Calculations Calculate the amount of sodium acetate needed to make 30 mL of Procedure Make sure to wear goggles and gloves. Transfer the sodium acetate into a 50 mL conical tube.
Add about 20 mL of DI water into the conical tube. Secure the cap on the tube and invert to mix the contents until the solute is completely dissolved.
Pour out all of the solution into a 50 mL graduated cylinder. Add DI water to bring the total volume to Transfer all of the solution back into your 50 mL conical tube and secure the cap.
Invert the tube several times to thoroughly mix the contents. Label the tube with contents Verify your work by creating a buffer solution Pipette exactly 5. Pipette exactly 5. Secure the cap on the conical tube or a piece of parafilm over the test tube opening.
Invert several times to thoroughly mix the 10 mL of solution into an acetate buffer. Measure the pH of the test buffer solution using a calibrated pH meter.
If you were accurate in all of your work, the test buffer should have a pH of 4. Check in with your instructor and report the pH of your test buffer. If your test buffer pH is within the expected range , then congratulations!
You have verified that the sodium acetate solution you made earlier has a concentration of Give your 50 mL tube of remaining sodium acetate solution to your instructor to save for use in a future lab. Part II: Preparation of a Standard Curve In this part of the lab, we will be preparing solutions of known concentrations.
This new percentage concentration is equivalent to 5. Very accurately pipette Secure the cap on the tube and invert repeatedly to thoroughly mix the solution. Pipette 2. Seal the tube and invert repeatedly to mix.
What is the concentration of your new solution? Pipette 4. Pipette 6. Pipette 8. Measuring Absorbance of Methylene Blue Working Solutions Turn on the spectrophotometer and let it warm up for at least 10 minutes. Place 1 mL of DI water into a clean cuvette. This is your blank. Place 1 mL of your methylene blue solutions into clean cuvettes. These are your samples. Set the wavelength of the spectrophotometer to nm. Place the blank into the spectrophotometer.
Place your first sample into spec and record the absorbance reading. Do not press any buttons. Repeat with each sample and record into lab notebook. To cultivate a known target organism, E. Then, cover the tube and incubate it over night at 37 degrees Celsius. To evaluate the regions of a Winogradsky Column, add approximately one gram of material from the aerobic zone to T1 and resuspend by vortexing.
Then, repeat this process with one gram of material from the anaerobic zone. Remove the tube containing solution zero inoculated with E. Then, pipette one milliliter of the solution into a T1 test tube and vortex to mix. Remove one milliliter of solution from T1 and transfer it to T2, vortexing to mix. Repeat this process through tube T To evaluate the aerobic and anaerobic zones of the Winogradsky Column, remove one milliliter of solution from each of the previously prepared T1 tubes and transfer it to the appropriate T2 tubes.
Then, continue the serial dilutions through the T10 tubes as previously demonstrated. To spread plate, pipette microliters of the diluted sample from each T3 tube on to the corresponding petri dish. Then, use a sterile spreading rod to gently distribute the sample on to the petri dish and replace the plate lid.
Repeat this process for the T6 and T9 dilutions, as previously demonstrated. Incubate the plates containing aerobic organisms in a 37 degree Celsius incubator for 24 hours. Incubate the plates containing anaerobic organisms in an anaerobic chamber set to 37 degrees Celsius for 24 hours.
The next day remove the T3, T6, and T9 dilution plates from the incubator and the anaerobic chamber and transfer them to the bench top. Working with one plate at a time, glide a sterile inoculating loop across the top of the media in a zig-zag pattern. Then, replace the petri dish lid. Repeat this streaking method for the remaining plates, as previously shown.
Then, place the streaked plates containing aerobic organisms in a 37 degree Celsius incubator overnight and the streaked plates containing anaerobic organisms in an anaerobic chamber set to 37 degrees Celsius overnight. Cultures were harvested from the aerobic and anaerobic zones of a seven day Winogradsky Column. Then, the cultures were serially diluted prior to streaking and spreading on LB agar plates.
Streaking revealed a mixed population from each of the evaluated Winogradsky zones, and the spread plates produced similar results. A plate streaked from a mixed population will result in bacterial colonies of different shapes, sizes, textures, and colors. In contrast, the streaked and spread plates containing the known organism, E. Generally, it is best to calculate CFUs per milliliter using the average colony count of three plates spread with the same sample and dilution factor.
Multiply the average number of colonies by the dilution factor and divide by the amount aliquoted. Finally, isolated colonies chosen from each plate can be used in further enrichment assays to determine species identity.
Bacterial enumeration and strain isolation by plating requires manageable concentrations of target organisms. Successful plating is therefore contingent upon serial dilution.
As such, the aforementioned techniques remain the cornerstone of microbiological examination and experimentation. Though simple by design, dilution factors and plating technique can be modified to by the experimenter to bolster outcomes without compromising the integrity of each method. Plotting the four phases of bacterial growth can be helpful when characterizing desired microbes. These phases, lag, log, stationary, and death, are marked by changes in bacterial replication.
The lag phase features slow growth due to physiological adaptation, the log phase is the period of maximum proliferation featuring an exponential rise in viable cells, stationary phase is then reached due to environmental limitations and accumulations of toxins, before the death phase where cell counts begin to fall.
This can be accomplished by serially diluting or 1-step diluting to avoid confusion Solution 0 every hour for a total of 8 hours, beginning at Time 0 Solution 0 should be returned to a shaking incubator after each dilution. Repeat until each time Time 1 -Time 8 are plotted on the X-axis. Serial Dilutions and Plating: Microbial Enumeration.
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Login processing Previous Video Next Video. Overview Source: Jonathan F. Corbo 1 1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, Quantitative assessment of prokaryotes can be onerous given their abundance, propensity for exponential proliferation, species diversity within a population, and specific physiological needs.
Set-up 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. 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.
Prepare 90 mL of 0. Using a clean graduated cylinder, measure 90 mL of sterile water and transfer it to a clean Erlenmeyer flask labeled 0. Swirl repeatedly until no solute remains visible. 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.
Media Preparation 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. 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. 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.
Since we wish to prepare mL, use mL or larger Erlenmeyer flasks to prevent boil-over when autoclaving. Label one "Broth" and the other "Agar. Our volume of mL requires a solution of 6. Since it is not supplemented with a solidifying agent, it will not harden when cooled. Weigh the media and mix it with water in proportions consistent with manufacturer recommendations. Add 6. Once the appropriate temperature has been reached, pour the contents of the flask labeled "Broth" into a mL Erlenmeyer, or round-bottom flask.
Label the mL flask "solution 0 ". Obtain 10, mm 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.
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 becomes a much greater advantage when the span of the calibration standards must cover several orders of magnitude in concentration. Sean Lancaster has been a freelance writer since Lancaster holds a Doctor of Philosophy in chemistry from the University of Washington.
Definition of Endpoint Titration. How to Calculate Concentration With a Spectrophotometer. How to Calculate With the Taylor Series. How to Calculate the pH Effect of Dilution. Differences in Lab Glassware. How to Find the Midpoint of the Interval. How to Solve for Range.
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