- 1 why does this exercise call for an older culture of bacillus
- 2 Why do you need to use older cultures of Bacillus for Endospore staining?
- 3 Why was a 48 hour culture of B subtilis used rather than a fresh culture?
- 4 How might the age of the culture affect the results of the endospore stain?
- 5 What is the relationship between age of a culture and number of endospores?
- 6 Why is an old culture necessary for finding spores?
- 7 How does the culture affect the results of an Endospore stain quizlet?
- 8 Why does the Endospore stain exercise call for an older 48 hour or 5 day culture of Bacillus?
- 9 What is the function of the Z ring in binary fission?
- 10 Why would older gram positive cultures stain incorrectly?
- 11 How might an old culture affect the results of a Gram stain?
- 12 How does the age of the culture affect the results of the Gram stain for gram-positive bacteria?
- 13 What is the function of mordant?
- 14 What does endospore mean?
- 15 What does the endospore do?
- 16 Why is it important to determine the location of the endospore within the bacterial cell?
- 17 What is the purpose of Bacillus structure?
- 18 Why do bacteria produce spores?
- 19 Which of the following are characteristic of the genus Bacillus?
- 20 How does an endospore containing cell look after Gram staining quizlet?
- 21 How does an endospore containing cell look after Gram staining?
- 22 Why is a Gram stain generally done prior to an endospore stain?
- 23 Why is endospore staining important?
- 24 What does a positive result for the endospore stain indicate about the organism quizlet?
- 25 What types of bacteria does an endospore stain differentiate between?
- 26 What is the function of the Z ring in binary fission quizlet?
- 27 What factors regulate the formation of the Z ring at mid cell site during binary fission?
- 28 Why do bacteria have different generation times?
- 29 Why a bacterium that should be gram-positive might turn out gram-negative?
- 30 Why do Gram positive bacteria and gram negative bacteria appear different in Gram staining?
- 31 Why is crystal violet used in Gram staining?
- 32 Why is gram stain called a differential stain?
- 33 What is the result of Gram staining?
- 34 Why is culture Age important in the Gram reaction quizlet?
- 35 BACILLUS TRONG BEC BZT SINH KHỐI NHƯ THẾ NÀO?
- 36 Welcome to Microbugz – Endospore Stain
- 37 Difference Between Gram-Positive and Gram-Negative Bacillus
- 38 Characteristics of Gram-Positive Bacilli
- 39 Types of Gram-Positive Bacilli
- 40 Treatment of Gram-Positive Bacilli
- 41 Gram-Negative Bacteria
- 42 Risks for Developing Gram-Negative Bacterial Infections
- 43 How Are Gram-Negative Bacteria Treated?
- 44 Preventing and Controlling Antibiotic Resistance
- 45 Lab 6: Gram Stain and Capsule Stain
- 46 PERFORMANCE OBJECTIVES LABORATORY 6
- 47 Contributors and Attributions
- 48 2.4 Staining Microscopic Specimens – Microbiology
why does this exercise call for an older culture of bacillus
What causes the gram stain to behave in the manner that it does? … What is the reason that an older (5-day) culture of Bacillus species is required for the endospore stain? Because older bacteria are more nutritionally stressed, there will be a higher number of endospores present. Which characteristics of the organism do a positive result for the spore stain reveal about it? A dormant, tough, and non-reproductive structure formed by certain bacteria belonging to the phylum Firmicutes, an endospore is a structure that does not reproduce.
Endospores allow bacteria to remain inactive for long periods of time, perhaps even centuries.
Why do you need to use older cultures of Bacillus for Endospore staining?
Endospore staining is referred to as a structural stain since it is used to draw attention to a specific structure within a cell. … Most techniques recommend using cultures that are 18-36 hours old in order to increase the likelihood that spore formers will be discovered. As cultures age, they become more nutrient deprived, which results in sporulation.
Why was a 48 hour culture of B subtilis used rather than a fresh culture?
Why is a 48-hour culture necessary for an endospore stain to be effective? Endospores are formed as a result of environmental stress. You will be able to tell if the bacterium is starving if you wait 48 hours.
