Serial Dilutions and Plating: Microbial Enumeration

1. Set-up

1. A flow chart listing all materials, stepwise experimental protocol and method for discarding supplies should be written in a laboratory notebook and kept near the experimental workspace.
2. Workspaces should be sterilized with an appropriate antiseptic (70% ethanol) and the experimenter should mitigate contamination risk by wearing clean laboratory garments that also protect them from exposure anomalies. Suitable garments include but are not limited to a lab coat, latex or nitrile gloves, googles, respirators, and closed shoes. It is critical to maintain aseptic technique at all times.
3. Prepare 90 mL of 0.45% saline. Using a clean graduated cylinder, measure 90 mL of sterile water and transfer it to a clean Erlenmeyer flask labeled 0.45% saline. Weigh .405 g of sodium chloride (Sigma-Aldrich NaCl S9888) and add it to the flask labeled 0.45% saline. Swirl repeatedly until no solute remains visible.
4. Upon completion, the experimenter should re-sterilize all surfaces and discard any unwanted organisms, diluent stocks, petri dishes, or disposable inoculating loops according to OSHA guidelines. Laboratory garments can be removed before washing hands.

2. Media Preparation

1. Select media that is appropriate for cultivation of a desired organism. In most scenarios, a broth would enable sufficient bacterial growth. Since organisms from a Winogradsky protocol are desired here, a column consisting of calcium carbonate, sulfur, cellulose and mud was assembled and left undisturbed for 7 days. The forenamed column is separated into aerobic, microaerophilic, and anaerobic sections.
2. Choose a medium appropriate for plating the organism of interest. Supplementation of liquid media with microbiology grade agar is typically employed as a solidifying agent. LB medium/agar is sufficient when harvesting samples from aerobic, microaerophillic and anaerobic regions of the forenamed column. Note: Samples from the microaerophillic region were not harvested for this procedure. However, these organisms should be cultivated in candle jars. Introducing a candle to this cultivation chamber before sealing creates a low oxygen environment that is suitable for microaerophilic proliferation.
3. Since we wish to prepare 250 mL, use 500 mL (or larger) Erlenmeyer flasks to prevent boil-over when autoclaving. Label one "Broth" and the other "Agar."
4. Determine the amount of media required to create each solution by following the manufacturer concentration recommendations. LB Agar, used here, is prepared by combining 25g/L with ultrapure water. Our volume of 250 mL requires a solution of 6.25 LB Agar/250 mL water. Similarly, LB Broth is prepared by combining the same ratio of LB Broth and water. Since it is not supplemented with a solidifying agent, it will not harden when cooled.
5. Weigh the media and mix it with water in proportions consistent with manufacturer recommendations. Add 6.25g of LB Agar to a flask labeled "Agar", and 6.25g of LB Broth to a flask labeled "Broth." Add 250 mL ultrapure water to each flask.
6. Wrap aluminum foil over each flask and use an autoclave to sterilize media for a minimum of 15 minutes at 121°C, 15 psi.
7. Using a heat resistant glove or pad, remove the flasks from the autoclave when the cycle is complete and place them in a 40-50°C water bath.
8. Once the appropriate temperature has been reached, pour the contents of the flask labeled "Broth" into a 250 mL Erlenmeyer, or round-bottom flask. Label the 250 mL flask "solution₀".
9. Obtain 10, 100mm x 15 mm sterile petri dishes and label them with the date, name, the type of media used and the Winogradsky column zone that organisms will be harvested from.
10. Remove the flask labeled "Agar" from the water bath and begin pouring into each of the 10 petri dishes. No more than 15 mL should be added to each dish. This may also be performed with a pipettor and 25 mL serological pipette to improve accuracy. Use a sterile pipette tip to remove any bubbles present, and then cover with the plate lids and leave to solidify overnight.

3. Diluent Preparation

1. Prepare ten test tubes capable of storing 20 mL or more in a rack and label them T1-T10. Each tube number is consistent with the dilution factor it corresponds to (i.e., T4 = 1x10^-4 or 0.0001 or 1/10,000th of stock concentration).
2. Pipet 9 mL of 0.45% saline into each of the 10 test tubes.
3. Saline blanks are now ready to be sterilized by autoclave. Use aluminum foil to cover each of the 10 test tubes and then transfer them to an autoclave compatible test tube rack. Sterilize for a minimum of 15 minutes at 121°C, 15 psi.
4. Remove blanks using heat resistant gloves and allow to cool. Cover and store at 4°C until needed when tubes have reached room temperature, or when cool to the touch.

