Mic 224 Lab 6

Streak Plate Technique and Antibiotic Sensitivity

Streak Plate

• Pure culture technique –allows you to isolate colonies from a mixed culture

• Technique (pg 52 of lab manual)

Streak Plate Results

Isolated colonies

Antibiotic Sensitivity• Most microbiologists distinguish two groups of antimicrobial agents

used in the treatment of infectious disease:– antibiotics, which are natural substances produced by certain groups

of microorganisms, and – chemotherapeutic agents, which are chemically synthesized.– A hybrid substance is a semisynthetic antibiotic, wherein a molecular

version produced by the microbe is subsequently modified by the chemist to achieve desired properties.

• The modern era of antimicrobial chemotherapy began in 1929, with Fleming's discovery of the powerful bactericidal substance, penicillin, and Domagk's discovery in 1935 of synthetic chemicals (sulfonamides)

• Natural antibiotics produced: penicillin (fungus), streptomycin (bacteria) that a, and bacitracin (bacteria)

• Artificially produced: sulfonamides

Antibiotic Sensitivity

• Most of the natural antibiotics that are being used in agriculture and medicine are produced by three unrelated groups of microbes, including eucaryotic molds and two types of spore-forming bacteria. – Penicillium and Cephalosporium molds produce beta-lactam

antibiotics such as penicillin and cephalosporin and their relatives.

– 2. Actinomycetes, mainly Streptomyces species, produce tetracyclines, aminoglycosides (streptomycin and its relatives), macrolides (erythromycin and its relatives), chloramphenicol, ivermectin, rifamycins, and most other clinically-useful antibiotics that are not beta-lactams.

– 3. Bacillus species, such as B. polymyxa and B. subtilis, produce polypeptide antibiotics (e.g. polymyxin and bacitracin)

• Artificially produced antibiotics: sulfonamides

Antibiotic Sensitivity

• Antimicrobial drugs are classified as to their effect on an organism. – Broad spectrum antibiotics are effective

against a wide range of bacteria including both Gram positives and Gram negatives

– Narrow spectrum: kill only a few species of bacteria or either Gram positives or Gram negatives but not both

Antibiotic Sensitivity

• Antibiotics can be bacteriostatic (bacteria stopped from multiplying) or bactericidal (bacteria killed).

• To perform either of these functions, antibiotics must be brought into contact with the bacteria.

• It is believed that antibiotics interfere with the surface of bacteria cells, causing a change in their ability to reproduce.

• Testing the action of an antibiotic in the laboratory shows how much exposure to the drug is necessary to halt reproduction or to kill the bacteria.

• Although a large amount of an antibiotic taken at one time might kill the bacteria, it could also cause the patient to die

Methods of Testing Antibiotic Sensitivity: Tube Dilution

• Tube Dilution: a series of tubes are filled with a given amount of growth medium inoculated with the bacteria being tested

• An antibiotic is placed in the first tube at a high concentration and then and then halved and placed into the next tube. This continues until the last tube contains a very low concentration of the antibiotic

• The first tube that exhibits no turbidity (no growth) in the series is the minimum inhibitory concentration (MIC) necessary to kill the pathogen

Methods of Antibiotic Sensitivity Testing: Kirby Bauer

• The Kirby-Bauer test, known as the disk-diffusion method, is the most widely used antibiotic susceptibility test in determining what treatment of antibiotics should be used when treating an infection.

• For this test, a culture medium, specifically the Mueller-Hinton agar, is uniformly and aseptically inoculated with the test organism and then filter paper discs, which are impregnated with a specific concentration of a particular antibiotic, is placed on the medium. 

• The organism will grow on the agar plate while the antibiotic “works” to inhibit the growth.  If the organism is susceptible to a specific antibiotic, there will be no growth around the disc containing the antibiotic.  Thus, a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism.

• The measurement is compared to the criteria set by the National Committee for Clinical Laboratory Studies (NCCLS).  Based on the criteria, the organism can be classified as being Resistant (R), Intermediate (I) or Susceptible (S)

Kirby Bauer Procedure• Using a asceptic technique, place a sterile swab into the broth

culture of a specific organism and then gently remove excess liquid by gently pressing the swab against the inside of the tube.

• Using the swab, streak the Mueller-Hinton agar plate.  To obtain uniform growth, streak the plate with the swab in one direction and then rotate the plate 90° and streak the plate again in that direction.  Repeat the rotation 3 times.  Allow the plate to dry for approximately 5 minutes.

• Antibiotic discs can be dispensed onto the agar using an Antibiotic Disc Dispenser. 

• Using flame-sterilized forceps, gently press each disc to the agar to ensure that the disc is attached to the agar. 

• Plates should be incubated overnight at an incubation temperature of 37°.

Kirby Bauer Results

• After the plates have been incubated, there should be a noticeable “clearing” zone around each of the antibiotic discs.  The diameter of each zone should be measured and recorded in millimeters (mm).

• Each measurement can be compared to a zone-size interpretive chart.  Using the chart, the organism can be characterized as being resistant, intermediate or susceptible to the specific antibiotic.  Intermediate susceptibility means that some inhibition from the antibiotic occurred but not sufficiently enough to inhibit the growth of the organism in the body.

Kirby Bauer Results

• See Zone of Inhibition Evaluation Tableon pg 50.