How might the age of the culture affect the results of the endospore stain?
What effect does the age of the culture have on the findings of a spore stain? Old cultures contain a higher concentration of spores, and bacteria generate more endospores as they become older.
What is the relationship between age of a culture and number of endospores?
Which relationship exists between the age of a culture and the quantity of endospores present? The younger cultures included more vegetative cells, whereas the older cultures contained endospores. The more advanced the age, the more likely it is that the vegetative cell will die and release the spore. – Counterstain for one minute with safranin stain.
Why is an old culture necessary for finding spores?
Bacterial bodies and flagella will discolor the surface of the Material. Why was it required to utilize an old Bacillus culture for the spore stain, rather than a new one, in this experiment? It is because endospores are forming in an older culture that the needed nutrients are not accessible that the endospores are being created. 2.
How does the culture affect the results of an Endospore stain quizlet?
What role does culture have in the outcome of an endospore staining experiment? For example, if the culture is kept in a hostile environment, there will be more viable spores than viable cells. The same is true in the other direction. What color are endospores after they have been stained with a Gram stain?
Why does the Endospore stain exercise call for an older 48 hour or 5 day culture of Bacillus?
What causes the gram stain to behave in the manner that it does? … What is the reason that an older (5-day) culture of Bacillus species is required for the endospore stain? Because older bacteria are under more nutritional stress, there will be a higher concentration of endospores in the environment. Which characteristics of the organism do a positive result for the spore stain reveal about it?
What is the function of the Z ring in binary fission?
What is the role of the Z ring in the process of binary fission?
It is in charge of the replication of DNA. It contracts at the septum, forming a contractile ring.
Why would older gram positive cultures stain incorrectly?
What causes gram-positive cultures that are more than a decade old to stain incorrectly? When gram-positive cultures become older, they lose their capacity to maintain the crystal-violet iodine complexes and provide misleading gram-negative readings. What component of a bacterial cell wall allows an organism to be classified as gram positive?. Is it possible that the culture smear was not heat fixed?
How might an old culture affect the results of a Gram stain?
Cells that have burst or died in older cultures are more prevalent. Even though the bacteria in the culture are Gram positive, cells from old cultures may stain Gram negative. To yet, the specific mechanism of the Gram stain response has not been determined in detail.
How does the age of the culture affect the results of the Gram stain for gram-positive bacteria?
The age of a culture has an effect on the gram stain response, but how? a.Old cultures have the potential to change to gram-variable or gram-negative, resulting in incorrect findings. If done for an excessive amount of time, gram-positive can seem gram-negative, and if done for an insufficient amount of time, gram-negative can appear gram-positive.
What is the function of mordant?
Unbinding dyes on fabrics is accomplished with the use of a mordant or dye fixative. The mordant or fixative works by establishing a coordination complex with the dye, which then adheres to the cloth (or tissue). It may be used to color materials as well as to increase stains in cell or tissue preparations, among other things.
What does endospore mean?
Endospore — A differentiated cell generated within the cells of some Gram-positive bacteria that is particularly resistant to heat as well as other hazardous circumstances and agents, such as pathogens. Endopsore are the dormant stage of certain bacteria’s life cycle that develops as a result of exposure to harsh environmental circumstances.
What does the endospore do?
It enables the bacteria to develop a dormant and extremely resistant cell that is capable of preserving the genetic material of the cell under harsh conditions. It is possible for endospores to withstand attacks from the environment that would typically kill the bacteria.
Why is it important to determine the location of the endospore within the bacterial cell?
Location. The location of the endosporediffers among bacterial species and how they differ from one another is important for identification. Bacillus cereus is one of the bacteria that have an endospore that is centrally located. The endospore can grow to such an enormous size that the cell becomes distended around the endospore.
What is the purpose of Bacillus structure?
It is this structure on the outside of the cell that serves as a second barrier between the bacteria and the environment, while also maintaining the rod shape and withstanding the pressure created by the bacterium’s turgor (turgor means “pressure”).
