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BTEC101, Day 5
Prokaryotes (bacteria) andGram Staining
Gram positive andGram negative
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BTEC101, Day 5
Why are we learning this? We will be monitoring our bioreactors
for bacterial contamination. We sometimes are culturing E.coli as
the organism of choice and in this casewe want to make sure it is notcontaminated.
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BTEC101, Day 5
Mycoplasmas Mycoplasmas small prokaryotes without
a cell wall. Huge contamination problem in industry
and research laboratories doing Cellculture
Treatment of contaminated cultures isquite problematic, usual solution isabandoning the culture.
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BTEC101, Day 5
Mycoplasma haemofelis in ablood film from an infected cat.
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BTEC101, Day 5
Methods of Detection forMycoplasma
PCR (Polymerase Chain Reaction)Test
Culture on Selective media Hoechst DNA stain
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BTEC101, Day 5
Hoechst DNA stain test
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BTEC101, Day 5
Colonies of Mycoplasma exhibit a distinctive "friedegg" morphology when viewed under a platemicroscope. The assay requires a 28 day testinterval before a definitive result can be obtained.
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BTEC101, Day 5
Generalized bacterialinformation Single celled organisms No nucleus or membrane bound
organelles Used extensively in biotechnology Also a major contaminate of cultures
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BTEC101, Day 5
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BTEC101, Day 5
Terms
Cytoplasm: Cellular contents inside the plasmamembrane- water, nutrients, building blocks for thecell.
Outer membrane: Seen in gram negativebacterial cells. Endotoxins are lipopolysacchridesfound in this membrane.
Capsule (Slime Layer): this is a polysacchridelayer around the cell. Some evidence that theyhelp in avoiding the immune system.
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BTEC101, Day 5http://www.lhup.edu/smarvel/seminar/fall_2004/dolan-ward/index.htm
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BTEC101, Day 5
Terms Cell Wall: The function of the cell wall is to
give shape and rigidity to the cell. Here wefind peptidoglycan and structurally similarmolecules.
Plasma membrane: This is the membranethat surrounds the cell keeping thecytoplasm in and the external environmentout.
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BTEC101, Day 5
Terms Ribosomes: Protein complex and RNA which
makes other proteins in the cell. Nucleoid: The region inside the cell where the
bacterial chromosome is housed. Mesosome: folding of the plasma membrane. Flagellum: Method of bacterial locomotion.
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BTEC101, Day 5
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Bacterial anatomy and physiology
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Bacteria can have different shapes
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BTEC101, Day 5
Four shapes of bacteria Rod (bacillus) Circular (coccus) Spiral (spirillium) Corkscrew (spirochete)
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BTEC101, Day 5
Genetic Material Bacterial chromosome is circular and
attaches to the plasma membrane ofthe bacterial cell.
Plasmids small DNA loops transfercharacteristics such as antibioticsresistance
In eukaryotes the chromosomes arelinear and are contained in the nucleus.
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BTEC101, Day 5
Binary Fission
Bacterial chromosomes are attached tothe plasma membrane.
The DNA is replicated and the secondchromosome is attached to themembrane.
Membrane is added between the tworeplicated chromosomes.
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BTEC101, Day 5
Membrane begins to pinch in andbegins to divide.
End up with two bacterial cells that aregenetically identical.
Binary Fission
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BTEC101, Day 5
Bacterial Growth Bacterial growth is very rapid. E. coli divides can divide every twenty
minutes. The bacterial culture starts out slow this
is called the lag phase.
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BTEC101, Day 5
Next the bacteria start to grow rapidly,which is the logarithmic phase.
Finally the bacterial culture runs out ofroom and nutrition, this phase of growthis called the stationary phase. This iswhen the culture does not continue toincrease in number.
Bacterial Growth
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BTEC101, Day 5
Bacterial Growth
Time
Num
ber o
f Bac
teria
lag
logarithmic
stationary
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BTEC101, Day 5
Bacteria can live in differentenvironments Acidophiles Thermophiles Anaerobes Aerobes Halophiles
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BTEC101, Day 5
Cardinal temperatures
Minimum growth temperature Lowest temperature at which an organism will grow Below this temp.nutrient transport difficulty due to the fact that
membrane gels and transport too slow
Optimum growth temperature Temperature at which an organism grows best Metabolic enzyme reactions occurring at maximum rate
Maximum growth temperature Highest temperature at which an organism will grow Above this temp.protein denaturation; membrane collapse, and
lysis
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BTEC101, Day 5
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BTEC101, Day 5
Different Bacteria havedifferent antibiotic sensitivity Cell membrane Cell wall synthesis Folic acid biosynthesis DNA gyrase DNA polymerase Protein synthesis, 30S inhibitors Protein synthesis, 50S inhibitors
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BTEC101, Day 5
antibiotics Many antibiotics such as
penicillin work by disruptingcell wall synthesis
The cell walls become weakand eventually allow the cell toburst
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BTEC101, Day 5
Gram Stain This test allows scientists to
differentiate between gram negativeand gram positive bacteria
Gram negatives will stain a red Gram positives will stain a purple
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Gram Stain
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BTEC101, Day 5
Gram Positive Bacteria Gram Negative Bacteria
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BTEC101, Day 5
Bacterial Cell wall types Gram Negative vs.
Gram Positive
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BTEC101, Day 5
Gram negative Thinner cell wall
Less peptidoglycan Two membranes Many are pathogenic Examples:
E.Coli Salmonella
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Gram negative bacteria
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BTEC101, Day 5
Gram Negative Cell Wall
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Gram Positive Bacteria
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BTEC101, Day 5
Gram positive Thicker cell wall Only one membrane
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BTEC101, Day 5
Comparison of Cell Walls
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BTEC101, Day 5
Difference between Grampositive and Gram negative Cell wall is thicker in a gram positive
bacteria. Gram positive bacteria has only the
inner plasma membrane and no outermembrane.
Gram positive bacteria stain blue whilegram negative stain red.
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BTEC101, Day 5
General Summary of Bacteria Lack organelles Nucleus is lacking Smaller ribosomes Endotoxins are problematic Linked transcription and translation Can make simple proteins
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BTEC101, Day 5
Control of BacterialContamination
Cleaning- removing debris and residues thatcan be nutrients for microbes
Chemical disinfectants such as 70% IPA orEtOH, bleach
Treatment with Gamma Irradiation or UltraVioletlight (UV) irradiation
Treatment with Gases Ethylene oxide orpropylene oxide (common in spice trade,hospitals for equipment)
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BTEC101, Day 5
The Three Main Cell Types used inFermenation
Tools of the Trade: Bacteria (E. coli-Prokaryote):
Used for insulin and growth hormone, DNA products We will use it for GFP.
Yeast (P. pastoris, S. cerevisiae-Eukaryote): Use in food processing ( brewing , baking) We will use it for HSA.
Mammalian Cells (CHO, BHK, Hybridomas -Eukaryote): Antibody products , large complex glycosylated proteins Too expensive for us to use, however extensively used in the industry.
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BTEC101, Day 5
Fermentor In the fermentor we will try and duplicate
optimum conditions. Increase density as far as possible. Will also be inducing the formation of the
GFP protein by using arabinose.
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BTEC101, Day 5
The advantages of E.coli orprokaryotes as a host cell
Can be grown to high numbers in shorttime
Can be grown in economical simpledefined media
Robust organisms withstand high agitationwithout shearing
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BTEC101, Day 5
Disadvantages of Prokaryotes Inability to glycosylate proteins Proteins are sometimes expressed in
refractile bodies that needresolubilisation and refolding
Inherent endotoxin contamination Folding of complex proteins is often
problematic