Animation link: https://vlab.amrita.edu/repo/BIOTECH/CEL/CellOrganizationAndSubcellular_prokaryotic_Eukaryotic/index.swf
Online procedure
- Choose any sample A or B.
- Clean the slide using soap and water. Dry the slide with the help of a tissue paper.
- Take the culture sample.
- Place a drop of water on the slide.
- Heat the inoculation loop till red hot. Allow it to cool sufficiently.
- Hold the Petri plate in the left hand, open it and pick an isolated colony.
- Mix with the water on the slide.
- Air dry the spot. And fix the smear by waving it over a flame.
- Pour 4 or 5 drops of stain over the smear.
- Keep for a minute.
- Wash off the excess stain.
- Blot dry.
- Place the slide on the stage of the microscope.
- Observe first at low power i.e., at 10x Objective lens.
- Adjust the diaphragm to the largest diameter, allowing the greatest amount of light to pass through.
- Adjust the coarse adjustment knob until the specimen is in focus.
- Adjust the fine focus.
- Observe the image.
- Then adjust the diaphragm to get the best lighting. Start with the most light and gradually lessen it until the specimen image has clear, sharp contrast.
- Observe the image formed.
- Rotate nose piece to 40x objective.( Lens with the blue band.)
- Then adjust the diaphragm to get the best lighting. Start with the most light and gradually lessen it until the specimen image has clear, sharp contrast.
- Observe the image formed.
- Partially rotate nose piece so that 40x and 100x objectives straddle the specimen.
- Place a small drop of oil on the slide in the center of the lighted area.
- Rotate nose piece so that the 100x oil immersion objective touches the oil.
- Focus only the fine adjustment.
- Identify the shape of the given bacteria and write down the answer.
- Choose the other sample and continue the procedure.
Real Experiment Scenario
Materials required
Clean glass slides, light microscope, Bacterial culture, inoculation loop, Bunsen burner, any of the basic dye(crystal violet, Carbol fuchsin or Methylene blue), dropper and wash bottle.
Procedure
- Clean the slide using soap and water. Dry the slide with the help of a tissue paper.
- Take the culture sample.
- A drop of water is placed on the slide.
- Heat the inoculation loop till red hot. And is sufficiently allowed to cool.
- Hold the Petri plate in the left hand, open it and pick an isolated colony.
- Mix with the water on the slide.
- The spot is air dried. And fix the smear by waving it over a flame.
- Pour 4 or 5 drops of crystal violet (or the selected stain) stain over the smear.
- Keep for a minute (the time period varies with the dye used).
- The excess stain is washed off.
- Blot dry.
- The slide is placed on the stage of the microscope.
- Observe at 10x, 40x and 100x.
Difference Encountered in a Real Laboratory
In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:
1. While inoculating agar media (plates and agar slants), make sure not to gouge or tear the surface of the agar with the loop.
2. Always label the slide and plates with:
1. The name of the organism
2. The type of media
3. Your initials
4. The date
3. Make sure to place agar plates in the incubator upside down. This prevents water condensation from dripping onto the agar surface and spreading over the colonies. This will also prevent contamination and aid in the formation of isolated colonies.
4. 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.
5. 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.
6. 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 inoculum 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.
7. 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.
8. Double-checking the name of the organism on the stock culture from which you are collecting your inoculums.
9. When removing the caps from tubes, always keep the caps in your hand. Never set them on the table, as they could pick up contaminants.
10. Always handle open tubes 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 inoculum, as this will help prevent environmental contamination.
11. Always flame the lip of a culture tube when you open it and before you replace the cap.
12. As soon as you are done inoculating, flame your loop or needle. Never place a contaminated tool on your workbench.
13. Discard all contaminated materials properly and return your supplies to the proper storage locations, and clean up your working area.
14. Always disinfect your work area when you are finished.