Why do bacteria produce spores?
Forming spores to shield themselves against environmental degrading chemicals is one of the most typical coping methods for bacteria. When the surrounding environmental circumstances are favorable for bacterial growth and reproduction, endospores germinate and return to the state of vegetative cells (an active bacterial cell that conducts metabolism).
Which of the following are characteristic of the genus Bacillus?
Bacillus species are rod-shaped, endospore-forming, aerobic or facultatively anaerobic Gram-positive bacteria; in some species, cultures may become Gram-negative with age.
Bacillus species are Gram-positive bacteria that produce endospores. Each of the several species in the genus have a diverse set of physiological properties that enable them to survive in a variety of different environmental environments.
How does an endospore containing cell look after Gram staining quizlet?
What does an endospore look like when it is gram stained? Sporulated cells are purple or pink in color, with circular holes in the center of the cell.
How does an endospore containing cell look after Gram staining?
When using the Gram staining process, it is possible to obtain two distinct colored stains. Finally, when endospore staining is performed, vegetative cells will be stained red due to the presence of the Safranin counterstain. If endospores are present in the sample, the malachite green stain will be retained by the endospores, which will look bluish-green in color.
Why is a Gram stain generally done prior to an endospore stain?
Prior to the use of the endospore staining process, the gram staining procedure was often used to categorize isolated bacteria into the widest possible groupings. Alternative stains, on the other hand, provide for a far better degree of accuracy in the determination.
Why is endospore staining important?
What is the significance of endospore staining?. It aids in the identification of the few medically significant genera that generate endospores, which are rare. It is used to distinguish between bacteria and protozoans under a microscope. It aids in the identification of the few medically significant genera that generate endospores, which are rare.
What does a positive result for the endospore stain indicate about the organism quizlet?
What does a positive result for the endospore stain tell you about the organism it was used to stain? … because an endospore stain is a sort of staining process that is differentiated from others It enables us to view both spores and vegetative cells at the same time. As a result, it is not required to include a separate negative control comprised solely of vegetative cells in the experiment.
What types of bacteria does an endospore stain differentiate between?
The primary goal of endospore staining is to distinguish bacterial spores from other vegetative cells as well as to distinguish spore formers from non-spore formers in a bacterial culture.
What is the function of the Z ring in binary fission quizlet?
What is the role of the Z ring in the process of binary fission? It contracts at the septum, forming a contractile ring.
What factors regulate the formation of the Z ring at mid cell site during binary fission?
During the vegetative growth of bacilli that divide by binary fission, the Z ring develops near the midway of the cell wall. Neither the Min proteins nor the condition of the chromosome are able to positively influence this medial location.
Why do bacteria have different generation times?
The cells divide at a consistent pace, which is determined by the content of the growth medium and the circumstances of incubation in which they are grown. The generation time of a bacterial culture, as well as the doubling time of the bacterial population, is used to represent the rate of exponential growth of a bacterial culture.
Why a bacterium that should be gram-positive might turn out gram-negative?
When the skin has been over-decolorized, either by extended contact to the decolourizer or through the use of acetone alone. It occurs when the cell wall is destroyed by exposure to lysozyme or antibiotics that act on the cell wall such as Penicillin.
Why do Gram positive bacteria and gram negative bacteria appear different in Gram staining?
It has been found that Gram positive bacteria (which have a thicker peptidoglycan layer in their cell membrane) retain crystal violet stain during the decolorization process, while Gram negative bacteria (which have a thinner peptidoglycan layer) lose the crystal violet stain and turn yellow during the decolorization process.
Why is crystal violet used in Gram staining?
Crystal violet is used as the principal stain in the gram staining procedure.
Because this basic dye is positively charged, it clings to the cell membranes of both gram negative and gram positive cells. … Gram’s Iodine is used as a mordant. This forms a huge complex with the crystal violet, which clings to the cell membrane as a result of the binding.
Why is gram stain called a differential stain?
