CHAPTER TWO
Storage of Bacteria and Yeast Cheryl M. Koh1 Department of Pathology, The 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 5. Protocol A Cryopreservation of Bacterial Cultures 5.1 Preparation 5.2 Duration 6. Step 1A Grow Bacterial Cultures 6.1 Overview 6.2 Duration 7. Step 2A Freeze the Bacterial Cells 7.1 Overview 7.2 Duration 8. Protocol B Cryopreservation of Yeast 8.1 Preparation 8.2 Duration 9. Step 1B Grow Yeast Cultures 9.1 Overview 9.2 Duration 10. Step 2B Freeze the Yeast Cells 10.1 Overview 10.2 Duration References
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Abstract Yeast and bacteria can be cryopreserved and stored almost indefinitely. It is useful to prepare stocks for archival purposes.
1. THEORY Glycerol is used as a cryoprotectant as it disrupts the hydrogen bonding between water molecules, hence preventing the formation of ice Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00002-7
#
2013 Elsevier Inc. All rights reserved.
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Cheryl M. Koh
crystals during freezing. This protects yeast and bacterial cells from ice crystal damage, and maintains their viability for future reculture. Cell viability decreases with each freeze–thaw cycle; thus, to recover yeast or bacterial cells from frozen stocks, avoid thawing the stock completely. Instead, scrape off a small amount of frozen culture and inoculate directly into liquid media.
2. EQUIPMENT Autoclave Shaking incubator Roller drum (optional) Incubator (37 C for bacteria, 30 C for yeast) Vortex mixer 80 C freezer Liquid nitrogen freezer (optional) Petri dishes (10 cm, sterile) 13-ml sterile polypropylene culture tubes Micropipettors Micropipettor tips Cryovials Freezer storage boxes
3. MATERIALS Glycerol Sterile deionized water Storage of bacteria: Yeast extract Tryptone Sodium chloride (NaCl) Storage of yeast: Sucrose Yeast extract Peptone Bacto Agar
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Storage of Bacteria and Yeast
3.1. Solutions & buffers Step 1A LB Broth Component
Amount
Yeast Extract
5g
Tryptone
10 g
NaCl*
0.5–10 g
Add deionized water to 1 l. Sterilize by autoclaving *The salt-sensitivity of bacteria varies between strains. If antibiotics are to be added to the media, a low salt concentration (0.5 g) is preferable
Step 1B 20% sucrose
Dissolve 20 g sucrose in 80 ml deionized water. Adjust the volume to 100 ml. Pass through a 0.2-mm filter to sterilize
YPD Medium Component
Amount
Yeast extract
1g
Peptone
2g
Add deionized water to 900 ml and mix until no clumps remain. Sterilize by autoclaving. Add 100 ml sterile 20% sucrose
YPD Plates Component
Amount
Yeast extract
1g
Peptone
2g
Bacto Agar
20 g
Add deionized water to 900 ml and mix until no clumps remain. Sterilize by autoclaving and then cool to 55–60 C. Add 100 ml sterile 20% sucrose. Pour into sterile 10-cm Petri dishes, 10 ml per dish. Cover and allow agar to solidify
Step 2(A and B) 80% Glycerol
Mix 80 ml glycerol and 20 ml deionized water. Sterilize by autoclaving
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Cheryl M. Koh
4. PROTOCOL For a list of common culture media for bacteria and yeast, see Growth Media for E. coli and Saccharomyces cerevisiae Growth Media. For preparation of plates and isolation of single colonies, see Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies.
5. PROTOCOL A CRYOPRESERVATION OF BACTERIAL CULTURES 5.1. Preparation Prepare the appropriate liquid media. Have ready a freshly grown plate with colonies or a fresh overnight culture of the bacterial strain to be frozen.
5.2. Duration Preparation
1 day
Protocol
Overnightþ 10 min
See Fig. 2.1 for the flowchart of Protocol A.
Figure 2.1 Flowchart of Protocol A.
Storage of Bacteria and Yeast
19
6. STEP 1A GROW BACTERIAL CULTURES 6.1. Overview Inoculate liquid media with bacterial cells from an isolated colony on a freshly grown plate or an existing culture and grow cells overnight.
6.2. Duration About 10 min þ overnight (to grow the bacteria) 1.1a Inoculate 5 ml of LB broth with bacteria from an isolated colony or an existing liquid culture. 1.2a Incubate overnight in a shaking incubator at 37 C, shaking at 250 rpm.
7. STEP 2A FREEZE THE BACTERIAL CELLS 7.1. Overview Bacteria are transferred into a glycerol-based medium for freezing and longterm storage.
7.2. Duration 10 min 2.1a Label a sterile cryovial with the name of the bacterial strain to be frozen and the date. 2.2a Transfer 812 ml of the bacterial culture into a cryovial. Add 188 ml of sterile 80% glycerol (to give a final concentration of 15% glycerol). Mix well. 2.3a Put cryovials in a freezer storage box at 80 C. For long-term storage, transfer the vials to a liquid nitrogen freezer.
