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Acetamide Utilization Test: Principle, Procedure, and Results
Continue ReadingAcetamide Utilization Test Introduction
Many of the biochemical tests are used to determine the ability of the micro-organism to utilize the chemicals and their reaction to certain enzymes. One such biochemical test is Acetamide utilization test.
This test is completely based on the acetamide agar, which is used to detect the ability of the organism to use acetamide by performing deamidation in the culture medium. This medium also contains carbon as its sole source along with inorganic ammonium salts.
What is Acetamide Utilization Test?
Acetamide utilization test is one of the biochemical tests which helps us to identify the aerobic organisms.
This test is based on the utility of acetamide by the organism during the process of deamidation.
This process occurs only during the presence of an enzyme acalamidase. This acetamide utilization test is performed to differentiate the group of fermentative organisms from the oxidative group of species.
This test is most commonly used for differentiating the Gram-negative, non-fermentative group of bacteria on the basis of their capability to utilize acetamide.
A medium containing acetamide as the sole source of carbon is used for utilizing the acetamide by the organism present in the growth medium.
Thus, the change in color in the growth medium of the medium, detects the growth of the organism present in the medium.
Acetamide Utilization Test Objective
The main aim of the test is to differentiate the bacteria based on their ability to use the acetamide as the sole source of carbon.
To identify the species of Aeruginosa from the other non-glucose-fermenting, Gram negative rods.
Acetamide Utilization Test Principle
As mentioned above, Acetamide utilization test is usually used to determine the ability of the specific organism to utilize acetamide as a source of carbon. This process usually takes place by deamidation.
The medium consists of acetamide. As a sole source of carbon along with the inorganic ammonium salts which serves as a sole of nitrogen.
The growth of organisms in the medium of acetamide agar indicates the positive Test for utilization of utilization.
During the metabolism of acetamide by the organism, the enzymatic action of acetamidase breaks the ammonium salts into ammonia. The ammonia thus released increases the alkalinity in the medium and results in change in the pH.
The change in the pH in the medium causes bromothymol blue indicator present in the medium to turn the color from green to blue, and it indicates the positive test.
When there is no release of ammonia in the medium, the color remains same and it indicates the negative result. In some cases, assimilation of the acetamide results in a formation of yellow color and it is mistaken for a positive result.
But the digestion of the acetamide by a deamination is limited to only some of the organisms. Hence this test is only performed for differentiating Pseudomonas aeruginosa from the other non-glucose fermenting, Gram-negative rods.
Acetamide Utilization Test Reagents
Media: Acetamide agar, it can be purchased commercially or it can also be prepared in laboratory in the following composition.
Ingredients Gram/liter Acetamide 3.0 Dextrose 0.2 Yeast extract 0.5 Potassium dihydrogen phosphate 1.0 Phenol red 0.03 Bacteriological agar 15.0 Sodium agar 5.0 Lab Supplies:
• Sterile inoculating loops or sticks
• Incubator
Acetamide Utilization Test Procedure
About 24.7 grams of the dehydrated powder or a lab prepared medium is added in a beaker containing about 1000 milli liters of distilled water.
The suspension is heated until it boils; so that the solution dissolves completely.
The dissolved medium is then dispersed into the tubes and it is sterilized in an autoclave at a temperature of about 121ºC for about 15 minutes.
After completing the autoclave process the tubes are taken out and they are cooled at a slanted position until it forms a butt at a depth of about 1.5 to 2.0 cm.
Acetamide Utilization Test
Here, a well isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating loop or a needle.
Then the acetamide agar inoculated tubes are stroked in the slant in the direction of back and froth using the sterile inoculating loops or sticks.
After streaking the caps of the tubes are closed loosely and adequate aeration is ensured.
Then the tubes are incubated at a temperature of 35 to 37ºC for 7 days.
After incubating they are examined daily for 4 days and at the 7th day before discarding in order to ensure the negative result.
Acetamide Utilization Test Result
Positive Result: In case of positive result, there will be change in change and a growth is detected in the medium
Negative Result: Here, negative result is indicated by no change in color and the slant remains green and there will be no change in growth
Controlled Organism
In case of controlling the quality for the acetamide utilization test, two different organisms are considered here as negative and positive controls.
Control Incubation Results Pseudomonas aeruginosa It is incubated in an aerobic incubation of about 24 to 48 hours in a temperature of 33 to 37ºC It results in Acetamide positive with a growth and change in color to blue Escherichia coli In an aerobic incubation for about 24 to 48 hours in a temperature of about 33 to 37ºC It results in Acetamide negative, hence there will be no growth and there will also be no change in color Acetamide Utilization Test Uses
• Acetamide utilization test is usually used to detect the ability of the particular organism to utilize the acetamide as the only source of carbon.
• It is also used as qualitative test for differentiating Gram-negative bacteria into the fermentative and the oxidative group bacteria into fermentative and the oxidative group bacteria. Acetamide agar is used in the selective medium for isolating P. aeruginosa
• This medium has can also be used as a modification of Simmon Citrate agar to determine the ability of acetamide in acting a carbon source without the presence of peptone and other protein sources.
Acetamide Utilization Test Citations
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Bile Esculin Test: Principle, Procedure, and Results
Continue ReadingBile Esculin Test Introduction
Generally Biochemical tests are used to identify the species of bacteria by differentiating the species on the basis of their biochemical activities.
The factors such as protein and fat metabolisms and the enzyme production and ability of the organism to utilize the compounds in the mediums helps us in differentiating and identifying the specific species of the bacteria.