This type of stain is known as a differential stain because it distinguishes between Gram-positive and Gram-negative bacteria. Bacteria that stain purple when subjected to the Gram staining method are referred to as Gram-positive bacteria, and bacteria that stain pink are referred to as Gram-negative bacteria.
What is the result of Gram staining?
What exactly do the results of a gram stain mean?. Gram-positive bacteria will look purple under a microscope as a result of the staining procedure utilized, whereas gram-negative bacteria would appear pink under a microscope. Additionally, the shape, size, and number of germs present will reveal information regarding your infection.
Why is culture Age important in the Gram reaction quizlet?
For a correct gram response, what is the optimal culture age that should be used? Because older cells do not retain stain as well as younger cells, older cultures of gram positive cells may provide false negative findings (for example, pink cells). The finest cultures are those that last 16-18 hours.
BACILLUS TRONG BEC BZT SINH KHỐI NHƯ THẾ NÀO?
Why is it necessary to use an older culture of Bacillus quizlet for this exercise? Why was it required to use an old bacillus culture for the spore stain? Why was it necessary to use an old bacillus culture for the spore stain? Endospores do not readily stain with a variety of dyes. Maybe you’ve seen them before. Some oral bacteria create extracellular capsules, which are neutrally charged and are produced by other oral bacteria. If this is the case, how is it possible to watch flagella in action?
See more entries in the FAQ category.
Welcome to Microbugz – Endospore Stain
Endospore Stain is a stain that is used to identify endospores. The endospore stain is a differential stain that is used to see bacterial endospores in their natural environment. Endospores are generated by bacteria belonging to a few species, such as Bacillus. Bacteria can live in adverse environments because they produce spores. Spores are resistant to high temperatures, desiccation, chemicals, and radiofrequency radiation. Following exposure to harsh circumstances, bacteria can produce endospores within 6 to 8 hours of being subjected to the same conditions.
- Spores are biologically inert and dehydrated at the time of harvest.
- After being exposed to suitable circumstances for 90 minutes, spores can germinate and develop into a vegetative cell.
- They can be classified as either central, subterminal, or terminal.
- Terminal endospores are found near the tip of the vegetative cell’s apical segment.
- Endospores can also have a diameter that is more or less than that of the vegetative cell.
- These endospore traits are similar among spore-forming species, and they may be used to identify the organism from which they are originating.
- Heating causes malachite green, the principal dye used in the technique for staining endospores, to be absorbed into the cells.
Moreover, because malachite green is a water-soluble dye that does not stick well to cells, and because heat has ruptured the vegetative cells, the malachite green rinses easily off the vegetative cells, allowing them to rapidly absorb the counterstain. The staining process may be found here.
Difference Between Gram-Positive and Gram-Negative Bacillus
Bacterial classification can be accomplished in a variety of methods. One strategy is based on the membrane of the cell. An American bacteriologist by the name of Christian Gram developed a test in 1884 that could tell whether or not a bacterium was covered in an extracellular matrix called peptidoglycan. Gram positive bacteria are those that have a thick layer of peptidoglycan on their surface. When the peptidoglycan coating is thin, the organism is characterized as gram negative.
Characteristics of Gram-Positive Bacilli
A gram-positive bacillus doesn’t have an exterior cell wall beyond the peptidoglycan membrane. As a result, it becomes more absorbent. Gram-negative bacilli have a peptidoglycan coating that is much thicker than that of gram-positive bacteria. Microorganisms that are Gram positive are shaped like rods. They can also be classified according to whether or not they produce spores and whether or not they require oxygen to exist.
Types of Gram-Positive Bacilli
Flora that grows in the area. The majority of gram-positive bacilli are safe and do not cause any effects on your body. These are referred to as “resident flora.” Your body has the following locations where you can find them. Gram-positive bacteria that are pathogenic, which implies they cause disease in humans, include gram-positive bacilli. Anthrax is one of the most dangerous gram-positive bacteria that may cause sickness, and it is caused by anthrax. Yersinia pestis is a potentially lethal illness that often affects the skin or lungs, although it can also infect the gastrointestinal tract in rare cases.
