1. Microbiology lab (BIO 3126)

2. Teaching Staff Lab coordinator: John Basso Lecturer: Benoît Pagé
Office: Bioscience 102
Tel.: Ext. 6358
Lecturer: Benoît Pagé

3. 2 bonus points for 100% on 4/8 quizzes
Course Evaluation
Quiz
2 bonus points for 100% on 4/8 quizzes
Assignments
20%
Midterm exam
30%
Final practical exam
10%
Final theoretical exam
40%

4. Working in the microbiology lab

5. At the beginning of the lab
Wash your hands as soon as you enter the lab
Helps to avoid contamination of the cultures with microorganisms from your natural flora

6. Before starting –At the end
Disinfect you work area
Helps prevent contamination of cultures with microorganisms from the environment

7. Before leaving the lab Wash your hands before leaving the lab
Helps prevent contamination of the environment

8. Working with solutions

9. Definitions Solution Mixture of 2 or more substances in a single phase
Solutions include two constituents
Solute
Part that is being dissolved or diluted – Usually smaller amount
Solvent (or Diluent)
Part of solution in which solute is dissolved – Usually greater volume

10. Concentrations Concentration = Quantity of solute
Quantity of solution (Not solvent)
Four ways to express concentrations:
Molar concentration (Molarity)
Percentages
Mass per volume
Ratios

11. Molarity No of Moles of solute/Liter of solution
Mass of solute/MW of solute = Moles of solute
Moles of solute/vol. in L of solution = Molarity

12. Percentages Percentage concentrations can be expressed as either:
V/V – volume of solute/100 ml of solution
m/m – mass of solute/100g of solution
m/V – mass of solute/100ml of solution
All represent fractions of 100

13. Percentages (Cont’d) %v/v %m/v % m/m
Ex. 4.1L solute/55L solution =7.5%
Must have same units top and bottom!
%m/v
Ex. 16g solute/50mL solution =32%
Must have units of same order of magnitude top and bottom!
% m/m
Ex. 1.7g solute/35g solution =4.9%

14. Mass per volume A mass amount per a given volume Ex. 1kg/L
Know the difference between an amount and a concentration!
In the above example 1 litre contains 1kg (an amount)
What amount would be contained in 100 ml?
What is the percentage of this solution?

15. Ratios
A way to express the relationship between different constituents
Expressed according to the number of parts of each component
Ex. 24 ml of chloroforme + 25 ml of phenol + 1 ml isoamyl alcohol
Therefore 24 parts + 25 parts + 1 part
Ratio: 24:25:1
What is the total number of parts?

16. Reducing a Concentration A fraction
Dilutions
Reducing a Concentration
A fraction

17. Dilutions Dilution = making weaker solutions from stronger ones
Example: Making orange juice from frozen concentrate. You mix one can of frozen orange juice with three (3) cans of water

18. Dilutions (cont’d)
Dilutions are expressed as a fraction of the number of parts of solute over the total number of parts of the solution (parts of solute + parts of solvant)
In the orange juice example, the dilution would be expressed as 1/4, for one can of O.J. ( 1 part) for a TOTAL of four parts of solution (1 part juice + 3 parts water)

19. Another example:
If you dilute 1 ml of serum with 9 ml of saline, the dilution would be written 1/10 or said “one in ten”, because you express the volume of the solution being diluted (1 ml of serum) per the TOTAL final volume of the dilution (10 ml total; 10 parts).

20. Another example:
One (1) part of concentrated acid is diluted with 100 parts of water. The total solution volume is 101 parts (1 part acid parts water). The dilution is written as 1/101 or said “one in one hundred and one”.

21. Dilutions (cont’d)
Dilutions are always a fraction expressing the relationship between ONE part of solute over a total number of parts of solution
Therefore the numerator of the fraction must be 1
If more than one part of solute is diluted you must transform the fraction

22. Example
Two (2) parts of dye are diluted with eight (8) parts of solvent
The total number of parts of the solution is 10 parts (2 parts of dye + 8 parts of solvent)
The dilution is initially expresses as 2/10
To transform the fraction in order to have a numerator of one, use an equation of ratios:
The dilution is expressed as 1/5.

23. Problem Two parts of blood are diluted with five parts of saline
What is the dilution?
10 ml of saline are added to 0.05 L of water

24. Problem: Multiple ingredients
One part of saline and three parts of sugar are added to 6 parts of water
What are the dilutions?
How would you prepare 15mL of this solution?

25. Serial Dilutions Dilutions made from dilutions
Dilutions are multiplicative
Ex.
A1:
A2:
A3:
The final dilution of the series = (A1 X A2 X A3) =
Change pipettes between each dilution to avoid carryover

26. The Dilution Factor Represents the inverse of the dilution
Expressed as the denominator of the fraction followed by “X”
EX. A dilution of 1/10 represents a dilution factor of 10X
The dilution factor allows one to determine the original concentration
Final conc. multiplied by the dilution factor = initial conc.

27. Determining the required fraction (the dilution)
What I have
What I want
Determine the reduction factor
(The dilution factor) =
Ex. You have a solution at 25 mg/ml and want to obtain a solution at 5mg/ml
Therefore the reduction factor is: mg/ml
5mg/ml =
5 (Dilution factor)
The fraction is equal to 1/the dilution factor = 1/5 (the dilution)

28. Determining the amounts required
Ex. You want 55 ml of a solution which represents a dilution of 1/5
Use a ratio equation:
1/5 = x/55 = 11/55
Therefore 11 ml of solute / (55 ml – 11 ml) of solvent
= 11 ml of solute / 44 ml of solvent

29. Problem Prepare 25mL of a 2mM solution from a 0.1M stock
What is the dilution factor required?
What is the dilution required?
What volumes of solvent and solute are required?

30. Problem
What volume of a 10M solution of HCl should you add to 18mL of water to obtain a 1M solution?
What is the dilution required?
What is the volume of one part?
What volumes of solvent and solute are required?

31. Tonicity and Osmolarity
Terms used to describe the relationship between the relative concentrations of solute particles on both sides of a semi-permeable membrane and the movement of water
Tonicity only takes into consideration the concentration of impermeable solutes particles
Osmolarity takes into consideration the total concentration of all solute particles
Permeable et non permeables

32. Tonicity and Osmolarity

33. Tonicity Impermeable solute Isotonic solution Hypotonic solution
Hypertonic Solution
Impermeable solute

34. Tonicity Impermeable solute Permeable solute Isotonic solution
Hypotonic solution
Isotonic solution
Hypertonic Solution

35. Osmolarity Impermeable solute Isosmotic solution Hyperosmotic solution
Hyposmotic solution
Isosmotic solution
Hyperosmotic solution
Impermeable solute

36. Osmolarity Impermeable solute Permeable solute Hyperosmotic solution
Hyposmotic solution
Hyperosmotic solution
Isosmotic solution
Impermeable solute
Permeable solute

37. Tonicity and Osmolarity
Determine the concentration of solute particles
Expressed as the number of osmoles (Osm) or osmolarity (Osm/L: OsM)
Ex. 1 molar (1M) NaCl = 1 mole of NaCl per liter of solution
1 molecule of NaCl = 2 particles (1 Na + 1 Cl)
Therefore 1M NaCl = (1 moles Na + 1 moles Cl)/L
Which is equal to 2 OsM