Binary Fission

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Binary Fission
Binary Fission 1 ? 2 ? 4 ? 8 ? 16 ? 32 ? 64 ?
128 ? 256 ? 512 ? 1024 ? 2048 ? 4096 ? 8192 ?
16384 ? 32768 ? 65536 ? 131072 ? 262144 ? 524288
? 1048576 ? 2097152 ? 4194304 ? 8388608 ?
16777216 ? 33554432 ? 67108864 ? 134217728 ?
268435456 ? 536870912 ? 1073741824 ? 2147483648 ?
4294967296 ? 8589934592 (see it with your own
eyes!)
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Binary Fission
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Generation Time

• The time it takes for one bacterium to become two
  bacteria time for number of bacteria in a
  population to double
• Mostly dependent on temperature and nutrients
• Typically 1-3 hr, some take 24 hrs
• E. coli in a lab can double in 20 mins!

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Generation Time
Its a logarithmic function!! Number of
cells 2number of generations example 1
bacterium growing for 10 generations now
210 1024 bacteria for E. coli that
takes 3.5 hr
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In theory, the size of the earth
Theoretical example if a lab culture of E.
coli starting with 1 cell grows for 24 hr,
doubling every 20 mins, it will produce 72
generations 72 generations how many
cells? One E. coli cell weighs 1x10-12 g How
much would 72 generations worth of E. coli weigh?
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In reality, not.
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Phases of growth

• Lag Phase
• Exponential Growth
• Stationary Phase
• Decline / Death

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Serial Dilutions
How do you know how many bacteria you
have? serial dilutions reduce cell number to a
countable one
1101 1102 1103 1104
1105
Example 5x106/ml 5x105/ml
5x104/ml 5x103/ml 5x102/ml
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Pour Plates
What do you do with your serial dilutions? Pour
plates
2 potential disadvantages?
serial dilution
(not watermelon)
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Spread Plates(hockey sticking)
What do you do with your serial
dilutions? Spread plates
2 advantages?
hockey stick
serial dilution agar plate(solidified)
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Back Calculating
Count colonies on plates with gt30 and lt300
colonies multiply colony count on plate
x the dilution factor (for the serial dilution)
x dilution factor for amount spread cells /
ml in original sample
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Filtration
often done with water and air samples - why?
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Direct Count
use a calibrated microscope slide --
advantage? -- disadvantage?
multiply count per square times a dilutionfactor
count bacteriausing microscope
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Turbidity

• widely used to monitor growth in research labs
  -- 3 advantages?
• as a culture grows in broth, it gets cloudy
• what instrument is used to measure cloudiness?
• how do you get a growth curve?

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Turbidity
no bacteria -all light istransmitted bacteria
scatter light - less light istransmitted
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Weight

• each bacterial cell has a certain amount of
  macromolecules and so has a defined weight
• when would you use weight?
• what should you do to a sample before weighing
  it?

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Bacterial Cell Growth
Requirements Physical temperature, pH,
osmotic pressure Chemical sources of
required elements and organic compounds
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Temperature
Temperature all bacteria have a minimum,
maximum and optimal temperature for growth
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Temperature
Temperature and Bacteria - why do we care?
Food Spoilage (food poisoning) Not Real
Problems most pyschrophiles - require lt
room temp thermophiles - require gt45?C,
other issue with them? extreme thermophiles
- require gt65?C
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Temperature
Food Spoilage (food poisoning) Real
Problems pyschrophiles why? mesophiles
why?
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Temperature
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Cool it fast!!!
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pH
pH all bacteria have a minimum, maximum and
optimal pH for growth Most bacteria grow best at
what pH? Acidity is usually a preservative
(why?) - pickles, sauerkraut Acidophiles -
tolerant of acid pH Alkaliphiles - tolerant of
basic pH
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Osmotic Pressure
Osmotic Pressure bacteria grow in an
environment of essentially water Hypertonic
solutions preserve food -- high salt (salt
pork) -- high sugar (honey) Halophilic
bacteria (salt loving) - can grow in up to 30
salt (Dead Sea)
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Chemical Requirements
Chemical requirements carbon, nitrogen, sulfur,
phosphorus, trace elements, oxygen (sometimes),
and organic molecules carbon, nitrogen, sulfur,
and phosphorus together account for almost 70 of
bacterial weight
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Chemical Requirements
Chemical requirements Carbon 50 of
bacterial weight, mostly from organic material
but can be from CO2Nitrogen 14 of bacterial
weight, mostly from catabolism of proteins --
needed for which macromolecules? Sulfur and
Phosphorus 4 of bacterial weight, from
nucleic acids, proteins, phosphate ions --
needed for which macromolecules?
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Chemical Requirements
Chemical requirements Trace elements iron,
copper, magnesium, zinc, etc. -- often
function as what for enzymes? -- typically
obtained from what source? Organic Growth
Factors often vitamins, amino acids, purines
and pyrimidines - bacterial specific Oxygen
not always required for life what always is
required for active life?
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Oxygen
Obligate Aerobes must have oxygen(Pseudomonas
aeruginosa, Mycobacterium tuberculosis) Obligate
Anaerobes oxygen is toxic to them (Clostridium
and Bacteroides species)
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Oxygen
Facultative Anaerobes can use oxygen when
present but can use fermentation when not present
(E. coli) Microaerophiles prefer to grow in
reduced (but not absent) amounts of oxygen
(Campylobacter species) Aerotolerant Anaerobes
dont use oxygen but can tolerate it
(Lactobacillus species)
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Oxygen
what kind of media is used?
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Oxygen
Singlet Oxygen O2 in high energy state - highly
reactive Superoxide Radicals small amounts
formed during normal respiration - highly
unstable and take electrons from neighboring
molecules all organisms growing in the
presence of oxygen have superoxide
dismutase (SOD) to neutralize them O2- O2-
2H ? H2O2 O2
SOD
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Oxygen
Peroxide H2O2 (O22- anion) produced by SOD is
also toxic so cells have catalase or peroxidase
to neutralize it 2H2O2 ? 2H2O
O2 H2O2 2H ? 2H2O Obligate
anaerobes often dont have SOD or catalase so
they cant neutralize superoxide radicals and die
catalase
peroxidase
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Bacterial Growth Media

