Counting Microorganisms
Methods
•
•
•
•
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
3
Turbidity measurements
• Spectrophotometer (A600):
– Measuring optical density
Light
Detector….reading
600nm
Different reading
4
Turbidity measurements
O.D. 600nm
2.0
1.0
0
% Transmission
0
Inverse relationship
Cellular density
50
100
5
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
6
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
What if there were 100 ml in the flask?
7
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
?
– CFU
one bacteria
?
=
=
• Ex. One CFU of Streptococcus  one of E.coli
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
Using a hemacytometer
Using a hemacytometer (Cont’d)
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
13
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
14
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?
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
31
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
34
Summary
Gram Positive
Gram Negative
Fixation
Primary staining
Crystal violet
Wash
Destaining
Counter staining
Safranin
35
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)