- This organism has the potential to be used as a biological weapon since its spores may be disseminated through the air and ingested by humans.
- This infection can be avoided with vaccination.
- Infections caused by the enterococcal bacteria.
- There are more than 17 distinct species to choose from.
- It is possible for them to spread to other parts of your body, such as your bloodstream, heart valves, or skin, where they can cause serious infections.
- Skin infection caused by handling infected animals that generally occurs as a result of a scrape or puncture wound on the skin.
- It is associated with symptoms like as fever, chills, nausea, vomiting, and diarrhea.
Treatment of Gram-Positive Bacilli
Antibiotics are used to treat illnesses caused by Gram-positive bacilli. Penicillin, cloxacillin, and erythromycin are effective antibiotics that treat more than 90 percent of gram-positive bacteria. The development of antibiotic resistance, on the other hand, is becoming a severe concern with gram-positive infections. New medications are being developed to assist in the treatment of this condition.
In most cases, antibiotics should only be taken when absolutely necessary. In order to avoid the development and spread of antibiotic-resistant diseases, it is essential that infection control guidelines are strictly adhered to.
Gram-negative bacteria have a hard, protective outer shell that protects them from the environment. The peptidoglycan layer of gram-negative bacteria is much thinner than that of gram-positive bacteria. Gram-negative bacteria have a tougher cell wall than Gram-positive bacteria, which makes them more difficult to destroy. Gram-negative bacteria emit endotoxins when their cell wall is disrupted, which might exacerbate your symptoms. A wide range of dangerous illnesses can be caused by Gram-negative bacteria, including:
- Cholera is a dangerous intestinal infection that can be fatal. E. coli bacterium An illness of the lymph nodes and lungs that is known as the plague. Cat scratch illness (also known as cat scratching disease). An infection caused by Campylobacter bacteria that mainly affects the digestive system
- Legionnaire’s disease is a lung ailment caused by Legionella bacteria. Infection with Salmonella, a digestive infection brought on by contaminated food An infection of the urinary or respiratory tract caused by Klebsiella, which is more common in long-term care facilities
- There are several distinct types of infections caused by the bacteria Pseudomonas that affect different regions of the body. A disease caused by wild animals, tularemia, is spread by contact with them. Typhoid fever, a disease that is more prevalent in impoverished nations than in developed countries
Risks for Developing Gram-Negative Bacterial Infections
Cholera is a major intestinal infection that can be fatal; cholera is a serious intestinal infection that can be fatal; Bacillus coliformes An infection of the lymph nodes and lungs that is known as the plague Infection caused by scratching a cat’s paw. An infection caused by the bacteria Campylobacter, which mainly affects the digestive system a lung illness known as Legionnaire’s disease Ailment with Salmonella, a digestive infection brought on by contaminated food; An infection of the urinary or respiratory tract caused by Klebsiella, which is more common in long-term care facilities.
A disease caused by wild animals, tularemia, is spread around the world.
- Being on a ventilator because of renal illness
- Undergoing dialysis
- Etc. Having a compromised immune system
- Having just had surgery
- Inflicting war wounds
- Making use of a catheter
How Are Gram-Negative Bacteria Treated?
Antibiotic resistance in Gram-negative bacteria is quite prevalent. They are considered to be one of the most important public health problems in the world today. Gram-negative bacteria are capable of inflicting a wide range of illnesses on human beings. They have the ability to reach nearly all of the organ systems. It is possible that your doctor will need to try numerous drugs to successfully treat the illness. It’s possible that older antibiotics are more effective.
Preventing and Controlling Antibiotic Resistance
Antibiotic resistance is exacerbated as a result of the widespread usage of antibiotics. You can assist regulate and prevent antibiotic resistance by doing the measures listed below:
- Antibiotics should only be taken as directed by your healthcare practitioner. Follow the recommendations of your healthcare practitioner when it comes to antibiotic use. Don’t re-use or distribute antibiotics that have expired. Hands should be washed
- Maintain a safe sexual environment
- Maintain your immunization records
- It is best to avoid contact with ill persons whenever possible. Prepare your meals in a safe manner.