8. PROTOCOL B CRYOPRESERVATION OF YEAST 8.1. Preparation Prepare the appropriate liquid media or agar plates. Have ready a freshly grown plate with yeast colonies of the strain to be frozen.
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Cheryl M. Koh
Figure 2.2 Flowchart of Protocol B.
8.2. Duration Preparation
1 day
Protocol
1–2 daysþ 10 min
See Fig. 2.2 for the flowchart of Protocol B.
9. STEP 1B GROW YEAST CULTURES 9.1. Overview Yeast cells from an isolated colony are grown either on a plate or in liquid culture.
9.2. Duration About 10 min þ 1–2 days to grow the yeast cells 1.1b To grow cells on a plate, streak the yeast cells from a single colony on a YPD plate. Invert the plate and incubate at 30 C for 2 days. 1.2b To grow cells in liquid culture, inoculate 5 ml of YPD media in a sterile culture tube with yeast cells from a single colony. Place on a roller drum or in a shaking incubator, and incubate overnight at 30 C.
Storage of Bacteria and Yeast
21
10. STEP 2B FREEZE THE YEAST CELLS 10.1. Overview Yeast cells are transferred into a glycerol-based medium for freezing and long-term storage.
10.2. Duration 10 min 2.1b Label a sterile cryovial with the name of the yeast strain to be frozen and the date. 2.2b If starting with cells grown on a plate, add 812 ml sterile deionized water and 188 ml sterile 80% glycerol to each cryovial, for a final concentration of 15% glycerol. Use a sterile micropipette tip to scrape up the yeast cells from the streak and transfer to the cryovial. Screw the cap onto the vial and vortex it to obtain an even distribution of cells. 2.3b If starting with cells in liquid culture, transfer 812 ml of the yeast culture into a cryovial. Add 188 ml of sterile 80% glycerol. Screw the cap onto the vial and vortex it to obtain an even distribution of cells. 2.4b Put the cryovials in a freezer storage box at 80 C. For long-term storage, transfer to a liquid nitrogen freezer.
REFERENCES Referenced Protocols in Methods Navigator Growth Media for E. coli. Saccharomyces cerevisiae Growth Media. Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies.
Storage of Bacteria and Yeast Cheryl M. Koh1 Department of Pathology, The 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 5. Protocol A Cryopreservation of Bacterial Cultures 5.1 Preparation 5.2 Duration 6. Step 1A Grow Bacterial Cultures 6.1 Overview 6.2 Duration 7. Step 2A Freeze the Bacterial Cells 7.1 Overview 7.2 Duration 8. Protocol B Cryopreservation of Yeast 8.1 Preparation 8.2 Duration 9. Step 1B Grow Yeast Cultures 9.1 Overview 9.2 Duration 10. Step 2B Freeze the Yeast Cells 10.1 Overview 10.2 Duration References
15 16 16 17 18 18 18 18 19 19 19 19 19 19 19 19 20 20 20 20 21 21 21 21
Abstract Yeast and bacteria can be cryopreserved and stored almost indefinitely. It is useful to prepare stocks for archival purposes.
1. THEORY Glycerol is used as a cryoprotectant as it disrupts the hydrogen bonding between water molecules, hence preventing the formation of ice Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00002-7
#
2013 Elsevier Inc. All rights reserved.
15
16
Cheryl M. Koh
crystals during freezing. This protects yeast and bacterial cells from ice crystal damage, and maintains their viability for future reculture. Cell viability decreases with each freeze–thaw cycle; thus, to recover yeast or bacterial cells from frozen stocks, avoid thawing the stock completely. Instead, scrape off a small amount of frozen culture and inoculate directly into liquid media.
