- Email: [email protected]
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
CHAPTER ONE
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies Elizabeth Eyler1 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Preparation of LB-Agar Solution 5.1 Overview 5.2 Duration 5.3 Caution 5.4 Tip 5.5 Tip 5.6 Tip 5.7 Tip 6. Step 2 Pouring Agar Plates 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Caution 6.5 Tip 6.6 Tip 7. Step 3 Streaking for Individual Colonies 7.1 Overview 7.2 Duration 7.3 Tip 7.4 Tip 7.5 Caution 8. Step 4 Spreading Cells on Agar Plates 8.1 Overview 8.2 Duration Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
4 4 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 8 9 9 9 9 9 9 10 11 11 11 12 12 12 #
2013 Elsevier Inc. All rights reserved.
3
4
Elizabeth Eyler
8.3 Tip 8.4 Tip 8.5 Caution References
13 13 13 14
Abstract Agar plates, composed of growth media solidified with agar, are commonly used to culture yeast and bacteria. Cultures may be spread or streaked across the surface of these plates to facilitate titer calculations or to permit isolation of single, genetically identical colonies.
1. THEORY Although the protocol provided contains instructions for preparing LB agar plates, different kinds of agar plates may be prepared in a similar fashion using other rich media or minimal media (see Growth Media for E. coli or Saccharomyces cerevisiae Growth Media). Antibiotics or supplements may also be added to the media/agar mixture immediately before pouring to permit the selection of particular strains. Several methods are included for streaking or spreading yeast or bacterial cultures on agar plates. These methods distribute cells evenly over the surface of the plates and also permit the calculation of cell titer or the isolation of single colonies for further analysis. For example, a researcher might transform bacteria with a DNA plasmid containing a gene of interest and a selectable marker, such as ampicillin resistance. When these bacteria are spread on LB-Amp plates, only those bacteria that have retained the selectable marker will be able to grow on the plates. Ideally, the resistant bacteria will also contain the entire plasmid of interest. However, occasionally the desired insert is lost even though the selectable marker is retained. Undesirable mutations can also be introduced during the transformation process. Isolation of single colonies allows researchers to screen for only those bacteria containing the correct construct and to grow pure cultures for diagnostic testing and future experiments.
2. EQUIPMENT Autoclave 1-l graduated cylinder 2-l Erlenmeyer flask Sterile Petri dishes
5
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
Incubator (optional) Bunsen burner (optional) 25-ml serological pipettes (optional) Inoculation loop (optional) Sterile toothpicks (optional) Micropipettor (optional) Sterile micropipettor tips (optional) Spreader or glass Pasteur pipette (optional) Autoclaved glass beads, 4 mm (optional)
3. MATERIALS Tryptone Yeast extract Sodium chloride (NaCl) Sodium hydroxide (NaOH, optional) Distilled water Agar Antibiotics (ampicillin, kanamycin, chloramphenicol, tetracycline, carbenicillin, gentamycin, or an another as appropriate) Supplements (IPTG, X-Gal, etc.) 70% ethanol (optional)
3.1. Solutions & buffers Step 1 LB-agar Component
Amount
Tryptone
10 g
Yeast extract
5g
NaCl
10 g
Agar
15 g
Add water to 1 l. Optional: adjust the pH to 7.5 with 1 ml of 1 N NaOH
4. PROTOCOL 4.1. Preparation Assemble equipment and materials for mixing LB-agar solution and Petri dishes for pouring plates. Assemble appropriate equipment for spreading or streaking plates, if desired.
6
Elizabeth Eyler
Figure 1.1 Flowchart of the complete protocol, including preparation.
4.2. Duration Preparation
Variable
Protocol
2.5 h
See Fig. 1.1 for the flowchart of the complete protocol.
5. STEP 1 PREPARATION OF LB-AGAR SOLUTION 5.1. Overview LB-agar is mixed and autoclaved in order to sterilize it and to permit the agar to dissolve. The solution is then cooled and any supplements or antibiotics are added prior to pouring plates.
