Today

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Today

• Assignments
• Pre-lab for 9
• 2nd submission Bacterial Growth Report Due
• Worksheet 8 (filled out during lab)
• Exercise 9
• Antimicrobials and antibiotics
• Exercise 8
• Record Results of Bacterial Transformation
  Experiment
• Environmental Isolate

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Exercise 9 Antimicrobials
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Antimicrobials History

• Mid 1800s Semmelweis
• Insisted on washing hands between patients in
  hospitals, particularly maternity ward
• Mortality rate (from peurperal fever) reduced
  from 13 to less than 1
• Mid to late 1800s Lister
• Sterilized surgical instruments
• Mortality rate (from surgery) reduced from 50 to
  15

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Antimicrobials History

• First attempts to use antibiotics on people
• Bad side-effects
• Determined to be too dangerous
• Ehrlich's magic bullet (1909)
• Found dyes which could penetrate microorganisms,
  but not animal tissue
• Reasoned that chemicals could attack pathogens,
  but not hosts magic bullets
• Discovered a chemical to treat syphilis
• Salvarsan or preparation 606

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Antimicrobials History

• Ehrlich stressed selective toxicity as key factor
  in success
• Specific to the pathogen
• Do not harm the host
• The search for antibiotics was refined
• Penicillin was introduced in 1945

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Antimicrobials History

• Germ Theory Microorganisms can invade other
  organisms and cause disease
• Growing acceptance of the germ theory at the end
  of the 19th century led to the search for
  antimicrobial compounds.

www.molbio.princeton.edu
Alexander Fleming, who discovered that the mold
Penicillium produced a compound that lysed
Staphylococcus cells he had streaked on a plate.
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Antimicrobials

• Microbial growth is never truly controlled
• Try to discourage harmful microorganisms
• Microorganisms that cause disease, spoil food,
  corrode machinery
• Try to encourage beneficial microorganisms
• Microorganisms that ferment beer or cheese,
  produce antibiotics or insulin

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Antimicrobials

• Antimicrobials chemotherapeutic agents that
  inhibit or kill microbes
• Antibiotics antimicrobials that are produced
  naturally by other microorganisms

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Antimicrobials

• Antibacterial spectrum
• Broad spectrum affects a wide variety of Gram
  and Gram- bacteria
• Narrow spectrum affects limited variety of
  bacteria

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Antimicrobials

• Sterilizing Agents
• Kill everything
• Autoclave (heat and pressure), radiation
• Disinfectants
• Kill most things, too strong for living tissues
• Lysol, ammonia

• Antiseptics
• Prevent growth, can be used topically
• Alcohol, iodine, hydrogen peroxide
• Chemotherapeutics
• Kill microbes or prevent growth, can be ingested
• Penicillin, sulfa drugs

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Antimicrobials

• Bacteriocidal kills microorganisms
• Minimum Bacteriocidal Concentration (MBC)
• lowest concentration that kills 99.9 of
  organisms in standardized dilution

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Antimicrobials

• Bacteriostatic inhibits growth of the organism
• Minimum Inhibitory Concentration (MIC) lowest
  concentration that inhibits growth in
  standardized dilution (used to determine best
  antimicrobial dosage)

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Antibiotics

• Antibiotics can attack several parts of the cell
• Membrane (lipids)
• Proteins
• Cell wall
• DNA replication

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Antibiotics Sources

• Mold-type fungi
• Penicillin from Penicillium species
• Cephalasporin from Cephalosporium species
• Certain strains of Bacillus
• Bacitracin, Neosporin, Polysporin
• Many strains of Actinomycetes
• Streptomycin from Streptomycetes species
• Majority of antibiotics come from these organisms

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Antibiotics

• Antibiotic synergism
• Combination of two or more antibiotics which have
  enhanced activity when used together
• Helps cut down on resistance to specific drugs
• Antibiotic antagonism
• Activity of one antibiotic interferes with the
  activity of another antibiotic

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Antibiotics

• An organism can acquire resistance in several
  ways
• Transformation
• Conjugation
• Transduction

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Antibiotics

• Several ways to be resistant
• Pump it out (efflux pump)
• Degrade it with an enzyme
• Alter it with an enzyme

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Antibiotics

• Antimicrobial susceptibility testing (AST) is
  routine procedure in clinical laboratories
• Susceptibility to antimicrobials may change
• Microorganisms acquire drug resistance
• Potential pathogen isolated from a patient
• Determine susceptibility to antimicrobial drugs
• Determine treatment of patient

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Antibiotics

• AST methods
• Minimum inhibitory concentration (MIC)
• Uses serial dilutions
• Kirby-Bauer
• Uses agar plates, lawn cells, and antibiotic disks

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Antibiotics

• Minimum inhibitory concentration (MIC)
• Uses serial dilutions
• Determines minimum concentration that inhibits
  growth
• This is that point at which bacterial growth is
  bacteriostatic

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Antibiotics

• Kirby-Bauer
• Uses agar plates, lawn cells, and antibiotic disks

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Antimicrobials Purpose

• Use Kirby-Bauer method
• Compare sensitivity of Gram-negative and
  Gram-positive organisms to antibiotics
• Gram-negative organisms
• Escherichia coli and Pseudomonas aeruginosa
• Gram-positive organism
• Staphylococcus epidermidis

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Antimicrobials Purpose

• Determine antibacterial spectrum
• Broad-spectrum inhibits a wide variety of
  Gram-positive and Gram-negative bacteria
• Narrow-spectrum inhibits a limited variety of
  bacteria

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Antimicrobials Procedure

• Prepare Mueller Hinton agar plates
• 12 mL of melted top agar
• 0.2 mL of bacterial culture (lawn cells)
• Escherichia coli
• Pseudomonas aeruginosa
• Staphylococcus epidermidis
• Mix agar and culture
• Pour agar and culture on plate
• Quickly rotate plate to cover surface

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Antimicrobials Procedure

• Allow plates to cool and set
• Apply antimicrobial impregnated disks
• Flame sterilize forceps
• If necessary, clean stains off forceps first
• Refer to pattern in lab manual
• Incubate at 37?C for 24 hours

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Antimicrobials Procedure
GM 10
P 10
K 30
TMP 5
TE 30
SXT 25
PB 300
C 30
S 10
E 15
Cl 10
Figure 9.2. Antibiotic disk placement.
G 300
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Antimicrobials Procedure

• Next week
• Measure zones of inhibition
• Determine susceptibility (see Table 9.1)
• Complete antimicrobials worksheet

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Antimicrobials Procedure

• Figure 9.1. The size of a zone of inhibition
  around a high potency disk reflects the
  resistance or sensitivity of the bacterium.
  Bacteria may also exhibit an intermediate
  reaction.

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AntimicrobialsProcedure
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Next Week

• Assignments
• Pre-lab 10
• Environmental Isolate Report Due for extra credit
• Worksheet 9 (filled out during lab)
• Exercise 9
• Discuss Data and turn in Worksheet
• Exercise 10
• UV light
• Extra Credit Practical
• Finish any last minute work on Environmental
  Isolate