Aseptic Laboratory Techniques: Plating Methods

[Narrator] This protocol incorporates aseptic technique in plating methods used to isolate, propagate, or enumerate microorganisms like bacteria and phage. Procedures include streak plating bacterial cultures to isolate single colonies. Pour plating to determine concentration of bacteria. And spread plating to enumerate viable bacterial colonies. Soft agar overlays are used to isolate phage and enumerate plaques while replica plating transfer cells from one plate to another in an identical spatial pattern. Ultimately, the practical applications of these techniques to culture microorganisms range from identification of bacteria in environments to technological advances in moleculare genetics and high throughput bioassays.

- Generally, individuals new to these plating methods may struggle because the manipulations require careful and coordinated movements avoiding contact with non-sterile surfaces. Learning these routine procedures calls for training and practice. Demonstrating the procedures will be my laboratory coordinator, Dr. Kris Reddi.

- [Narrator] Begin work with microorganisms by knowing the laboratory rules and safety precautions including biohazard classification, waste decontamination, and disposal. Confirm that all instruments, solutions, and media for plating procedures are sterile. Also establish a clear workspace. Clean it with disinfectant. Set up a Bunsen burner with a tube attached to a gas line, arrange the necessary supplies with materials labeled properly. Proceed to wash hands thoroughly with antiseptic soap and warm water. First, wet hands with warm, running water then apply and thoroughly distribute soap. Vigorously rub hands generating friction on all surfaces including thumbs, backs of fingers, backs of the hands, and beneath the fingernails. Then rinse thoroughly to remove residual soap and dry using paper towels dispensed from a holder. Finally, with a fresh paper towel, shut off the faucet. The Streak Plate Procedure is designed to isolate pure cultures of bacteria, or colonies, from mixed populations by simple mechanical separation. Remove an agar plate from a four degrees Celsius cold box and pre-warm to room temperature. Select a tool from the array of instruments for streaking the plate. Make sure the plate is dry without condensation on the lid. Label the bottom of an agar plate around the edge. When proficient, use a single plate for multiple samples. Then light a Bunsen burner to flame the metal loop. Start three to four inches from the metal loop with the wire in the tip of the blue cone, the hottest part of the Bunsen burner flame. When the metal becomes red hot, move the wire so the flame approaches the loop. To cool the loop, touch the edge of the agar medium generating a sizzling sound. Continue to pick up the inoculum on the loop. Lift the bottom half of the plate. Move the loop back and forth from the rim to the center in the first quadrant of the plate. Set down the inverted plate back onto the lid. Re-flame the metal loop. Turn the petri dish 90 degrees. Touch the loop near the end of the last streak using the back and forth pattern, crossover the last half of the streaks in the first quadrant, then move into the empty second quadrant. Once the second quadrant is filled, set down the plate. Repeat streaking procedure for the third and fourth quadrants while avoiding contact with the first quadrant. For streaking with a sterile, flat toothpick, hold the narrow end gently between thumb and ring finger at a 10 to 20 degree angle to the medium, use the wide end to streak the quadrants. Invert the plate back onto the lid between quadrants and dispose of the toothpick appropriately. Proceed to incubate the streaked plates upside down. The Pour Plate Procedure enumerates the total number of colony forming units on the surface and within the agar of a single plate. Set a timer for 10 minutes then transfer 18 milliliters of melted agar medium from a 55 degrees Celsius water bath to a 48 degrees Celsius heat block and equilibrate for 10 minutes. Label the bottom of a sterile petri dish. If applicable, include dilution factor. Now dispense one milliliter of sample into the middle of the petri dish. Close the lid. Remove the cap from the tube of melted agar and pass the rim of the open tube through a flame. Pour the agar carefully into the petri dish. Close the lid then mix the sample with the agar by gently swirling the plate. Wait for 30 minutes to allow the agar to solidify. Once the agar has solidified, invert and place the plate in a warm room. Spread-plating aims to separate microorganisms contained within a small sample volume distributing resulting colonies evenly across the agar surface. Equilibrate a plate to room temperature and label appropriately. Position the plate on the turntable. Pipette 0.1 milliliters of sample onto the center of the agar and close the lid. Eject the tip into a waste container. Dip the metal rod into a beaker of 70% ethanol covering the entire bottom portion of the spreader and the first inch of the stem and then drain. Ignite excess ethanol by passing through the flame of a Bunsen burner. Next, open the lid of the agar plate and cool the spreader by touching it to the agar along the edge near the rim. Spin the turntable slowly. Holding the spreader gently on the surface of the agar, gradually spread the sample evenly over the entire plate using a back and forth motion while the turntable is spinning. Close the lid and allow the sample to absorb thoroughly for at least five minutes. Then, incubate the inverted plate. First, label the agar plate appropriately. Open the lid of the agar plate. Then open the container of pre-sterilized glass beads and flame the rim. Carefully dispense 10 to 12 sterile glass beads onto an agar plate. Close the lid of the plate and flame the rim of the glass bead container before replacing the cap. Now, pipette the sample onto the center of the agar. In a horizontal motion, gently shake the beads across the surface of the agar seven times. If done properly, the procedure sounds like shaking maracas. Rotate the plate 60 degrees and horizontally shake again seven times. Again, rotate the plate 60 degrees and repeat shaking. Verify that the sample is absorbed. Then, pour off the contaminated beads into a marked collection beaker containing 10% chlorine bleach. Incubate the inverted plate. The plaque assay is commonly used to detect and quantify bacteria phage. Culture the indicator