Bile Esculin test is one of the biochemical tests which is used widely used for differentiating the Enterococci from the group D viridians group of Streptococci.
What is Bile Esculin Test?
Bile esculin test is one of the biochemical tests which is performed in order to differentiate Enterococci and the group D streptococci from non-group-D viridians group Streptococci, based on their ability to hydrolyze esculin.
Many organisms have the capability to hydrolyze esculin, but only few species have the capability to hydrolase esculin in the presence of bile.
This property is used in the biochemical tests to differentiate the organisms.
The bile esculin test is usually performed in a medium of a selective agar known as bile esculin test that contains bile and esculin as their vital substances.
As agar consists of various types of bile salts, that inhibits the growth of other Growth-positive organisms and it also allows the selective isolation of the Enterococci and the Group D Streptococci.
Esculin is one of the derivatives of the glycoside-coumarin, which is also considered as the fluorescent compound as its hydrolysis, and it can be differentiated on the absence of fluorescence.
As years passed, bile esculin tests are modified accordingly and in recent times they are performed using bile esculin disks and they are performed more widely.
Bile-esculin disks are usually used for rapid differentiation of the Group D Streptococci and the non-Group D streptococci.
Bile Esculin Test Procedure
The main aim of the test is to identify the Enterococci and the Group D Streptococci on the basis of their ability to hydrolyze esculin in the presence of the bile.
To detect the difference between the members of the family Enterococci and Group D streptococci from other species of Non-Group D Streptococci.
Bile Esculin Test Principle
The basis of the esculin test is to hydrolyze the esculin that is hydrolyze at the presence of bile salts due to the enzymatic activity of enzyme esculinase.
Esculin is a glucoside containing glucose and hydroxycoumarin which are linked together by an ester bond with the help of an oxygen.
This test helps us to selects the organisms initially on the basis of their ability to grow in the medium containing about 4% of bile salts and they are detected on the basis of their ability to hydrolyze esculin.
Thus, the hydrolysis of esculin results in glucose and another compound known as esculetin.
On degrading esculin, Esculetin is produced which on hydrolysis reacts with the iron ions present in the medium and forms a compound known as phenolic iron complex, and results in the formation of dark brown or black color.
On the other hand, Esculin is a fluorescent compound, which on hydrolysis can be observed as a loss of fluorescence.
Once a bile is added into a medium, the micro-organisms get the capability to grow and it starts hydrolyzing the esculin.
The bile in turn inhibits the growth or production of the other Gram-positive organisms which in turn makes the medium more selective.
Availability of 4% of bile in the esculin medium inhibits most of the strains of the Streptococci excluding the Species S. bovis. Whereas it does not inhibit Enterococci or Listeria.
Bile Esculin Test Reagent
Media:
Bile esculin agar that slants with iron citrate is used.
Agar plate media is usually composed of Enterococcosel agar, or any other similar formulation.
Bile-esculin aide agar or a broth with iron citrate and azide is used. Azide inhibits most of the Gram-negative bacteria.
Peptone-yeast esculin broth, but it is used mostly in the anaerobic atmosphere.
Esculin agar is used without bile or aside but it usually contains iron citrate.
Lab Supplies:
Long wave-UV light
1% of ferric ammonium citrate.
Bile Esculin Test Procedure
About 64.5 grams of the dehydrated powder is added in a beaker containing 1000 milli liters of the distilled or deionized water.
The prepared solution is heated till it boils to dissolve the solution completely.
The dissolved medium is then dispensed into the tubes.
Further the tubes are sterilized in an autoclave at a temperature of about 121ºC for about 15 minutes.
After the completion of the autoclave processing the tubes are taken out and they are cooled in a slanted position at a temperature of about 40 to 45ºC.
The same position is maintained until the butts are formed at a depth of about 1.5 to 2.0 cm.
Esculate Hydrolysis
Esculate hydrolysis is detected either by using tube test or by disk test. Disk test is considered as one of the rapid tests.
I. Bile Esculin Tube Test
An isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating needle.
The bile inoculating tubes are then inoculated by streaking the surface of the slant by using light inoculum from the agar plate and the tubes are inoculated with 10µl loopful of 0.5 McFarland standard suspension using a sterilized water.
Then the cap of the test tubes is left loosened in order to ensure aeration.
Then the tubes are left to incubate aerobically at a temperature of about 35 to 37ºC for about 24 hours and for a maximum of 7 days until the color changes are observed.
For esculin broth without iron citrate, the tubes are observed daily for the decent of fluorescence.
In the absence of the fluorescence 2 to 3 drops of 1.0% ferric ammonium citrate is added to the esculin tube and the color change is observed.
I. Bile Esculin Disk Test
In disk test, esculin test is moistened with the one drop of distilled water.
And it is ensures, that it is not saturated. By using a sterile loop, two to three isolated colonies are picked from 18 to 24-hour culture.
Then the disk is observed for the development of a dark brown or black color for about 10 minutes in a room temperature.
Bile Esculin Test Result: Tube Test
A positive result in a tube test is determined in the medium containing ferric ammonium citrate is usually detected by blackening of the medium.
A negative result in the tube test is detected by absence of change in colors. On the other hand, the medium will fluorescence under the UV light.
For esculin broth without iron citrate, a positive test is usually observed by blackening of the medium on addition of ferric reagent or by the loss of fluorescence in the medium.
Whereas here, the negative tests occurs when the bile esculin medium losses its ability to let grow the organisms on the medium containing bile.
