Streaking is a technique used to isolate a pure strain from a single

species of microorganism, often bacteria. Samples can then be taken from the
resulting colonies and a microbiological culture can be grown on a new plate so that
the organism can be identified, studied, or tested.

Objective: To obtain isolated microbial colonies from an inoculum by creating areas of increasing
dilution on an agar petriplate.
Principle: The resulting diminution of the population size ensures that, following inoculation,
individual cells will be sufficiently far apart on the surface of the agar medium to effect a separation of
the different species present. Although many type of procedures are performed, the four ways or
quadrant streak is mostly done.

Procedure in 4- quadrant streaking:

1. Loosen the cap of the bottle containing the inoculum.

2. Hold an inoculation loop in your right hand.
3. Flame the loop and allow it to cool.
4. Lift the test tube containing the inoculum with your left hand.
5. Remove the cap/ cotton wool plug of the test tube with the little finger of your right hand.
6. Flame the neck of the test tube.
7. Insert the loop into the culture broth and withdraw. At all times hold the loop as still as possible.
8. Flame the neck of the test tube again.
9. Replace the cap/ cotton wool plug of the test tube using the little finger of your right hand. Place the test tube in a
rack. For a liquid culture, dip the loop into the broth, or for solid media, lightly touch a colony with the loop.
10. Partially lift the lid of the Petri dish containing the solid medium.
11. Place a loopful of the culture on the agar surface on the area 1. Flame the loop and cool it for 5 seconds by touching an
unused part of the agar surface close to the periphery of the plate, and then drag it rapidly several times across the surface of
area1.
12. Remove the loop and close the Petri dish.
13. Reflame and cool the loop, and turn the petri dish 90C then touch the loop to a corner of the culture in area1 and drag
it several times across the agar in area 2, hitting the original streak a few times. The loop should never enter area 1 again.
14. Remove the loop and close the Petri dish.
15. Reflame and cool the loop and again turn the dish 90C anticlockwise. streak area 3 in the same manner as area 2,
hitting last area several times.
16. Remove the loop and close the Petri dish.
17. Flame the loop, again turn the dish 90C and then drag the culture from a corner of a area3 across area 4, contacting
area 3 several times and drag out the culture as illustrated. Using a wider streak. Do not let the loop touch any of the previously
streaked areas. The flaming of the loop at the points indicated is to effect the dilution of the culture so that fewer organisms are
streaked in each area, resulting in the final desired separation.
18. Remove the loop and close the Petri dish.
19. Tape the plate closed and incubates the plate in an inverted position in an incubator for 24-48 hours.
20. Flame the loop before putting it aside.
 Single-tube-agar test
(Slant)
Simmons Citrate Agar is used for the differentiation of microorganisms on the
basis of citrate utilization
Procedure in preparing:

1. Dissolve above salts in deionized water.

2. Adjust pH to 6.9.
3. Add agar and Bromothymol blue.
4. Gently heat, with mixing, to boiling until agar is dissolved.
5. The medium may be used either as slopes in test tubes or as a plate medium in petri dishes. In both cases the
surface of the medium is lightly inoculated by streaking and, where slopes are used, the butt of medium is inoculated by
stabbing.
6. For tubes, dispense 4.0 to 5.0 ml into 16-mm tubes.
7. Autoclave at 121 degree C under 15 psi pressure for 15 minutes.
8. Cool in slanted position (long slant, shallow butt).
9. Tubes should be stored in a refrigerator to ensure a shelf life of 6 to 8 weeks.
10. The uninoculated medium will be a deep forest green due to the pH of the sample and the bromothymol blue.

(Butt Slant )
Procedure for Triple Sugar Iron Agar (TSI) Test
1. With a sterilized straight inoculation needle touch the top of a well-isolated colony
2. Inoculate TSI Agar by first stabbing through the center of the medium to the bottom of the tube and then streaking on the
surface of the agar slant.
3. Leave the cap on loosely and incubate the tube at 35C in ambient air for 18 to 24 hours.

( eto yung straight di ko alam tawag )

SIM (Sulfide Indole Motility) a combination differential medium that tests three different parameters, which
are represented by the three letters in the name: Sulfur Reduction. Indole Production. Motility.

Procedure:

1. Consult current editions of appropriate references for the recommended

procedure for sample preparation, inoculation, testing, and interpretation.
2. Lightly inoculate SIM Medium from a pure, 18-24 hour culture of the test
isolate. Using an inoculating needle, stab down the center of the medium to
within the bottom of the tube.
3. Incubate tube in ambient air with loosened cap at 33-37C for 18-24 hours.
4. Examine tube for H2S production and motility (see Interpretation).
5. To detect indole production, add 3-4 drops of Kovacs' Reagent (REF R21227)
or Ehrlichs Reagent (REF R21213) and observe medium for a red color
development.