Lab 6: Gram Stain and Capsule Stain
THE GRAM STAIN (PART A)
Bacteriology students and professionals both employ the Gram stain as the most common staining process. It is referred to as adifferential staining because it is capable of distinguishing between Gram-positive and Gram-negative bacteria. Bacteria that stain purple when subjected to the Gram staining method are referred to as Gram-positive, whereas bacteria that stain pink are referred to as Gram-negative. There is no connection between the labels positive and negative in terms of electrical charge; rather, they are used to denote two separate morphological categories of bacteria.
- The bacterial cell wall is responsible for the size and form of the organism, as well as for preventing osmotic lysis from occurring.
- Cell walls that are Gram-positive look as a wide, dense wall that is 20-80 nm thick and is made up of several interconnecting layers of peptidoglycan when seen under an electron microscope (Figures1Aand1B).
- Teichoic acids are teichoic acids that are found in the cell wall of Gram-positive bacteria.
- Some of them have a lipid connected to them (lipoteichoic acid).
- On the other hand, the Gram-negative cell wall comprises just 2-3 layers of peptidoglycan and is surrounded by an outer membrane that is formed of phospholipids, lipopolysaccharide, lipoprotein, and proteins, as opposed to the Gram-positive cell wall (Figures2Aand2B).
- The phospholipids and lipoproteins that link the outer membrane to the peptidoglycan are mostly found in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan.
- Lipopolysaccharides are found in the outer layer of the outer membrane and are found in the outer layer of the outer membrane.
The outer membrane of the bacteria also contains a number of proteins that vary depending on the strain and species of the bacterium in question. In your Lecture Guide, you’ll find the following Learning Objects that provide further information about the Gram-negative and Gram-positive cell walls:
- The Prokaryotic Cell Wall (Unit 1, Section IIB2)
- The Gram-Positive Cell Wall (Unit 1, Section IIB2a)
- The Gram-Negative Cell Wall (Unit 1, Section IIB2b)
- The Gram-Negative Cell Wall (Unit 1, Section IIB2c)
- The Gram-Negative Cell Wall (Unit 1, Section IIB2d)
- The Gram-Negative Cell Wall (Unit 1, Section IIB2e)
The Gram staining technique is comprised of four fundamental steps: 1. The bacteria are first dyed with crystal violet, which is a fundamental dye. During this stage, both Gram-positive and Gram-negative bacteria are directly stained with a purple dye and look purple. 2. Gram’s iodine solution is used to treat the germs once they have been killed. This improves the stain’s retention by producing an insoluble crystal violet-iodine complex, which is insoluble in water. After this stage, both Gram-positive and Gram-negative bacteria retain their purple color.
- This is the stage when the differences are made.
- Final step is to apply the counterstainsafranin (which is also a basic dye).
- A colorless Gram-negative bacterium becomes directly stained by the safranin, which is now present in the culture.
- Gram staining is hypothesized to be caused by a reaction between the crystal violet and iodine in Gram-positive bacteria, which results in the formation of a bigger molecule that precipitates out of the cell.
- Gram-negative bacteria are susceptible to the effects of the alcohol/acetone combination because it is a lipid solvent that dissolves the outer membrane of the cell wall and may also cause damage to the cytoplasmic membrane, which is connected to the peptidoglycan.
- In order to understand Gram-positivity (the capacity to preserve the purple crystal violet-iodine complex), it is necessary to understand that it is not an all-or-nothing phenomena but rather a question of degree.
- Overheating during heat fixation, excessive decolorization with alcohol, and even too much washing with water between processes can cause Gram-positive bacteria to lose their crystal violet-iodine complex, which is essential for their survival.
Cultures that have been incubated for more than 24 hours may lose their capacity to maintain the crystal violet-iodine combination, which is harmful.
There are some Gram-positive bacteria that are better at retaining the crystal violet-iodine combination than other Gram-positive bacteria.
