Cell - The Unit of Life
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For each phosphate that ATP loses, it results in release of 33.5 kJ/mol of energy (stored in the bonds between the phosphate atoms). ATP -> ADP -> AMP However, when AMP (which is like 'an ATP with only one phosphate') loses its last phosphate, it only results in release of 13.5 kJ/mol of energy.
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Eukaryotic cells contain a true nucleus and include cells in humans, animals, plants, fungi and algae that reproduce sexually.
The very exterior of a cell is the cell membrane. This is a semi-permeable barrier that allows only a small number of molecules to move back and forth through it. The cell membrane has a double layer to separate the inner parts of a cell from the outside, but it also allows transport of different substances between the cell and surrounding cells.
Cytoplasm is a fluid that is held inside the cell by the cell membrane. Its job is to support all of the cell structure and shape as well as support the organelles or tiny organs that have specific functions for normal cellular operation.
The nucleus is often called the brain center of the cell. It contains the genetic material or DNA and RNA. It has a nuclear membrane surrounding it with pores to enable protein movement both into and out of it. The nucleolus is inside of the nucleus, and it holds the ribosomes for a cell.
Ribosomes synthesize protein for normal cell functioning. They can be suspended in the cytoplasm or they may be attached to the endoplasmic reticulum. The endoplasmic reticulum is basically the transportation department of a cell and is the means by which proteins move about.
Lysosomes contain digestive enzymes to help break down any waste and remove it from the cell. Lysosomes have a circular shape.
Centrosomes are located near the nucleus of a cell. The centrosome makes microtubules, which aid in cell division of tissues in mitosis by moving the chromosomes to opposite poles of the cell.
Vacuoles are contained by a membrane and are small organelles that store substances and help transport waste out of a cell.
Golgi bodies are also called the Golgi apparatus or the Golgi complex. They form an organelle that packs substances in preparation for transport out of a cell.
Mitochondria are the energy sources of cells. They have a double membrane and take the shape of a sphere or rod. They are located in the cell's cytoplasm, and their function is to convert nutrients and oxygen into energy sources for the cell.
The cell's cytoskeleton helps maintain its shape, using microtubules and fibers. Cilia and flagella are hair-like structures that are present on the cell membrane. These two types of appendages help the cells to move from one place to another.
In a typical bacterial cell, there are three layers :
The innermost layer is the plasma membrane which directs what enters and exits the cell; phospholipid bilayer with some protein scattered between it
The middle layer is the Cell wall that provides structural support to the cell and is made up of peptidoglycan or polysaccharide. The cell wall of bacteria Gram-positive and negative bacteria differ in their thickness. It is an important layer to understand the structure and difference between Gram-positive and negative bacteria, which we will understand later in this write-up.
The third layer is the Capsule which is the sticky outer layer for attachment and protection.
The major differences between Gram-positive and Gram-negative bacteria can be understood with the structural differences and Gram staining differences between them.
Similarity
It possesses an inner plasma membrane.
It also possesses an inner plasma membrane.
Cell Wall
It has a thickly layered peptidoglycan cell wall.
It has a thin layered peptidoglycan cell wall.
Second Phospholipid Plasma Membrane
It is absent in Gram-positive bacteria.
It has an additional outer phospholipid plasma membrane.
Similarity
Outer Capsule Present
Outer Capsule Present
Treatment Possibility
It is more easily treatable with Antibiotics due to the absence of a second phospholipid bilayer.
It is harder to treat with antibiotics as it is difficult for antibiotics to enter the Gram-negative cell.
Gram-staining results
It appears purple/violet under a microscope after performing a gram stain experiment.
It appears red/pink underneath the microscope after performing a Gram stain experiment.
Examples of Gram-positive and Gram-negative Bacteria
Staphylococcus and Streptococcus
Escherichia and Salmonella.
Toxins Produced
Exotoxins
Endotoxins/Exotoxins
Lipid Content
Very Low
It has 20-30% of lipid content in the cell.
Flagella Structure
It consists of two rings in the basal body
It has four rings in the basal body
Teichoic Acids
Present
Absent
Mesosome
More Prominent
Less Prominent
Morphology
Cocci/spore-forming rodes are present
Non-spore forming rodes are present.
