Materials Required:
- Nutrient broth
- Sterile petriplates
- Micropipettes
- Cuvette
- Conical flask
- Sterile tips
- Culture – Overnight culture of Staphylococcus aureus
- Colorimeter
Procedure:
- An isolated colony of the organism (Staphylococcus aureus) was inoculated into 15 ml nutrient broth and kept for overnight incubation
- Following day, the OD of this culture was measured and confirmed.
- In order to adjust the OD of the inoculum to the standard value (0.05) the following dilution formula was used
OD1V1 = OD2V2
Where,
OD1 = OD of the broth culture, inoculated the previous day.
V1 = volume of this broth culture to be added to the inoculums
OD2 = OD of the inoculum (as a standard, this value was adjusted to 0.05)
V2 = volume of the inoculums (in this experiment, 50 ml)
- Substitute the values in the equation and V1 was calculated.
- That much amount (V1) of the inoculums was pipetted out before adding an equivalent amount of the broth to it, so that the net volume remains constant.
- The OD was checked at every 30 minutes interval and recorded.
- Using this OD value, a standardized growth curve of the organism was plotted. (Absorbance verses time).
- Generation time was calculated.
Differences encountered in a Real Laboratory:
In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:
- Always wear lab coat and gloves when you are in the lab. When you enter the lab, switch on the exhaust fan and Laminar Air Flow.
- Make sure media required for the experiment are available. If it is not available, prepare the media and autoclave it.
- Always label the plates and conical flask with:
1. The name of the organism
2. The type of media
3. Your initials
4. The date
- Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone; it is not a large plume, nor is it orange.
- While flaming the loop (or needle), be sure that each segment of metal glows orange/red-hot before you move the next segment into the flame. Be careful; the metal will get extremely hot.
- Once you have flamed your loop (or needle), do not lay it down, blow on it, touch it with your fingers, or touch it to any surface other than your inoculums or the sterile media. If you do touch the tip to another surface or blow on it, you will have to re-flame the loop before you proceed with your inoculation.
- Allow your loop or needle to cool before you try to pick up your organism. If you pick up organism with a hot tool, your cells will be killed. To cool your loop or needle quickly, place it on a section of agar that is uninoculated or is at least different from the area from which you will remove cells.
- Ensure that you are transferring the correct organism into your labeled conical flask by double-checking the name of the organism on the stock culture from which you are collecting your inoculums.
- When removing the caps (cotton plug) from conical flask, always keep the caps (cotton plug) in your hand. Never set them on the table, as they could pick up contaminants.
- Always handle open flasks at an angle; never let them point directly up, since airborne or other environmental organisms could fall into the tube and cause contamination. Also, keep the lid over a plate when removing inoculums, as this will help prevent environmental contamination.
- As soon as you done inoculating, flame your loop or needle. Never place a contaminated tool on your workbench.
- Always flame the lip of the conical flask when you open it and before you replace the cap.
- As soon as you are done inoculating, flame your loop or needle. Never place a contaminated tool on your workbench.
- While operating the colorimeter, make sure that water droplets do not enter the slot of the colorimeter as it will damage the instrument. So wipe the cuvette using a lint free tissue paper before inserting it into the slot.
- Do not touch the part of the cuvette that gets inserted into the colorimetric slot with hands and ensure that the cuvette is free from water droplets.
- Discard all contaminated materials properly and return your supplies to the proper storage locations, and clean up your working area.
- Always disinfect your work area when you are finished.
Help:
Unfortunately, this Virtual Lab requires Adobe Flash player. Please see additional information if this does not work on your computer
• For Google Chrome - https://support.google.com/chrome/answer/6258784?co=GENIE.Platform%3DDesktop&hl=en
• For Microsoft Edge - https://support.microsoft.com/en-in/help/4532571/microsoft-edge-turn-on-flash
• For Mozilla Firefox - https://support.mozilla.org/en-US/kb/install-flash-plugin-view-videos-animations-games
On GNU/Linux machines, please see appropriate online help. For example,
• on Ubuntu - https://help.ubuntu.com/stable/ubuntu-help/net-install-flash.html.en
• on Fedora - https://docs.fedoraproject.org/en-US/quick-docs/using-adobe-flash/