5.2. Duration 90 min 1.1 Mix all components for LB-agar in a 2-l Erlenmeyer flask. Note that the agar will not dissolve completely before autoclaving. However, the solution should still be thoroughly mixed to prevent clumping. 1.2 Cover the top of the flask with foil and place into an autoclavable container with 1–2 cm of water covering the bottom of the container. 1.3 Autoclave for at least 20 min at 121 C on the liquid cycle. Liquid cycles vent slowly to prevent liquids from boiling over during autoclaving while dry cycles vent rapidly.
7
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
1.4 Remove LB-agar solution from autoclave and allow to cool to 55 C on a bench top or in a water bath. At this temperature, the flask will still be warm, but can be comfortably picked up with bare hands. 1.5 If needed, add antibiotics or supplements to LB-agar mixture after the solution has cooled to 55 C (Table 1.1). Mix solution thoroughly.
5.3. Caution Use heat-resistant autoclave gloves when removing flasks from the autoclave. The solution will be very hot and should be handled with care.
5.4. Tip If preparing plates from minimal media, a 2 media solution should be autoclaved separately from a 2 agar solution and the two solutions should be mixed together after autoclaving. An insoluble precipitate will form if the minimal media and agar are autoclaved together.
5.5. Tip A stir bar can be added to the bottom of the flask before autoclaving to facilitate thorough mixing of the solution after autoclaving. This will allow you to maintain the sterility of your solution as you will not have to add a nonsterile stir bar to your solution after autoclaving.
5.6. Tip If plates will not be poured soon after autoclaving, the flask can be stored in a 55 C incubator or water bath until you are ready to pour. Table 1.1 Common additives and final concentrations Antibiotic/supplement
Final concentration
Ampicillin
50–100 mg ml1
Kanamycin
50 mg ml1
Chloramphenicol
15–34 mg ml1
Tetracycline
15–35 mg ml1
Carbenicillin
50–100 mg ml1
Gentamycin
25 mg ml1
IPTG
0.1 mM
X-Gal
40 mg ml1
8
Elizabeth Eyler
Figure 1.2 Flowchart of Step 1.
5.7. Tip Many antibiotics and chemicals will degrade at high temperatures. It is essential to allow the solution to cool to 55 C before adding them to your mixture. See Fig. 1.2 for the flowchart of Step 1.
6. STEP 2 POURING AGAR PLATES 6.1. Overview After the LB-agar solution is prepared and cooled, it is poured into Petri dishes and allowed to solidify. After solidifying and drying, these plates can be used to culture yeast or bacterial strains of interest.
6.2. Duration 15 min (pouring); 30 min to overnight (drying) 2.1 Remove the sterile Petri dishes from the sleeve and place on bench top. Save the sleeve. 2.2 Pour LB-agar mixture into each Petri dish until the dish is about half full. 2.3 Alternatively, pipette 20–25 ml of LB-agar into each plate. If using 10-cm Petri dishes, 1 l of LB-agar should yield 40–50 plates. 2.4 Allow the plates to cool and solidify on the bench top. 2.5 Leave the plates on bench top to dry overnight or for up to 2–3 days. Alternatively, dry the plates for 30 min in a 37 C incubator with the
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
9
lids off. Freshly poured, wet plates will not absorb any liquids spread on them and may release moisture under cells streaked on them, causing the cells to float away. 2.6 Invert the dried plates (agar side up), stack, and reseal in original Petri dish sleeve. Label, date, and store at 4 C for 3–4 weeks.
6.3. Tip If bubbles are present on the surface of your plates, they can be removed by briefly passing a Bunsen burner flame over the top of the agar before the plates solidify. Small bubbles can mimic the appearance of colonies on a solidified plate.
6.4. Caution Care should always be taken in the presence of an open flame. Work quickly to avoid melting the plastic of the Petri dishes.
6.5. Tip Antibiotics will degrade after a few weeks and therefore limit the length of time that plates can be stored and still be used. However, plates that do not contain an antibiotic can potentially be stored longer than 3–4 weeks at 4 C, as long as they have not dried out or become contaminated. Examine the plates carefully for small colonies or mold before using.