Bile Esculin Test Result: Disk Test
In disk test, the positive result is determined by development of dark color or black color
Where as the negative disk test is determined by absence of color.
Bile Esculin Test Citations
- Esculin hydrolysis by Enterobacteriaceae. J Clin Microbiol . 1977 Aug;6(2):111-6.
- The use of bile – esculin agar for the taxonomic classification of the family Enterobacteriaceae. Antonie Van Leeuwenhoek . 1977;43(1):31-5.
- Presumptive identification of group D streptococci: the bile-esculin test. Appl Microbiol . 1970 Aug;20(2):245-50.
- Bile-esculin test for presumptive identification of enterococci and streptococci: effects of bile concentration, inoculation technique, and incubation time. J Clin Microbiol . 1998 Apr;36(4):1135-6.
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Bacitracin Susceptibility Test: Principle, Procedure, and Results
Continue ReadingBacitracin Susceptibility Test Introduction
Many types of biochemical and antimicrobial tests are being performed in a laboratory in order to detect the specific characteristic of a micro-organism especially a pathogen and its reactions to the certain enzymes.
One such biochemical test is Bacitracin susceptibility test. Bacitracin susceptibility test is one of the antimicrobial tests which are used for identifying a group A streptococcus from the other Beta-hemolytic species of Streptococci.
What is Bacitracin Susceptibility Test?
Bacitracin susceptibility test is considered as an antimicrobial disk tests used foe identifying the Group A Streptococci from the other species of Beta-hemolytic Streptococci.
Antimicrobial susceptibility tests are usually performed in order to differentiate a particular species from another specific genus.
Here, antimicrobial tests are used in order to determine the susceptibility of a bacterial species to a specific antibiotic.
Bacitracin
Bacitracin is one of the bactericidal drugs which is used to treat the superficial skin infections. But these are rarely injected for systemic uses.
Bacitracin is obtained from the Bacillus subtilis which is one of the polypeptide antibiotics.
Bacitracin often interferes with the peptidoglycan and help in producing the bacteria and also in inhibiting their growth.
Bacitracin blocks the bactoprenol from transporting the sugars such as NAM and NAG sugars across the cell membranes, which further inhibits the production of peptidoglycan.
The growth of Group A Beta-hemolytic Streptococcus is inhibited by the Bacitracin. Where as the other species of beta-hemolytic streptococci are not inhibiting.
This helps us to differentiate the two different group of organisms. However, Bacitracin disks are mainly used against the Streptococcus pyrogens which helps in inhibiting the growth of an organism.
Bacitracin results in forming the zone of inhibition of about 12mm against the S. pyrogens, hence it helps us to determine the positive control of the organism while testing.
Bacitracin Susceptibility Test Objective
The main aim of the Bacitracin susceptibility test is to differentiate the Group A Beta-hemolytic Streptococci form the other species of Beta-hemolytic Streptococci.
To detect the pattern of antibiotic susceptibility in various organisms against Bacitracin.
Bacitracin Susceptibility Test Principle
Generally, the growth of Group A Beta-hemolytic streptococci on the blood agar is inhibited by using 0.04 units of Bacitracin disks.
Whereas the other similar species like Micrococci and Streptococci are also inhibited but this 0.04-unit disc, whereas the other Coagulate-negative Staphylococci are resistant to this.
Bacitracin susceptibility test discs are simply a filter paper disc, which are impregnated with 0.04 units of Bacitracin.
These impregnated discs are then placed on an agar, which allows the antimicrobial to diffuse along with the medium thus inhibiting the growth of the organisms.
This test result is evaluated after incubating based on the zone of inhibition that are formed around the discs.
If the growth is seen at the edges of the disk, the it is deemed as resistant, in other case if the zone is present in a circular zone around the stick, then it represents the inhibition and susceptibility of the organisms.
Usually, Bacitracin disks are time saving experiments along with minimum labor and materials, if they are used in the form of screening test before serological grouping.
It is also said that Group A Streptococci are more sensitive to Bacitracin than the Beta-Hemolytic strains of the other groups.
Hence it is advisable to perform Bacitracin susceptibility test through antimicrobial disks for a rapid diagnosis, especially for Group A Streptococci.
Micro Organisms Tested
This test is generally used for Penicillin-susceptible test or for stick colonies of the Gram-positive cocci, which are found in groups and catalyzes the positive and negative coagulates.
Here lemon-Yellow colored colonies are not detected as they are assumed as Micrococcus.
Bacitracin Susceptibility Test Reagents
Media Used:
Blood agar or
Muller Hinton Agar
Supplies Used:
Bacitracin 0.04-unit discs
Sterile forceps
Swab
Inoculation broth.
Bacitracin Susceptibility Test Procedure
Usually, two different kinds of methods are used for Bacitracin Susceptibility test based on the kind of culture media they are used.
The test can be performed either by using pure culture of an organism or directly via clinical samples. The methods of Bacitracin susceptibility tests are listed below.
1. Hebert’s Method Using Blood Agar Plates
Initially 0.1 McFarland suspension of the organism is performed using a over night culture of the organism.
Different sections of blood agar plate are inoculated which results in forming a lawn culture.
Here each section is inoculated in one specific direction and the area of inoculation should be at a separation of 10mm between each of the discs that were placed.
These are then left to dry for about 10 minutes and after drying they are placed on the agar using sterile forceps.
Then the disc is tapped using the sterile stick and adherence is ensured.
The plates are then incubated at a temperature of about 35 to 37ºC for about 24 hours.
After a period of incubation, the zone of incubation is observed thus results are measured.
