Measuring Absorbance in Streptococcus

Day 1: Streaking Bacteria From Freezer Stocks

• Refer to the strain database to determine which strain you plan to remove from the -80 degree Celsius freezer stock.

1.) Practicing Bio safety 2 protocols, bring an ice box to the -80 degree Celsius freezer (located in S223 instrument room). Obtain desired strains and put in the ice box.

2.) Under a sterile hood, use an inoculating loop to pick the desired strain. Streak out onto pre-labeled blood plate (date, strain number, and strain code).

3.) Dispose of contaminated inoculating look in 5% virkon S solution.

4.) Place streaked blood plates face down in the 37 degree Celcius incubator and allow them to incubate overnight.

Day 2: Overnight Culture

• All work done in the hood while practicing sterile technique.

1.) Obtain autoclaved empty culture tubes and Todd Hewitt Broth (THB).

2.) Pipette 3ml of THB into culture tubes. One tube will be used as a control/blank and all other tubes will be used to culture individual strains overnight.

3.) Remove culture plate from the 37-degree Celsius incubator. Place under a sterile hood.

4.) Label each culture tube with the corresponding strain number on the blood plate.

5.) Using an inoculating loop, pick a single colony from the blood plate. Place loop in corresponding culture tube and move loop up and down in the THB media to mix. Place loop in the Virkon S for disposal.

6.) Repeat step 5 for all other samples.

7.) Place culture tubes/ plates back into the 37-degree Celsius incubator and allow to sit overnight.

Day 3: Spectrophotometry, Absorbance (Abs), and Dotting

1.) Turn on the WP-120 Spectrophotometer. Wait 15 minutes for the lamp to fully warm up.

2.) Set wavelength to 600 and make sure the machine is in Abs Mode.

3.) While waiting for the lamp, remove several blood plates from the fridge. These plates will be used to dot serial dilutions at different time intervals for each sample. Label each plate with the time interval and sample code.

4.) Make 1X PBS from 10X stock. a. For a 200ml solution: 180ml MilliQ water and 20ml of 10X PBS.

5.) Begin prepping the serial dilutions. Using sterile 2ml 96 well-plate, add 900uL of 1X PBS into each well.

6.) After 15 minutes, vortex the “blank” culture tube (containing just THB) and insert it into the spectrophotometer. Press the up arrow (0A/100% T). This will blank the machine.

7.) Vortex samples, insert into the spectrophotometer, and take readings. Record online in Google Sheets the Abs recording and paste copy of the final results into your lab notebook for later analysis. a. Note: Want initial Abs to be 0.05. Overnight culture tubes will be saturated from 18-24hrs of growth. Thus, you may have to make fresh culture tubes with 3mL THB. Add in 100-200uL increments from original overnight culture to fresh culture tube until Abs is approximately 0.05. Record the Abs.

8.) To dot onto the blood plate, pipette 10uL from culture tubes and plate in corresponding location (Figure 1). Remember to vortex culture tube before pipetting!

9.) Remove 100uL from fresh culture tube and place in the corresponding location on 96-well plate. This will create a 10-1 dilution. Remember to vortex culture tube before!

10.) Place culture tubes back into the 37-degree incubator and let sit for another hour.

11.) Now, finish creating the serial dilutions. To create the next serial dilution, pipette 100uL from the 10-1 dilution and place it into the next well with 900uL 1X PBS. This will create a 10-2 dilution. Repeat this process to end with a 10-6 dilution.

12.) Take 10uL from each dilution and pipette onto corresponding location on the culture plate.

13.) Once plate dries, place it into the 37-degree Celsius incubator.

14.) After an hour, repeat the Abs measurements and dotting procedures.

15.) Once compete, put the red autoclave waste bag into the “to be autoclaved container” (the bag should be full of pipette tips).

16.) Clean 96-well plate in a beaker full of Virkon S. Additionally pour out each culture tube into a separate beaker full of Virkon S. Make sure that all culture tubes are fully submerged in the Virkon S to ensure that all parts of the tubes are disinfected. After an hour, rinse the 96-well plate really well with water and dispose of the glass culture tubes in the glassware container.

Alternative Dotting Procedure: Strains on Individual Blood Plates

1.) To create a dilution of the strain under Abs examination, remove 100uL from the fresh culture tubes and place in the corresponding location in the 96-well plate (containing 900uL of 1X PBS). This will create a 10^-1 dilution. Remember to vortex culture tube or mix sample before pipetting.

2.) To create the other serial dilutions, pipette 100uL from the 10^-1 dilution and place in the next well with 900uL of 1x PBS. This will create a 10^-2 dilution. Repeat this process and end with desired dilution.

3.) Place 6-8 autoclaved beads in the blood agar plate.

4.) Pipette 100 ul from the selected dilution and place on labeled blood plate.

5.) Shake beads in a perpendicular motion to distribute sample throughout the plate.

• • • Note: Some strains had ambiguous initial CFU counts and may require two blood plates with samples taken from two different dilutions. This will ensure that a reasonable number of CFUs grow overnight. Additionally, it is not required to place the control when plating onto individual blood plates.

6.) Wait 10-20 min for sample to absorb in agar.

7.) Turn blood plate upside down (Lid is facing the ground). Give small taps to the plate in order to get all the small beads to rest on the lid.

8.) Incubate the blood plates upside down in the 37°C incubator overnight.

Experiment should be replicated in triplicate. However, due to time constraints, we only performed the experiment once under the assumption that the results would be reproducible.

Day 4: Counting CFUs

1.) Obtain strains from the 37 degree Celsius hood.

2.) Place all plates under a sterile hood.

3.) Open the lid to one of the blood plates and count the number of CFUs. Record on the Laboratory Ipad.

a. For CFUs plated in a dilution series (on the SAME blood plate) count CFUs up to ~50 CFUs. 
b. For CFUs plated onto individual blood plates, an ideal number of CFUs is in the range of 20-200 CFUs. For anything above ~200 CFUs, you can count the CFUs by counting one quadrant and multiplying by 4. 
c. If cells are too abundant, record as TMTC (Too Many to Count). 

3.) Repeat step 3 until ALL CFUs have been recorded for each sample.

4.) Dispose of blood plates in the red bio-safety containers.