Generation of C. Strain ATCC was used for this experiment. In industrial fermentations increases in osmotic pressure due to product accumulation are detrimental to bacterial growth and formation of product [ 19 ]. While strategies employed by C. An essential place for poly P in the regulation of responses to nutritional deficiencies, environmental stresses and survival in the stationary phase has been established in Escherichia coli , [ 20 ]. Though some corynebacteria were among the first organisms to be studied with regard to poly P and its enzymes, knowledge on the occurrence and role of poly P in C.
In consequence, we have previously established that this organism is able to accumulate up to mM of poly P expressed in P-units under aeration, with concomitant decrease of poly P within minutes under anoxic conditions [ 5 ]. In the present paper, we show that C. To obtain good visibility of stained granules, cells are usually grown on special media, such as Loeffler medium. Thus, strain C. Samples for both Neisser and toludine staining were taken after 48 h of growth. Neisser staining gave more consistent results than toluidine staining.
Globules were comparable in number and size to those of C. Microscopic evidence for poly P granules in C. Formation of metachromatic granules in C. Cells were mounted on slides and stained according to Neisser see Section 2. For details see text. Therefore we further investigated the nature of the metachromatic granules in C.
The large intracellular electron-dense granules Fig. They were tentatively assigned to volutin granules. Occasionally, equally large yet less electron-dense globules could be observed Fig. They resembled the granular centers in M.
TEM ultrastructure of C. Samples were fixed with OsO 4 , but not contrasted. Bar represents 0. It has been reported that Mg is present in poly P granules [ 22 , 23 ]. Therefore, the elemental composition of large granules and cytoplasmic areas in C. Several samples and cells were analyzed. The large electron-dense bodies accumulated phosphorus fold in comparison to the cytoplasm.
Magnesium, and to a lesser extent aluminum, appeared to be slightly enriched in the dark large globules, while chloride and silicium were equally distributed between granules and background Fig. Electron dispersive X-ray analysis of C. Unstained and unfixed cells taken from CGIII-medium were examined for the presence of electron-dense granules.
The elemental compositions of a large granule inset, TEM, g and a cytoplasmic area inset, c were analyzed by electron dispersive X-ray analysis. It has been reported that Mg ions stimulated the formation of volutin granules in Chlorella cells [ 22 ]. Therefore, we studied the effect of Mg on granule formation in C. We used fluorescence microscopy to find out whether i the C. A comparable effect of MgCl 2 was observed with C.
Thus, Mg ions stimulated the poly P formation in C. Besides poly P, protein is a major component of volutin granules in other microorganisms [ 23 ]. The pyrophosphate and poly P rich acidocalcisomes detected in Agrobacterium tumefaciens and Rhodospirillum rubrum [ 25 ] also contain protein. To obtain further evidence that the electron-dense and P-rich granules in C.
Granular fractions from disrupted cells were obtained as described in Section 2. Both micrographs were taken with DAPI as fluorescence marker. Volutin granules green in intact cells appeared predominantly at the cell poles, while the remainder showed the typical blue fluorescence of DAPI—DNA complexes. Granules isolated from these cells see Section 2 , aggregated to larger agglomerizations, as seen in Fig. They resembled in size and appearance those aggregates shown in TEM pictures of volutin granular fractions from cells of M.
NMR spectroscopy was used to corroborate that poly P was a major constituent in volutin granules. The same signal was also observed in the poly P standard Fig.
A comparison with an orthophosphate standard Fig. Granular fractions obtained from M. To see whether proteins were also present in the C. The arrow in Fig. S, sample; M, molecular mass marker.
Docampo and coworkers find pyrophosphatase PPase in acidocalcisomes of A. We cannot rule out that one of the other proteins in the C. However, this enzyme in C. Yet there remains a striking coincidence: i C. Acidocalcisomes in A. Some of the less electron-dense globular subcellular structures in C. Currently we do not know whether a membrane also surrounds the similar structures in C. Finally, we tried to answer the following question: it has been shown above that MgCl 2 stimulated poly P formation in C.
We identified genes differentially expressed in response to growth in the presence of high MgCl 2 concentrations as follows: cells of C. Under high-Mg conditions, operons encoding putative uptake systems for Mn, Zn, iron siderophores and alkanesulfonates showed increased expression RNA ratios higher than 3. These genes code for a transcriptional AraC-like regulator, a putative ATPase, and a putative secreted hydrolase Table 1.