Further the results are confirmed using serological testing.
2. Muller-Hinton Agar Method
This method is also used to observe the susceptibility of the fast-growing organisms. Here 0.5 Mc Far land suspension of the organisms is prepared using an over night culture of the organism.
The MHA plates disks are inoculated using a suspension with sterile swabs to form a bacterial lawn on the agar.
Then the medium is allowed to dry. After drying the antibiotic disks are placed on the agar placed using the sterile forceps and the discs are placed by maintaining a distance of about 10mm.
Then further the discs are tapped using sterile sticks.
Then the inverted plates are incubated at a temperature of about 35 to 37ºC for about 24 hours.
After incubation the zone of inhibition is observed and measured.
Bacitracin Susceptibility Test Results
Zone of Inhibition Media Result Zone of inhibition is 6mm or less than that Blood agar or Muller Hinton Agar Resistant Zone of inhibition is greater than 10mm Blood agar or Muller Hinton Agar Susceptible Zone of inhibition is between 6mm to 10mm Blood agar or Muller Hinton Agar It indicates probable susceptibility so the tests should be repeated Bacitracin Susceptibility Test Citations
- The bacitracin sensitivity test for identifying beta-haemolytic streptococci of Lancefield group A. J Postgrad Med . 1981 Apr;27(2):86-9.
- Variations in bacitracin susceptibility observed in Staphylococcus and Micrococcus species. J Clin Microbiol . 1986 May;23(5):963-4.
- The bacA gene, which determines bacitracin susceptibility in Streptococcus pneumoniae and Staphylococcus aureus, is also required for virulence. Microbiology (Reading) . 2000 Jul;146 ( Pt 7):1547-1553.
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Acetate Utilization Test: Principle, Procedure, and Results
Continue ReadingAcetate Utilization Test Introduction
In the modern world, apart from life style changes, we come across many medical terms as a part of our daily activities, such as Lab test, diagnosis, vaccine, chemicals and so on, which became most common terms as a part of other things we hear.
Many tests are performed in a laboratory and in biochemical labs to detect the nature of the pathogen or a disease. Acetate utilization test is one such test which is used to determine whether the organism has the ability to use acetate as a sole source of carbon.
What is Acetate Utilization Test?
The acetate utilization test is generally used to test the ability of the organism to utilize the acetate as the single source of carbon.
This test is also performed as a qualitative test for the differentiation of the Gram-negative bacteria into fermentative and the oxidative group of bacteria.
This test is also being used to differentiate the species of Shigella from E. coli and the non-fermentative negative bacteria.
Acetate agar which contains sodium acetate as the sole source of nitrogen is used for inoculating the organism. Growth is indicated for the positive test of acetate utilization test.
When the bacteria metabolize acetate, the ammonium salts are broken down into ammonia, which increases the pH medium of the culture, this increase in pH turns the bromothymol blue indicator in the medium changes from green to blue.
Acetate Utilization Test Principle
In acetate utilization test, Acetate agar is employed as a test organism. This acetate agar has the ability to utilize acetate.
The culture medium is composed of Sodium acetate which acts as a sole carbon source and the inorganic ammonium salts as the source of the nitrogen. Where the growth of organisms suggests the positive results for the utilization of acetate.
During the metabolism process of acetate, by the bacteria, the ammonium salts are broken into ammonium, that elevates alkalinity.
The shift in the pH changes bromothymol blue indicator in the medium from green to blue. This medium is generally used for differentiating the Shigella spp from Escherichia coli.
As Shigella spp doesn’t have the capability to metabolize the acetate, However, approximately of about 94% of Escherichia coli plays a major role in utilizing the acetate.
Acetate Utilization Test Reagents
Media: The culture media is composed of Sodium acetate agar.
Ingredients Gram/liter Sodium Chloride 5.0 Magnesium sulfate 0.1 Ammonium phosphate-monobasic 1.0 Potassium phosphate-dibasic 1.0 Sodium acetate 2.0 Agar 20.0 Bromothymol blue 0.08 Sterilized sticks and inoculating loops
Sterile pipette
Incubator
Sterile saline.
Acetate Utilization Test Procedure
Procedure for Acetate utilization test involves two steps;
1. Preparation of media
2. Utilization test
1. Preparation of Media
For the preparation of media, 69.1 grams of the dehydrated powder is added in beaker along with 1000 milliliters of the deionized or the distilled water.
Instead of dehydrated powder lab-prepared media can also be used.
The prepared suspension is then heated till boiling; so that the medium is dissolved completely.
Then the dissolved medium is dispensed into tubes and they are sterilized in an autoclave at 121ºC for about 15 minutes.
After completing the process of autoclaving the tubes are taken out and cooled at a slanted position to a temperature of about 40 to 45º.
The position is then maintained in order to obtain butts of depth 1.5 to 2.0 cm.
2. Utilization Test
The isolated colony is taken from an 18 to 24-hour culture with the help of a sterile inoculating needle.
A turbid suspension of saline is prepared by using 18-to-24-hour culture from a noninhibitor plate of culture.
The acetamide agar tube is inoculating by streaking the surface of a slant with the light inoculum which is picked from the culture.
The slant is then streaked back and froth with the loop or using an inoculating stick.
The cap or the test tubes are loosened to ensure whether the inoculum is getting sufficient aeration.
The tubes are then incubated aerobically at the temperature of about 35 to 37ºC for about seven days.
As incubation at 35 to 37ºC is not sufficient for thee Enterobacteriaceae, incubation at 30ºC is followed for seven days for the non-fermentation of Gram-negative rods.
