Basic techniques of microbiology
This week for a change Dr Fairolniza is our lecturer until the our last week of basic techniques of microbiology class. My first impression about Dr Fairol is strict. But is actually opposite to my impression, Dr Fairol is so friendly, nice and also beautiful like Dr Adelene. Dr Fairol give some briefing and explanation about the experiment 11. We did our experiment 11 by our ownself without teaching by Encik Hussain and Encik Zainuddin. On wednesday, as usual we observe the slide we are smear and stain. And also we did our hand's on test. Hand's on test like the demo gave us one unknown bacterial cultural and we have to isolate by using streak plate method and staining using Gram staining method. After that, we have to observe the slide under microscope and write the observation in the paper that provided to us. Lastly, after we finished observing our experiments, our demo gave some examples how to write the references for lab reports.
EXPERIMENT 11: Differential staining for visualization of bacterial cell structures
Part A: Spore stain
Certain bacterial species, most commonly gram-positive bacilli such as those of the genera Bacillus and Clostridium, undergo a complex developmental cycle that produces a resting endospore when faced with environmental adversity. The process of sporulation allows the bacteria to survive in harsh environmental conditions such as low nutrients, high temperatures, UV radiation, acids and toxic chemicals. If conditions improve, the spore may germinate to form a new vegetative cell and growth will resume. These cells have the capacity to undergo sporogenesis and give rise to a new intracellular structure called the endospore, which is surrounded by imprevious layers called spore coats. Endospores are very dehydrated structures that are not metabolically active. They possess a protein coat, called an exosporium, that forms a barrier around the spore. Since endospores are not easily destroyed by heat or chemicals, they define the conditions necessary to sterility. For example, to destroy endospores by heating, they must be exposed for 15-20 minutes to steam under pressure, which generates temperatures of 121° C. As conditions continue to worsen, the endospore is released from the degenerating vegetative cell and becomes an independent cell called a free spore. With the return of favourable environmental conditions, the free spore may revert to a metabolically active and less resistant vegetative cell through germination.
Primary stain
Malachite green used as the primary stain. Malachite green unlike most vegetative cell types that stain by common procedures, the free spores, because of its impervious coats, will not accept the primary stain easily. For further penetration, the application of heat is required. After the primary stain is applied and the smear is heated, both the vegetative cell and spore will appear green.
Decolorizing Agent
Water is used as the decolorizing agent. Once the spore accepts the malachite green, it cannot be decolorized by tap water, which removes only the excess primary stain The spore remains green. On the outer hand, the stain does not demostrate a strong affinity for vegetative cell components; the water removes it, and these cells will be colorless.
Counterstain
Safranin is used as the counterstain. This contrasting red stain is used as the second reagent to color the decolorized vegetative cells, which will absorb the counterstain and appear red. The spores retain the green of the primary stain.
Part B: Capsule Stain
For capsule staining, the capsule stain
employs an acidic stain and a basic stain to detect capsule production. A
capsule is a gelatinous outer layer that is secreted by the cell and that
surrounds and adheres to the cell wall. Capsules are formed by
organisms such as Klebsiella pneumoniae . Most capsules are
composed of polysaccharides, but some are composed of polypeptides. The capsule
differs from the slime layer that most bacterial cells produce in that it is a
thick, detectable, discrete layer outside the cell wall. Some capsules have well-defined
boundaries, and some have fuzzy, trailing edges. Capsules protect bacteria from
the phagocytic action of leukocytes and allow pathogens to invade the body. If
a pathogen loses its ability to form capsules, it can become avirulent. Bacterial
capsules are non-ionic, so neither acidic nor basic stains will adhere to their
surfaces. Therefore, the best way to visualize them is to stain the background
using an acidic stain and to stain the cell itself using a basic stain. We use
India ink and Gram crystal violet. This leaves the capsule as a clear halo
surrounding a purple cell in a field of black. Capsule staining is more
difficult than other types of differential staining procedures because the
capsular materials are water-soluble and may be dislodged and removed with vigorous
washing. Smears should not be heated because the resultant cell shrinkage may
create a clear zone around the organism that is an artifact that can be
mistaken for the capsule.
Primary stain
Decolorizing agent
Copper sulfate (20%) is used as decolorizing agent because the capsule is nonionic,
unlike the bacterial cell, the primary stain adheres to the capsules but does
not bind to it. The copper sulfate washes the purple primary stain out of the
capsular material without removing the stain bound to the cell wall. At the
same time, the decolorized capsule will now appear blue in contrast to the deep
purple color of the cell.
