Pour-Plate and Subculture Techniques

An alternative method for using agar plates to obtain isolated colonies, other than streaking their surfaces, is to prepare a “pour plate.” In this case, an aliquot of the specimen to be cultured is placed in the bottom of an empty, sterile petri dish, then melted, cooled agar is poured over it. Quickly, before the agar cools, the plate is gently rocked to disperse the inoculum. When the agar has solidified and the plate is incubated, any bacteria present in the specimen will grow wherever they have been embedded within the agar layer or localized on its surface. Their colonies will be isolated and can be removed from subsurface positions by inserting the inoculating loop or a straight wire into the agar. To prepare pour plates, the inoculum must be a liquid specimen or culture. If it is not, it must be suspended in sterile fluid before being placed in the petri dish.

Another method for preparing a pour plate is to inoculate the specimen or culture directly into the tube of melted, cooled, but not yet solidified agar. Mix it by rolling it back and forth between the outstretched fingers of both hands, and pour the inoculated agar into a sterile petri dish. These steps must be performed quickly before the agar cools enough to harden.

When primary isolation plates have been properly poured or streaked, individual colonies can be picked up on an inoculating loop or straight wire and inoculated to fresh agar or broth media. These new pure cultures of isolated organisms are called subcultures. If they are indeed pure and do not contain mixtures of different species, they can be identified in stepwise procedures.

Purpose	A. To learn the pour-plate technique for obtaining isolated colonies B. To obtain isolated colonies from streaked plate cultures and grow them as pure subcultures
Materials	Tubed nutrient agar (10 ml per tube) Sterile petri dishes Sterile 1-ml pipettes (cotton plugged) Mixed broth culture containing Escherichia coli and Staphylococcus epidermidis Nutrient agar plates Nutrient agar broth Nutrient agar plate cultures streaked in, containing isolated colonies of three bacterial species

Procedures

1. Pour-Plate Technique
1. Place a tube of sterile nutrient agar in a boiling water bath. (A simple water bath can be set up by placing a glass beaker or tin can half filled with water on a tripod over a Bunsen flame. An asbestos mat must be used under glass vessels. The water should be kept at a steady but not rapid boil. Keep the water level at the halfway mark. An electric burner may be used instead).
2. When the agar is liquefied, remove the tube and allow it to cool to about 50°C.
3. Place an empty sterile petri dish before you, top side up.
4. Remove a sterile 1-ml pipette from its container, keeping your fingers on the plugged mouth end.
5. Pick up the mixed broth culture in the other hand, remove its closure with the little finger of the hand holding the pipette (do not touch the pipette to anything), and insert the pipette into the broth.
6. Holding the tube and pipette vertically, poise your index finger over the pipette mouth. Allow the pipette to fill to the level of the broth in the tube and then close off its mouth with your finger (there should be about 0.3 to 0.4 ml of culture in the pipette).
7. Keeping your finger pressed on its top, raise the pipette until the tip is free of the broth and then slowly allow the material in the pipette to run back into the tube until only the last 0.1 ml remains. Now press your finger tightly to close the pipette’s mouth and prevent further dripping (fig. 10.1). Never use your mouth to draw fluid into a pipette.
8. Before you withdraw the pipette from the tube, touch its tip against the dry inner wall to remove any drop hanging from it.
9. Withdraw the closed pipette, replace the tube closure, and put the tube down in the rack.
10. Now, with your free hand, remove the top of the petri dish (do not put it down), place the tip of the pipette against the bottom of the dish, release your finger from the mouth, and let 0.1 ml of broth culture run into the plate bottom.
11. Replace the dish top and discard the pipette into a container of disinfectant.
12. Pick up the tube of melted, cooled agar, remove its closure, and put it down on the bench top.
13. With your free hand, remove the top of the petri dish (again, do not put it down). Quickly pour the agar into the dish.
14. Replace the petri dish cover (the tube may be set aside for washing). Gently rock the closed dish, or rotate it in circular fashion on the bench top, being careful not to allow the still melted agar to wave up over the edge of the bottom half or onto the cover.
15. Let the agar solidify without further disturbance. When it is quite firm (about 30 minutes), invert the plate and place it in the 35°C incubator.
2. Subculture Technique (Picking Isolated Colonies for Pure Culture)
1. Look again at figure 2.6. This figure illustrates the correct method of picking a single colony from the surface of a streaked plate.
2. Now open the nutrient agar plate from a mixed broth culture containing three organisms. Hold the exposed agar surface in good light so that you can see all facets of individual isolated colonies.
3. With your sterilized, cooled loop held steady in your other hand, bring the loop edge down against the top surface of one isolated colony you have selected for pure subculture. Withdraw the charged loop (don’t touch it to anything!) and close the streaked plate.
4. Inoculate a fresh, sterile nutrient broth by gently rubbing the charged loop against the inner wall of the tube, just beneath the fluid surface. When you bring the loop out of the tube, be sure it holds some of the broth.
5. Now use the loop to inoculate a fresh nutrient agar plate. Rub the inoculum onto a small area near the edge, sterilize the loop, and then go back and complete the streaking of the plate by using the technique illustrated in figure 9.1.
6. Inoculate two more agar plates, each with a different type of colony picked from your previous plate culture.
7. Incubate your new plate cultures (inverted) and broth cultures at 35°C.

Results

1. Examination of Pour Plate
   Diagram the distribution of colonies you can see in your pour-plate culture (surface and subsurface locations, separation). Indicate any colonial distinctions you can recognize.


2. Examination of Streaked Plate Subcultures
   1. Examine your streaked nutrient agar plate subcultures and determine whether you have obtained pure cultures. In the following table, indicate the size, shape, and pigmentation of colonies on each plate. Make Gram stains of colonies on each subculture plate and record Gram-stain reactions in the table.
   2. Examine your nutrient broth subcultures. Make Gram stains to determine whether they are pure. Describe your microscopic observations of each broth subculture.
   3. Are the organisms recovered in your plate and broth cultures the same as those you originally recorded in Streaking Technique to Obtain Pure Cultures?
      If not, specify the differences: