MCB3020L FINAL

REVIEW
Thursday June 14 during class
LAB MATH: Conversions
Reminders:
■ 1 L = 1000 mL
■ 1 mL = 1000 microliters
■ Assume 1 g = 1 mL
■ : and / are used interchangeably for describing dilution ratios
 LAB MATH: Weight/Volume%
mass of solute (g)
w/v% concentration = x 100
volume of solution (mL)

EXAMPLE: How many grams of salt would you need to make 1L of a 0.9% NaCl
solution?
1000 𝑚𝐿
1𝐿 𝑥 = 1000 mL
1𝐿
𝑥 𝑔𝑟𝑎𝑚𝑠
𝑥 100 = .9%
1000 𝑚𝐿
𝑥 𝑔𝑟𝑎𝑚𝑠
= .009
1000 𝑚𝐿

𝑥 = 9 g NaCl
LAB MATH: Molar Concentration
moles (mol) grams
Molarity (M)= Molecular weight (MW)=
liter (L) 1 mole

EXAMPLE: How much arginine do you need to make 100 mL of a 0.01M

solution of arginine (MW = 174.20)?
1𝐿
100 𝑚𝐿 𝑥 = .1 𝐿
1000 𝑚𝐿 x mol
= .01 M
.1 L

x = .001 moles arginine

174.20 𝑔
.001 𝑚𝑜𝑙 𝑥 = .174 g arginine
1 𝑚𝑜𝑙
 LAB MATH: Dilution Ratios
EXAMPLE: In the following dilutions, what is the concentration of the final sample?
Part
Dilution ratio =
Whole

1 1/10 1/100 1/1000 1/10000

1 1 1 1 1 1 .5 3 1 1 2
𝑥 𝑥 𝑥 = 𝑥 𝑥 𝑥 𝑥 𝑥 =
10 10 10 10 10,000 10 5 30 100 10 20

Serial dilution! 1 1 1 1 1 1 1
𝑥 𝑥 𝑥 𝑥 𝑥 =
10 10 10 100 10 10 10,000,000
EXAMPLE: How would you make 240mL of a 1:3 dilution of dye?

1 x
=
3 240 𝑚𝐿

x = 80 mL dye in 160 mL of water = 240 mL total volume

LAB MATH: Dilution Factor
Initial concentration (IC)
Dilution Factor (DF) =
Final concentration (FC)

EXAMPLE: What is the dilution factor?

𝐼𝐶 1
𝑥 = 10,000
𝐹𝐶 1/10,000

1 1/10 1/100 1/1000 1/10000

 LAB MATH: Diluting Solutions
C1V1 = C2V2
C = concentration (%/M)
V = volume
Make sure units are same on each side!

EXAMPLE: How would you prepare 50 mL of a 3M solution from a 4M stock

solution? Give your answer in liters.

C1V1 = C2V2

3M (50 mL) = 4M (x mL)

x = 37.5 mL = .0375 L of 4M solution + add .0125 L of water = .05 L 3M solution

EXAMPLE: What percent of dextrose is prepared when 3 mL of a 10%
dextrose solution is mixed with 12 mL of broth?
C1V1 = C2V2

10% (3 mL) = x% (15 mL)

x = 2% dextrose solution
LAB MATH: Diluting Solutions (Unspecified Volumes)
EXAMPLE: How do you dilute a solution that has an initial concentration of
solute of 10% down to 2%?
Assume 1 L of initial
10% x 1 L = 2% x X L solution. OR:
𝐼𝐶 10%
AMOUNT of solute in 1 L 𝐷𝐹 = = =5
10% x 1 L = 2% x 5 L of 10% is equal to 𝐹𝐶 2%
AMOUNT in 5L of 2%.

Perform a 1:5 dilution. Add 1 volume of 10% to 4 volumes of diluent to make

5 volumes of 2%.
LAB MATH: Enumeration
𝐶𝐹𝑈 𝑐𝑜𝑢𝑛𝑡 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
=
𝑚𝐿 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛 (𝑚𝐿)

EXAMPLE: 3 mL of bacterial culture is added to 27 mL of diluent, then 4

consecutive 1/10 dilutions are made. .01 mL of the last sample is added
to a plate. After incubation, you count 83 colonies on the plate. What is
the CFU/mL of the original bacterial culture?
3 1 1 1 1
𝑥 𝑥 𝑥 𝑥 =
30 10 10 10 10
1 1 1 1 1 1 83 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑥 100000
𝑥 𝑥 𝑥 𝑥 = = 830,000,000
10 10 10 10 10 100000 .01 𝑚𝐿

Dilution factor = 100,000

Week 1
Microscopy
Reminders:

