Isolation of Pure Cultures by Dilution Techniques and Gram Staining Method

The bacterial cell wall is comprised of glycan strands of repeating disaccharide derivatives N-acetylglucosamine acid and N-acetylmuramic acid attached to a cross-link of amino acids, including L-alanine, D-alanine, D-glutamic acid, and either L-lysine or diaminopimelic acid (DAP). This mesh-like structure is called the peptidoglycan. It serves as an exoskeleton for the bacterial cell and is critical to bacterial cell’s ability to survive environmental conditions [1]. In gram-positive bacteria, the peptidoglycan is the cell’s outermost structure, while in the gram-negative bacteria, this structure is sandwiched between an outer membrane and the inner membrane called the periplasm.

The first result of importance was the result of the Gram stain. The observations of the unknown bacteria from the slant culture after Gram staining showed that the unknown bacteria were Gram negative bacilli (Image 1). After determining the unknown bacteria was Gram negative, an oxidase test was conducted on a sample from the slant culture. The cotton swap with the sample of bacteria did not change color when the oxidase reagent was applied, thus providing a negative result. With a negative oxidase test, further tests were conducted to determine various characteristics of the unknown bacteria. A MR-VP broth was inoculated with a sample from a slant culture of unknown bacteria. After incubation, the methyl red reagent was added to the broth, and the broth turned red, providing a positive result (Image 2). An EMB agar streak plate was inoculated with a sample from a slant culture of the unknown bacteria, and after incubation, growth was found on the plate, providing a positive result (Image 3). A Citrate agar slant was inoculated, and after incubation, growth was found on the media, providing a positive result (Image 4). A Urea agar slant was inoculated, and after incubation, the agar had changed from a peach color to a bright pink color, providing a positive result (Image 5). Using the flowchart (Figure 1) developed from the Table of Expected Results, the lab partners started at the oxidase test. Given the negative result of the oxidase test, the flowchart is

The Gram stain was used to determine if the bacteria was gram positive or negative. A negative test shows a pink color and a positive test is a purple color. When a bacterium is negative it is because it has an outer membrane and a thin layer of peptidoglycan that is much harder to stain. A positive bacterium has a thick layer of peptidoglycan and no outer membrane that can be penetrated by crystal violet.

The next step of the project included preparing a Gram stain to discover the cell shape, arrangement, and if the bacteria is gram positive or

Next I performed a KOH test to further confirm that my organism was a Gram-negative species. For the KOH test, I added 3 drops of 10% potassium hydroxide (KOH) to a small drop of distilled water onto a clean microscope slide, transferred a visible clump of organism to the KOH solution using my inoculating loop. I than mixed the cells into the solution using small, circular motions for 60 seconds and then lifted up the loop to look for what appears to be a “stringing” affect which means it’s confirmed that it is gram- negative species. Next, I created a streak plate using nutrient agar so that I could see pure culture of my organism. I aseptically obtained a loop full of my organism and gently inoculated one quarter of the nutrient agar plate by running the loop back and forth across the surface. I then flame sterilized the inoculating loop, allowing it to cool for 10 seconds, and then streaked the organism from quadrant I into quadrant II using a zigzag motion technique. I repeated those steps streaking from quadrant II to quadrant III and then streaking from quadrant III to quadrant IV. Once completed, I put the streak plate in the incubator at 37° for 24-48 hours. 48 hours later, I check my streak plate and it had a lot of growth on it. I was able to determine that the organism was definitely an off white color, opaque. The IV quadrant was the quadrant that best

This means that the bacteria for this lawn have cells that are surrounded by a cell wall which includes a thin layer of peptidoglycan and surrounding that is an outer lipid membrane that does not contain the Gram strain. Yet at the same time while Kanamycin was the most effective the Ampicillin and Penicillin had reactions as well. The Ampicillin would kill both Gram-positive and Gram-negative so this could indicate that there are Gram-negative bacteria in the lawn, but the fact that the Penicillin worked as well was what was interesting. This means that this bacterial lawn contains both gram-positive and gram-negative bacteria. Gram-positive are bacteria with cell walls that contain a thick layer of peptiodoglycan that retains the Gram strain. For plate 2, trashcan lid, only the Kanamcyin worked which indicates that the only bacteria in this lawn are Gram-negative, but if that is true why didn't the Ampicillin work at all? Ampicillin works to kill both positive and negative grams, but there was no reaction this time. Could it be that it needs more

Escherichia coli cells would be pink following a gram stain. E. coli is a gram negative cell due to its thin peptidoglycan layer and its outer membrane structure. In the gram staining process, the outer membrane does not play a role in staining but the peptidoglycan

