Food microbiology

EXPERIMENT 3

Determination Of Antibiotic Sensitivity

  1. DISC DIFFUSION METHOD One of the most frequently used techniques of determining the sensitivity of bacteria to antibiotics is the disc-diffusion technique. In this method a disc impregnated with antibiotic is placed on an agar plate seeded with bacteria. The antibiotic will diffuse into the agar and inhibit sensitive bacteria which show a zone of inhibition of growth on the agar. Either single discs are used or, as here, a multodisc containing 8 different antibiotics can be used. Determine the antibiotic sensitivity of the E. coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive)cultures by the following method:
  2. Spread 0.1 ml of each culture onto the surface of nutrient-agar (NA) plates and allow to dry.
  3. Using forceps (sterilized either by flaming and allow to cool). Place one of the Mastring discs on the plate.

Press gently onto the surface of the agar making sure that the whole of the disc is in contact with the agar.

Resterilize your forceps.

  1. Incubate plates at 37° (inverted). (Plates will be incubated for 24h then refrigerated). Do not forget to record what type of multodisc was used e.g. antibiotics and concentrations etc.
  2. Next class -examine plates for an area of inhibition around the antibiotic discs. The diameter of the zone of inhibition is related to the sensitivity to the antibiotic.

 

 

 

  1. MINIMUM INHIBITORY CONCENTRATION

 

The susceptibility of an organism to an antibiotic can be defined by two parameters: the concentration required to inhibit growth (minimum inhibitory concentration MIC) and the concentration required for killing action (minimum bactericidal concentration, MBC). These values are usually measured by inoculating a series of doubling dilutions of concentration of antibiotic and examining for growth and viability, after incubation. A simpler method for determining MIC using a strip impregnated with a concentration gradient of antibiotic, the E-test has recently been introduced

Microtitre plate assay for Minimum Inhibitory and Minimum Bactericidal Concentrations (MIC and MBC)

  1. You are provide with sterile single and double strength iso-sensitest broth and a 2 mg ml-1 solution of the test antibiotic.
  2. Add 100 μl double strength Iso-sensitestbroth to the first well in each row in a microtitre plate.
  3. Add 100 μl single strength broth to the remaining wells.
  4. Add 100 μl of 2mg ml-1 test compound to the first well and mix (= 1mg ml-1). Remove 100 μl and add to the next well and mix. (=0.5 mg ml-1)
  5. Removed 100 μl from this well with a fresh pipette tip and add to the next well and mix. Repeat dilution and mixing to give dilution series of 1, 0.5, 0.25, 0.125, 0.0625, 0.3125, 0.016125, 0.008, 0.004, 0.002, 0.001 mg ml-1.
  6. Discard 100 μl from the last row.
  7. Inoculate each well with 10 μl of bacterial culture adjusted to 107 cells ml-1, one organism per row. Each well therefore receives 105 cells.
  8. Incubated the plates at 30 or 37°C (as appropriate for the test organisms) for 24 h.
  9. Record the wells which are turbid (= growth). The last well showing no growth is taken as the MIC.
  10. Take a loopful was from each well showing no growth, spot onto nutrient agar and incubate at 30 or 37°C overnight. The lowest concentration showing no growth is taken as the MBC.
  11. Examine the prepared plated with the E-strips and E. coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive).

Interpret the results as described by the demonstrator.

STUDY QUESTIONS

  1. What antibiotics were the cultures sensitive to?
  2. What were the MIC’s
  3. Was there a difference between the two cultures? If yes, why?

 

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