New findings challenge current view on origins of Parkinson's disease
It was found that the bulk of the damage to neurons with damaged mitochondria stems from a related but different source -- the neighbouring maze-like endoplasmic reticulum (ER). The ER has the important job of folding proteins so that they can do the vast majority of work within cells. Misfolded proteins are recognized by the cell as being dangerous. Cells halt protein production if there are too many of these harmful proteins present. While this system is protective, it also stalls the manufacture of vital proteins, and this eventually results in the death of neurons. To find out if ER stress might be at play in Parkinson's, a team led by Dr Miguel Martins analyzed fruit flies with mutant forms of the pink1 or parkin genes. Mutant forms of pink1 and parkin are already known to starve neurons from energy by preventing the disposal of defective mitochondria. These genes are also mutated in humans and result in hereditary versions of the disease. Much like Parkinson's patients, flies with either mutation move more slowly and have weakened muscles. The insects struggle to fly and they lose dopaminergic neurons in their brains -- a classic feature of Parkinson's. Compared to normal flies, Miguel's team found that the mutants experienced large amounts of ER stress. The mutant flies did not manufacture proteins as quickly as the non-mutants. They also had elevated levels of the protein-folding molecule BiP, a telltale sign of stress.
Lysosomes
Macrophage Lysosome Damage Crucially Contributes to Fungal Virulence
Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans–induced lysosome damage was observed in infected murine bone marrow–derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans–containing lung cells compared with C. neoformans–free cells. Among C. neoformans–containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-g abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.
Capsule staining of Enterobacter aerogenes using nigrosin
Capsule staining of Klebsiella pneumoniae using nigrosin
Spore staining of Bacillus cereus
Capsule staining of Klebsiella pneumoniae using crystal violet
Capsule staining of Enterobacter aerogenes using crystal violet
Microbiology week 8 ( Eukaryotic cell)
Endoplasmic reticulum
New findings challenge current view on origins of Parkinson's disease
It was found that the bulk of the damage to neurons with damaged mitochondria stems from a related but different source -- the neighbouring maze-like endoplasmic reticulum (ER). The ER has the important job of folding proteins so that they can do the vast majority of work within cells. Misfolded proteins are recognized by the cell as being dangerous. Cells halt protein production if there are too many of these harmful proteins present. While this system is protective, it also stalls the manufacture of vital proteins, and this eventually results in the death of neurons. To find out if ER stress might be at play in Parkinson's, a team led by Dr Miguel Martins analyzed fruit flies with mutant forms of the pink1 or parkin genes. Mutant forms of pink1 and parkin are already known to starve neurons from energy by preventing the disposal of defective mitochondria. These genes are also mutated in humans and result in hereditary versions of the disease. Much like Parkinson's patients, flies with either mutation move more slowly and have weakened muscles. The insects struggle to fly and they lose dopaminergic neurons in their brains -- a classic feature of Parkinson's. Compared to normal flies, Miguel's team found that the mutants experienced large amounts of ER stress. The mutant flies did not manufacture proteins as quickly as the non-mutants. They also had elevated levels of the protein-folding molecule BiP, a telltale sign of stress.
Lysosomes
Macrophage Lysosome Damage Crucially Contributes to Fungal Virulence
Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans–induced lysosome damage was observed in infected murine bone marrow–derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans–containing lung cells compared with C. neoformans–free cells. Among C. neoformans–containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-g abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.
Peroxisome
Antifungal activity of Saccharomyces cerevisiae peroxisomal
3-ketoacyl-CoA thiolase
Peroxisomes play an important role in cellular defense systems and generate secondary messengers for cellular communication. Saccharomyces cerevisiae containing oleate-induced peroxisomes were subjected to buffer-soluble extraction and two chromatographicprocedures, and a protein with antifungal activity was isolated. The results of MALDI-TOF analysis identified the isolated protein as peroxisomal 3-ketoacyl-CoA thiolase (ScFox3). Purified yeast ScFox3 exhibited thiolase activity that catalyzed the thiolytic cleavage of 3-ketoacyl-CoA to acetyl-CoA and acylCoA. ScFox3 protein inhibited various pathogenic fungal strains, with the exception of Aspergillus flavus. Using ScFox3-GFP and PTS2 signal-truncated ScFox3M-GFP, we showed that only ScFox3-GFP, with an intact PTS2 peroxisome signal sequence, was able to translocate into peroxisomes. Yeast ScFox3 is a natural antifungal agent found in peroxisomes.
