Streak Plate Method – Patterns, Procedure, Principle

Streak Plate Method Overview

There are two types of microbial culture such as mixed culture and pure culture. The mixed culture contains two or more species while the pure culture contains only a single species. 

During the identification of a microorganism, the first and important step is to isolate the individual species from a mixed sample. This is mainly done by the streak plate method, therefore streak plate method is an isolation technique.

The streak plate method is widely employed and vital laboratory technique used to obtain discrete colonies and pure culture. This method was first developed by 2 bacteriologists Loeffler and Gaffkey in the laboratory of Robert Koch.

In this method a sterile inoculating loop is first dipped into a diluted bacterial culture; then the culture-containing loop is streaked on the surface of a solidified agar plate to make a series of parallel, non-overlapping streaks.

As the culture is diluted before streaking on solid agar, the organism number will decrease by the third or fourth quadrant. Therefore only a few bacterial cells are transferred on the solidified agar medium as a result it will give discrete colony forming units (CFUs).

Because some colonies form from individual cells and others from pairs, chains, or clusters of cells, the term colony-forming unit (CFU) is a more correct description of the colony origin.

During the streaking an agar plate different patterns are used, depends on the source of inoculum and the microbiologist’s preference.the patterns of streaking patterns range from simple to more complex, these are designed to separate deposited cells (CFUs) on the agar surface so individual cells (CFUs) grow into isolated colonies.

A quadrant streak pattern is used for samples suspected of high cell density, while a simple zigzag pattern used for samples containing lower cell densities.

Objective of Streak Plate Method

The main purpose of this practice is to obtain colonies of microorganisms that are pure. i,e. growth derived from a single cell/spore.

Principle of Streak Plate Method

The streak plate method is a rapid qualitative isolation method. The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inoculums be reduced. It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. 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. 

In the streaking procedure, a sterile loop or swab is used to obtain an uncontaminated microbial culture. The process is called “picking colonies” when it is done from an agar plate with isolated colonies and is transferred to a new agar or gelatin plate using a sterile loop or needle.

The inoculating loop or needle is then streaked over an agar surface. On the initial region of the streak, many microorganisms are deposited resulting in confluent growth or the growth of culture over the entire surface of the streaked area.

The loop is sterilized by heating the loop in the blue flame of the Bunsen burner, between streaking different sections, or zones and thus lesser microorganisms are deposited as the streaking progresses. The streaking process will dilute out the sample that was placed in the initial region of the agar surface.

Material Required for Streak Plate Method

• Mixed Culture
• Inoculation loop
• A striker/lighter
• Bunsen burner
• Lysol (10%v/v)
• Agar plate (nutrient agar or any other agar medium)
• Paper towels

Streak Plate Procedure

There are different streaking patterns among them two streak patterns are mostly used such as a three-sector “T streak” and four-quadrant streak methods.

General Procedure

1. Labeled the Petri plate at the bottom rather than on the lid. Write the organism name, type of agar, date, and the plater’s name or initials.
2. Sterilize the wire loop or inoculating loop by holding it in the light blue area of a Bunsen burner just above the tip of inner flame of the flame until it is red-hot. Then allow to cool it.
3. While holding the loop between the thumb and forefinger, grasp the mixed bacterial culture in the other hand. With the hand holding the loop, curl the little finger around the tube cap and remove it. Do not set the cap down. Continue to hold in it in the curled finger.
4. Heat the opening of the culture tube by briefly passing it through the flame of the gas burner or, if using a Bacti-Cinerator, by holding the tube mouth next to the incinerator opening for 5-10 seconds.
5. Insert the cooled loop into the broth culture and withdraw it. The loop should contain a drop of liquid.
6. Again, heat the end of the mixed culture tube, then replace the culture cap being held in the opposite hand. Place the culture tube in a rack.

Note: Make sure the loop is cool, first touch it to the inside part of the glass tube above the medium. If the loop causes the medium to sizzle/hiss, it is too hot still. If this occurs, go back to step 3 and begin again.

T-Streak or Three Sector Streak Method

This method can be done by sterile tool, i.e. a cotton swab or commonly an inoculation loop.

1. Sterilize the loop by holding in a blue flame; allow to cool it; dip the sterile loop into an inoculum containing many species of bacteria (Follow the general procedure for detail).
2. Hold the swab or inoculum containing loop comfortably in your dominant hand and lift the lid of the Petri dish with the other. Use the lid as a shield to protect the agar from airborne contamination. Alternatively, the plate can be held in the air.
3. Lightly drag the cotton swab or loop across the agar surface in a zigzag pattern. Be careful not to cut the agar surface.
4. Re-sterilized  the loop.
5. 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.
6. Repeat 4 and 5 step again untill the remaining area of the plate is filled with zig-zag formation. Avoid to touch any of the areas you previously streaked.
7. Each time the loop aggregates fewer and fewer bacteria until it gathers just single bacterial cells that can grow into a colony.
8. Replace the lid.
9. Label the base of the plate with your name, date, and sample.
10. Incubate the plate in an inverted position for the assigned time at the appropriate temperature.
11. The plate should show the heaviest growth in the first section. The second section will show less growth and a few isolated colonies, while the final section will contain the least amount of growth and many isolated colonies.