6.6. Tip If using antibiotic-containing plates that are older than 3–4 weeks, or if using plates that did not originally contain an antibiotic, fresh antibiotic can be added prior to plating. Estimate the total volume of LB-agar in the plate (20–25 ml if using 10-cm dishes) and add antibiotic to the appropriate final concentration (Table 1.1). Dilute antibiotic in dH2O to a total volume of 100–150 ml to facilitate even spreading, and allow dishes to stand for 30–60 min before using so that the antibiotic can be absorbed into the agar. See Fig. 1.3 for the flowchart of Step 2.
7. STEP 3 STREAKING FOR INDIVIDUAL COLONIES 7.1. Overview Bacteria or yeast can be streaked on solidified and dried agar plates to isolate individual colonies for further experiments such as colony PCR and plasmid purification (see Colony PCR and Isolation of plasmid DNA from bacteria) (see Video 1: Streaking a plate). http://dx.doi.org/10.1016/B978-0-12420067-8.00001-5
10
Elizabeth Eyler
Figure 1.3 Flowchart of Step 2.
7.2. Duration 5 min 3.1 Using a sterile inoculating loop, remove an inoculum of cells from the culture to be streaked or pick a single colony from a previously streaked plate. Use the loop to transfer the cells to an agar plate. Alternatively, a micropipettor can be used to transfer a small volume of cells to the plate. For dense cultures, cells may need to be diluted in dH2O to a total volume of 100 ml. For more dilute cultures, this may not be necessary. 3.2 With the sterile inoculating loop, streak cells back and forth across one quadrant of the plate. 3.3 After streaking, sterilize the loop by dipping into 70% ethanol (or a higher percentage of ethanol) and lighting in a Bunsen burner flame. The loop should become red while flaming. Allow the ethanol to burn off completely. 3.4 Cool the loop by touching briefly to an unstreaked portion of the plate. A faint sizzling sound may be heard as the loop cools. When the loop is cool, streak back and forth as before through a second quadrant of the plate, making sure to pass at least once through the cells streaked in the first quadrant.
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
11
3.5 Repeat Steps 3.3 and 3.4 once or twice more in fresh quadrants of the plate. Cells should become progressively more dilute in each successively streaked quadrant (Fig. 1.4). 3.6 Invert streaked plate(s) (agar side up) and place in a 37 C incubator overnight to allow colonies to grow.
7.3. Tip If too many cells are streaked or plated, you may end up with a lawn of cells and be unable to isolate individual colonies from your plate.
7.4. Tip Alternatively, a sterile toothpick can be used for streaking instead of an inoculating loop. Use a fresh toothpick to streak each quadrant.
7.5. Caution Care should always be taken in the presence of an open flame. Do not place beakers or flasks containing ethanol too close to the flame, and avoid splattering or spilling ethanol while working. See Fig. 1.5 for the flowchart of Step 3.
Figure 1.4 An example of a streaked plate. An agar plate has been streaked for isolation of individual colonies. Note that the cells become progressively more dilute in each successively streaked region.
12
Elizabeth Eyler
Figure 1.5 Flowchart of Step 3.
8. STEP 4 SPREADING CELLS ON AGAR PLATES 8.1. Overview Bacteria or yeast can be spread on plates to distribute cells evenly across a plate and to permit calculations of cell titer (see Video 2: Spreading a plate). http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
8.2. Duration 5 min 4.1 Use a sterile inoculating loop to transfer an inoculum of cells from the culture to be spread to the agar plate. Alternatively, a micropipettor can be used to transfer a small volume of cells to the plate. For dense cultures, cells may need to be diluted in dH2O to a total volume of 100 ml. For more dilute cultures, this may not be necessary. 4.2 Sterilize spreader by dipping in 70% ethanol (or a higher percentage of ethanol) and lighting in a Bunsen burner flame. Allow the ethanol to burn off completely, and then cool by touching briefly to a portion of the plate that is free of cells. 4.3 Spread cells by passing the spreader back and forth through the cells and across the entire plate to spread evenly across the dish (Fig. 1.6). The plate should not be too wet after spreading. A good test is to tilt the plate slightly to make sure that excess liquid does not run across the plate after tilting. 4.4 Invert spread plate(s) (agar side up) and place in a 37 C incubator overnight to allow colonies to grow.
