The procedure for making a smear is as follows:. If more than one culture is to be examined using the same stain, it is possible to prepare up to 6 smears on the same slide.
Before preparing the slide, divide it into the appropriate number of sections and clearly label each section on the underside of the slide. If your culture has been grown on a agar slant or agar plate.
Place a small drop of water on a clean, grease-free slide. Next, using a sterile loop or straight wire needle, transfer a bit of the growth to the drop of water and rub the needle around until the material is evenly emulsified. Spread the drop over a portion of the slide to make a thin film. The suspension should be only slightly turbid. If you are using a broth culture, the broth culture must have clearly visible turbidity. Transfer a loopful of culture from the broth onto a clean grease free slide.
Allow the film to air-dry. To get a good stain, it is important to let the smear dry completely. Basic stains, having a positive charge, bind strongly to negatively charged cell components such as bacterial nucleic acids and cell walls. Stain by covering the smear completely with methylene blue. This should be done over a sink with a slide holder. Tilt the slide to allow the stain to drain off.
Now, rinse the remaining dye off with a gentle stream of water from a faucet or wash bottle. Blot the slide dry with bibulous paper. Remove excess water from the slide by touching one corner of the slide to the blotting paper, then place the slide between clean sheets of paper in the blotting pad and blot dry.
Be sure you do not rub off the smear. Examine under the microscope using first the 10X and then the X oil-immersion objective.
Check your "test plates" from Lab1: Exercise I, part D ubiquity of microorganisms for isolated single colonies to be candidates for your test plate isolate. Attempt a simple stain of your candidate test plate isolate as described in part B of Exercise II. Record your observations. Show an instructor your isolated single test plate isolate and the slide of the simple stain of the specimen you have chosen.
This is to ensure you will be working with a bacterial culture instead of a fungal isolate. If you did not obtain any single colony bacterial isolates on your "test plates" use one from another student in the lab, but make sure you choose a different isolate than that student has chosen, or use one from the instructor's plates.
After picking a candidate for your test plate isolate it is necessary to make sure it is a pure culture. Use a new Nutrient agar plate to re-streak for single colonies using the technique from Exercise I, part C the "T" streak.
Make certain you try and touch only one colony, the one you are interested in using as your unknown. Save your original plate. Incubate until the next laboratory period. You have just prepared slides of Bacillus a rod shape and Micrococcus a coccus.
We have set up a slide to demonstrate a third major cellular shape seen in bacteria. Spirillum volutans is a spiral shaped microorganism found in fresh water. View the demonstration slide and record your observations in the Results page.
Watch Video 2: on Gram Staining. Video filmed at NC State. Learning Outcomes Prepare microorganisms for microscopic observation. Observe the difference in size between bacteria and other unicellular microorganisms. Perform a simple stain and a Gram stain. Observe stained microorganisms and identify their size, shape, and staining properties.
Materials Clean microscope slides Staining trays and newspaper Water bottle for rinsing Bacterial cultures: Escherichia coli , Staphylococcus aureus , Micrococcus luteus , Pseudomonas aeruginosa ,. How to make a Bacterial Smear 1.
Gently mix the bacteria into the water drop. Staining Bacteria. Photographic guides to differential stains. Bacterial smear after heat fixing and before staining. Positive bacterial control in circle on the left, negative control on right, and unknown bacteria in center circle. Science Prof Online.
Virtual Microbiology. MICRO 8wk. MICRO 15wk. Instructor's Corner. Cell Biology.
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