In microbiology, 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.The Aim of this method is to obtain the discrete, well-developed colonies of the microorganism that are pure, i.e. the growth derived from the single bacteria cell/spore.
The modern streak plate method has progressed from the efforts of Robert Koch and other microbiologists to obtain microbiological cultures of bacteria in order to study them. The dilution or isolation by streaking method was first developed by Loeffler and Gaffky in Koch’s laboratory, which involves the dilution of bacteria by systematically streaking them over the exterior of the agar in a Petri dish to obtain isolated colonies which will then grow into quantity of cells, or isolated colonies. If the agar surface grows microorganisms which are all genetically same, the culture is then considered as a microbiological culture.
Streak Plate Method is done by diluting a comparatively large concentration of bacteria to a smaller concentration. The decrease of bacteria should show that colonies are sufficiently spread apart to affect the separation of the different types of microbes. Streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. Aseptic techniques are used to maintain microbiological cultures and to prevent contamination of the growth medium. There are many different types of methods used to streak a plate. Picking a technique is a matter of individual preference and can also depend on how large the number of microbes the sample contains.
The three-phase streaking pattern, known as the T-Streak, is recommended for beginners. The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. The inoculation loop is first sterilized by passing it through a flame. When the loop is cool, it is dipped into an inoculum such as a broth or patient specimen containing many species of bacteria. The inoculation loop is then dragged across the surface of the agar back and forth in a zigzag motion until approximately 30% of the plate has been covered. The loop then is re-sterilized and the plate is turned 90 degrees. Starting in the previously streaked section, the loop is dragged through it two to three times continuing the zigzag pattern. The procedure is then repeated once more being cautious to not touch the previously streaked sectors. Each time the loop gathers fewer and fewer bacteria until it gathers just single bacterial cells that can grow into a colony. The plate should show the heaviest growth in the first section. The second section will have less growth and a few isolated colonies, while the final section will have the least amount of growth and many isolated colonies.
Reagents And Material Required
- 24 hours old nutrient broth culture of two or more bacteria (Mixed Culture) or Sample/Specimen.
- Nutrient Agar Medium
- Six 9 ml Sterile Water Blanks
- Sterile Petri plates
- Graduated pipette (1ml or 1000 ml)
- Bunsen burner or Spirit lamp
Serial Dilutions of the Specimen / Sample
- Label the 6 Sterile Water blanks (9ml sterile water in each tube) as number 1 to 6 with the help of Marker. Also, label the Sterile Petri plates as number 1 to 6. Pour the Agar media in the plates and Kept aside to Solidify.
- Place the labeled tubes in the test tube rack. Mix well the 24 hours old broth culture to equally distribute the bacterial cells in the tube.
- After mixing, Remove the Cotton plug and aseptically transfer the 1 ml of the bacterial suspension from the tube of culture to sterile water blank tube no. 1 using a graduated pipette.
- Shake the tube no. 1 to mix well the content to uniformly distribute the bacterial cells. Transfer the 1 ml of this to the water blank tube no. 2 by using the graduated pipette.
- In this way, make serial dilutions till the six water blanks (no. 1 to no. 6).
Inoculating / Streaking the Specimen on Media plates
There are various methods or patterns of streaking have been developed for inoculating the specimen on the media plates for the isolation of microorganisms.They include:
- Parallel Streaking method (Quadrant)
- Zigzag streaking method (Quadrant)
- T streak Method (Three sectors)
The parallel Streak Quadrant Method & Zigzag Streak Quadrant Method are the Quadrant methods whereas the T- Streak method is the three sector method.
Three Sector Streak (T streak)
- Sterilize the wire loop.
- Cool the loop by touching it on the edge of the sterile agar plate.
- Dip the loop into the broth culture containing the mixture of bacteria.
- Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface of the top one-third of the plate back and forth in a “zig-zag” formation.
- The loop has picked up thousands of bacteria which are spread out over the surface of the agar.
- Sterilize the loop in the flame.
- Turn the plate 90 degrees and drag the loop through the area you have just streaked two to three times and continue to drag the loop in a “zig-zag” formation in the remaining half of the plate without touching that area again.
- Sterilize the loop in the flame.
- Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three times through the area you just streaked, and fill in the remaining area of the plate (zig-zag formation), being very careful not to touch any of the areas you previously streaked.
- Incubate the plate for 24 hours. If you streaked correctly, you will see isolated colonies in the third sector. The heaviest growth will be in the first sector. There will be less growth and some isolated colonies in the second sector. The third area should have the least growth with isolated colonies.
Streak Quadrant Method
- Hold the Specimen tube or Broth culture tube of mixed culture or the Diluted Specimen tube in the left hand.
- Now, sterilize the inoculating loop in the flame of the Bunsen burner or Spirit lamp, holding in the Right hand. Remove the cotton plug of the tube and immediately flame the mouth of the tube.
- Insert the Sterilized inoculating loop into the broth culture tube and withdraw one loopful of culture.
- Promptly, flame the mouth of the tube, replace the cotton plug and place the tube in the test tube rack.
- Now, hold the solidified sterile media plate in your left hand at an angle of 60° and place the inoculum (the loop containing the droplet of the specimen) on the agar surface in area 1 (at the edge farthest from you).
- Flame the inoculating loop and cool for 5-10 seconds or promptly by touching to the unused area of solidified agar medium in the media plate. Streak the inoculum from side to side in parallel lines across the surface area 1.
- Remove the loop and close the inoculated media plate. Reflame and cool the loop and turn the media plate 90° anti-clockwise and touch the sterilized inoculating loop to a corner of the culture in area 1 and drag it several times across the agar in area 2, hitting the original streak a few times. Remember that the loop should never enter area 1 again.
- Remove the loop and close the inoculated media plate. Reflame and cool the loop and turn the media plate 90° anti-clockwise. Now Streak the area 3 in the same manner as area 2, hitting the last area several times.
- Reflame and Cool the loop and turn the Media plate to 90°. Touch the loop to the corner of the culture streak in Area 3 and streak the inoculum across the agar.
- Finally, the last streak is made in a zigzag manner by touching the corner of the culture streak in area 4 and streaking it in a zigzag manner making a tail in the culture. Remember not to overlap this closing tail with any of the Area of Quadrant.
- Replace the Lid of the Inoculated Media plate and sterilize the inoculating loop in the flame of Bunsen burner or spirit lamp.
- Incubate all the plates at optimum temperature, usually at 37 °C, in an inverted position for 24-48 hours.
Typically, a confluent growth will be observed where the initial streak was made, the growth is less dense away from the streak, and discrete colonies will be there in the last area and tail streak.
If the diluted specimen was used to inoculate the media plates, the colonies in the cultures media plates will shows the lesser and lesser no. of the colonies with the increase in dilution factor that means the highest no. of colonies will develop in Plate no. 1 and least no. of colonies in plate no. 6 which will be distributes more or less sparsely along the streak lines.
Precautions Of Streak Plate Method
- While flaming the inoculation loop be sure that each segment of metal glows orange/red-hot before you move the next segment into the flame.
- Always handle open tubes at an angle near to the flame of the burner; never let them point directly up, since airborne or other environmental organisms could fall into the tube and cause contamination.
- As soon as the inoculation is completed, flame your loop or needle. Never place a contaminated tool on your workbench.
- Rotate the plate counter clockwise 90 degrees and cross the prior streaks to pick up some bacteria and spread them into the next quadrant (Repeat in all the four quadrants).
- Open the lid of the plate sufficiently (45 degrees) to introduce an inoculation loop and only for the time it takes to obtain inoculums.
- Turn the inoculated petriplate upside down while keeping it in the incubator.