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
13
Figure 1.6 An example of a spread plate. An agar plate with cells spread evenly across the surface. Note the uniform distribution of colonies across the plate. Also note the colony density, with numerous but nonoverlapping colonies that can be isolated for future experiments.
8.3. Tip Although metal and plastic cell spreaders can be purchased, a disposable spreader can easily be fashioned from a 900 glass Pasteur pipette. Hold the narrow tip over a Bunsen burner flame and allow the heat to bend the glass into a triangular shape (see Video 3: Making a cell spreader). http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
8.4. Tip When processing multiple plates, cells can also be spread using sterile 4-mm glass beads. Pour approximately six to ten beads onto each plate, replace lids, stack the plates, and shake side to side to distribute cells. Shake beads out of dishes after spreading. Beads can be reused by washing thoroughly in deionized water and 70% ethanol, drying, and autoclaving to sterilize. This method is efficient when handling large batches of plates and results in a very homogenous distribution of cells (see Video 4: Spreading with glass beads). http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
8.5. Caution Care should always be taken in the presence of an open flame. Do not place beakers or flasks containing ethanol too close to the flame, and avoid splattering ethanol while
14
Elizabeth Eyler
Figure 1.7 Flowchart of Step 4.
working (see Video 5: Ethanol flame risks). http://dx.doi.org/10.1016/B9780-12-420067-8.00001-5 See Fig. 1.7 for the flowchart of Step 4.
REFERENCES Referenced Protocols in Methods Navigator Growth Media for E. coli. Saccharomyces cerevisiae Growth Media. Colony PCR. Isolation of plasmid DNA from bacteria.
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies Elizabeth Eyler1 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Preparation of LB-Agar Solution 5.1 Overview 5.2 Duration 5.3 Caution 5.4 Tip 5.5 Tip 5.6 Tip 5.7 Tip 6. Step 2 Pouring Agar Plates 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Caution 6.5 Tip 6.6 Tip 7. Step 3 Streaking for Individual Colonies 7.1 Overview 7.2 Duration 7.3 Tip 7.4 Tip 7.5 Caution 8. Step 4 Spreading Cells on Agar Plates 8.1 Overview 8.2 Duration Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
4 4 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 8 9 9 9 9 9 9 10 11 11 11 12 12 12 #
2013 Elsevier Inc. All rights reserved.
3
4
Elizabeth Eyler
8.3 Tip 8.4 Tip 8.5 Caution References
13 13 13 14
Abstract Agar plates, composed of growth media solidified with agar, are commonly used to culture yeast and bacteria. Cultures may be spread or streaked across the surface of these plates to facilitate titer calculations or to permit isolation of single, genetically identical colonies.
1. THEORY Although the protocol provided contains instructions for preparing LB agar plates, different kinds of agar plates may be prepared in a similar fashion using other rich media or minimal media (see Growth Media for E. coli or Saccharomyces cerevisiae Growth Media). Antibiotics or supplements may also be added to the media/agar mixture immediately before pouring to permit the selection of particular strains. Several methods are included for streaking or spreading yeast or bacterial cultures on agar plates. These methods distribute cells evenly over the surface of the plates and also permit the calculation of cell titer or the isolation of single colonies for further analysis. For example, a researcher might transform bacteria with a DNA plasmid containing a gene of interest and a selectable marker, such as ampicillin resistance. When these bacteria are spread on LB-Amp plates, only those bacteria that have retained the selectable marker will be able to grow on the plates. Ideally, the resistant bacteria will also contain the entire plasmid of interest. However, occasionally the desired insert is lost even though the selectable marker is retained. Undesirable mutations can also be introduced during the transformation process. Isolation of single colonies allows researchers to screen for only those bacteria containing the correct construct and to grow pure cultures for diagnostic testing and future experiments.
