Microbiology lab (BIO 3126) 1 Slide 2 My Coordinates Instructor
: Benot Pag Email : [email protected] Office : Bioscience 102 Web
page: http://mysite.science.uottawa.ca/jbasso/microlab/home.htm 2
Slide 3 My Availability 3 By e-mail: All week including weekends
Office hours: Monday to Tuesday : 10h00am 12h00pm Thursday to
Friday : 2h00pm 4h30pm Also available by appointment Slide 4 Course
Evaluation Quiz 2 bonus points for 100% on 4/8 quizzes Pre and post
labs5% Assignments20% Midterm Exam30% Final Exam*45% *The final
exam will consist of a practical and theoretical component Slide 5
Overview of web page
http://mysite.science.uottawa.ca/jbasso/microlab/home.htm 5 Slide 6
Microbiology Working in a microbiology lab Slide 7 At the beginning
of the lab As soon as you enter the lab wash your hands Helps avoid
the contamination of cultures with microorganisms from your natural
flora Slide 8 Before starting and at the end of the lab Disinfect
you work area Helps prevent the contamination of cultures with
microorganisms from the environment Slide 9 Before leaving the lab
Wash your hands before leaving the lab Helps prevent the
contamination of the environment Slide 10 Working in a Microbiology
Lab Sterile Technique Slide 11 The Material The material used for
the growth and handling of microorganisms must be sterile and
remain sterile Growth media Tubes Petri dishes Inoculation loop Etc
Slide 12 Maintaining Sterility Use sterile technique for all
transfers of microorganisms Prevents the contamination of your
cultures Prevents the contamination of the environment Prevents
self contamination All bacteria are opportunistic Slide 13
Transfers Using Sterile Technique Test tube to test tube Sterilize
the inoculation loop with the Bunsen burner The entire length of
the wire must become Red/Orange Do not deposit the loop on the
table! Allow it to cool down Boucle densemencement Slide 14
Transfers Using Sterile Technique Test tube to test tube Remove the
cap with the small finger from the hand holding the inoculation
loop Do not put the cap on the table! Slide 15 Transfers Using
Sterile Technique Test tube to test tube Heat the mouth of the test
tube with the Bunsen burner Keep the test tube as close to
horizontal as possible Keep the opening of the cap downward Flame
mouth of tube Slide 16 Transfers Using Sterile Technique Test tube
to test tube Use the sterile loop to remove inoculum Liquid from
broths Solid from plates Solid from slants Slide 17 Transfers Using
Sterile Technique Test tube to test tube Heat the mouth of the tube
once again Keep the test tube as close to horizontal as possible
Flame mouth of tube Slide 18 Transfers Using Sterile Technique Test
tube to test tube Put the cap back on the pure culture test tube
(test tube containing the inoculum) Return the test tube to the
rack Slide 19 Transfers Using Sterile Technique Test tube to test
tube Repeat the same steps to inoculate a new tube Remove cap Flame
mouth of tube Inoculate Flame mouth of tube Close tube Inoculation
Slide 20 Transfers Using Sterile Technique All transfers should be
done using sterile technique Test tube to plate Plate to test tube
Plate to plate Etc Under certain circumstances such as transfers
done from plates (or to plates), the sterile technique should be
slightly modified Slide 21 Working in a Microbiology Lab Working
with solutions Slide 22 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 (volume or mass) Solvent (OR Diluent) Part of
solution in which solute is dissolved Usually greater volume Slide
23 Concentrations Concentration = Quantity of solute Quantity of
solution (Not solvent) Four ways to express concentrations: Molar
concentration (Molarity) Percentages Mass per volume Ratios Slide
24 Molarity # of Moles of solute/Liter of solution Mass of solute:
given in grams (g) Molecular weight (MW): give in grams per mole
(g/mole) Slide 25 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 represented as a fraction of 100 Slide 26 Percentages
(Contd) %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!