The test tubes are examined regularly for at least 7 days before discarding the samples.
Acetate Utilization Test Result
For a positive result, the growth is represented as a change of color green to intense blue along the slant. Where as for negative result, the growth is absent and there will be no color change and the slant remain green as same.
Bacterial Control
Mostly two different organisms are taken for the positive and negative controls as the form of quality control for the acetate utilization test.
Control Incubation Results Shigella flexneri Aerobic incubation is followed for 24 to 48 hours at temperature of about 33 to 37ºC Acetate negative, where there is no growth and no change in color and the medium remains green Escherichia coli Aerobic incubation is followed for 24 to 48 hours at temperature of about 33 to 37ºC Acetate positive where the growth is shown and change of color to intense blue from green Acetate Utilization Test Uses
• This test is usually used to test the ability of the organism to utilize acetate as the source of carbon.
• This test is also used in the form of qualitative test for differentiation of Gram-negative bacteria for fermentation and oxidative group of bacteria.
• Acetate agar can also be used as a selective media for the isolation of Escherichia coli.
Acetate Utilization Test Citations
- Acetate tolerance and the kinetics of acetate utilization in diabetic and nondiabetic subjects. Am J Clin Nutr . 1990 Jan;51(1):112-8.
- Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies. PLoS One. 2014; 9(10): e109255.
- Acetate utilization is inhibited by benzoate in Alcaligenes eutrophus: evidence for transcriptional control of the expression of acoE coding for acetyl coenzyme A synthetase. J Bacteriol . 1995 Oct;177(20):5826-33.
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Mitochondria: Function, Definition, Structure, & Facts
Continue ReadingMitochondria
o Mitochondria are the powerhouses of the eukaryotic cell. According to the endosymbiont theory, mitochondria may have evolved from a symbiotic relationship between ancient prokaryotes and eukaryotes.
o Like prokaryotes, mitochondria have their own circular DNA that replicates independently from the eukaryotic cell.
o This DNA contains no histones or nucleosomes.
o Sections of DNA that aren’t in use are wrapped tightly around globular proteins called histones.
o Eight histones wrapped in DNA form a nucleosome.
o Nucleosomes, in turn, wrap into coils called solenoids, which wrap into supercoils.
o Mitochondrial DNA is passed maternally (from the mother) even in organisms whose male gamete contributes to the cytoplasm.
o The genes in the mitochondrial DNA code for mitochondrial RNA that is distinct from the RNA in the rest of the cell.
o Thus mitochondria have their own ribosomes with a sedimentation factor of 55-60 S.
o Antibiotics that block translation in prokaryotes also seem to work in the mitochondria.
o Mitochondria also have some different codons (presenting an exception to the universal genetic code).
o However, most proteins used by mitochondria are coded for by nuclear DNA, not mitochondrial DNA.
Mitochondria Structure
o Mitochondria are surrounded by two phospholipid bilayers.
o The inner membrane invaginates to from the cristae.
o It is the inner membrane that holds the electron transport chain.
o Between the inner and outer membrane is the intermembrane space (lower pH and higher [H+] than the matrix).
o Organelles with two membranes: nucleus, mitochondria, and chloroplast.
Mitochondria Function
o TCA / Krebs Cycle: (aerobic, occurs in the cytoplasm for prokaryotes, mitochondrial matrix for eukaryotes).
Citations
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Cell Junctions: Definition, Types, and Function
Continue ReadingAbout PhD In Psychology
o Certain cells need to be able to contact other nearby cells. Cell junctions adhere membranes of adjacent cells.
Types of Cell Junctions
There are three different types of cellular junctions:
I. Gap Junctions
o Connect the cytoplasm of adjacent cells, which allows for the movement of molecules between the cells.
o Gap junctions are found in the heart muscle. Gap junctions allow for rapid spread of information between cells.
o Ex. Electrical synapse
Diagram Representing Types of cell Junctions
II. Tight Junctions
o Provide waterproof seals between adjacent cells to prevent leakage of fluid.
o Tissues held together by tight junctions may act as a complete fluid barrier.
o Tight junctions also act as a barrier to protein movement between the apical and basolateral surface of a cell.
o The portion of the cell exposed to the lumen is called its apical surface.
o The rest of the cell (i.e., its sides and base) make up the basolateral surface.
o The lumen is the space enclosed by an organ/organelle.
o Tight junctions perform two vital functions:
o They prevent the passage of molecules and ions through the space between cells.
o So materials must actually enter the cells (by diffusion or active transport) in order to pass through the tissue.
o This pathway provides control over what substances are allowed through.
o They block the movement of integral membrane proteins (red and green ovals) between the apical and basolateral surfaces of the cell.
o Thus the special functions of each surface, for example o receptor-mediated endocytosis at the apical surface o exocytosis at the basolateral surface can be preserved.
III. Adhering Junctions (Desmosomes)
o Join two cells at a single point and they directly attach the cytoskeleton of each cell.
o They don’t prevent fluid from circulating around all sides of a cell.
o Are the types of junctions found cells that experience a lot of stress and they often accompany tight junctions.
o Ex. in epithelial cells which allow for stretching.
o Desmosomes (adhering junctions) act like spot welds.