2. EQUIPMENT Autoclave 1-l graduated cylinder 2-l Erlenmeyer flask Sterile Petri dishes
5
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
Incubator (optional) Bunsen burner (optional) 25-ml serological pipettes (optional) Inoculation loop (optional) Sterile toothpicks (optional) Micropipettor (optional) Sterile micropipettor tips (optional) Spreader or glass Pasteur pipette (optional) Autoclaved glass beads, 4 mm (optional)
3. MATERIALS Tryptone Yeast extract Sodium chloride (NaCl) Sodium hydroxide (NaOH, optional) Distilled water Agar Antibiotics (ampicillin, kanamycin, chloramphenicol, tetracycline, carbenicillin, gentamycin, or an another as appropriate) Supplements (IPTG, X-Gal, etc.) 70% ethanol (optional)
3.1. Solutions & buffers Step 1 LB-agar Component
Amount
Tryptone
10 g
Yeast extract
5g
NaCl
10 g
Agar
15 g
Add water to 1 l. Optional: adjust the pH to 7.5 with 1 ml of 1 N NaOH
4. PROTOCOL 4.1. Preparation Assemble equipment and materials for mixing LB-agar solution and Petri dishes for pouring plates. Assemble appropriate equipment for spreading or streaking plates, if desired.
6
Elizabeth Eyler
Figure 1.1 Flowchart of the complete protocol, including preparation.
4.2. Duration Preparation
Variable
Protocol
2.5 h
See Fig. 1.1 for the flowchart of the complete protocol.
5. STEP 1 PREPARATION OF LB-AGAR SOLUTION 5.1. Overview LB-agar is mixed and autoclaved in order to sterilize it and to permit the agar to dissolve. The solution is then cooled and any supplements or antibiotics are added prior to pouring plates.
5.2. Duration 90 min 1.1 Mix all components for LB-agar in a 2-l Erlenmeyer flask. Note that the agar will not dissolve completely before autoclaving. However, the solution should still be thoroughly mixed to prevent clumping. 1.2 Cover the top of the flask with foil and place into an autoclavable container with 1–2 cm of water covering the bottom of the container. 1.3 Autoclave for at least 20 min at 121 C on the liquid cycle. Liquid cycles vent slowly to prevent liquids from boiling over during autoclaving while dry cycles vent rapidly.
7
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
1.4 Remove LB-agar solution from autoclave and allow to cool to 55 C on a bench top or in a water bath. At this temperature, the flask will still be warm, but can be comfortably picked up with bare hands. 1.5 If needed, add antibiotics or supplements to LB-agar mixture after the solution has cooled to 55 C (Table 1.1). Mix solution thoroughly.
5.3. Caution Use heat-resistant autoclave gloves when removing flasks from the autoclave. The solution will be very hot and should be handled with care.
5.4. Tip If preparing plates from minimal media, a 2 media solution should be autoclaved separately from a 2 agar solution and the two solutions should be mixed together after autoclaving. An insoluble precipitate will form if the minimal media and agar are autoclaved together.
5.5. Tip A stir bar can be added to the bottom of the flask before autoclaving to facilitate thorough mixing of the solution after autoclaving. This will allow you to maintain the sterility of your solution as you will not have to add a nonsterile stir bar to your solution after autoclaving.
5.6. Tip If plates will not be poured soon after autoclaving, the flask can be stored in a 55 C incubator or water bath until you are ready to pour. Table 1.1 Common additives and final concentrations Antibiotic/supplement
Final concentration
Ampicillin
50–100 mg ml1
Kanamycin
50 mg ml1
Chloramphenicol
15–34 mg ml1
Tetracycline
15–35 mg ml1
Carbenicillin
50–100 mg ml1
Gentamycin
25 mg ml1
IPTG
0.1 mM
X-Gal
40 mg ml1
8
Elizabeth Eyler
Figure 1.2 Flowchart of Step 1.