Slide 27 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? Slide
28 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 are there in this solution? Slide 29 Dilutions Reducing
a Concentration Slide 30 Dilutions Dilution = making less
concentrated solutions from more concentrated ones Example: Making
orange juice from frozen concentrate. You mix one can of frozen
orange juice with three (3) cans of water. Slide 31 Dilutions
(contd) 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) Slide 32 A Second 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). Slide 33 A third 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. Slide 34 Dilutions
(contd) 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 Slide 35
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. Slide
36 Problem 1.Two parts of blood are diluted with five parts of
saline What is the dilution? 2.10 ml of saline are added to 0.05 L
of water What is the dilution? 2/(2+5) = 2/7 =1/3.5 10/(10+50) =
10/60=1/6 Slide 37 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 Slide 38 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 Note: Change pipettes between each dilution
to avoid carryover Slide 39 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. * the dilution factor = initial
conc. Note: The denominator is the dilution factor only when the
numerator is 1. Slide 40 Determining the Required Fraction (The
Dilution) Ex. You have a solution at 25 mg/ml and want to obtain a
solution at 5mg/ml The fraction is equal to 1/the dilution factor =
1/5 (the dilution) What I have What I want Determine the reduction
factor (The dilution factor) = Therefore the reduction factor is:
25mg/ml 5mg/ml = 5 (Dilution factor) Slide 41 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 Slide 42 Problem #1 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 Slide 43 Solution
#1 Fractions : 1) 2mM = 0.002M (what I want) Stock = 0.1M (what I
have) Dilution factor = (what I want)/(what I have) Dilution factor
= 0.1/0.002 = 50x 2) Required dilution = 1/Dilution factor = 1/50
3) Volume of a part = (Final volume)/(# of parts) Volume of a part
= 25mL/50 parts = 0.5mL/part Volume of solute = 1 part * 0.5mL/part
= 0.5mL Volume of solvent = (50 1) parts * 0.5mL/part Volume of
solvent = 24.5mL Slide 44 Solution #1 (contd) C 1 V 1 = C 2 V 2 1)
See previous slide 2) See previous slide 3) C 1 = 0.1M; C 2 =
0.002M; V 1 = ?; V 2 = 25mL; C 1 V 1 =C 2 V 2 V 1 = C 2 V 2 /C 1 =
0.002M * 25mL / 0.1M = 0.5mL Volume of solute = V 1 = 0.5mL Volume
of solvent = V 2 V 1 =25mL0.5mL=24.5mL Slide 45 Problem #2 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 Solvent (water) = 18mL and solute
(HCl) = 2mL Slide 46 Solution #2 Fractions: 1) What I want = 1M
What I have = 10M Dilution factor = (what I have) / (what I want)
Dilution factor = 10/1 = 10x Required dilution = 1/10 2) Volume
solvent = 18mL Dilution = 1/10 = 1/ (9 parts solvent + 1 part
solute) Volume 1 part = Volume Solvent / # of parts solvent Volume
1 part = 18mL / 9 parts = 2mL/part Volume of solute = # of parts
solute * (volume/part) Volume of solute = 1 part * 2mL/part = 2mL
Slide 47 Solution #2 (contd) C 1 V 1 = C 2 V 2 1) See previous
slide 2) C 1 = 10M; C 2 = 1M; V 1 = ?; V 2 = 18mL + V 1 ; C 1 V 1
=C 2 V 2 10M * V 1 = 1M * (18mL + V 1 ) 10V 1 = 18 + V 1 10V 1 V 1
= 18 9V 1 = 18 V 1 = 18/9 = 2mL Slide 48 Osmolarity Number of
osmoles (Osm, solute particles) per litre of solution (Osm/L = OsM)
Ex. 1 molar (1M) NaCl = 1 mole of solute molecules (NaCl) per liter
of solution 1 osmolar (OsM) NaCl = 1 mole of solute particles Na +
Cl) per liter of solution 1 molecule NaCl = 2 particles (1 Na + 1
Cl) Therefore 1 OsM NaCl = (0.5 moles Na + 0.5 moles Cl)/L 1 Molar
NaCl is equal to what osmolarity? Slide 49 Microbiology The Study
of Microorganisms Slide 50 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 50 Slide 51 Microorganisms in the Lab
Growth Media Slide 52 Goals Growth under controlled conditions
Maintenance Isolation of pure cultures Metabolic testing Slide 53
Types Liquid (Broths) 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
Slide 54 Growth in Broths Non inoculated clear Turbid + sediment
Clear + sediment Turbid Slide 55 Growth on Agar Growth on solid
surface Isolated growth Allows isolation of single colonies Allows
isolation of pure cultures Single colony Slide 56 Solid Media
(Contd) Slants Growth on surface and in depth Different
availabilities of oxygen Long term storage Stab Semi-solid medium
Long term storage Low availability of oxygen