Cell Junctions Citations
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Cytoskeleton: Description, Structure, and Function
Continue ReadingCytoskeleton
o The cytoskeleton is a cellular “scaffolding” or “skeleton” contained within the cytoplasm.
o The cytoskeleton is present in all cells; it was once thought this structure was unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton.
o It is a dynamic structure that maintains cell shape, protects the cell, enables cellular motion (using structures such as flagella, cilia and lamellipodia), and plays important roles in both intracellular transport (the movement of vesicles and organelles, for example) and cellular division.
Cytoskeleton Component
Types of Cytoskeleton
There are two types of filaments in the cytoskeleton;
I. Microtubules
o Microtubules are built from subunits-molecules of tubulin-each one of which is itself a dimer composed of two very similar globular proteins called a-tubulin and b-tubulin bound tightly together by noncovalent bonds.
o Although tubulin is globular it can polymerize into long straight filaments.
o Thirteen of these filaments lie alongside each other to form the microtubule.
o In nine triplet sets (star-shaped), they form the centrioles.
o Centrioles function in the production of flagella and cilia, but are NOT necessary for microtubule production.
o The major portion of each flagellum and cilium, called the axoneme, contains nine pairs of microtubules forming a circle around two lone microtubules.
o The latter formation is commonly referred to as a “9+2” arrangement, wherein each doublet is connected to another by the protein dynein.
o The mitotic spindle is made from microtubules.
o Mitotic spindle provides the machinery that will segregate the chromosomes equally into the two daughter cells.
o Microtubules have a + and a – end.
o This is because of the polarity of the a and b tubulin, thus making the microtubule polar.
o The – end attaches to a microtubule-organizing center (MTOC) in the cell.
o The major MTOC in animals is the centrosome.
o A microtubule grows away from an MTOC at its + end.
o Centrosomes are composed of two orthogonally arranged centrioles Centrosomes are often associated with the nuclear membrane during interphase of the cell cycle.
o In mitosis the nuclear membrane breaks down and the centrosome nucleated microtubules can interact with the chromosomes to build the mitotic spindle.
o The centrosome is copied only once per cell cycle so that each daughter cell inherits one centrosome, containing two centrioles.
o The centrosome replicates during the S phase of the cell cycle.
II. Microfilaments
o Microfilaments are the thinnest filaments of the cytoskeleton found in the cytoplasm of all eukaryotic cells.
o These linear polymers of made of actin subunits are flexible and relatively strong.
o Microfilaments produce the contracting force in muscle as well as being active in cytoplasmic streaming, phagocytosis, and microvilli movement, cytokinesis, and muscle movement.
o In fungi, the Septa is usually perforated to allow exchange of cytoplasm between cells, called cytoplasmic streaming.
o Both microtubules and microfilaments are involved in intracellular movements in eukaryotic cells.
o In both cases the movements are generated by motor proteins, which bind to microfilaments and microtubules and use the energy derived from repeated cycles of ATP hydrolysis to travel steadily along the microtubule or microfilaments in a single direction.
o The motor proteins that move along cytoplasmic mircrotubules are: kinesins ( – ⇒ + ) and dyneins ( + ⇒ – ).
o Actin uses myosin as its motor protein.
Eukaryotic Flagella and Cilia
o Eukaryotic flagella and cilia are specialized structures also made from microtubules.
o The major portion of each flagellum and cilium, called the axoneme, is arranged in nine doublets oriented about two additional microtubules (wheel-shaped) they form cilia and flagella.
o This formation is commonly referred to as a “9+2” arrangement, wherein each doublet is connected, via a crossbridge, to another by the protein dynein.
o The cross bridges cause the microtubule pairs to slide along their neighbors creating a whip action in cilia causing fluid to move laterally, or a wiggle action in flagella causing fluid to move directly away from the cell.
o Both cilia and flagella grow from a basal body.
o In humans cilia are only found in the fallopian tubes and the respiratory tract and in cerebrospinal fluid in ependymal cells.
o Bacterial flagella are long, hollow, rigid, helical cylinders made from a globular protein called flagellin, these shouldn’t be confused with eukaryotic flagella which are made up of microtubules.
o They rotate counterclockwise. When they are rotated clockwise, the bacterium tumbles.
o This tumbling acts to change the orientation of the bacterium allowing it to move forward in a new direction.
o A basal body is an organelle formed from a centriole, a short cylindrical array of microtubules.
o It is found at the base of a eukaryotic cilium or flagellum and serves as a nucleation site for the growth of the axoneme microtubules.
Cytoskeleton Citations
- The cytoskeleton and cancer. Cancer Metastasis Rev . 2009 Jun;28(1-2):5-14.
- Cytoskeleton-a crucial key in host cell for coronavirus infection. J Mol Cell Biol . 2020 Jul 1;12(12):968-979.
- Mechanical Properties of the Cytoskeleton and Cells. Cold Spring Harb Perspect Biol . 2017 Nov 1;9(11):a022038.
- Overview of the Cytoskeleton from an Evolutionary Perspective. Cold Spring Harb Perspect Biol . 2018 Jul 2;10(7):a030288.
- The Cytoskeleton as a Modulator of Aging and Neurodegeneration. Adv Exp Med Biol . 2019;1178:227-245.
- Cell mechanics and the cytoskeleton. Nature . 2010 Jan 28;463(7280):485-92.
