In this case, the endospores are seen as clear oval or spherical areas within the stained cell. Endospores can also be directly observed in cells by using phase contrast microscopy, as shown in Figure 8. Because many differential staining methods require several steps and take a long time to complete, we will not be performing all of the differential staining methods discussed above.
Pre-stained slides will be used to visualize bacterial capsules, metachromatic granules, and acid-fast bacilli. Obtain one slide of each of the three bacteria listed in the table below.
Your environmental isolate may have one or more of these cellular features, and learning to recognize them will aid in identification. These should all be viewed using the oil immersion objective lens.
All staining procedures should be done over a sink. The Gram stain procedure will be demonstrated, and an overview is provided in Table 1. A volunteer from your lab bench should obtain cultures of the bacteria you will be using in this lab, as directed by your instructor. One of the cultures will be a Gram positive bacterium, and the other will be Gram negative. Below, write the names of the bacteria you will be using, along with the BSL for each culture:.
Obtain two glass slides, and prepare a smear of each of the two bacterial cultures, one per slide, as demonstrated. Stain both smears using the Gram stain method.
Observe the slides with a light microscope at 1,X and record your observations in the table below. Heat fix the smear and Gram stain it. You should be able to determine the Gram stain reaction, cellular morphology and arrangement of BOTH bacteria in this mixed smear. Your instructor may ask to see this slide and offer constructive commentary. Only a few genera of bacteria produce endospores and nearly all of them are Gram-positive bacilli.
Most notable are Bacillus and Clostridium species, which naturally live in soil and are common contaminants on surfaces. The growth of Clostridium spp. Endospore-forming bacteria are distinct from other groups of Gram positive bacilli and distinguishable by their endospores.
After staining, endospores typically appear as light green oval or spherical structures, which may be seen either within or outside of the vegetative cells, which appear pink. The shape and location of the endospores inside the bacterial cells, along with whether the sporangium is either distending D or not distending ND the sides of the cell, are important characteristics that aid in differentiating among species see Figure 9.
A volunteer from your lab bench should obtain bacterial cultures for endospore staining, as directed by your instructor. Note that these will all be species of Bacillus. Prepare smears and stain each using the endospore staining technique. Observe the slides and note the shape and location of the endospore and the appearance of the sporangium swollen or not swollen in the table below:. In addition, choose ONE of the cultures from above and Gram stain it.
Record your results below in the spaces provided:. Are endospores visible in the Gram stained smear? Skip to content Viewing Bacterial Cells The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Figure 1. Negative stain of Cyptococcus neoformans , an encapsulated yeast Figure 2. Positive stain of Staphylococcus aureus. Figure 3.
Possible bacterial cell arrangements for cocci. Figure 4. Possible bacteria cell arrangements for bacilli. Gram positive bacterium: Gram negative bacterium: Morphology Arrangement. Figure 6. Acid-fast bacilli in sputum. If your slide is wet and you heat fix it, the bacteria will boil and the cellular morphology will be lost.
If your slide is wet and fix it in methanol, it will most likely wash off the slide. Smears that are too thick will most likely wash off the slide regardless of the fixation method. Broth cultures are usually easier to work with because the cells are already diluted in the broth. Be sure to carefully mix the culture tube to suspend the bacteria in the broth. You can scoop a lot of organisms off with your loop.
You may want to use an inoculating needle to transfer your organism to the slide. Be sure to use sterile water to dilute your samples. Regular tap water or the de-ionized water in your rinse bottles are often contaminated with bacteria. The fixation procedure is the same regardless of smear source, plate or broth.
There are two methods of adhering your bacteria to the slide, heat fixation or methanol fixation. Heat fixing is only used with BSL1 organisms. The organisms we will be working with are BSL2, so you will need to use the methanol fixation technique.
Heat fixing the slide can create aerosols and with BSL2 organisms, we need to prevent this as much as possible.
Methanol fixation causes fewer changes in cellular morphology and creates no aerosols. Simple stains are just that - add one stain to a fixed smear slide, let it sit, rinse it off, let it dry, and view.
It is a quick procedure for determining the presence and morphology of bacteria in clinical samples such as stool and discharges. Methylene blue is used to determine the morphology of fusiform and spirochetes in oral infections.
It is also the stain of choice for identifying the metachromatic granules in Corynebacterium diphtheriae. The granules will stain a distinctly deeper blue than the surrounding blue bacteria. Other species of Corynebacterium do not have the metachromatic granules. Any basic dyes, such as methylene blue, crystal violet, malachite green, or safranin work well.
Basic cationic or positively charged dyes bind to negatively charged components in the cell membrane and cytoplasm. Staining is part art and part science. There are no hard and fast rules for staining and rinsing times. The times listed are suggestions that usually work well.
You will need to experiment with what works for the bacteria you have and the techniques you use. It is essential that you record exactly what you do and the results you observe in your lab book.
It would be useful for each lab bench member to pick a different stain so you can see what they all look like. Negative stains are even simpler than simple stains because you do not have to make a smear. A drop of cells is spread on a slide and viewed without fixation. The stain is a suspension of carbon, found in India ink or nigrosin. The carbon particles are negatively-charged, as is the cell membrane. The background looks black or sepia colored and the cells remain clear, since they repel the dye.
Some positively charged inclusion bodies, such as sulfur, may stain. This stain gives accurate information on cell morphology and capsule presence because the cells are not fixed.
Cell size appears slightly larger because any extracellular coatings or secretions on the outside of the cell membrane also do not stain. Negative stains are useful for rapid determination of the presence of Cryptococcus neformans , the causative agent of cryptococcisis, in cerebral spinal fluid.
This technique is also used when you stain for endospores and capsules. Just as in preparing a smear, you only need a small amount of organism. It is also important not use too much nigrosin. If it is too thick, the background will have a cracked appearance similar to mud puddles drying in the sun. You want to get a light film.
Your instructor will demonstrate this technique for you. Nigrosin comes off the slide and onto your oil immersion lens very easily. Be sure to thoroughly clean your oil lens when you are finished. Then clean it again. Once it dries on the lens it is very difficult to remove and will impair your ability and the other micro students using that scope to see clearly out of the lens. The Gram stain is the most common differential stain used in microbiology.
Differential stains use more than one dye. The unique cellular components of the bacteria will determine how they will react to the different dyes. The Gram stain procedure has been basically unchanged since it was first developed in Almost all bacteria can be divided into two groups, Gram negative or Gram positive. Be sure that you properly time the decolorization step. Flood the smear with decolorizing agent evenly over the smear.
You can use 2 slides, 1 for each bacterium, or you can divide one slide in half and smear each bacterium on the divided slide. There are also prepared, gram stained slides of bacteria of different shapes and sizes of bacteria to look at.
Make the bacterial smear from broth, slant, or plate. If taken from a broth , use loopfuls of the broth solution. If taken from a solid agar medium plate or slant , suspend the inoculum in a drop of water on the slide and mix it well.
Spread the suspension on the slide so that it covers an area at least the size of a nickel, preferably a quarter. You might think about marking the smear area with a surrounding wax pencil mark so that you can find the smear under the microscope easily.
Place a piece of tape on the side of the smear so you know which way is UP. Let the slide air-dry totally before proceeding on with the procedure. Heat-fix the slide by holding the slide at one end with your fingers and quickly moving it back and forth a few times over the flame. Perform the Stain procedure as stated below. Flood the smear with drops of crystal violet, leaving in for 1 minute.
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