counting microorganisms. methods turbidity measurements viable counts most probable number direct...
TRANSCRIPT
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Counting Microorganisms
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Methods
• Turbidity measurements• Viable counts• Most probable number• Direct counts
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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 0.001
3 2 1 150
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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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Determining the Direct Count
7
• 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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Microscopy
Differential Staining
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Differential StainingGram Stain
Divides bacteria into two groupsGram 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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Rule 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
15
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
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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
safranin
Vegetative cells(actively growing)
Spores(resistant
structures used for survival under
unfavourable conditions.)
Endospore(spore within
cell)
Sporangium(cell +
endospore)
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Pathogens
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19th Century: Robert Koch
• Studies anthrax disease which kills cows• Grows in pure culture bacteria obtained from
the blood of diseased animals– Bacillus anthracis
• Observations:– Blood of diseased animals transmits the disease– The microorganisms is found only in diseased
animals– The microorganism grown in the lab transmits the
disease to healthy animals26
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Robert Koch (Cont’d)
• Conclusion: Microorganisms are responsible of diseases– Pathogens
• These results lead Robert Koch to formulate guidelines to associate a microorganism to a disease– Koch’s postulates
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Koch’s Postulates
• The microorganism must be present in each diseased case but absent from healthy individuals
• The microorganism must be isolated and grown in pure cultures
• The disease must develop when the isolated microorganism is inoculated in a healthy host
• The same microorganism must be isolated again from the diseased host
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