• Agarose Gel Electrophoresis
• Bacterial Streak Plate
• Bacterial Transformation
• DNA Ligation
• PCR
• Pipettors
• Plasmid DNA Isolation and Restriction Enzyme Digests
• Preparation of Agar Plates

• Laboratory Citizenship & Performance
• BioSciences Lab Program Honor Code
• Molecular Biology Tips
• Writing Up Methodology

• *LAB SAFETY*
• Laboratory notebook

• Biuret Protein Assay
• Experimental Biosciences

Bacterial Streak Plate

Growth and Check of Bacterial Strains

Bacteria can be propagated on liquid or solid media. The use of liquid allows large quantities of bacteria to be harvested but does not permit easy selection or determination of phenotype of single cells. The technique of "streaking" cells onto a solid media provides simple isolation of colonies arising from single cells. Colonies selected for the desired phenotype are then used to inoculate liquid broth. A single colony inoculum is preferred because bacteria can undergo many types of mutations naturally. The instability of some of the mutations, especially transposons and phages, can allow some cells to lose characteristics important to the selection scheme and may complicate the analysis. It is always wise to check the parent strain for proper phenotype that reflects the genotype and then use "picks" from single colonies to start liquid cultures.

Aseptic removal of toothpicks

PROTOCOL (dispose of toothpicks in biohazard bag, not in regular trash)

NOTE: plates have been prepared for you; the information about the media is included here so you will know the contents.
Recipe for Luria-Bertani (LB) Agar (per liter):
1% Bacto-Tryptone = 10 g
0.5% Yeast extract = 5 g
85 mM NaCl = 5 g
1.5% Bacto Agar = 15 g

*adjust pH to 7.0 with NaOH and bring to 1 L; sterilize by autoclaving for 20 minutes at 121°C at 15 psi
**autoclaved media is always cooled to 50-60°C prior to the addition of antibiotics and some salts that are inactivated or precipitated by autoclaving; holding the agar at this temperature prevents congealing

Antibiotic for selection:
STOCK = 20-50 mg/ml in water (stored at - 20°C) was added to the LB agar in a 1:1000 ratio (1 µl of antibiotic for every 1 ml of solution) just before the plates were poured

Obtain a plate. The first thing you should do is label your plate with the plasmid ID, the bacterial strain, today's date, and "who" streaked the plate--put your "Team #" on each plate in your group. Label the bottom, since lids can readily be separated and mixed up.

Single colonies can be produced in the following manner.

Touch a clean toothpick to the surface of the culture solution and lightly streak the culture onto surface of the plate (LB-kan) as indicated in the figure.  Many passes in a zigzag motion are recommended but do not overlap from line to line.
Remove a sterile toothpick from the tube and lightly pull the end of the pick through a single portion of the preceding streak.  Continue a zigzag pattern on the plate being careful NOT to overlap with the previous streak.
Repeat the above step lightly crossing a single portion of the second streak as indicated in the figure.
Invert the plates and incubate at 37°C overnight. Stack all of the plates for your team.

Examine your “streak” plates and record your observations in your notebook.  Did you get single, well-isolated colonies?  If not, how might you modify your technique next time to obtain single colonies?

We would like to thank New England Biolabs for their generous support of our laboratory program