1. Counting Microorganisms

2. Methods Turbidity measurements: Optical density Viable counts MPN
Direct counts

3. 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

4. Turbidity measurements
Spectrophotometer (A600):
Measuring optical density
Light
600nm
Detector….reading
Different reading

5. Turbidity measurements
2.0
1.0
O.D. 600nm
% Transmission
100 50
Cellular density
Inverse relationship

6. 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

7. Serial Dilutions 63 CFU/0.1ml of 10-5 630 CFU/1.0ml of 10-5
Bacterial
culture
CFU
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 sample
What if there were 100 ml in the flask?

8. = = Viable Counts Advantages: Limits: ? ?
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
Can differentiate between different microorganisms = b/c they are single colonies can count how many of each distinct morphology, colour etc.
No universal media = if you have a mix of bacteria, not all will be happy on one media type.
Requires growth = if you have live, but non reproducing bacteria, they won’t show up. = i.e. recall bacteriostatic antibiotics. = ? = ?

9. 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

10. Using a hemacytometer

11. Using a hemacytometer (Cont’d)

12. Using a hemacytometer (Cont’d)

13. Determining the Direct Count
Count the number of cells in three independent squares
8, 8 and 5
Determine the mean
( )/3 =7
Therefore 7 cells/square

14. 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

15. 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?
2x10^9 cells/ml squares is relevant.
Recall for cell culture:
1mmx1mmx0.1mm
=0.1x0.1x0.01cm
=0.0001cm3
10^-4
10^4 dil factor

16. Most probable Number: MPN
Based on Probability Statistics
Presumptive test based on given characteristics
Broth Technique

17. Most Probable Number (MPN)
Begin with Broth to detect desired characteristic
Inoculate different dilutions of sample to be tested in each of three tubes
Dilution
3 Tubes/Dilution
1 ml of Each Dilution into Each Tube
After suitable incubation period, record POSITIVE TUBES (Have GROWTH and desired characteristics)

18. 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:
Positive tubes:
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. tubes
MPN/g (mL)
0.10
0.01
0.001 3 2 1
150

19. Microscopy
Staining

20. Simple Staining Positive staining Negative staining Stains specimen
Staining independent of the species
Negative staining
Staining of background

21. Method Simple stain: One stain
Allows to determine size, shape, and aggregation of bacteria

22. 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

23. 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

24. Cell Shapes (Cont’d) Rods: Division along one axis only
Types of aggregations are typical of different bacterial genera

25. If it doesn’t end in cocci, it’s probably a rod.
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.

26. Differential Staining
Microscopy
Differential Staining

27. Differential Staining Gram Stain
Divides bacteria into two groups
Gram Negative & Gram Positive

28. Gram Positives Stained Purple Rods Coccus
Genera Bacillus and Clostridium
Coccus
Genera Streptococcus, Staphylococcus and Micrococcus
-Rule of thumb: cocci and not Neisseria, Moraxella and Acinetobacter = G+ cocci (sphere)
-Genus is Bacillus or Clostridium then = rod G+
-All others are rod G-

29. Gram Negative Stained Red Rods: Coccus:
Genera Escherichia, Salmonella, Proteus, etc.
Coccus:
Genera Neisseria, Moraxella and Acinetobacter

30. 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

31. Cell Wall Gram + Vs Gram - Peptidoglycan wall Plasma Membrane
Lipopolysaccharide
layer
Absent
Plasma
Membrane

32. Method – Primary staining+
Staining with crystal violet
Addition of Gram’s iodine (Mordant) + +
Wall:peptidoglycan
LPS
Plasma membrane
Gram positive
Gram negative

33. Method – Differential step
Alcohol wash
Wall is dehydrated
– Stain + iodine complex is trapped
Wall is not dehydrated
– Complex is not trapped
Wall: peptidoglycan
LPS
Plasma membrane + +
Gram positive
Gram negative

34. Method – Counter Stain Staining with Safranin + + +
Wall:peptidoglycan
LPS
Plasma membrane +
Gram positive
Gram negative

35. Summary Gram Positive Gram Negative Fixation Primary staining
Crystal violet
Wash
Destaining
Counter staining
Safranin

36. Acid Fast Staining Diagnostic staining of Mycobacterium
Pathogens associated with Tuberculosis and Leprosy
Cell wall has mycoic acid
Waxy, very impermeable

37. Method
Basis:
High level of compounds similar to waxes in their cell walls, Mycoic acid, makes these bacteria resistant to traditional staining techniques

38. 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

39. 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

40. Malachite Green Staining
Spores
(resistant structures used for survival under unfavourable conditions.)
Sporangium
(cell + endospore)
Permeabilization of spores with heat
Primary staining with malachite green
Wash
Counter staining with safranin
Endospore
(spore within cell)
Vegetative cells
(actively growing)