Transcription
Certain bacterial structures can be examined using stains specific for the structure. Endospores are produced by several genera such as Bacillus and Clostridium and can be identified by the endospore stain. Endospores and intracellular inclusions such as lipids or volutin don't stain in the Gram stain.
We will be working with a previously made fixed smear of the genus Bacillus. Cover the smear with a small piece of paper towel to prevent it from drying out. Endospores are difficult to stain, so heat will be used to drive the stain into the endospore.
Place the slide on a boiling water bath. Cover the paper towel with malachite green. Steam the slide for five minutes. You may need to stain the slide longer depending on the reagents used in your lab. Add more stain as needed to keep the paper towel covering the slide wet.
Use forceps to gently lift the paper towel from the slide. Discard the piece of paper towel properly. Remove the slide from the steam and let the slide cool for approximately 1 minute. After the slide has cooled, wash the smear with distilled water. Hold the slide at an angle and run the water over the slide until the water runs clear.
Cover the smear with safranin for 30 seconds to stain the cells. You may need to stain the smear for longer than 30 seconds depending on the reagents used in your lab. Wash the smear with distilled water. Hold the slide at an angle and run the water over the slide until the water runs clear. Gently blot the slide dry. Do not rub the slide, just gently blot.
Examine the slide under the microscope. Endospores will be green structures inside red cells. Some endospores may have been released from cells. The endospores will be uniform in size and shape. Green debris will be various shapes. When you have finished viewing your results, gently blot the oil from the slide and store the slide in a slide box.
Bacterial capsules are not usually visible by simple staining but can be seen using negative staining procedures. Place a loop full of Congo red on a slide. Make a thick smear of the test bacterium in the Congo red. Flame the loop. Flame the mouth of the tube of bacteria and obtain a loop full of inoculum. You make an emulsion on the slide with the bacteria and the Congo red. Let the smear air dry. This slide is not heat fixed.
Fix the smear with acid alcohol for approximately 15 seconds. The Congo red will change color with the decreased pH of the acid. Wash the smear with distilled water until the water runs clear. Cover this smear with acid fochsin for one minute to stain the cells. Wash the smear with distilled water. Gently blot the slide dry. Do not wipe, just gently blot the slide.
Examine the slide under the microscope. The Congo red is a negatively charged dye and the bacterial surface is also negatively charged, so the dye is repelled. The background stains a dark color and capsules will be visible as a white halo around a red cell. Using the acid fochsin stains the cell red, but not the capsule. The capsule remains as a clear boundary surrounding the cell against a dark background. When you have finished viewing your results, gently blot the oil from the slide and store the slide in a slide box.
For non-pathogenic bacteria, motility is most easily determined by observing moving cells in a wet mount. Place a drop of broth culture on the slide. Slam the loop. Flame the mouth of the tube of inoculum and obtain two or three loops of broth to make a good-sized drop of broth on the slide. Gently lower a coverslip on to the broth.
On another slide, make a culture on a solid medium. Flame the loop and place two or three loops of water on the slide. Flame the loop again and obtain a loop full of bacteria from the plate on the slide. Make a thick suspension of bacteria in the water. Gently lower a coverslip on to the slide.
Examine the slide under the microscope. Note that this Brownian movement is not motility; it is caused by molecules in the liquid. The cells all vibrate but retain their relative positions. Bacterial motility is most easily determined by observing moving cells in a wet mount. When you have finished examining the slides, properly discard them in disinfectant.
Bacterial flagella are too thin to see at the microscope, but they can be visualized using special staining techniques. Handle the cells carefully because flagella are easily broken off of the cells. Dip a scalpel in alcohol. Use your flame to ignite the alcohol to burn it off. Hold the scalpel pointing down while the alcohol is burned. Repeat this procedure to sterilize your forceps.
Using the sterile scalpel and forceps, cut out a piece of agar on which your test bacterium is growing. Gently place the agar culture side down on a slide. You then carefully remove the agar from the slide and put the piece of agar back into the Petri plate. Replace the cover on the Petri plate. Sterilize the scalpel and forceps before putting them down. Sterilize the instruments by dipping them in alcohol and passing them through the flame.
Let the slide air dry, but do not fix it. Cover the slide with flagella mordant for 10 minutes. The mordant will adhere to the flagella, making them appear wider. Gently rinse the slide with distilled water. Allow the slide to air dry. Do not blot it.
Examine the slide under the microscope. The cells and flagella will be stained brown. Observe the arrangement of the flagella on a cell. When you have finished viewing your results, gently blot the oil from the slide and store the slide in a slide box.