AST - DITCH PLATE METHOD
AIM :
To determine the sensitivity profile of different bacterial isolates to the given insoluble antibiotic using the ditch plate method.
THEORETICAL BACKGROUND :
In addition to the discovery of penicillin, Alexander Fleming made two significant contributions to the field of antibiotic testing in the 1920's. In 1924, he introduced the use of the ditch plate technique for evaluating the antimicrobial qualities of a test solution. In 1929, this technique was modified by Reddish, who cut wells into the agar and filled the wells with the test solution. In 1929, Fleming introduced a broth dilution method that could be considered a definite forerunner to contemporary MIC methodology.
Using the first agar diffusion technique, the "ditch plate", Fleming excavated a strip of agar in the shape of a ditch from a Petri dish and replaced it with medium containing the extract of potential antimicrobial activity (for Fleming, the mold extract of penicillin). At right angles, he streakad various strains across the ditch. Fleming did not observe the "radial zones" of inhibition that we recognize today. Instead, he saw lanes of inhibition, and concluded that the lane in the streak of microorganisms that was furthest away from the antimicrobial-laden ditch represented activity against that particular strain. Fleming was more interested in isolating "B. infuenzae" than inhibiting the other susceptible microorganisms.
The ditch plate technique is used to determine the antimicrobial potential of a new drug i.e. it is a part of the secondary screening to determine its antimicrobial potential and is specifically used for insoluble drugs such as sulfonamides.
The original antibacterial sulphonamides (sometimes simply called sulpha drugs) are systemic antimicrobial agents that contains the sulphonamide group.
Some representative sulfonamides include:
- sulfacetamide,
- sulfadimethoxime,
- sulfadoxine,
- Sulfamethoxazole,
- sulfasalazine,
- sultiame
- sumatriptan.
Sulfonamides target a bacterial metabolic pathway as competitive inhibitors of the enzyme dihydropteroate synthetase (DHPS). DHPS activity is vital in the synthesis of folate and folate is required for cells to make nucleic acids such as DNA or RNA. So, if DNA molecules cannot be built, the cell cannot divide and the effect is bacteriostatic rather than bactericidal. Sulfa drugs are used to treat some types of bacterial pneumonia, urinary tract infections, shigellosis and Nocardia infections.
The medium used for ditch plate technique is Mueller and Hinton agar for the following reasons -
1 . it is simple and transparent,
2. lacks heat labile material,
3. the starch in the medium acts as a protective colloid,
4. lacks thymine / thymidine that are antagonistic to sulphonamide and trimethoprim
5. contains casamino acids which makes the media enriched - allowing growth of even gonococci and meningococci.
A homogenous preparation of the drug in molten nutrient agar at 40°C is poured into a ditch cut in a MH agar plate. The test organisms are then streaked across the ditch. Inhibition / growth of the test organisms is noted - on the ditch containing the antibiotic, the agar immediately adjacent to the ditch and farther from the ditch.
Requirements:
1. St. MH agar plate x 1,
2. St. MH agar butt (5 ml) x 1,
3. Powdered insoluble antibiotic (100 mg Septran - a sulfa drug),
4. Saline suspension of the Test organism (e.g. E. coli, S. typhi, S. aureus, C. diphtheriae),
5. Scalpel
6. Aicohol
Procedure:
1. Cut a ditch 10 mm wide in a sterile MH agar plate usinga sterile (alcohol flamed and cooled) scalpel.
2. Add the antibiotic powder to the 5 ml molten MH agar butt at 40°C. Mix well and pour the mixture into the ditch until the surface of the molten agar is flush, i.e. at the same level, with the surface of solid agars on either sides.
3. Allow the antibiotic containing agar in the ditch to solidify and set.
4. Streak the test organisms perpendicular to the ditch.
5. Incubate the plates in inverted position at 37°C for 24 hours. Observe for inhibition of growth on ditch as well as on the agar surface adjacent to the ditch.
