Counting Microorganisms
Methods
•
•
•
•
Turbidity measurements
Viable counts
Most probable number
Direct counts
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
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
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
7
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
8
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?
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
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
Gram +
Cell Wall
Vs
Gram -
Peptidoglycan
wall
Plasma
Membrane
Absent
Lipopolysaccharide
layer
15
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
18
Summary
Fixation
Primary staining
Crystal violet
Wash
Destaining
Counter staining
Safranin
19
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)
Pathogens
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 animals
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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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