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Endoplasmic Reticulum: Definition, Function, and Structure
Continue ReadingEndoplasmic Reticulum
o The endoplasmic reticulum (ER) by the nucleus has many ribosomes attached to it on the cytosolic side, giving its granular appearance, hence the name granular or rough ER.
o Translation on the rough ER propels proteins into the ER lumen as they are created.
o These proteins are tagged with a signal sequence of amino acids and sometimes glycosylated (carbohydrate chains are added – doesn’t occur in the cytosol).
o A 20 amino acid sequence called a signal peptide near the front of the peptide is recognized by protein-RNA signal-recognition particles (SRPs) that carries the entire ribosome complex to a receptor on the ER.
o The peptide is actually pulled through the membrane through an ATP driven process.
o The signal peptide is also usually removed by an enzyme.
o Some proteins have an ER retention signal which tells the ER that it is destined to function there, so if that protein escapes to the Golgi it will be returned to the ER.
o Rough ER has ribosomes attached to its cytosol side, and it synthesizes virtually all proteins not used in the cytosol.
o Proteins synthesized on the rough ER are pushed into the ER lumen and sent to the Golgi.
o The ER lumen is contiguous in places with the space between the double bilayer of the nuclear envelope.
Endoplasmic Reticulum Structure
Features of Endoplasmic Reticulum
o The newly synthesized proteins are moved through the lumen toward the Golgi apparatus or Golgi complex.
o The Golgi apparatus is a series of flattened, membrane bound sacs (cisterna) which are piled like stacks of plates.
o The Golgi apparatus is usually located close to the nucleus, and in animal cell is often close to the centrosome.
o Each Golgi stack has two distinct faces: an entry, or cis, face and an exit, or trans, face.
o The cis face is adjacent to the ER and the trans face is facing the cytosol.
o Small transport vesicles bud off from the ER and carry the proteins across the cytosol to the Golgi.
o Proteins are distinguished based upon their signal sequence and carbohydrate chains.
o Those proteins not possessing a signal sequence are packaged into secretory vesicles and expelled from the cell in a process known as bulk flow.
o The Golgi may change proteins chemically by glycosylation or by removing amino acids.
o The end product of the Golgi is a vesicle full of proteins.
o These protein filled vesicles may either be expelled from the cell as secretory vesicles, released from the Golgi to mature into lysosomes, or transported to other parts of the cell.
o The Golgi is like the post office it sorts and packages proteins.
o It can also alter proteins.
o Usually where proteins are modified and these modifications of the proteins determine where they will go.
Secretory Vesicles
o Secretory vesicles (sometimes called zymogen granules) may contain enzymes, growth factors, or extracellular matrix components.
o Secretory vesicles release their contents through exocytosis.
o Since exocytosis incorporates vesicle membranes into the cell membrane, secretory vesicles also act as the vehicle with which to supply the cell membrane with its integral proteins and lipids, and as the mechanism for membrane expansion.
o Secretory vesicles are continuously released by most cells in a process called constitutive secretion.
o Some specialized cells (ex. neuronal cells) can release secretory vesicles in response to a certain chemical (ex. Ca+2) or electrical stimulus in a process called regulated secretion.
o Ex. Neural synapses
o Some proteins are activated within secretory vesicles.
o (Ex. Proinsulin ⇒ insulin).
Lysosomes
o Lysosomes contain acid hydrolases (hydrolytic enzymes) such as proteases, lipases, nucleases, and glycosidases.
o Together, these enzymes are capable of breaking down EVERY major type of macromolecule within the cell, including old organelles.
o Lysosomes generally have a pH of 5.
o They fuse with endocytotic vesicles and digest their contents.
o Any material not degraded by the lysosome is ejected from the cell through exocytosis.
o Under certain conditions lysosomes will rupture and release their contents into the cytosol killing the cell in a process called autolysis.
o This is useful in the formation of certain organs and tissues, like the destruction of the tissue between the digits of a human fetus in order to form fingers.
o Contain hydrolytic enzymes that digest substances taken in by endocytosis.
Smooth Endoplasmic Reticulum
o Smooth ER lacks ribosomes and contains an enzyme which is used in the liver, intestinal epithelial cells, and renal tubule epithelial cells, to make glucose from glycogen.
o Rough ER tends to resemble flattened sacs, whereas smooth ER tends to be tubular.
o Glucose ⇒ Glycogen = glycogenesis
o Glycogen ⇒ Glucose = glycogenolysis
o Triglycerides are produced in the smooth ER and stored as fat droplets.
o Adipocytes are cells containing predominately fat droplets.
o Such cells are important in energy storage and body temperature regulation.
o Adipocytes, also called fat cells, are specialized cells whose cytoplasm contains almost nothing but triglycerides.
o The Smooth ER and the cytosol share in the role of cholesterol formation and its conversion to various steroids.
o Most of the phospholipids in a cell membrane are originally synthesized in the smooth ER.
o The phospholipids are all synthesized on the cytosol side of the membrane and then some are flipped to the other side by proteins called phospholipid translocators located EXCLUSIVELY in the smooth ER.
o Smooth ER oxidizes foreign substances, detoxifying drugs, pesticides, toxins, and pollutants.
o The Smooth ER is the site of lipid synthesis, including steroids, detoxifying drugs, and useful for converting glycogen to glucose.
o Peroxisomes are vesicles in the cytosol that grow by incorporating lipids and proteins from the cytosol.
o Rather than budding off membranes like lysosomes from the Golgi, peroxisomes are self-replicating.
o They are involved in the production and breakdown of hydrogen peroxide.
o Peroxisomes inactivate toxic substances such as alcohol, regulate oxygen concentration, play a role in the synthesis and break of lipids, and in the metabolism of nitrogenous bases and carbohydrates.
Endoplasmic Reticulum Citations
- Endoplasmic reticulum stress signalling – from basic mechanisms to clinical applications. FEBS J . 2019 Jan;286(2):241-278.