4. Cultivation of Target Organism

1. Inoculate "solution₀" with a single colony from a previously streaked plate or 50 µL of a frozen stock. Allow the target organism time to replicate by placing inoculated "solution₀" into a 37°C incubator overnight with shaking (if necessary). (Note: The flask should be covered to prevent contamination. If the target organism is aerobic, use sterile gauze and cotton plugs to prevent contamination. If evaluating regions of Winogradsky column, simply remove 1 gram from each desired zone (aerobic and anaerobic for the purposes of this study) and resuspend in T1 before proceeding to step 5.3.

5. Serial Dilution

1. Obtain the flask labeled "Nutrient broth" from the incubator and shake vigorously.
2. Pipet 1 mL of "solution₀" into the test tube labeled T1. Vortex T1. If evaluating Winogradsky explants, weigh 1 gram of the desired zone and add it to T1 prior to vortexing. (Note: 1 mL is used here for simplicity- smaller or larger volumes of diluent may also be used).
3. Remove 1 mL from test tube T1 and add it to test tube T2. Vortex T2.
4. Remove 1 mL from test tube T2 and add it to test tube T3. Vortex T3.
5. Remove 1 mL from test tube T3 and add it to test tube T4. Vortex T4.
6. Remove 1 mL from test tube T4 and add it to test tube T5. Vortex T5.
7. Remove 1 mL from test tube T5 and add it to test tube T6. Vortex T6.
8. Remove 1 mL from test tube T6 and add it to test tube T7. Vortex T7.
9. Remove 1 mL from test tube T7 and add it to test tube T8. Vortex T8.
10. Remove 1 mL from test tube T8 and add it to test tube T9. Vortex T9.
11. Remove 1 mL from test tube T9 and add it to test tube T10.

6. Spread Plating

1. Pipet 100 µL of a diluted sample from T1 directly onto a petri dish. This step can be, but does not need to be, repeated for each tube.
2. Obtain a sterile, disposable spreading rod or flame sterilize a glass spreading rod. In a clockwise/counterclockwise motion, glide the horizontal portion of the spreading rod to equally distribute the sample within the petri dish.
3. Repeat for each zone of the Winogradsky column that is to be evaluated.
4. Incubate plates in a 37°C incubator for 24 hours. For anaerobic organisms, use an anaerobic chamber.

7. Streaking

1. Select an appropriate dilution of your target organism. For example, solution₄ will yield a 1/10,000th dilution of your initial concentration. Typically, dilutions of 1/1,000^th (T3/Solution), 1/1,000,000^th (T6/Solution₆) and 1/1,000,000,000^th (T9/Solution₉) are evaluated to enumerate microbes.
2. Using a plastic sterile disposable inoculating loop or a reusable metal inoculating loop that has been under fire for no less than 10 seconds, immerse in the desired solution from step 5. Calibrated inoculating loops should transfer 0.01 mL. (Caution: Do not allow flamed loop to contact bacteria immediately after removing from heat)
3. Slightly raise the petri dish cover on one side so that only the inoculating loop can access the agar. Glide the inoculating loop across the top of the media in a zig-zag fashion being careful not to compromise the agar. Lower the petri dish lid.
4. Use a new disposable inoculating loop or re-sterilize your reusable loop.
5. Rotate the plate by roughly 1/3^rd (~118°) and reduce the frequency of zig-zag motion.
6. Again, use a new disposable loop or re-sterilize a metal loop before rotating a final time and reduce zig-zag frequency once more. Lower the petri dish lid.
7. Repeat steps 7.2 - 7.6 until at least three petri dishes have been streaked for three different dilutions, using a new disposable loop or by re-flaming a reusable loop (Figure 2).
8. Place streaked petri dishes in a 37°C incubator overnight. For anaerobic organisms, use an anerobic chamber.

8. Data Analysis and Results

1. Cultures were harvested from the oxic and anoxic zones of a 7-day Winogradsky column. These zones are suitable for heterotrophic and iron-oxidizing anaerobes, respectively.
2. Column explants were serially diluted prior to streaking or spreading on LB Agar plates.
3. Streaking revealed a mixed population from each of the Winogradsky zones evaluated. Spread plates produced similar results.
4. To calculate CFU/mL or CFU/g, average the number of counted colonies from three plates. Multiply the average number of colonies by the dilution factor and divide by the amount aliquoted. For example, if an average of 65 colonies were counted on plates inoculated with 0.1 mL of solution₆ (T6) the formula described previously would equal 650,000,000 CFU/mL.
5. Isolated colonies can now be chosen from each plate for use in enrichment assays to determine species identity.