Four Quadrant Streak or Quadrant method

1. Sterilize the loop by holding in a blue flame; allow to cool it; dip the sterile loop into an inoculum containing many species of bacteria (Follow the general procedure for detail).
2. Leave the sterile agar plate on the table and lift the lid slightly, using it as a shield from airborne contamination.or, Place the plate lid down on the table. Then remove the base and hold it in the air on an angle
3. Place a loopful of the culture on the agar surface.
4. Starting at the edge of the plate lightly drag the loop back and forth across the agar surface. Be careful not to cut the agar surface.
5. Remove the loop and replace the lid.
6. Sterilize your loop as before. It is especially important to flame it from base to tip now because the loop has lots of bacteria on it.
7. Rotate the plate a little less than 90°.
8. Let the loop cool for a few moments (or you can touch an open part of the agar), then perform another streak with the sterile loop beginning at one end of the first streak pattern. Intersect the first streak only two or three times.
9. Sterilize the loop, then repeat with a third streak beginning in the second streak.
10. Sterilize the loop, then perform a fourth streak beginning in the third streak and extending into the middle of the plate. Be careful not to enter any streaks but the third
11. Sterilize the loop.
12. Label the plate’s base with your name, date and sample inoculated.
13. Incubate the plate in an inverted position for the assigned time at the appropriate temperature.

Quadrant Streak Method B

1. Streak one loopful of organisms over Area 1 near edge of the plate. Apply the loop lightly. Don’t gouge into the medium.
2. Flame the loop, cool 5 seconds, and make 5 of 6 streaks from Area 1 through Area 2. Momentarily touching the loop to a sterile area of the medium before streaking insures a cool loop.
3. Flame the loop again, cool it, and make 6 or 7 streaks from Area 2 through Area 3.
4. Flame the loop again and make as many streaks as possible from Area 3 into Area 4, using up the remainder of the plate surface.
5. Flame the loop before putting it aside.

Other Methods of Streak Plate

RADIANT STREAK

1. Spread a loopful of organisms in small area near the edge of the plate in Area 1. Don’t gouge medium.
2. Flame the loop and allow it to cool for 5 seconds. Touching a sterile area of the medium will insure coolness.
3. From the edge of Area 1 make 7 or 8 straight streaks to the opposite side of the plate.
4. Flame the loop again, cool it sufficiently, and cross streak over the last streaks, starting near Area 1.
5. Flame the loop again before putting it aside.

CONTINUOUS STREAK

1. Starting at the edge of the plate (Area A) with a loopful of organisms, spread the organisms in a single continuous movement to the center of the plate. Use light pressure and avoid gouging the medium. 
2. Rotate the plate 180 degrees so that the uninoculated portion of the plate is away from you.
3. Without flaming the loop, and using the same face of the loop, continue streaking the other half of the plate by starting at Area B and working toward the center. 
4. Flame your loop before putting it aside.

Importance of Streak Plate Method

1. Used for isolating specific bacteria from a sample containing a mixture of different microorganisms.
2. This method is used to grow bacteria on a growth media so we can isolate and sample individual bacteria.
3. Used to identify, study or test the microorganism.
4. Used to identify the causative agent of a bacterial disease.

Disadvantage of Streak Plate Method

• Obligate anaerobes can not be used in this method.
• Only organisms that were alive in the original sample are able to be grown.

References

1. MICROBIOLOGY Laboratory Theory & Application by Michael J. Leboffe and Burton E. Pierce
2. Benson, H. J. (2005). Benson’s microbiological applications: Laboratory manual in general microbiology. Boston: McGraw-Hill Higher Education.
3. James G. Cappuccino, Chad T. Welsh (2017). Microbiology: A Laboratory Manual, 11th Edition. Pearson Publications.
4. Western Nevada College Biology 251 Laboratory Manual;”Three Streaks for Bacterial Isolation”; Dr. Steve Carman; 2009
5. http://crcooper01.people.ysu.edu/microlab/streak-plate.pdf
6. http://www.practicalbiology.org/areas/advanced/health-and-disease/hygiene/title-making-a-streak-plate,108,EXP.html
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8. http://microbiology.suite101.com/article.cfm/streak-plate-technique-for-isolating-bacteria
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10. http://www.microbelibrary.org/edzine/details.asp?id=2808
11. https://microbeonline.com/streak-plate-method-principle-purpose-procedure-results/
12. http://www2.fiu.edu/~makemson/MCB2000Lab/Exp3MediaPrep.pdf
13. http://www.nuffieldfoundation.org/practical-biology/making-streak-plate
14. http://www.talron.co.il/index.php?module=pagemaster&PAGE_user_op=view_page&PAGE_id=17&MMN_position=18:2
15. https://www.jove.com/video/3064/aseptic-laboratory-techniques-plating-methods
16. http://www.answers.com/Q/What_is_the_advantages_of_streak_plate_method
17. https://hk.answers.yahoo.com/question/index?qid=20060718085954AAM7266&guccounter=1