• Start on 4X and 10X

first
• Prokaryotes visible on
100X
• Oil on 100X lens ONLY
Week 1
Gram Stain

■ Gram positive: appear purple

■ Gram negative: appear pink
Week 2
1: More concentrated w/ bacteria
Streaking for Isolation
■ Purpose: produce pure culture
(only one species of microbe) and
isolated colonies

Reminders:
■ Never “redip” from your original
plate!
■ Reflame between each quadrant
■ Wait until loop cools/stops glowing
red
■ Use Bunsen burner/aseptic
technique

3: Less concentrated w/ bacteria, isolated colonies

 Lack isolated colonies

Good!
Contamination (not a pure culture)
Week 2
Pipetting
Reminders:
■ Volumes should only be set from
X/10 – X microliters
– Example: P100 should only
be set from 10-100
microliters
■ Keep pipette upright when
there’s liquid inside
■ Check for bubbles in pipette tip
Week 2
Bacterial Growth
 𝑡𝑖𝑚𝑒
Generation time =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠
𝑡2 −𝑡1 𝑥 log(2)
Generation time = 𝑂𝐷𝑡2
log( )
𝑂𝐷𝑡1

• Generation time is the time taken for the population

to divide.
• Calculated from LOG PHASE
Week 2
Biochemical Diagnostics
■ Selective media: only allows one group of organisms to grow,
inhibits others
■ Differential media: distinguishes between two groups of organisms
■ Mannitol Salt Agar ■ MacConkey Agar
– Selective
– Selective
■ Only high-salt withstanding
organisms grow ■ Only bile-salt and crystal
■ Mainly gram positive violet tolerating bacteria
staphylococcus grow (gram-negative)
– Differential – Differential
■ Yellow color: mannitol ■ Red/pink color: lactose-
fermenter
fermenter
■ Pink color: non-mannitol
fermenter ■ Yellow color: non-lactose
fermenter
-Tolerant of high salt conditions
-Mannitol fermenter
■ Blood Agar ■ DNAase Test
– Differential – Differential
■ Clear color: beta ■ Green color: negative for
hemolysis/complete deoxyribonuclease activity
■ Green color: alpha ■ Colorless: positive for
hemolysis/incomplete deoxyribonuclease activity
■ Red color: gamma
hemolysis/none
■ Phenol Red Broth ■ SIM Agar
– Differential
■ Yellow color:
carbohydrate fermenter
■ Red color: non-
carbohydrate fermenter
– Differential
■ Bubbles in Durham tube:
gas production
(heterofermentative)
■ No bubbles: no gas
production
(homofermentative)
– Differential
■ Black color: sulfide production
■ Yellow color: no sulfide
production
– Differential
■ Red color after using Kovac’s
reagent: indole production
■ Yellow color: no indole
production
– Differential
■ Growth radiating from stab:
motile
■ No growth radiating: non-
motile
■ Catalase Test ■ Oxidase Test
– Bubbles: catalase produced – Blue/purple color: oxidase
– No bubbles: no catalase produced
– No color: no oxidase
 Week 2
DNA Extraction + Purification
Lyse cells with
Cell pellet DNA in the supernatent
chemglass beads

Solution 5 (high salt) DNA bound to silica

Wash away all salt +

Solution 6 (wash) other cell contents DNA still bound to silica
(proteins,
carbohydrates, etc.) DNA unbinds
Solution 7 (low salt) from silica, now
in supernatent
Week 2
PCR Denaturation Annealing
95° C 45° C
Heat to break Primers specific for 16S
hydrogen bonds of rRNA gene attach
double helix
amplification

MasterMix:
Elongation TAQ Polymerase
72° C dNTPs
dNTPs added 16S rRNA primers
Sterile PCR water
 Cathode (-)

Week 3
Gel Electrophoresis
■ Check PCR product
■ Smaller DNA: move faster
■ Larger DNA: moves slower

Anode (+)
Week 3
Bioinformatics
■ BLAST
– Identify organism by nucleotide sequence
– Use FASTA format
■ Gideon
– Identify organism by biochemical test results
– Input both positive/negative results!
■ Phylogeny.fr
– Know how to read relationships from tree
Week 4
MRSA
■ Methicillin Resistant Staphylococcus aureus
■ 25-30% population colonized, but not necessarily infected
■ Transmitted in hospital + community
■ Produce beta-lactamase  resistance to penicillin class drugs
■ Infection starts off as small pimple-like bump + often develops pus
drainage; causes boils, abscesses, and furuncles
Week 4
ELISA (Enzyme-Linked Immunosorbent Assay)
Week 5
Antibiotics