1. Microbiology lab (BIO 3126)

2. My coordinates Instructor : John Basso :
Office : Bioscience 102
Tel. : Ext. 6358
Web page:

3. My Availability  :
All week before 5
Tel. :
Mon - Fri : 9-5

4. Course Evaluation Quiz Assignments 20% 2 Reports 10% Midterm Exam 25%
2 bonus points for 100% on 4/8 quizzes
Assignments 20%
2 Reports 10%
Midterm Exam 25%
3MT presentations 10%
Practical Exam
In lab 5%
Job Interview 5%
Final Exam 25%

5. Overview of web page

6. Working in the microbiology lab

7. 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

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

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

10. Working in Microbiology
Sterile Technique

11. The Material
The material used for the growth and handling of microorganisms must be sterile and maintained sterile
Growth media
Tubes
Petri dishes
Inoculation loop
Etc.

12. Use sterile technique for all transfers of microorganisms
Prevents contamination of your cultures
Prevents contamination of the environment
Prevents contamination of self
All bacteria are opportunistic

13. Transfers Using Sterile Technique
Sterilize the inoculation loop with the Bunsen burner
The whole length of the wire must become Red
Do not deposit on the table!
Allow to cool down
Boucle
d’ensemencement

14. Transfers Using Sterile Technique
Remove cap with your small finger of the inoculation loop hand
Do not put the cap on the table!

15. Transfers Using Sterile Technique
Heat the mouth of the tube with the Bunsen burner
Keep the orientation of the tube as close as possible to the horizontal
Keep the opening of the cap downward
Flame mouth of tube

16. Transfers Using Sterile Technique
Use the sterile loop to remove inoculum
Liquid from broths
Solid from plates
Solid from slants

17. Transfers Using Sterile Technique
Heat once again the mouth of the tube!
Keep the orientation of the tube as close as possible to the horizontal
Flame mouth of tube

18. Transfers Using Sterile Technique
Put the cap back on the tube of pure culture
Return the tube to the rack

19. Transfers Using Sterile Technique
Repeat the same steps to inoculate a new tube
Remove cap
Flame mouth of tube
Inoculate
Close tube
Inoculation

20. Working with solutions

21. Definitions Solution Mixture of 2 or more substances in a single phase
Solutions are composed of 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

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

23. Molarity # 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

24. 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

25. 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%

26. Mass per volume A mass amount per a 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 100ml?
What is the percentage of this solution?
100g
100%

27. 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
How many parts total? 50

28. Reducing a Concentration A Fraction
Dilutions
Reducing a Concentration
A Fraction

29. 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.

30. 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)

31. 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).

32. 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”.

33. 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

34. 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 dye + 8 parts 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.

35. 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
2/(2+5) = 2/7 =1/3.5
10/(10+50) = 10/60=1/6

36. Problem : More than one ingredient
One part of saline and three parts of sugar are added to 6 parts of water
What are the dilutions?
Saline: 1/(1+3+6) = 1/10
Sugar: 3/(1+3+6) 3/10 = 1/3.3
How would you prepare 15mL of this solution?
Express each component being diluted over the same common denominator!
Saline: 1/10 + Sugar 3/10
= 1.5/ /15

37. Serial Dilutions Dilutions made from dilutions
Dilutions are multiplicative
Ex.
A1: 1/10
A2: 1/4
A3: 0.5/1.5 = 1/3
The final dilution of the series = (A1 X A2 X A3) = 1/120

38. 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. X the dilution factor = initial conc.

39. 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)

40. 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

41. Problem Prepare 25mL of a 2mM solution from a stock of 0.1M
What is the dilution factor required?
What is the dilution required?
What volumes of solvent and solute are required? 50
1/50
Solute 0.5ml
Solvent 24.5ml

42. Problem
How much of a 10M solution of HCl would you add to 18mL of water to obtain a 1M solution?
What is the dilution required?
What volumes of solvent and solute are required?
1/10
1 part Solute / 10 parts of Solution
1 part Solute / 1 part Solute = 9 parts Solvent
Therefore 9 parts solvent = 18mL or 1 part = 2mL