ORGANISMS Escherichia coli (a tiny, Gram-negative bacillus) and Staphylococcus epidermidis plate cultures on trypticase Soy agar were used to study the effects of trypticase on their growth (a Gram-positive coccus with a staphylococcus arrangement).
Follow these steps to heat-fix an Escherichia coli smear: a.
An alternative method is to pour a drop of deionized water on the slide using your sterilized inoculating loop.
Gram staining requires a high-quality smear with the appropriate number of germs to be effective.
- If there are too many bacteria on the slide, the decolorization will be inadequate
- If there are too few, the decolorization will be excessive.
3.Incinerate any residual bacteria on the inoculating loop that hasn’t been consumed. The water will not show stained individual bacteria if too much culture is introduced, and the Gram stain may not be as reliable when too much culture is applied. Following the cooling of the inoculating loop, distribute the suspension over about half of the slide to produce a thin coating. A properly produced smear with the appropriate amount of microorganisms should look somewhat like Fig. 3. 5. Allow this thin suspension to completely dry out in the open air (Figure 4).
- Sixth, to heat-fix the bacteria to the slide, take the air-dried slide in your hands using coverslip forceps and hold it for 10 seconds at the aperture of the microincinerator, as explained by your teacher.
- If the bacterium becomes overheated, the structural integrity of the bacteria may be compromised.
- Gently wash your hands with water (Figure 7).
- In a separate container, stain with Gram’s iodine solution for one minute (Figure 8) and gently rinse with water.
- To ensure a consistent color distribution across the smear and that you are not under- or over-decolorizing, check the following: Wash with water as soon as possible
E. Stain for one minute with safranin (Figure 10). Wash the excess safranin away with care and caution, since it is possible to wash off part of the sarfanin present in the bacteria if you do not wash gently and quickly enough. f.Blot dry (as shown in Figure 11) and examine with an oil immersion microscope. 2.Staphylococcus epidermidis is a kind of bacteria. The following steps should be used to heat-fix a smear of Staphylococcus epidermidisas follows: To begin, apply 1/2 of a typical sized drop of deionized water on a clean slide by contacting the dropper to the slide with the tip of the dropper bottle that you located in the staining rack (Figure 1).
Use your sterile inoculating loop to carefully take a little bit of the culture off the agar surface and gently contact it 2 – 3 times to the drop of water until the water becomes noticeably hazy (see image below) (Figure 2).
- If there are too many bacteria on the slide, the decolorization will be inadequate
- If there are too few, the decolorization will be excessive.
3.Incinerate any residual bacteria on the inoculating loop that hasn’t been consumed. The water will not show stained individual bacteria if too much culture is introduced, and the Gram stain may not be as reliable when too much culture is applied. Once the inoculating loop has cooled, distribute the suspension over about half of the slide to create a thin layer (Figure 3). 5. Allow this thin suspension to completely dry out in the open air (Figure 4). Before the slide can be heat attached, the smear must be entirely dried on the surface.
Because if the slide is not heated sufficiently, all of the microorganisms will be removed.
Stain using Hucker’s Crystal Violet for one minute after cleaning (Figure 6).
It is not necessary to wipe dry in between procedures; instead, shake off any extra water.
In a separate container, stain with Gram’s iodine solution for one minute (Figure 8) and gently rinse with water. To decolorize, take up the slide and run it down the slide until the purple only slightly stops flowing at the bottom of the slide, then set the slide aside (Figure 9).
- To ensure a consistent color distribution across the smear and that you are not under- or over-decolorizing, check the following: Wash with water as soon as possible
E. Stain for one minute with safranin (Figure 10). Wash the excess safranin away with care and caution, since it is possible to wash off part of the sarfanin present in the bacteria if you do not wash gently and quickly enough. f.Dry the area and examine it with an oil immersion microscope. 3.Make certain that you properly pour the spent dye from the staining tray into the waste dye collecting container, rather than into the drain, to avoid contamination. B.DISCUSSION OF THE CAPSULE STAINING Bacteria that produce a slimy, sticky coating known as an acapsule or a glycocalyx are common.
