1. The Study of Microorganisms
Microbiology
The Study of Microorganisms

2. Definition of a Microorganism
Derived from the Greek: Mikros, «small» and Organismos, “organism”
Microscopic organism which is single celled (unicellular) or a mass of identical (undifferentiated) cells
Includes bacteria, fungi, algae, viruses, and protozoans

3. Microorganisms in the Lab
Growth Media

4. Goals Growth under controlled conditions Maintenance
Isolation of pure cultures
Metabolic testing

5. Types Liquid (Broths) Solid media Allows growth in suspension
Uniform distribution of nutrients, environmental parameters and others
Allows growth of large volumes
Solid media
Same as liquid media + solidification agent
Agar: Polysaccharide derived from an algae

6. Growth in Broths Non inoculated clear Turbid + sediment Turbid
Clear + sediment

7. Growth on Agar Growth on solid surface Isolated growth
Allows isolation of single colonies
Allows isolation of pure cultures
Easier for counting colonies and observing morphology of colonies.
Plates are used to:
grow and culture bacteria, fungi, animal tissues, or plant tissues
obtain separated pure cultures of bacteria (plate streaking)
count colonies from serial dilutions
test for growth and reactions on certain materials (such as manitol-salt, or blood agar)
test for bacteria viruses (bacteriophages)
test for resistance to materials (such as antibiotics) or nutritional needs (this is also used to select for bacteria with certain properties or favor the growth of one type/strain over others)
Single colony

8. Solid Media (Cont’d) Slants Stab Growth on surface and in depth
Different availabilities of oxygen
Long term storage
Stab
Semi-solid medium
Low availability of oxygen

9. Counting Microorganisms

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

11. 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

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

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

14. 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

15. Direct Counts Advantages: Limits: Quick Growth is not required
No information about organism required
Limits:
Does not discriminate between live and dead
May be difficult to distinguish bacteria from detritus

16. Using a hemacytometer

17. Using a hemacytometer (Cont’d)

18. Hemacytometer
This slide has 2 independent counting chambers

19. Using a hemacytometer (Cont’d)

20. 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

21. 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

22. Problem
A 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

23. Viable Counts
A viable cell: a cell which is able to divide and form a population (or colony)
A viable cell count is done by diluting the original sample
Plating aliquots of the dilutions onto an appropriate culture medium
Incubating the plates under appropriate conditions to allow growth
Colonies are formed
Colonies are counted and original number of viable cells is calculated according to the dilution used

24. 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

25. Dilution of Bacterial Sample

26. Dilution of Bacterial Sample

27. Dilution of Bacterial Sample

28. Dilution of Bacterial Sample

29. Plating of Diluted Samples
5672 57 4

30. Viable Counts
The total number of viable cells is reported as Colony-Forming Units (CFUs) rather than cell numbers
Each single colony originates from a colony forming unit (CFU)
A plate having colonies is chosen
Calculation:
Number of colonies on plate X reciprocal of dilution (dilution factor) = Number of CFU/mL
Ex. 57 CFU/0.1mL X 106 = 5.7 X 107 CFU/mL

31. 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?

32. = = 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
Only living cells develop colonies
Clumps or chains of cells develop into a single colony
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. = ? = ?

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

34. 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)

35. 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

36. Microscopy
Staining

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

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

39. 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

40. 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

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

42. 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.

43. Differential Staining
Microscopy
Differential Staining

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

45. 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-

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

47. 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

48. Gram Staining- Principal
Uses a combination of two stains
Primary stain - Crystal violet
Purple
Secondary stain – Safranin
Red
Gram positive
The cell wall traps the 1o stain
Gram negative
Cell wall does not allow 1o stain to be trapped

49. Cell Wall

50. Method – Primary Stain Staining with crystal violet+
Staining with crystal violet
Add Gram’s iodine (Mordant) + +
Wall: peptidoglycan
LPS
Plasma membrane
Gram positive
Gram negative

51. Method – Differential Step
Alcohol wash
Cell wall is dehydrated and less permeable
– Stai + iodine complex is trapped
LPS layer is dissolved
Cell wall is dehydrated, but permeable
– Complex is not trapped
Wall: peptidoglycan
LPS
Plasma membrane + +
Gram positive
Gram Negative

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

53. Summary Fixation Primary stain Crystal violet Wash Decolorization
Counter stain
Safranin

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

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

56. 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

57. 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

58. 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)