5.7. Tip Many antibiotics and chemicals will degrade at high temperatures. It is essential to allow the solution to cool to 55 C before adding them to your mixture. See Fig. 1.2 for the flowchart of Step 1.
6. STEP 2 POURING AGAR PLATES 6.1. Overview After the LB-agar solution is prepared and cooled, it is poured into Petri dishes and allowed to solidify. After solidifying and drying, these plates can be used to culture yeast or bacterial strains of interest.
6.2. Duration 15 min (pouring); 30 min to overnight (drying) 2.1 Remove the sterile Petri dishes from the sleeve and place on bench top. Save the sleeve. 2.2 Pour LB-agar mixture into each Petri dish until the dish is about half full. 2.3 Alternatively, pipette 20–25 ml of LB-agar into each plate. If using 10-cm Petri dishes, 1 l of LB-agar should yield 40–50 plates. 2.4 Allow the plates to cool and solidify on the bench top. 2.5 Leave the plates on bench top to dry overnight or for up to 2–3 days. Alternatively, dry the plates for 30 min in a 37 C incubator with the
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
9
lids off. Freshly poured, wet plates will not absorb any liquids spread on them and may release moisture under cells streaked on them, causing the cells to float away. 2.6 Invert the dried plates (agar side up), stack, and reseal in original Petri dish sleeve. Label, date, and store at 4 C for 3–4 weeks.
6.3. Tip If bubbles are present on the surface of your plates, they can be removed by briefly passing a Bunsen burner flame over the top of the agar before the plates solidify. Small bubbles can mimic the appearance of colonies on a solidified plate.
6.4. Caution Care should always be taken in the presence of an open flame. Work quickly to avoid melting the plastic of the Petri dishes.
6.5. Tip Antibiotics will degrade after a few weeks and therefore limit the length of time that plates can be stored and still be used. However, plates that do not contain an antibiotic can potentially be stored longer than 3–4 weeks at 4 C, as long as they have not dried out or become contaminated. Examine the plates carefully for small colonies or mold before using.
6.6. Tip If using antibiotic-containing plates that are older than 3–4 weeks, or if using plates that did not originally contain an antibiotic, fresh antibiotic can be added prior to plating. Estimate the total volume of LB-agar in the plate (20–25 ml if using 10-cm dishes) and add antibiotic to the appropriate final concentration (Table 1.1). Dilute antibiotic in dH2O to a total volume of 100–150 ml to facilitate even spreading, and allow dishes to stand for 30–60 min before using so that the antibiotic can be absorbed into the agar. See Fig. 1.3 for the flowchart of Step 2.
7. STEP 3 STREAKING FOR INDIVIDUAL COLONIES 7.1. Overview Bacteria or yeast can be streaked on solidified and dried agar plates to isolate individual colonies for further experiments such as colony PCR and plasmid purification (see Colony PCR and Isolation of plasmid DNA from bacteria) (see Video 1: Streaking a plate). http://dx.doi.org/10.1016/B978-0-12420067-8.00001-5
10
Elizabeth Eyler
Figure 1.3 Flowchart of Step 2.
7.2. Duration 5 min 3.1 Using a sterile inoculating loop, remove an inoculum of cells from the culture to be streaked or pick a single colony from a previously streaked plate. Use the loop to transfer the cells to an agar plate. Alternatively, a micropipettor can be used to transfer a small volume of cells to the plate. For dense cultures, cells may need to be diluted in dH2O to a total volume of 100 ml. For more dilute cultures, this may not be necessary. 3.2 With the sterile inoculating loop, streak cells back and forth across one quadrant of the plate. 3.3 After streaking, sterilize the loop by dipping into 70% ethanol (or a higher percentage of ethanol) and lighting in a Bunsen burner flame. The loop should become red while flaming. Allow the ethanol to burn off completely. 3.4 Cool the loop by touching briefly to an unstreaked portion of the plate. A faint sizzling sound may be heard as the loop cools. When the loop is cool, streak back and forth as before through a second quadrant of the plate, making sure to pass at least once through the cells streaked in the first quadrant.
