prokaryotes (bacteria) and gram staining · prokaryotes (bacteria) and gram staining gram positive...

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Prokaryotes (bacteria) andGram Staining

Gram positive andGram negative

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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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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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Mycoplasma haemofelis in ablood film from an infected cat.

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Methods of Detection forMycoplasma

PCR (Polymerase Chain Reaction)Test

Culture on Selective media Hoechst DNA stain

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Hoechst DNA stain test

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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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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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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.

BTEC101, Day 5http://www.lhup.edu/smarvel/seminar/fall_2004/dolan-ward/index.htm

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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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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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Bacterial anatomy and physiology

Bacteria can have different shapes

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Four shapes of bacteria Rod (bacillus) Circular (coccus) Spiral (spirillium) Corkscrew (spirochete)

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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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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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Membrane begins to pinch in andbegins to divide.

End up with two bacterial cells that aregenetically identical.

Binary Fission

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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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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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Bacterial Growth

Time

Num

ber o

f Bac

teria

lag

logarithmic

stationary

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Bacteria can live in differentenvironments Acidophiles Thermophiles Anaerobes Aerobes Halophiles

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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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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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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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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

Gram Stain

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Gram Positive Bacteria Gram Negative Bacteria

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Bacterial Cell wall types Gram Negative vs.

Gram Positive

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Gram negative Thinner cell wall

Less peptidoglycan Two membranes Many are pathogenic Examples:

E.Coli Salmonella

Gram negative bacteria

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Gram Negative Cell Wall

Gram Positive Bacteria

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Gram positive Thicker cell wall Only one membrane

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Comparison of Cell Walls

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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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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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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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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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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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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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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