Vacuoles
Light Shielding in Blue-Green Algae
Gas vacuoles are small, cylindrical, gas-filled vesicles
which occur in certain procaryotic cells. They
are found in numerous blue-green algae, many
photosynthetic bacteria, some halophilic bacteria,
and some planktonic freshwater bacteria. Because gas-vacuolate blue-green algae
are often observed floating at the surface of water
where high light intensity may damage cells,
Lemmerman first suggested that gas vacuoles
may function as light-shielding organelles. Recently,
it has been suggested that such light shielding
(if it occurred) might be due to the optical
properties and the intracellular distribution of the
gas vacuoles. Being gas filled, these
vacuoles have a refractive index much lower than
that of the cytoplasm which surrounds them, and
therefore they scatter light. A suspension of gas vacuolate
cells becomes visibly less milky and more
transparent when the gas vacuoles are collapsed.
Similarly, when a milky-white, opalescent suspension
of isolated gas vacuoles is subjected to sudden
pressure, a completely transparent suspension
results. These changes are due to a decrease in
light scattering when gas vacuoles collapse. But,
although light scattering by gas vacuoles could
protect cells by scattering away a large portion of
the incident radiation, it could also lead to increased
rather than decreased irradiation of some
cellular components if the light were back-scattered
into these components. Whether gas vacuoles function
as light shields would depend upon the distribution
of the gas vacuoles inside the cells.
Chloroplast
Toward mosquito control with a green alga: Expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas.
We are developing Chlamydomonas strains that can be used for safe and sustainable control of mosquitoes, because they produce proteins from Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast. Chlamydomonas has a number of advantages for this approach, including genetic controls that are not generally available with industrial algae. The Bti toxin has been used for mosquito control for > 30 years and does not engender resistance; it contains three Cry proteins, Cry4Aa (135 kDa), Cry4Ba (128 kDa) and Cry11Aa (72 kDa), and Cyt1Aa (25 kDa). To express the Cry proteins in the chloroplast, the three genes were resynthesized and cry4Aa was truncated to the first 700 amino acids (cry4Aa700 ); also, since they can be toxic to host cells, the inducible Cyc6:Nac2-psbD expression system was used. Western blots of total protein from the chloroplast transformants showed accumulation of the intact polypeptides, and the relative expression level was Cry11Aa > Cry4Aa700 > Cry4Ba. Quantitative western blots with purified Cry11Aa as a standard showed that Cry11Aa accumulated to 0.35% of total cell protein. Live cell bioassays in dH20 demonstrated toxicity of the cry4Aa700 and cry11Aa transformants to larvae of Aedes aegypti and Culex quinquefasciatus. These results demonstrate that the Cry proteins that are most toxic to Aedes and Culex mosquitoes, Cry4Aa and Cry11Aa, can be successfully expressed in the chloroplast of Chlamydomonas.
Mitochondria
Hydrogen Production. Green Algae as a Source of Energy
Hydrogen gas is thought to be the ideal fuel for a world in which air pollution has been alleviated, global warming has been arrested, and the environment has been protected in an economically sustainable manner. Hydrogen and electricity could team to provide attractive options in transportation and power generation. Interconversion between these two forms of energy suggests on-site utilization of hydrogen to generate electricity, with the electrical power grid serving in energy transportation, distribution utilization, and hydrogen regeneration as needed. A challenging problem in establishing H2 as a source of energy for the future is the renewable and environmentally friendly generation of large quantities of H2 gas. Thus, processes that are presently conceptual in nature, or at a developmental stage in the laboratory, need to be encouraged, tested for feasibility, and otherwise applied toward commercialization.
Nuclues
Regulation of eukaryotic DNA replication and nuclear structure
In eukaryote, nuclear structure is a key component for the functions of eukaryotic cells. More and more evidences show that the nuclear structure plays important role in regulating DNA replication. The nuclear structure provides a physical barrier for the replication licensing, participates in the decision where DNA replication initiates, and organizes replication proteins as replication factory for DNA replication. Through these works, new concepts on the regulation of DNA replication have emerged, which will be discussed in this minireview. Regulatory mechanisms for DNA replication are central to the control of the cell-cycle in eukaryotic cells. Recently, considerable progress has been made in our understanding of the relationship between regulation of eukaryotic DNA replication and nuclear structure. This review will briefly outline the progress and discuss some new concepts
appearing from the studies.
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