Basic Microbiological Techniques Used in Antibacterial Drug Discovery

Mary Motyl1, Karen Dorso1, John Barrett1, Robert Giacobbe1

1 Merck and Co., Inc., Rahway, New Jersey
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13A.3
DOI:  10.1002/0471141755.ph13a03s31
Online Posting Date:  January, 2006
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Abstract

In vitro assessment of activity is crucial to the development of antibacterial agents. Several basic clinical microbiology procedures for this evaluation are provided in this unit, including determination of the minimum inhibitor concentration (MIC), kill curves, and synergy testing. These techniques yield information on in vitro efficacy, cidal‐versus‐static activity, and the potential for synergy with other agents.

Keywords: MIC; kill curves; post‐antibiotic effect; synergy

     
 
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Table of Contents

  • Basic Protocol 1: Quadrant Plating on Agar for Isolating and/or Counting Bacteria
  • Alternate Protocol 1: Limited Dilution Spin Plating
  • Support Protocol 1: Preparation of Agar Isolation/Assay Plates Containing Medium
  • Support Protocol 2: Long‐Term Storage of Microorganisms Using Microbank Beads
  • Basic Protocol 2: Pour Plating for Isolating and/or Counting Bacterial Colonies
  • Basic Protocol 3: Determination of MIC and MBC by Microdilution Testing
  • Alternate Protocol 2: Determination of MIC50s, MIC90s of a Panel of Microorganisms
  • Basic Protocol 4: Time‐Kill Assays (Kill Curves) and Determination of Post‐Antibiotic Effects (PAE)
  • Basic Protocol 5: Checkerboard Microdilution Testing for Antimicrobial Synergy/Antagonism
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quadrant Plating on Agar for Isolating and/or Counting Bacteria

  Materials
  • Petri plates containing agar (see protocol 3)
  • Liquid bacterial culture containing a known organism, e.g., Staphylococcus aureus
  • Sterilizable, reusable bacterial loops or single‐use disposable inoculating loops
  • Bunsen burner or sterilizer
  • Incubator
NOTE: Generally, non‐fastidious test organisms are viable when maintained on agar slants for up to 1 month. The test organisms should be maintained as frozen stocks to maintain viability for longer periods (see protocol 4).

Alternate Protocol 1: Limited Dilution Spin Plating

  • Nutrient broth or non‐growth supporting diluents (e.g., 0.85% saline or water), sterile
  • 14‐ml capped tubes, sterile
  • Pipet, sterile
  • Sterile glass beads (4‐mm diameter; Fisher Scientific) or spreading rod and spin plater

Support Protocol 1: Preparation of Agar Isolation/Assay Plates Containing Medium

  Materials
  • Appropriate nutrient medium to support bacterial growth (see Tables 13.3.1 and 13.3.2)
  • Bacto agar (BD Biosciences)
  • Stir plate and magnetic bar
  • 55°C water bath
  • Petri plates (pre‐sterilized disposable or reusable, autoclavable glass)

Support Protocol 2: Long‐Term Storage of Microorganisms Using Microbank Beads

  Materials
  • Isolate streaked on agar plates (see protocol 1)
  • Microbank beads (Pro‐Lab Diagnostics) in cryo‐preservative fluid and stored at room temperature or 4°C
  • Medium for fastidious organisms (see recipe), optional
  • 2× skim milk medium (Remel), optional
  • Sterile swabs
  • Sterile pipets
  • Sterile loop or forceps
  • 2‐ml cryovials (Nalgene)
  • 35°C, 5% CO 2 humidified incubator

Basic Protocol 2: Pour Plating for Isolating and/or Counting Bacterial Colonies

  Materials
  • Nutrient medium components to support bacterial growth, e.g., Cation‐adjusted Mueller Hinton I broth (CAMHB; BBL or Teknova)
  • Agar
  • Microorganism, e.g., Staphylococcus aureus
  • Stir plate with magnetic stir bar
  • 25 × 150–mm borosiliate glass tubes and caps (Fisher), autoclaved
  • 55°C water bath or heating block
  • Variable speed vortexer
  • 100‐mm disposable petri plates

Basic Protocol 3: Determination of MIC and MBC by Microdilution Testing

  Materials
  • Microbank bead stock of microorganism(s), e.g., Staphylococcus aureus (see protocol 4)
  • Trypticase soy agar (TSA) II + 5% sheep blood agar plates (BBL)
  • Appropriate quality control (QC) strains for this assay, i.e., same genus and species or a pair of organisms, one of which is susceptible and the other of which is resistant to the class of compound being tested
  • Cation‐adjusted Mueller Hinton II broth (CAMHB; supplemented with DMSO, if required) (BBL or Teknova)
  • Antibiotic drug stock appropriate for assay (see recipe)
  • 0.5 McFarland barium sulfate standard (see recipe)
  • Sterile 5 N saline (BBL)
  • Incubator, set at 35° ± 2°C with 85% relative humidity
  • 96‐well microtiter plates (Thermo Labsystems)
  • Multichannel pipet (Matrix Technologies)
  • Perkin Elmer CETUS Pro/Pette diluter
  • Sterile inoculating loops (10‐µl; Remel)
  • Turbidity meter (550 nm; Dade Behring)
NOTE: Quality control (QC) is performed each time a new chemical entity is tested using Clinical Laboratory and Standards Institute (CLSI, previously known as the NCCLS) recommended ATCC strains and approved drugs. A list of quality control organisms can be found in the following current CLSI guidelines (see ).NOTE: Established antibiotics used as controls are usually chemically stable; stock solutions can be stored frozen up to 1 year at −80°C. Because of possible unknown solubility or stability issues, most experimental compounds are usually prepared the day of the assay and used immediately.

Alternate Protocol 2: Determination of MIC50s, MIC90s of a Panel of Microorganisms

  Materials
  • Microorganism, e.g., Staphylococcus aureus, maintained on Microbank beads at −80°C
  • Trypticase soy agar (TSA) II + 5% sheep blood agar plates (BBL)
  • 10 mg/ml test compound stock solution (see recipe)
  • 0.9% saline
  • Incubator, 35° ± 2°C with orbital shaker (50 rpm)
  • Sterile inoculating loops (10‐µl; Remel)
  • Sterile, disposable 25 × 150–mm glass culture tubes with caps (Fisher Scientific)
  • Sterile Pyrex glass beads (∼4‐mm diameter) or spreading rod and turntable
  • 2.0‐ml cryovials
  • Additional reagents and equipment for MIC determination (see protocol 6)
NOTE: It is important to select a medium that is optimum for the growth of the organism being tested (see protocol 3).NOTE: When doing kill‐curve studies, the addition of blood or blood components to media may affect the activity of antimicrobials that are highly bound to plasma proteins.

Basic Protocol 4: Time‐Kill Assays (Kill Curves) and Determination of Post‐Antibiotic Effects (PAE)

  Materials
  • Antimicrobial compound(s)
  • Medium to support organism growth (see protocol 3), e.g., cation‐adjusted Mueller Hinton II broth (for bacteria), with or without DMSO
  • Microbank bead stock of microorganism to be studied ( protocol 4), –80°C
  • Trypticase soy agar (TSA) II + 5% sheep blood agar plates (BBL) or other media
  • Sterile 0.85% saline
  • 0.5 McFarland barium sulfate standard (see recipe)
  • 35°C incubator
  • Sterile inoculating loops
  • 96‐well microtiter plates
  • Multichannel pipet (Matrix Technologies)
  • WellPro Pro/Pette diluter
  • Turbidity meter (550 nm; Dade Behring)
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Figures

Videos

Literature Cited

Key References
   Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. M100‐S15. CLSI 2005.
  Provides up‐to‐date information for drug selection, interpretation, and quality control.
  Lorian, V. ed. Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th ed. pp. 330‐338. Williams & Wilkins, Baltimore, Maryland.
  Provides detailed in vitro and in vivo methods for analyzing antimicrobial compounds.
   National Committee for Clinical Laboratory Standards, 2000. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard 5th ed. M7‐A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  Describes performance, applications, and limitations of antimicrobial susceptibility testing techniques, and includes a series of procedures to standardize the way the tests are done.
   National Committee for Clinical Laboratory Standards. 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline M26‐A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  Detailed procedures for kill‐curves and MBCs.
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