Microbiology Lab

1
Microbiology Lab

• Nov 13-14

2
Overview

• Assignments
• Work Sheet 8 (finish in lab today)
• Pre-lab for 9
• Hamburger Report
• 1st submission
• 2nd submission
• Rubric
• Exercise 9 Antimicrobials
• Complete Work on Environmental Isolate

3
Bacterial Transformation-Review

• SD5 (wild-type) vs. SD6 (mutant)
• SD5 has the ability to synthesize tryptophan SD6
  does not
• Tryptophan is an amino acid use to make proteins
• If an organism cannot synthesize an amino acid it
  must obtain it from its environment

4
Bacterial Transformation-Review

• Transformation is the process of taking up DNA
  from the environment and incorporating it into
  the genome
• Last week we gave SD6 (mutant) cells SD5
  (wild-type) DNA
• Ultimately, the SD6 cells we gave the DNA to
  gained the ability to synthesize tryptophan
• SD6 cell (the lawn cells) that did not receive
  DNA still cannot synthesize tryptophan

5
Bacterial Transformation-Procedure

• Examine plates for growth
• Where do you expect to see growth?
• Count all colonies and fill out worksheet on p.
  8-3

6
Transformation Plates
SD6 mutant lawn
Transformation Plate
Control Plate
7
Exercise 9 Antimicrobials
8
Antimicrobials

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

9
Antimicrobials

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

10
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

11
Antimicrobials

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

12
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)

13
History of Antibiotic Development

• Prior to the ability to harvest antibiotics from
  microorganisms diseases were treated in a variety
  of manners that were often harmful to the host as
  well
• Often dyes or metals (such as silver) were used
  to treat or prevent surface microbial infections
• Systemic bacterial infections however were often
  not able to be treated with these techniques

14
Antibiotics

• True antibiotics are substances produced by one
  organism (generally a bacteria or fungus) that
  inhibits or kills another
• Most antibiotics are produced by three genera of
  organisms
• the Streptomyces (bacteria)
• Over 500 compounds produced by this genera are
  known to have antibiotic properties
• It is suspected there are more
• the Actinomycetes (fungi)
• the Bacillus (bacteria)

15
Antibiotics-Function

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

16
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

17
Antibiotic Resistance

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

18
Antibiotics

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

19
Antibiotic Resistance

• The main reason for this phenomena is the
  inappropriate use of antibiotics
• World-wide, 20 of medical cases warrant
  antibiotic treatment however, 80 of cases are
  prescribed some type of antibiotic
• Additionally, half of the time the prescription
  is wrong in either recommended dose or length of
  treatment

Brock Biology of Microorganisms 11th ed.
20
Antibiotic Resistance

• This figure shows some common antibiotics and the
  percent of microorganism strains that are
  resistant to them

21
Antibiotics ResistanceCurrent Issues

• Methicillin resistant Staphylococcus aureus
  (MRSA)
• The only antibiotic left to treat some of these
  strains is vancomycin
• Vancomycin resistant Entercoccus faecium (VRE)
• This is a problem because these organisms can
  often reside near each other and S. aureus can
  gain resistance or at least reduced
  susceptibility to vancomycin

22
Antibiotics-The Future

• New drugs
• Developing novel drugs is often a difficult task
  however computer models has made progress in this
  field
• These programs allow researchers to design unique
  chemicals structures that will interact with
  microbial function without harming the host

23
Antibiotics

• In order to determine whether an antibiotic will
  work on a particular organism it has to be tested
• Antimicrobial susceptibility testing (AST) is
  routine procedure in clinical laboratories
• Susceptibility to antimicrobials may change
• Microorganisms acquire drug resistance
• In order to use this in a clinical setting the
  potential pathogen is isolated from a patient
• Determine susceptibility of that organism to
  antimicrobial drugs
• Determine treatment of patient

24
Antibiotics

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

25
Antibiotics

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

26
Antibiotics

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

27
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

28
Antimicrobials Purpose

• Determine antibacterial spectrum of the various
  drugs we are working with
• Broad-spectrum inhibits a wide variety of
  Gram-positive and Gram-negative bacteria
• Narrow-spectrum inhibits a limited variety of
  bacteria
• Additionally, look for examples of synergism and
  antagonism
• Which drugs have been combined?
• When combined do any of the drugs work more
  effectively? Less effectively?
• Keep this in mind for next week while you are
  reading your results and filling out your
  worksheet

29
Antimicrobials-Procedure

• Allergies
• If anyone has allergies to any antibiotics please
  wear gloves while you are working with them

30
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

31
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

32
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
33
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.

34
AntimicrobialsProcedure
35
Next Week

• Antimicrobials Worksheet
• Measure zones of inhibition
• Determine susceptibility (see Table 9.1)
• Complete antimicrobials worksheet
• Ex. 10-Effects of Ultraviolet Light