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
11
3.5 Repeat Steps 3.3 and 3.4 once or twice more in fresh quadrants of the plate. Cells should become progressively more dilute in each successively streaked quadrant (Fig. 1.4). 3.6 Invert streaked plate(s) (agar side up) and place in a 37 C incubator overnight to allow colonies to grow.
7.3. Tip If too many cells are streaked or plated, you may end up with a lawn of cells and be unable to isolate individual colonies from your plate.
7.4. Tip Alternatively, a sterile toothpick can be used for streaking instead of an inoculating loop. Use a fresh toothpick to streak each quadrant.
7.5. Caution Care should always be taken in the presence of an open flame. Do not place beakers or flasks containing ethanol too close to the flame, and avoid splattering or spilling ethanol while working. See Fig. 1.5 for the flowchart of Step 3.
Figure 1.4 An example of a streaked plate. An agar plate has been streaked for isolation of individual colonies. Note that the cells become progressively more dilute in each successively streaked region.
12
Elizabeth Eyler
Figure 1.5 Flowchart of Step 3.
8. STEP 4 SPREADING CELLS ON AGAR PLATES 8.1. Overview Bacteria or yeast can be spread on plates to distribute cells evenly across a plate and to permit calculations of cell titer (see Video 2: Spreading a plate). http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
8.2. Duration 5 min 4.1 Use a sterile inoculating loop to transfer an inoculum of cells from the culture to be spread to the agar plate. Alternatively, a micropipettor can be used to transfer a small volume of cells to the plate. For dense cultures, cells may need to be diluted in dH2O to a total volume of 100 ml. For more dilute cultures, this may not be necessary. 4.2 Sterilize spreader by dipping in 70% ethanol (or a higher percentage of ethanol) and lighting in a Bunsen burner flame. Allow the ethanol to burn off completely, and then cool by touching briefly to a portion of the plate that is free of cells. 4.3 Spread cells by passing the spreader back and forth through the cells and across the entire plate to spread evenly across the dish (Fig. 1.6). The plate should not be too wet after spreading. A good test is to tilt the plate slightly to make sure that excess liquid does not run across the plate after tilting. 4.4 Invert spread plate(s) (agar side up) and place in a 37 C incubator overnight to allow colonies to grow.
Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies
13
Figure 1.6 An example of a spread plate. An agar plate with cells spread evenly across the surface. Note the uniform distribution of colonies across the plate. Also note the colony density, with numerous but nonoverlapping colonies that can be isolated for future experiments.
8.3. Tip Although metal and plastic cell spreaders can be purchased, a disposable spreader can easily be fashioned from a 900 glass Pasteur pipette. Hold the narrow tip over a Bunsen burner flame and allow the heat to bend the glass into a triangular shape (see Video 3: Making a cell spreader). http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
8.4. Tip When processing multiple plates, cells can also be spread using sterile 4-mm glass beads. Pour approximately six to ten beads onto each plate, replace lids, stack the plates, and shake side to side to distribute cells. Shake beads out of dishes after spreading. Beads can be reused by washing thoroughly in deionized water and 70% ethanol, drying, and autoclaving to sterilize. This method is efficient when handling large batches of plates and results in a very homogenous distribution of cells (see Video 4: Spreading with glass beads). http://dx.doi.org/10.1016/B978-0-12-420067-8.00001-5
8.5. Caution Care should always be taken in the presence of an open flame. Do not place beakers or flasks containing ethanol too close to the flame, and avoid splattering ethanol while
14
Elizabeth Eyler
Figure 1.7 Flowchart of Step 4.
working (see Video 5: Ethanol flame risks). http://dx.doi.org/10.1016/B9780-12-420067-8.00001-5 See Fig. 1.7 for the flowchart of Step 4.
REFERENCES Referenced Protocols in Methods Navigator Growth Media for E. coli. Saccharomyces cerevisiae Growth Media. Colony PCR. Isolation of plasmid DNA from bacteria.