- The capacity to make a capsule is a hereditary trait of the organism, but the capsule is not a biological component that is absolutely necessary.
- Despite the fact that they are not required for life, capsules may aid bacteria in their efforts to survive in the wild.
- To keep bacteria from being eaten by protozoans, capsules are used in both soil and water environments.
- It also facilitates the formation of biofilms by several bacteria.
- In your Lecture Guide, you will find the following Learning Objects that provide further information about the bacterial capsules:
- The Glycocalyx (Capsule) and S-Layer
- Unit 1, Section IIB4a
- The Ability to Resist Phagocytic Engulfment
- Unit 3, Section B5b
- The Glycocalyx (Capsule) and S-Layer
- The Glycocalyx (Cap
ORGANISMS Enterobacter aerogenes cultured in milk broth (kim Milk broth culture). The skim milk offers necessary nutrients for capsule manufacture while also serving as a somewhat stainable backdrop for the capsules themselves. PROCEDURE TO BE FOLLOWED (to be done individually) To begin, stir the Skim Milk broth culture with your loop and place two or three loops of Enterobacter aerogene on a microscope slide. Use your inoculating loop to distribute the sample evenly around the slide, covering about one inch of the slide.
- Allow it to dry completely in the open air.
- Capsules adhere well to glass, and heat may cause the capsule to rupture.
- Leave the crystal violet stain on for one minute.
- 6.Shake off any excess copper sulfate solution and wipe the area dry as soon as possible.
- The organism and the milk dried on the slide will absorb the purple dye, however the capsule will stay colorless after exposure to it.
- The demonstration capsule stain of Streptococcus lactis, an encapsulated bacteria that is part of the normal milk flora, should be examined carefully.
THE RESULTSA. The Gram StainDraw pictures of each bacteria on the Gram stain preparation. Capsule StainPrepare a sketch of your capsule stain preparation ofEnterobacter aerogenes and the demonstration capsule stain ofStreptococcus pneumoniae to submit with your final paper.
PERFORMANCE OBJECTIVES LABORATORY 6
After successfully completing this lab, the student will be able to complete the following tasks and tasks: A. THE GRAM STAIN DISCUSSION 1. State why the Gram stain is said to be a differential stain. 2. Describe the differences between a Gram-positive and a Gram-negative cell wall. 3. Describe a theory as to why Gram-positive bacteria retain the crystal violet-iodine complex while Gram-negatives become decolorized. 4. Describe three conditions that may result in a Gram-positive organism staining Gram-negatively.
- State the procedure for the Gram stain.
- Perform a Gram stain when given all the necessary materials.
- Determine if a bacterium is Gram-positive or Gram-negative when microscopically viewing a Gram stain preparation and state the shape and arrangement of the organism.
- THE CAPSULE STAIN DISCUSSION 1.
Contributors and Attributions
- Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)
- Dr. Gary Kaiser
2.4 Staining Microscopic Specimens – Microbiology
Due to the widespread use of antibiotics in medical and agricultural settings, bacteria have evolved to become increasingly resistant to these medications. Methicillin-resistant S. aureus (MRSA), a bacterium strain that has evolved a high level of resistance to several antibiotics, is becoming an increasingly concerning problem. As a result, research is being conducted to discover new and more varied medications. Fluorescence microscopy can be useful in determining the efficacy of novel medicines against resistant types of bacteria, such as those seen in MRSA.
When using fluorescence microscopy to differentiate between dead and live cells, SYTOX Green is frequently utilized as a marker.
Researchers discovered that MRSA bacteria that had been exposed to MC21-A did, in fact, look green under a fluorescence microscope, leading them to believe that the antibiotic is an efficient MRSA antibacterial.
Antibiotic prescriptions are becoming increasingly discretionary among health care practitioners as a result of this trend, which is becoming more widespread.
A sick patient would fairly object to this frugal approach to antibiotic treatment if he or she is suffering from an infection.
However, this is not always the case. What is the tipping point at which the hazards of broad antibiotic usage outweigh the desire to utilize antibiotics in individual cases?