Micro Biology Lab Packet

profilead5221
Week4Day12DisinfectantsAntibioticSusceptibility2-6-18.ppt
Home>Biology homework help>Micro Biology Lab Packet

Disinfectants & Antiseptics
Antibacterial Susceptibility Test

  • Ex 2-14 Disinfectants & Antiseptics
  • Ex 7-3 Antimicrobial Susceptibility Test

Chemical Agents - General Principles

  • Sterilization

– killing or removal of all life forms

  • Disinfection/Antisepsis – removal of pathogens; reduction in the number of organisms, so they pose no danger of disease
  • Disinfectant – chemical agent used on an inanimate object

  • Antiseptic – chemical agent used externally or topically on living tissue

*

Effect of Chemical Agents on Bacteria

There are many different methods to assess effects of chemical agents on bacteria

  • Laboratory methods – Assess the properties of agents under laboratory conditions
  • E.g., methods to evaluate antibacterial potency
  • E.g., methods to evaluate –cidal or –static activity
  • In-use methods - Assess the effectiveness of agents under conditions similar to actual use
  • E.g., methods to evaluate performance for particular uses or in real-life situations
  • E.g., methods to compare performance of 2 agents

Chemical Agents – Tube Dilution Procedure

  • Determining the antibacterial potency of an agent against Staphylococcus aureus (S. aureus)

or Escherichia coli (E. coli)

  • Agents: Isopropanol, H2O2, Bleach, or Lysol
    at various concentrations
  • Inoculated with S. aureus or E. coli by swab technique with fresh broth culture

  • Determine the lowest concentration of that agent that inhibits growth of the test organism

1.

4.

2.

3.

5.

6.

8.

7.

1 min.

10 min.

dry for 10 min.

Effect of Chemical Agents

So which procedure is a laboratory method?

Beads procedure

Which procedure is an “in-use” method?

Fingertip antisepsis procedure

Chemical Agents

  • Active ingredient should be noted on the container
  • Class = the general group to which the active ingredient belongs:

Product Class

  • Isopropanol Alcohols
  • Hydrogen Peroxide Oxidizer
  • (H2O2)
  • Bleach Oxidizer (Sodium Hypochlorite)
  • Lysol Quaternary Ammonium

Compounds

Effectiveness
(Lowest Concentration of Reagent Needed)

Organism Bleach H2O2 Isopropanol Lysol
S. aureus
E. coli

Antibacterial Susceptibility

Ex 7-3 Antimicrobial Susceptibility Test

Why Study Antibacterial Susceptibility Testing?

  • Clinical labs routinely perform “culture & sensitivity” testing
  • Ideally, effective antibiotic treatment is directed against the specific pathogen infecting the patient
  • Some strains of a species have acquired resistance to antibacterial agents

*

Antibacterial Agents

  • Spectrum of activity
  • Broad spectrum – Active against a broad array of bacteria, including both Gram(+) & Gram(-) organisms
  • Narrow spectrum – Active against a limited range of organisms

The Sites of Activity in a Bacterial Cell for Various Antibiotics

Chloramphenicol inhibit protein synthesis

Ciprofloxacin: Iinhibit DNA gyrase

Trimethoprim: inhibits bacterial DNA synthesis

Penicillin: inhibit the formation of

peptidoglycan cross-links in the bacterial

cell wall

Kirby-Bauer method for determining antibacterial susceptibility

  • Disc-diffusion method

  • Antibiotic discs
  • Contain defined amount of a specific antimicrobial agent
  • Agent diffuses out of disc radially, into the medium
  • Forms a concentration gradient in the medium

  • Zone of inhibition
  • Zone size determined by concentration of the agent that inhibits bacterial growth

Assessing Antibacterial Activity

How to get from a zone of inhibition to effectiveness (or lack of effectiveness)?

  • Measure zone of inhibition
  • Bigger zone is better
  • Zone size alone doesn’t determine susceptibility or resistance

  • Must consider
  • Factors associated with the test method
  • Concentration of drug in the body after a usual dose

*

Kirby-Bauer method for determining antibacterial susceptibility

  • (Mueller-Hinton Agar used)
  • non-selective, non-differential medium (almost all organisms plated will grow)
  • has a few properties that make it excellent for antibiotic use
  • contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics.
  • it is a loose agar, which allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a truer zone of inhibition.

Interpretive Criteria

Interpretive criteria convert zone size (mm) to 1 of the following categories:

  • S = susceptible; pathogen with that zone size will likely be killed/inhibited by the concentration of drug in the bloodstream provided by the usual dose of that agent
  • This agent should provide effective therapy
  • R = resistant; pathogen with that zone size will NOT likely be killed/inhibited …
  • Select a different agent for therapy
  • I = intermediate; provides a buffer zone to prevent small differences in the test from having a major impact on interpretation
  • Select a different agent for therapy

Antibacterial Susceptibility – Day 2

  • Measure diameter of zone of inhibition with ruler (in mm)
  • Record data for all 4 agents
    & 2 different organisms (Worksheet 7-3 pg. 661)

  • Use interpretive criteria to convert zone size (mm) to a category (page 271):
  • S = susceptible
  • I = intermediate
  • R = resistant

Zone diameter (mm)

Interpretive Criteria

  • Convert zone size (mm) to S, I, or R
Agent Zone size (mm)
Susceptible Intermediate Resistant
Chloramphenicol - C30 Enterobacteriaceae & staphylococcus >18 13 - 17 <12
Ciprofloxacin - CIP Enterobacteriaceae & staphylococcus >21 16 - 20 <15
Trimethoprim – TMP5 Enterobacteriaceae & staphylococcus >16 11 - 15 <10
Penicillin – P10 Staphylococcus >29 <28

Expectations for Today: Work as table-teams

2 Mueller-Hinton Agar plates & 1 pkg swabs per table

Inoculate each plate with 1 organism:

Swab in 3 directions for confluent growth

Staphylococcus aureus Escherichia coli

Apply 4 different discs to each plate

Chloramphenicol - C30, Ciprofloxacin – CIP, Trimethoprim – TMP5, Penicillin – P10

Use flamed forceps; tap disc gently for good contact with agar

Incubate in the inverted position at 37°C

Disc will not fall off, even upside down in the plate

We will observe/interpret results next lab

C30

CIP

TMP5

P10

Disinfectants & Antiseptics
Antibacterial Susceptibility Test
Day 2: Interpretation of Results

Ex 2-14 Disinfectants & Antiseptics

Ex 7-3 Antimicrobial Susceptibility Test

Expectations for Today

  • Read & interpret antibacterial susceptibility test
  • Measure zone size (mm)
  • Use interpretive criteria to convert zone size to S,I,R

Summary of Class Data

Agent S. aureus E. coli
Chloramphenicol - C30 Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 =
Ciprofloxacin - CIP Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 =
Trimethoprim – TMP5 Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 =
Penicillin – P10 Table 1 = Table 2 = Table 3 = Table 4 = Table 1 = Table 2 = Table 3 = Table 4 =