The expression of the poly P metabolism genes ppgK, ppk2A, ppk2B, ppx1 and ppx2 [ 3 , 27 ] did not change significantly. Global gene expression changes in C. Thus, an influence of high MgCl 2 concentrations on the expression of poly P synthesizing genes was excluded. Stress dependent formation of poly P [ 1 ] from high concentrations of Mg or Cl seems unlikely: growth was not inhibited by mM MgCl 2 , and C.
Possibly, a similar mechanism was responsible in C. In conclusion, we revealed for the first time a poly P metabolizing enzyme, PPGK, in poly P granules, we also showed that the high concentrations of MgCl 2 enhanced granule formation in C.
It would also be tempting to investigate whether poly P occurs in high amounts in the related C. We thank F. Inorganic Polyphosphates: Biochemistry, Biology, Biotechnology. Progress in Molecular and Subcellular Biology.
Google Scholar. Google Preview. Sahm H. Eggeling L. Graaf A. Zhang H. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain iodine. Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink. Gram negative cell walls have an outer membrane also called the envelope that dissolves during the alcohol wash.
This permits the crystal violet dye to escape. Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram negative cells appear pink. When you interpret a Gram stained smear, you should also describe the morphology shape of the cells, and their arrangement. In Figure 5, there are two distinct types of bacteria, distinguishable by Gram stain reaction, and also by their shape and arrangement.
Below, describe these characteristics for both bacteria:. Some bacteria produce the waxy substance mycolic acid when they construct their cell walls.
Mycolic acid acts as a barrier, protecting the cells from dehydrating, as well as from phagocytosis by immune system cells in a host. This waxy barrier also prevents stains from penetrating the cell, which is why the Gram stain does not work with mycobacteria such as Mycobacterium , which are pathogens of humans and animals.
For these bacteria, the acid — fast staining technique is used. To perform the acid-fast stain, a heat-fixed smear is flooded with the primary stain carbol fuchsin, while the slide is heated over a steaming water bath. Then the slide is allowed to cool and a solution of acid and alcohol is added as a decolorizer. All other cell types will be decolorized.
Methylene blue is then used as a counterstain. In the end, acid-fast bacteria AFB will be stained a bright pink color, and all other cell types will appear blue. Capsule : The polysaccharide goo that surrounds some species of bacteria and a few types of eukaryotic microbes is best visualized when the cells are negative stained. In this method, the bacteria are first mixed with the stain, and then a drop of the mixture is spread across the surface of a slide in the thin film.
With this method, capsules appear as a clear layer around the bacterial cells, with the background stained dark. Metachromatic granules or other intracytoplasmic bodies : Some bacteria may contain storage bodies that can be stained.
Various staining methods are used to visualize intracytoplasmic bodies in bacteria, which often provide an identification clue when observed in cells. Endospores are dormant forms of living bacteria and should not be confused with reproductive spores produced by fungi. These structures are produced by a few genera of Gram-positive bacteria, almost all bacilli, in response to adverse environmental conditions. Two common bacteria that produce endospores are Bacillus or Clostridum.
Both live primarily in soil and as symbionts of plants and animals, and produce endospores to survive in an environment that change rapidly and often. The process of endosporulation the formation of endospores involves several stages. After the bacterial cell replicates its DNA, layers of peptidoglycan and protein are produced to surround the genetic material. Once fully formed, the endospore is released from the cell and may sit dormant for days, weeks, or years.
When more favorable environmental conditions prevail, endospores germinate and return to active duty as vegetative cells. Mature endospores are highly resistant to environmental conditions such as heat and chemicals and this permits survival of the bacterial species for very long periods.
Endospores formed millions of years ago have been successfully brought back to life, simply by providing them with water and food. Because the endospore coat is highly resistant to staining, a special method was developed to make them easier to see with a brightfield microscope. This method, called the endospore stain , uses either heat or long exposure time to entice the endospores to take up the primary stain, usually a water soluble dye such as malachite green since endospores are permeable to water.
Following a decolorization step which removes the dye from the vegetative cells in the smear, the counterstain safranin is applied to provide color and contrast. When stained by this method, the endospores are green, and the vegetative cells stain pink, as shown in Figure 7. Although endospores themselves are resistant to the Gram stain technique, bacterial cells captured in the process of creating these structures can be stained.
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.
Be sure to label the far edge of the slide. Do this consistently on the same end of the slide to help orient your slide.
Be patient and take the time to let your slide air dry before proceeding with adhering it to the slide. 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.
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