Microbiology lab (BIO 3126)
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My coordinates
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Instructor : John Basso
Email : jbasso@uottawa.ca
Office : Bioscience 102
Tel. : 613-562-5800 Ext. 6358
Web page:
http://mysite.science.uottawa.ca/jbasso/home.htm
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My Availability
•Email :
•All week before 5
•Tel. :
•Mon - Fri : 9-5
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Course Evaluation
• Quiz
– 2 bonus points for 100% on 4/8 quizzes
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Assignments
2 Reports
Midterm Exam
3MT presentations
Practical Exam
– In lab
– Job Interview
• Final Exam
20%
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Overview of web page
• http://mysite.science.uottawa.ca/jbasso/home.htm
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Microbiology
Working in the microbiology
lab
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
Before starting –At the end
• Disinfect your work area
– Helps prevent contamination of cultures with
microorganisms from the environment
Before leaving the lab
• Wash your hands
before leaving the
lab
– Helps prevent
contamination of the
environment
Working in Microbiology
Sterile Technique
The Material
• The material used for the growth and
handling of microorganisms must be sterile
and maintained sterile
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Growth media
Tubes
Petri dishes
Inoculation loop
Etc.
• 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
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
Transfers Using Sterile Technique
• Remove cap with your small
finger of the inoculation loop
hand
– Do not put the cap on the table!
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
Transfers Using Sterile Technique
• Use the sterile loop to remove
inoculum
– Liquid from broths
– Solid from plates
– Solid from slants
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
Transfers Using Sterile Technique
• Put the cap back on the tube of
pure culture
• Return the tube to the rack
Transfers Using Sterile Technique
• Repeat the same steps to inoculate a
new tube
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Remove cap
Flame mouth of tube
Inoculate
Flame mouth of tube
Close tube
Inoculation
Working with solutions
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
Concentrations
• Concentration = Quantity of solute
Quantity of solution (Not solvent)
• Four ways to express concentrations:
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Molar concentration (Molarity)
Percentages
Mass per volume
Ratios
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
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
Percentages (Cont’d)
• %V/V
– 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%
• Must have same units top and bottom!
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? 100g
100%
– What is the percentage of this solution?
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
Dilutions
Reducing a Concentration
A Fraction
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.
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)
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).
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 + 100 parts
water). The dilution is written as 1/101 or
said “one in one hundred and one”.
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
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.
Problem
1. Two parts of blood are diluted with five parts
of saline
– What is the dilution? 2/(2+5) = 2/7 =1/3.5
2. 10 ml of saline are added to 0.05 L of water
– What is the dilution? 10/(10+50) = 10/60=1/6
Problem : More than one ingredient
1. 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
2. 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 + 4.5/15
Serial Dilutions
• Dilutions made from dilutions
• Dilutions are multiplicative
– Ex.
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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
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.
Determining the required fraction
(the dilution)
Determine the reduction factor
(The dilution factor)
=
What I have
What I want
Ex. You have a solution at 25 mg/ml and
want to obtain a solution at 5mg/ml
Therefore the reduction factor is:
25mg/ml
5mg/ml
= 5 (Dilution factor)
The fraction is equal to 1/the dilution factor = 1/5 (the dilution)
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
Problem
• Prepare 25mL of a 2mM solution from a stock
of 0.1M
– What is the dilution factor required? 50
– What is the dilution required? 1/50
– What volumes of solvent and solute are required?
Solute 0.5ml
Solvent 24.5ml
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? 1/10
– What volumes of solvent and solute are required?
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