Understand The Difference Between Gram-Positive And Gram Negative Bacteria

To fully understand this lab we first need to know what gram-negative and gram-positive bacteria are and what it’s composed of. Gram-positive bacteria take up the class of bacteria that stain crystal violet in the method of bacterial differentiation. Where gram-negative bacteria are a class of bacteria that does not retain crystal violet. Instead they are counter-stained pink by Safranin, and because of this, identifying positive is possible. One of the many differences between gram-negative and gram-positive bacteria is their peptidoglycan. Which are attached polypeptides to form networks that surround and protect the entire cell. The peptidoglycan is pretty much what acts as their backbone. Knowing where to locate it and the amount of it
is one of the many ways to help determine if it’s gram-positive or gram-negative. Gram-positive’s peptidoglycan makes up about 90% of the cell wall, and is also the outermost structure of the gram-positive cells, which is also made up of enzymes. Gram-negative, on the other hand, is less complex where its peptidoglycan makes up only 5-20% of the cell wall and it is located in the periplasm, a gel-like fluid between the outer membrane and the plasma membrane. Another way to tell the difference between gram-positive and gram-negative bacteria is by their cell walls. Gram-positive cell wall contains teichoic acids that are made up of an alcohol and phosphate. The teichoic acids have two classes: lipoteichoic acid spans peptidoglycan layer and links to plasma membrane and wall teichoic acid that links to the peptidoglycan. These acids regulate the movement of cations that comes in and out of the cell and also prevents wall breakdown and cell lysis. In gram-negative cell wall, it has no teichoic acid. The periplasm contains degradative enzymes and transports protein. Their outer membrane consists of lipopolysaccharides (LPS), lipoprotein, and phospholipids. LPS contains lipids and carbohydrates and consists of lipid A, a core polysaccharide, and O polysaccharide. Lipid A is the lipid portion of the LPS and is embedded in the top layer of the outer membrane.
When gram-negative dies, they release lipid A and functions as endotoxin. Core polysaccharide is attached to lipid A and provides stability. Lastly, O polysaccharide is what extends outward from the core polysaccharide and functions as an antigen. O polysaccharide is useful for differentiating species of gram-negative bacteria. Lipoproteins are what connect the outer membrane to the peptidoglycan. The plasma membrane of these bacteria’s contains phospholipid molecules that are arranged parallel to each other called a lipid bilayer. The surface of the lipid bilayer are polar heads composed of a phosphate group and glycerol that is hydrophilic (water loving) and are soluble in water. Nonpolar tails are in the interior of the bilayer. These nonpolar tails are composed of fatty acids that are hydrophobic (water fearing) and not soluble in water. To contain, protect, and provide structure are the functions of the plasma membrane. It is selectively permeable and helps control what moves into and out of the cell. These are just the basic steps to know in order to understand this lab. It’s mostly like a puzzle that’s just waiting to be put
together.
The purpose of this lab experiment is to differentiate different kinds of microorganisms that identify as gram-positive and gram-negative bacteria.
One way to do this is through selective and differential media where they are used to isolate or identify particular organisms. Selective media allow certain types of organisms to grow and inhibit the growth of other organisms. Selective inhibition for some kind of microorganisms can be achieved by adding dyes, antibiotics, salts, or specific inhibitors that affect the metabolism or enzyme system of that organism. Differential media are used to differentiate closely related organisms or group of organisms. There are certain dyes or chemicals inside the media that bacteria will react to. These reactions can either be characteristic changes or growth patterns which are useful for identification or differentiation of microorganisms. Four kinds of cultured media are used in this lab experiment. The first one I will be talking about is the Eosin Methylene Blue agar or EMB for short. EMB is a selective and differential medium that inhibits the growth of gram-positive, but detects and isolate gram-negative enteric bacteria. Mythelene blue inhibits gram-positive. The selective part is used for inhibiting gram-positive and has methylene blue. The differential part is what ferments lactose. MacConkey agar is a selective and differential media used to isolate gram-negative bacteria and differentiate between lactose fermenting and
non-lactose fermenting. The selective part inhibits gram-positive and the differential shows if it’s going to lactose ferment or non-lactose ferment. It also has the ability to ferment lactose other than sugar. Triple sugar iron or TSI agar slant is a tubed differential medium that is used to differentiate enteric based on its ability to reduce sulfur and ferment carbohydrates. The carbohydrates can be metabolized aerobically or by fermentation through bacteria. Finally, tryptone broth that is a differential media. This is used to grow E. coli and has a large quantity of tryptone. Also, is used for detection of indole produced by coliforms and this is a key feature in differentiating bacteria.