It is a purpose that helps us to identify the type of infectious bacteria for proper diagnosis and treatment based upon outcome. It helps us to highlight the Gram positive and gram-negative bacteria differences.
Take a glass slide and smear bacteria samples from the culture on it with the help of an inoculation loop.
Fix the bacteria to the slide to avoid it getting washed away in the later steps, this is done by heat fixing the glass slide over a flame.
Add a few drops of crystal violet to the bacteria sample.
Add iodine molecules to the sample; it is golden brownish in colour.
Here, the crystal violet and iodine molecules bind together.
Now, wash the bacteria with alcohol which is a de-staining chemical. A major happening in the Gram positive bacteria takes place which is when the capsule of the cell gets washed away and shrinks the cell. And in Gram-negative bacteria, the second plasma membrane layer gets washed away along with the capsule due to the dehydrative nature of the alcohol. As the Gram -ve bacteria have a thin cell wall left, the crystal violet-iodine molecules also get washed away, and hence the cell loses its colour. On the other hand, Gram +ve bacteria has retained the colour from crystal violet dye and therefore appears purple violet colour under the microscope.
The next step is to apply safranin to the bacterial samples; safranin is a biological stain, pinkish in colour.
Results: The results for Gram positive vs. Gram-negative bacteria will be different as mentioned below.
Now under observation underneath the microscope, Gram-positive bacteria appear purple in appearance as the pinkish color of safranin is overshadowed by the deep color of crystal violet dye.
It is the first and foremost important step within the classification of bacteria. The morphology and arrangements of bacteria are often observed well once stained because it helps within the screening of pathogens in clinical specimens. While Gram staining is used to detect the differentiate bacteria there are many other microscopic organisms that can be found on the smear of a gram stain, which is subjected to the test.
Misinterpretation: Organisms that are gram-negative would appear red once they retain counterstain, even after getting washed out of the complex made of crystal violet and iodine in the heat fixed cell, by alcohol or acetone. Meanwhile, the gram-positive organisms, on over-decolourization after the loss of complex even though they retain the complex after decolourization and remain purple.
Side Effects: It is noted that blood samples would lead to side effects such as bruising due to the blood draw, even though some other types of samples would not entail certain side effects.
Limitations Under Environmental Microbiology: It is observed that in most medical conditions, this method is not solely recommended for bacterial identification since; it will not be able to identify bacteria to its specific level. Therefore, it is used in a combination of other molecular techniques in identifying bacteria.
Decolorization is one of the most prominent steps which is prone to be performed incorrectly, mishaps may occur more often.
Unbridled Heat During the Time of Fixation: It makes it easier to decolorize the cell once the cell morphology is altered once heat fixation is done in an excessive amount.
Overindulgent Washing between each step: CV stain is easily washable with water and it is recommended to not use more than 5 s water rinse during any stage of the procedure.
Prolonged Decolorization: It is recommended to decolorize no more than 15 seconds, and if the specimen is left for a longer period of time, the possibility of the positive cell losing Crystal Violet and turning red is higher.
Excessive Counterstaining: With overexposure, It is possible to replace the CV Iodine in the gram-positive cells with counterstain instead
Misread Stains: where the negative strains were misread as positive and positive stains as negative.
Mixed Culture
A minimal number of bacterial cells.
KOH String test The test has proven effective for food microorganisms and Bacillus spp, with a bit of mishap for some anaerobes. It has the advantage of simplicity and is effective on older samples/cultures.
Aminopeptidase Test L-alanine aminopeptidase is the enzyme that is localized in bacterial cell walls that bisects amino acid L-alanine from different peptides. The test is proven to be more useful in non-fermenters and gram-variable organisms, with faster results in the short span of 5 minutes.
Fluorescent Stains The test involves fluorescent nucleic acid binding dyes like hexidium iodide (HI)and SYTO 13. Gram-negative organisms turn green by SYTO 13 and gram-positive organisms are reddish-orange fluorescent by HI, overpowering the green colour of SYTO 13 when the dyes are used together in a single step.
LAL-based Assay This type of test uses the same reaction which is being used for the chromogenic LAL test. In the LAL test, clotting is formed due to the enzymes cleaving a peptide from the horseshoe crab coagulant. Gram-positive organisms lacking endotoxin do not trigger the colour change in this method, but gram-negative on the other hand is triggered.