- The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci . 2016 Jan;73(1):79-94.
- The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol . 2015;10:173-94.
- Endoplasmic Reticulum redox pathways: in sickness and in health. FEBS J . 2019 Jan;286(2):311-321.
- Endoplasmic reticulum stress: a key player in human disease. FEBS J . 2019 Jan;286(2):228-231.
- Endoplasmic reticulum quality control by garbage disposal. FEBS J . 2019 Jan;286(2):232-240.
- Form follows function: the importance of endoplasmic reticulum shape. Annu Rev Biochem . 2015;84:791-811.
- Endoplasmic Reticulum: Target for Next-Generation Cancer Therapy. Chembiochem . 2018 Nov 16;19(22):2341-2343.
- Endoplasmic reticulum in viral infection. Int Rev Cell Mol Biol . 2020;350:265-284.
- Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol . 2007 Jul;8(7):519-29.
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Endocytosis: Structure, Pinocytosis, and Phagocytosis
Continue ReadingCell Membrane and Endocytosis
o Besides transport across the membrane, cells can acquire substances from the extracellular environment through endocytosis.
o Endocytosis/exocytosis requires ATP hydrolysis.
o Prokaryotes’s membranes cant before endocytosis or exocytosis.
o Viruses enter eukaryotic cells via endocytosis.
Types of Endocytosis
There are several types of endocytosis:
I. Pinocytosis
o Pinocytosis – (“cell-drinking”) is a form of endocytosis in which small particles are brought into the cell suspended within small vesicles.
o It is primarily used for the absorption of extracellular fluids (ECF), and in contrast to phagocytosis, generates very small vesicles.
o Unlike receptor-mediated endocytosis, pinocytosis is nonspecific in the substances that it transports.
o The cell takes in surrounding fluids, including all solutes present.
o A portion of the plasma membrane is invaginated and pinched off forming a membrane-bounded vesicle called an endosome.
o This process, unlike phagocytosis, is nonselective.
II. Phagocytosis
o Phagocytosis is the cellular process of phagocytes (white blood cells) and protists of engulfing solid particles by the cell membrane to form an internal phagosome, which is a food vacuole.
o Phagocytosis is a specific form of endocytosis involving the vesicular internalization of solid particles, such as bacteria, and is therefore distinct from other forms of endocytosis such as pinocytosis, the vesicular internalization of various liquids.
o Phagocytosis is involved in the acquisition of nutrients for some cells, and in the immune system it is a major mechanism used to remove pathogens and cell debris.
o The impetus for phagocytosis is the binding of proteins on the particulate matter to protein receptors on the phagocytic cell.
o Organisms “eat” by stretching out pseudopodia and encircling any food particles they find in their paths.
o In humans, antibodies or complement proteins bind to particles and stimulate receptor proteins on macrophages and neutrophils to initiate phagocytosis.
III. Receptor Mediated Endocytosis
o Receptor mediated endocytosis refers to specific uptake of macromolecules such as hormones and nutrients.
o In this process, the ligand binds to a receptor protein on the cell membrane, and is then moved into a clathrin coated pit.
o Clatherin is a protein that forms a polymer adding structure to the underside of the coated pit.
o The coated pit invaginates to form a coated vesicle.
o One way this process differs from phagocytosis is that its purpose is to absorb the ligands, whereas the ligands in phagocytosis exist only to act as signals to initiate phagocytosis of other particles.
IV. Exocytosis
o Exocytosis is simply the reverse of endocytosis.
o In Eukaryotic cells, unlike prokaryotic, the membrane invaginates and separates to form individual, membrane bound compartments and organelles.
Endocytosis Citations
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Cell Nucleus: Definition, Function, Structure, and Facts
Continue ReadingCell Nucleus
o The major feature distinguishing eukaryotic cells from prokaryotic cells is the nucleus of the eukaryote.
o The aqueous ‘soup’ inside the nucleus is called the nucleoplasm.
o DNA is found in the nucleus and the mitochondria in eukaryotes.
o The nucleus is wrapped in a double phospholipid bilayer (two membranes) said the nuclear envelope or membrane.
Structure of Cell Nucleus
"DNA is found in the nucleus and the mitochondria in eukaryotes"
o The nuclear envelope is perforated with large holes called nuclear pores, through which all molecules enter or leave.
o Small molecules pass right though but larger molecules (such as RNAs and proteins) can’t pass through unless they carry an appropriate sorting signal.
o RNA can exit the nucleus through the nuclear pores, but DNA cannot.
o Proteins are transported in the their fully folded form in the nuclear pore and is actively transported in via GTP hydrolysis, proteins usually have to unfold to cross the membranes of other organelles.
o Within the nucleus is an area called the nucleolus where rRNA is transcribed and the subunits of the ribosomes are assembled.
o The nucleolus is not separated from the nucleus by a membrane.
o Both the nucleus and the nucleolus disappears during prophase of mitosis and meiosis.
"Both the nucleus and the nucleolus disappears during prophase of mitosis and meiosis"
o The eukaryotic cell nucleus.
o Visible in this diagram are the ribosome-studded double membranes of the nuclear envelope, the DNA (complexed as chromatin), and the nucleolus.
o Within the cell nucleus is a viscous liquid called nucleoplasm, similar to the cytoplasm found outside the nucleus.
o Chromatin is the complex combination of DNA, RNA, and protein that makes up chromosomes.
o It is found inside the nuclei of eukaryotic cells, and within the nucleoid in prokaryotic cells.
Cell Nucleus Citations
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