Download - Counting Microorganisms
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Counting Microorganisms
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Methods
• Turbidity measurements: Optical density• Viable counts• MPN• Direct counts
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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
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Light
600nm
Detector….reading
Different reading
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50
2.0
1.0
O.D. 600nm % Transmission
100
0
50
Cellular density
Inverse relationship
Turbidity measurements
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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
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• 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 sampleWhat if there were 100 ml in the flask?
Serial Dilutions
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• 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– CFU one bacteria
• Ex. One CFU of Streptococcus one of E.coli=?
Viable Counts
=?
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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
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Using a hemacytometer
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Using a hemacytometer (Cont’d)
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Using a hemacytometer (Cont’d)
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Determining the Direct Count
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• 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)
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• 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
1mm
1mmDepth: 0.1mm
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Problem
• A 500μl 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?
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Most probable Number: MPN
– Based on Probability Statistics– Presumptive test based on given characteristics– Broth Technique
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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 -2 -3 -4 -5 -6Dilution
3 Tubes/Dilution
1 ml of Each Dilution into Each Tube
After suitable incubation period, record POSITIVE TUBES (Have GROWTH and desired characteristics)
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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
– 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
Pos. tubesMPN/g (mL)
0.10 0.01 0.0013 2 1 150
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Microscopy
Staining
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Simple Staining
• Positive staining – Stains specimen– Staining independent of the species
• Negative staining– Staining of background– Staining independent of the species
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Method
• Simple stain: – One stain– Allows to determine size, shape, and aggregation
of bacteria
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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
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Cocci (Coccus)
Diplococcus
Streptococcus(4-20)
Tetrad
Staphylococcus
ArrangementsAxes of division
Hint: if name of genus ends in coccus, then the shape of the bacteria are cocci
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Cell Shapes (Cont’d)
• Rods:– Division along one axis only– Types of aggregations are typical of different
bacterial genera
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The Rods
Diplobacillus
Streptobacillus
ArrangementsAxes of division
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.
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Microscopy
Differential Staining
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Differential StainingGram Stain
• Divides bacteria into two groups
• Gram Negative & Gram Positive
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• Stained Purple – Rods
• Genera Bacillus and Clostridium– Coccus
• Genera Streptococcus, Staphylococcus and Micrococcus
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Gram Negative
• Stained Red– Rods:
• Genera Escherichia, Salmonella, Proteus, etc.
– Coccus: • Genera Neisseria, Moraxella and Acinetobacter
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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
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Cell Wall
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Peptidoglycanwall
PlasmaMembrane
Lipopolysaccharidelayer
Absent
Gram + Vs Gram -
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Method – Primary staining
1. Staining with crystal violet2. Addition of Gram’s iodine (Mordant)
+
Gram positive Gram negative
- - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall:peptidoglycan LPS
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Method – Differential step
3. Alcohol wash
Gram positive Gram negative
- - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall: peptidoglycan LPS
+ + + + + + + + + + + + + +
Wall is dehydrated – Stain + iodine complex is trapped
Wall is not dehydrated – Complex is not trapped
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Method – Counter Stain
4. Staining with Safranin
Gram positive Gram negative
- - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall:peptidoglycan LPS
+ + + + + + +
+
+ + + + + + + + + + + + + +
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Summary
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Fixation
Primary stainingCrystal violet
Counter stainingSafranin
WashDestaining
Gram Positive Gram Negative
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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
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Method
• Basis: – High level of compounds similar to waxes in their
cell walls, Mycoic acid, makes these bacteria resistant to traditional staining techniques
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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
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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
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Malachite Green Staining
• Permeabilization of spores with heat
• Primary staining with malachite green
• Wash• Counter staining with
safraninVegetative cells(actively growing)
Spores(resistant
structures used for survival under
unfavourable conditions.)
Endospore(spore within
cell)
Sporangium(cell +
endospore)