bacteria to exponential phase and store on ice. Then, label two sterile microcentrifuge tubes phage and control and add 50 microliters of phage sample or buffer respectively to each. Next, swirl the bacterial culture in the flask, then transfer an aliquot of bacteria to a sterile tube and gently vortex the indicator bacteria, then add 500 microliters of bacteria to each absorption tube. Mix by gently flicking the tubes. Never vortex or vigorously pipette phage samples. Incubate the phage bacteria mixture at a temperature appropriate for the indicator strain for 20 minutes. Meanwhile, transfer two soft agar tubes from a 55 degrees Celsius water bath to a 48 degrees Celsius heating block. Equilibrate for 10 minutes. Label two condensation-free nutrient hard agar plates pre-equilibrated to room temperature. Remove a soft agar tube from the heat block and check that the contents are not too hot to touch. Next, aseptically transfer the mixture from the phage absorption tube to the soft agar tube. Then, rapidly rotate between palms to mix the contents. With the tube in one hand, open the lid of the hard agar plate with the other hand. Immediately pour the entire contents of the tube onto the surface of a hard agar plate. Rock the plate swiftly and gently. Close the lid and place the plate on a level for 30 minutes until the soft agar solidifies. Next, repeat the procedure for the control absorption tube by transferring the control mixture into a soft agar tube. Mixing it, pouring the contents onto a hard agar plate, rocking that, and allowing the soft agar to solidify for 30 minutes. Inspect the plates for plaque formation. To isolate a plaque from a heterogeneous mixture, carefully punch the center with a sterile toothpick and transfer the inoculum into a sterile microcentrifuge tube containing 100 microliters of phage buffer. Continue to purify this lysate by repeating the plaque assay three to six times with serial dilutions as necessary. Replica plating exploits a selectable phenotype by permitting comparison of cell growth on a primary plate to secondary plates. Mark a grid on the bottom of the plate, label the primary plate, and number the resulting squares. Now, use aseptic technique to remove a pre-sterilized toothpick from the beaker, dab the center of each square with a cell sample, and dispose of the toothpick in an appropriate waste receptacle. Incubate the primary plate. Stack the primary plate and all the secondary plates. Place an orientation mark on the side of the bottom half of the plates. Now remove a sterile velveteen cloth from its wrapper and place it on the cylindrical block. Next, place the holder aligning the marks on the holder with those on the block. Note the orientation mark on the block and holder. Next, remove the lid from the primary plate. Line up the orientation marks on the plate and the block. Lower the plate so that the surface of the agar contacts the velveteen cloth. Using fingertips, lightly but evenly press down on the back of the primary plate and then carefully lift it off the block. Confirm that the imprint of the cells can be seen on the velvet. Replace the lid on the plate. Now, sequentially repeat the protocol on the velveteen cloth with each of the secondary plates. Use the velvet impression of cells from the primary plate to inoculate up to eight secondary plates ordered from least to most favorable substrate. Finally as a positive control, for the last plate in the series, use agar medium in which all strains tested should grow. Tape the inverted plates together and incubate them. Inspect the secondary plates for growth. Turn off the Bunsen burner then put away all supplies. Place contaminated lab wear, glass ware, and hazardous waste into the proper disposal receptacle. Clean the work area with disinfectant. Finally, wash hands thoroughly with antiseptic soap and warm water. Serratia Marcescens is a gram negative, rod-shaped proteobacterium that produces a reddish pigment called prodigiosin. It is commonly found growing in bathrooms and on shower curtains. In this example, the streak-plate technique generates single colonies in the fourth quadrant. This analysis of bacteria present in a water sample collected from a public drinking fountain uses the Pour Plate technique. Note the difference in appearance of the colonies. Surface colonies are large and circular in shape whereas some surface colonies are very small and irregularly shaped. The Spread Plate technique is an important component in enrichment, selection, and screening experiments. For instance, the copacabana method is a differentiating tool in the classical blue-white screen in recombinant DNA technology. Phage T4 is a virulent double-stranded DNA phage that infects its host as Escherichia coli to lyse and release progeny phage. This plaque assay on EHA agar shows phage in zones of clearing of approximately one millimeter in diameter. In the absence of infecting phage particles, bacterial growth results in a cloudy suspension of cells in the soft agar in which discrete colonies are not visible. In the soft agar overlay technique, plaque morphologies do vary. For instance, here the same mycobacterium host strain produces distinct plaque morphologies in the presence of microbacteria phage destroyers versus mycobacteria phage MSSS. The power of replica plating is in simultaneous screening of a large number of microorganisms. In this case, four pseudomonas strains were tested in duplicate for growth on three different carbon sources. Acetamide, lactose, and glycine. Here, the primary plate is a complete YTA medium inoculated with the four strains as indicated. The strains show variable growth patterns on replica plates of minimal MSA medium supplemented with a single carbon source acetamide, lactose, or glycine. All strains grow on the last positive control plate confirming cells were transferred to all secondary plates in this series. Tabulation os these replica plate results details simultaneous screening of the different wild type strains for characteristic growth requirements.

- After watching this video, you should have a good understanding of how to perform plating procedures without contaminating media or cultures and also to use the appropriate plating method for any given experimental task in the laboratory. Becoming proficient with these techniques takes practice. However, with proper training, the plating procedures described in this video will become second nature when working at the laboratory bench.