2. EQUIPMENT Autoclave Shaking incubator Roller drum (optional) Incubator (37 C for bacteria, 30 C for yeast) Vortex mixer 80 C freezer Liquid nitrogen freezer (optional) Petri dishes (10 cm, sterile) 13-ml sterile polypropylene culture tubes Micropipettors Micropipettor tips Cryovials Freezer storage boxes
3. MATERIALS Glycerol Sterile deionized water Storage of bacteria: Yeast extract Tryptone Sodium chloride (NaCl) Storage of yeast: Sucrose Yeast extract Peptone Bacto Agar
17
Storage of Bacteria and Yeast
3.1. Solutions & buffers Step 1A LB Broth Component
Amount
Yeast Extract
5g
Tryptone
10 g
NaCl*
0.5–10 g
Add deionized water to 1 l. Sterilize by autoclaving *The salt-sensitivity of bacteria varies between strains. If antibiotics are to be added to the media, a low salt concentration (0.5 g) is preferable
Step 1B 20% sucrose
Dissolve 20 g sucrose in 80 ml deionized water. Adjust the volume to 100 ml. Pass through a 0.2-mm filter to sterilize
YPD Medium Component
Amount
Yeast extract
1g
Peptone
2g
Add deionized water to 900 ml and mix until no clumps remain. Sterilize by autoclaving. Add 100 ml sterile 20% sucrose
YPD Plates Component
Amount
Yeast extract
1g
Peptone
2g
Bacto Agar
20 g
Add deionized water to 900 ml and mix until no clumps remain. Sterilize by autoclaving and then cool to 55–60 C. Add 100 ml sterile 20% sucrose. Pour into sterile 10-cm Petri dishes, 10 ml per dish. Cover and allow agar to solidify
Step 2(A and B) 80% Glycerol
Mix 80 ml glycerol and 20 ml deionized water. Sterilize by autoclaving
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Cheryl M. Koh
4. PROTOCOL For a list of common culture media for bacteria and yeast, see Growth Media for E. coli and Saccharomyces cerevisiae Growth Media. For preparation of plates and isolation of single colonies, see Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies.
5. PROTOCOL A CRYOPRESERVATION OF BACTERIAL CULTURES 5.1. Preparation Prepare the appropriate liquid media. Have ready a freshly grown plate with colonies or a fresh overnight culture of the bacterial strain to be frozen.
5.2. Duration Preparation
1 day
Protocol
Overnightþ 10 min
See Fig. 2.1 for the flowchart of Protocol A.
Figure 2.1 Flowchart of Protocol A.
Storage of Bacteria and Yeast
19
6. STEP 1A GROW BACTERIAL CULTURES 6.1. Overview Inoculate liquid media with bacterial cells from an isolated colony on a freshly grown plate or an existing culture and grow cells overnight.
6.2. Duration About 10 min þ overnight (to grow the bacteria) 1.1a Inoculate 5 ml of LB broth with bacteria from an isolated colony or an existing liquid culture. 1.2a Incubate overnight in a shaking incubator at 37 C, shaking at 250 rpm.
7. STEP 2A FREEZE THE BACTERIAL CELLS 7.1. Overview Bacteria are transferred into a glycerol-based medium for freezing and longterm storage.
7.2. Duration 10 min 2.1a Label a sterile cryovial with the name of the bacterial strain to be frozen and the date. 2.2a Transfer 812 ml of the bacterial culture into a cryovial. Add 188 ml of sterile 80% glycerol (to give a final concentration of 15% glycerol). Mix well. 2.3a Put cryovials in a freezer storage box at 80 C. For long-term storage, transfer the vials to a liquid nitrogen freezer.
8. PROTOCOL B CRYOPRESERVATION OF YEAST 8.1. Preparation Prepare the appropriate liquid media or agar plates. Have ready a freshly grown plate with yeast colonies of the strain to be frozen.
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Cheryl M. Koh
Figure 2.2 Flowchart of Protocol B.
8.2. Duration Preparation
1 day
Protocol
1–2 daysþ 10 min
See Fig. 2.2 for the flowchart of Protocol B.
9. STEP 1B GROW YEAST CULTURES 9.1. Overview Yeast cells from an isolated colony are grown either on a plate or in liquid culture.
9.2. Duration About 10 min þ 1–2 days to grow the yeast cells 1.1b To grow cells on a plate, streak the yeast cells from a single colony on a YPD plate. Invert the plate and incubate at 30 C for 2 days. 1.2b To grow cells in liquid culture, inoculate 5 ml of YPD media in a sterile culture tube with yeast cells from a single colony. Place on a roller drum or in a shaking incubator, and incubate overnight at 30 C.
Storage of Bacteria and Yeast
21
10. STEP 2B FREEZE THE YEAST CELLS 10.1. Overview Yeast cells are transferred into a glycerol-based medium for freezing and long-term storage.
10.2. Duration 10 min 2.1b Label a sterile cryovial with the name of the yeast strain to be frozen and the date. 2.2b If starting with cells grown on a plate, add 812 ml sterile deionized water and 188 ml sterile 80% glycerol to each cryovial, for a final concentration of 15% glycerol. Use a sterile micropipette tip to scrape up the yeast cells from the streak and transfer to the cryovial. Screw the cap onto the vial and vortex it to obtain an even distribution of cells. 2.3b If starting with cells in liquid culture, transfer 812 ml of the yeast culture into a cryovial. Add 188 ml of sterile 80% glycerol. Screw the cap onto the vial and vortex it to obtain an even distribution of cells. 2.4b Put the cryovials in a freezer storage box at 80 C. For long-term storage, transfer to a liquid nitrogen freezer.
REFERENCES Referenced Protocols in Methods Navigator Growth Media for E. coli. Saccharomyces cerevisiae Growth Media. Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies.