This experiment was given to us to utilized previous knowledge learned throughout the semester to identify a gram negative unknown bacterium. We had to first learn the difference between a gram negative and a gram positive organism. We started off with doing gram stains to determine whether it was positive or negative. Based on the gram stains, a gram positive stains purple and a gram negative stains pink. A gram positive stains purple because the cell walls is made of a thick peptidoglycan layer and doesn’t

Gram staining is a technique used to determine if the bacteria is Gram positive or Gram- negative. Gram staining procedure uses crystal violet stain, iodine moderator, alcohol decolorizer and safarin counter stain. In Gram- negative bacteria the primary stain will be washed out with the decolorizer and it will be stained with the counterstain. Whereas in Gram-positive bacteria the primary stain will not leave the cell wall. This difference comes from difference in the structure of the cell wall that retains the stain.

Gram negative and gram positive bacteria differ from each other in many ways especially in the composition and size of their cell walls. Unlike Gram-positive bacteria, Gram-negative bacteria have a thin peptidoglycan layer surround by an outer membrane. This outer membrane contains many proteins one of them being lipopolysaccharides (LPS), which contributes to the bacteria’s negative charge. One part of this protein is a lipid, called Lipid A, which is considered an endotoxin because this lipid triggers an immune response stimulating fever

Escherichia is a genus of aerobic gram-negative rod-shaped bacteria of the family Enterobacteriaceae that form acid and gas on many carbohydrates, such as dextrose and lactose, but not acetone, which include occasional pathogenic forms, including some strains of E. coli which are normally present in the human intestine as well as other forms which typically occur in soil and water (Webster). Escherichia coli is a gram-negative bacilli that rarely varies in shape and size and when stained often resemble safety pins because the ends of some bacilli stain more densely than does the middle; which is a characteristic called bipolar staining which is common in enteric gram-negative bacilli (ASM). Gram negative cells have a thin cell wall layer and will stain red to pink. The staining process is the same as Gram positive, requiring four steps: applying a primary stain, adding a mordant, then rapid decolorization and completing with a counter stain. Applying the alcohol for decolorization dissolves the outer membrane and leaves small holes in the thin peptidoglycan layer through which the crystal violet-iodine diffuse. The gram-negative bacteria is colorless after the decolorization; therefore adding safranin

A great deal of cultural tests and physiological procedures were performed on the unknown before the conclusion of P. aeruginosa was made. The first test preformed was Gram’s staining which involves the use of crystal violet, Gram’s Iodine, Ethyl Alcohol, and Safranin. Gram staining is known as a differentiating stain, since it can determine the presence or absence of peptidoglycan in the cell wall. The unknown bacteria were Gram stain negative. After that the motility of the cell was tested using a hanging drop slide. The slide was examined, but the results were inconclusive due to the inability to make a clear distinction between motility and Brownian motion. This test was not

One of the most prominent differences is the amount of peptidoglycan in the cell walls of both cells. In gram positive cells, PTG is abundant and numerous, creating a thick layer, and in gram negative cells, PTG is usually in small amounts or completely absent. In gram negative cells, teichoic acids are absent and present in gram positive cells, providing rigidity to the cell wall. Gram positive cells have no outer lipopolysaccharide membrane whereas the gram negative cells do giving the cells protection against certain chemicals as well as structural integrity. Conclusively, the flagella in both gram positive and gram negative cells have a different amount of rings in the basal body that anchor the flagellum to the cell. In gram positive cells, there are two basal bodies and four basal bodies in the gram negative

Gram negative bacteria have two layers of peptidoglycan which make import and export molecules (protein, enzymes and toxins) through them difficult, for that reason they have special mechanizes which known secretion systems .There are six type of these secretions type I, type II, type III, type IV, type V and type VI .The latter is the resent type has been discovered.

In all areas of biology, it is easy to see that structure is related to function. This statement holds true in microbiology as well, the study of microorganisms, including bacteria. One characterizing feature of bacteria is the cell wall, which can generally (although not in all situations) be categorized into one of two categories: either Gram positive or Gram negative. Gram positive bacteria’s cell walls are composed of a large peptidoglycan layer (up to 90% of their cell wall). Within this large peptidoglycan layer, one can find techoic acids, which contribute to the maintenance of cell wall structure, and lipotechoic acids, which attach to membrane lipids. Gram positive bacteria that act as pathogens can also potentially release exotoxins, which can have very dangerous effects on humans. Gram negative bacteria, on the other hand, have a very small layer of peptidoglycan in their cell wall, which is surrounded by an outer membrane. Within the outer membrane, one can find the lipopolysaccharide layer, which is one of the most distinguishing factors of Gram-negative bacteria. It is important to note that Gram negative bacteria fail to possess techoic