plates under appropriate conditions
to allow growth
•
Colonies are formed
4. Colonies are counted and original number of viable
cells is calculated according to the dilution used 23
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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Dilution of Bacterial Sample
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Dilution of Bacterial Sample
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Dilution of Bacterial Sample
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Dilution of Bacterial Sample
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Plating of Diluted Samples
5672
57
4
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Viable Counts
• The total number of viable cells is reported as
Colony-Forming Units (CFUs) rather than cell
numbers
– Each single colony originates from a colony
forming unit (CFU)
– A plate having 30-300 colonies is chosen
– Calculation:
• Number of colonies on plate X reciprocal of dilution
(dilution factor) = Number of CFU/mL
– Ex. 57 CFU/0.1mL X 106 = 5.7 X 107 CFU/mL
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Serial Dilutions
Bacterial
culture
• 63 CFU/0.1ml of 10-5
CFU CFU CFU
• 630 CFU/1.0ml of 10-5
• 630 CFU/ml X 105 = 6.3 x 107/ml in original sample
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What if there were 100 ml in the flask?
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
Only living cells develop colonies
Clumps or chains of cells develop into a single colony
?
?
CFU one bacteria
=
=
• Ex. One CFU of Streptococcus  one of E.coli
Most probable Number: MPN
– Based on Probability Statistics
– Presumptive test based on given characteristics
– Broth Technique
Most Probable Number (MPN)
• Begin with Broth to detect desired characteristic
• Inoculate different dilutions of sample to be
tested in each of three tubes
-1
Dilution
-2 -3 -4 -5 -6
3 Tubes/Dilution
1 ml of Each Dilution into Each Tube
After suitable incubation period, record POSITIVE TUBES
(Have GROWTH and desired characteristics)
MPN - Continued
• Objective is to “DILUTE OUT” the organism to zero
• Following the incubation, the number of tubes
showing the desired characteristics are recorded
• Example of results for a suspension of 1g/10 ml of soil
• Dilutions:
-1 -2 -3 -4
• Positive tubes: 3 2 1 0
– Choose correct sequence: 321 and look up in table
Pos. tubes
0.10 0.01 0.001
3
2
1
MPN/g (mL)
150
– Multiply result by middle dilution factor
» 150 X 102 = 1.5 X 104/mL
» Since you have 1g in 10mL must multiply again by 10
» 1.5 X 105/g
Microscopy
Staining
Simple Staining
• Positive staining
– Stains specimen
– Staining independent of the species
• Negative staining
– Staining of background
– Staining independent of the species
Method
• 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.
Microscopy
Differential Staining
Differential Staining
Gram Stain
• Divides bacteria into two groups
• Gram Negative
&
Gram Positive
• Stained Purple
– Rods
• Genera Bacillus and Clostridium
– Coccus
• Genera Streptococcus, Staphylococcus and Micrococcus
Gram Negative
• Stained Red
– Rods:
• Genera Escherichia, Salmonella, Proteus, etc.
– Coccus:
• Genera Neisseria, Moraxella and Acinetobacter
Rules of thumb
• If the genus is Bacillus or Clostridium
= Gram (+) rod
• If the genus name ends in coccus or cocci
(besides 3 exceptions, which are Gram (-))
= coccus shape and Gram (+)
• If not part of the rules above,
= Gram (-) rods
Gram +
Cell Wall
Vs
Gram -
Peptidoglycan
wall
Plasma
Membrane
Absent
Lipopolysaccharide
layer
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Method – Primary staining
1. Staining with crystal violet
2. Addition of Gram’s iodine (Mordant)
+ + +
Wall:peptidoglycan
Plasma membrane
+
+ + +
+ + +
+
+
+ + +
LPS
--------------Gram positive
--------------Gram negative
Method – Differential step
3. Alcohol wash
Wall is dehydrated
– Stain + iodine complex is trapped
Wall: peptidoglycan
Plasma membrane
Wall is not dehydrated
– Complex is not trapped
LPS
- - +- - -+- - +- - -+- - +
- - -+ +
Gram positive
- - +- - -+- - +- - -+- - +
- - -+ +
Gram negative
Method – Counter Stain
4. Staining with Safranin
+ + + + + + +
Wall:peptidoglycan
Plasma membrane
+
+ + + + + + +
LPS
- - +- - -+- - +- - -+- - +
- - -+ +
Gram positive
--------------Gram negative
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Summary
Gram Positive
Gram Negative
Fixation
Primary staining
Crystal violet
Wash
Destaining
Counter staining
Safranin
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Acid Fast Staining
• Diagnostic staining of Mycobacterium
– Pathogens associated with Tuberculosis and Leprosy
– Cell wall has mycoic acid
• Waxy, very impermeable
Method
• Basis:
– High level of compounds similar to waxes in their
cell walls, Mycoic acid, makes these bacteria
resistant to traditional staining techniques
Method (Cont’d)
• Cell wall is permeabilized with heat
• Staining with basic fuchsine
– Phenol based, soluble in mycoic layer
– Cooling returns cell wall to its impermeable state
• Stain is trapped
• Wash with acid alcohol
– Differential step
• Mycobacteria retain stain
• Other bacteria lose the stain
Spore Stain
• Spores:
– Differentiated bacterial cell
– Resistant to heat, desiccation, ultraviolet, and
different chemical treatments
• Thus very resistant to staining too!
– Typical of Gram positive rods
• Genera Bacillus and Clostridium
– Unfavorable conditions induce sporogenesis
• Differentiation of vegetative cell to endospore
– E.g. Anthrax
Malachite Green Staining
• Permeabilization of spores
with heat
• Primary staining with
malachite green
• Wash
• Counter staining with
safranin
Sporangium
(cell +
endospore)
Vegetative cells
(actively growing)
Spores
(resistant
structures used
for survival under
unfavourable
conditions.)
Endospore
(spore within
cell)