2.2: Procedure

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Make sure you follow aseptic procedures and label everything carefully!

A. Observation of bacterial growth characteristics on selective and differential media

Each pair of students: 1 MSA and 1 EMB plate

Bacteria used: Staphylococcus aureus, Escherichia coli, Micrococcus luteus, and Pseudomonas aeruginosa

Inoculation of selective and differential media

1. Divide the MS and EMB agar plates into 4 areas. Label each area with the type of bacteria that will be streaked there.

2. Use the inoculation loop to make a single streak of S. aureus, E. coli, M. luteus, and P. aeruginosa in each of the 4 areas (see below).

3. Incubate the plate until the next lab period

B. Inoculation for single colony isolation

Each pair of students: 4 TSA plates

1. Sterilize your inoculating loop.

2. Use your loop obtain some bacteria from your sample.

3. Streak the bacteria on a small portion of the plate.

4. Sterilize your loop.

5. Cool the loop on an uninoculated part of the agar surface, and then pass it once through the already-streaked region of the plate. Streak another small portion of the plate.

6. Sterilize your loop. Cool the loop, then pass it once through the second streak, and make a third streak. Depending on the density of the original sample, single colonies will usually appear by the second or third streak.

Use the procedure outlined above to streak S. aureus, E. coli, M. luteus, and P. aeruginosa for single colonies onto individual TSA plates. Remember that you are trying to isolate single cells or a cluster of cells to form individual colonies. Do not pick up too much bacteria or spread it evenly all throughout the plate! Incubate until the next lab period.

C. Isolation of single colonies from mixed cultures using the streak plate method

Each pair of students: 1 MSA and 1 EMB plate

Mixed cultures: Unknown A and Unknown B

1. Streak Unknown A for single colonies on an MSA plate.

2. Streak Unknown B for single colonies on an EMB plate.

3. Incubate until the next lab period.

D. Culturing Microorganisms from Environmental Samples

Each pair of students: 1 MSA plate, 1 EMB plate, 1 TSA plate; 1 tube of sterile water; 1 sterile swab.

1. Pick one part of the environment that you would like to sample. Possibilities include: bottom of shoe, floor, cell phone, sponge on table, or the bottom of your handbag or backpack. Be inventive—pick an area you would find interesting! Communicate with your lab partners so that different environments are represented at your table.

My environmental sample is from: ___________________________________

2. Moisten a swab in the sterile water; before the removing the swab, press it against the side of the tube to remove excess moisture.

3. Firmly swab the region you would like to sample.

4. Use the swab to inoculate the TSA plate, streaking the entire surface of the plate (see below). Use the same swab to streak the MS plate and the EMB plate.

5. Dispose of the swab in designated plastic container at your lab table. (DO NOT throw swabs in the regular garbage!)

6. Incubate the plates until the next lab period.

Figure 2.3.3: *Environmental sample plates*