• Bacterial Culture Media
• Sterile
• Inoculum
• Culture

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Agar

• Agar marine algae derived complex
  polysaccharide used to solidify media
• used at 1.5
• considering its temperature characteristics, why
  do we use it for making solid media?
• considering bacterial action, why do we use it?

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Chemically Defined
chemical composition of all ingredients is
known a simplemedia for E. coli
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Chemically Defined
Some bacteria are fastidious (picky like little
kids) a more complexmedia for N. gonorrhoeae
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Cheap and Easy
undefined nutrient medium is often used -- meat
or vegatable extracts cheap and easy example
for E. coli L-broth (add agar for plates)
tryptone 10 g yeast extract 5 g NaCl 5
g water 1 liter
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Anaerobes
Get the O2 out! Media use a chemical that
combines with O2, often thioglycolate (thio
sulfur) Air anaerobe packssodium bicarb
sodium borohydride water ? H2 and CO2 then
H2 O2 ? H2O no O2
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Anaerobes

• Glove Box
• inert atmosphere inside
• usually nitrogen gas (N2)
• lab handling a lot of anaerobic samples

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Special Atmosphere

• Many bacteria prefer to grow in ? CO2 and ? O2
• mimics many body tissues for pathogenic
  bacteria such as Neisseria
• Candle jars - low tech
• CO2 packs - low tech
• CO2 incubators

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Pure Cultures
Colony arises from a single bacterium or spore
or from a small group of bacteria attached to
each other (like Staph)
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Pure Cultures
Streak Plate Goal obtain an isolated
colony for identification or further study
-- bacteria desired has to be in relatively
high proportion in a sample --
if it is not, use which kind of medium first?
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Specimen Collection
http//www.hardydiagnostics.com/
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Selective Media

• suppresses growth of common bacteria you dont
  want
• enhances growth of desired bacteria

Bismuth Sulfate Agar suppresses Gram ()and most
Gram (-) other than Salmonella typhii
Cefsulodin-Irgasan-Novobiocin (CIN) Agar selects
for Yersinia
courtesy of BD Diagnostic Systems www.bd.com
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Differential Media

• contains a reagent that when acted upon will give
  a discernable change in the media (usually COLOR)
• LB Agar with x-gal
• colonies with b-galactosidase metabolize x-gal
  and turn blue-green
• colonies without the enzyme stay white

www.scientiis.com
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Differential Media
Blood Agar with hemolytic bacteria
lysed RBCs(clear-yellow)
intact RBCs(red)
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Selective and Differential

• MacConkey Agar w/sorbitol
• bile salts select against Gram ()
• pH indicator lactose fermentation red
  colonies

E. coli
P. mirabilis
courtesy of BD Diagnostic Systems www.bd.com
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Selective and Differential

• Mannitol Salt Agar
• 7.5 NaCl selects for halotolerant bacteria
  such as Staphylococcus aureus
• pH indicator mannitol fermentation acid
  yellow

Staphylococcus aureus(on mannitol salt
agar)mannitol fermenter?