Isolating and Examining Different Types of Bacteria
This experiment focuses on the idea that a gram of soil has the potential to contain 1010 different prokaryotic organisms. It uses a sample of sterilely collected soil and several common techniques in microbiology to isolate and examine different types of bacteria to not only determine the quantification of bacteria in the sample, but the different types of bacteria as well. One of the first methods used is morphology of the bacteria, where the size, shape, color, border, and elevation of certain colonies are examined and recorded. This is useful in not only determining the bacteria but can ensure that different types of bacteria are being tested. If the morphology of two different colonies is too similar, it’s likely that they are the same and only one should be carried out. The next step is to calculate the colony forming units per milliliter of diluted sample, or the CFU/mL. To isolate the colonies, they were streaked on nutrient agar and potato dextrose agar plates. These are both fairly common types of media but were chosen since they allow the growth of different types of bacteria. PDA allows the growth of yeasts and mold, and nutrient agar allows the growth of bacteria and fungi. These are both rich broths that keep the samples alive for long periods of time.
One of the first tests done after isolation is a gram stain. This test will determine of the bacteria is gram positive or gram negative. The main difference between these two bacteria is their cell wall composition. Gram positive bacteria have a thick layer of peptidoglycan that will cling onto the primary stain, crystal violet. Gram-negative cells will allow crystal violet in, but their thin walls and lipid content will not let it stick. Then, gram’s iodine is added, this is called the mordant, and will combine with the crystal violet in the cell walls. Then, ethanol is used as a decolorizing agent and will take the crystal violet in the gram-negative walls and wash it out by interacting with the lipids and dehydrating the layer around the cell. After the decolorizer, a counter stain, safranin, is added and will stain the gram-negative cells pink. This stain will not only reveal the kind of bacteria, but it also makes it easier to see the size and shape of the bacteria.
Another stain done for this experiment is an endospore stain. This stain will determine if bacteria are able to produce spores within their cells. The goal of this stain is to stain vegetative cells pink while staining the potential spores green. To do this, steam needs to be used as a mordant to open up the cells and allow the stain inside the cell. After an allotted time for steaming, the slide is removed from heat and rinsed with water. Then, a counter stain (safranin) is added to stain the vegetative cells. Most spore forming bacteria come from the Bacillus species and can usually survive in more extreme environments than other bacteria.
This experiment also utilizes selective and differential media to determine bacteria type. Selective media will only allow certain types of bacteria to grow and inhibit others, while differential media will determine the difference between closely related organisms. Mannitol salt agar is a selective media used. The media has a high salt content and utilizes phenol red to determine pH. On this media, usually only strains of staphylococci will be able to grow, and certain strains will ferment the mannitol, which produces acid and will change the pH of the media, taking it from a bright red color to a yellow color. There are two differential medias used, eosin methylene blue and MacConkey’s agar. In EMB, the eosin and methylene blue are used to differentiate between bacteria that can ferment lactose. Only gram-negative bacteria are able to grow on this media since methylene blue will inhibit gram-positive bacteria. E. coli colonies grow as a dark colony with a metallic sheen, and other types of enterobacteria grow with a pink color in large colonies. Salmonella is capable of growing on EMB but doesn’t ferment lactose or saccharose and will grow uncolored colonies. For MacConkey’s agar, it also allows the growth of only gram-negative bacteria since it contains crystal violet and bile salts. The differential agents are bile salts. Coliform bacteria will grow with a red color and will have a yellow ring around it due to the fermentation of lactose. Other types of bacilli won’t ferment lactose but will grow non-colored colonies on the medium.
Other media plates used are starch, gelatin, and blood plates. The starch plates will determine if bacteria can hydrolyze starch by using the enzyme amylase. Starch is a large molecule and needs to be split in order to go through the bacteria wall. Iodine is used to stain starch a dark blue color, and areas where the starch and sugar have been utilized will be clear and will yield a positive result. The gelatin plates identify plates that secrete protease as a result of gelatin digestion. Protease breaks down gelatin to form amino acids and can be seen by flooding the plate with HgCl2/HCl and looking for any clear zones. Blood agar plates are used to determine if bacteria can produce hemolysins, which can break down or damage red blood cells. This can be seen on an incubated plate, where there will be a clear zone around the bacteria that grew on the plate. Many types of bacteria can grow on blood plates, but not all of them will produce hemolysins so a positive result is only determined by the clear zone around the growth where the red blood cells have been destroyed.
Catalase and oxidase reactions are also utilized. Catalase is an enzyme that cells make to break down hydrogen peroxide into water and oxygen. Peroxide can oxidize a cell, so the ability to break it down or live in an environment with oxygen is a good test to determine if a cell is aerobic or not. Oxidase reactions are similar in the sense that they check to see if the cell is utilizing aerobic respiration. The reagent used will place an artificial electron acceptor in place of oxygen in the electron transport chain. This will turn the culture to a purple color when the last element in the chain is taken.
Nitrate reduction tests are done to determine if cells are capable of using nitrate as an electron acceptor. Certain bacteria are able to reduce nitrate into nitrite, and others are able to reduce nitrate to make other nitrite products. The broth is inoculated at room temperature, then two agents are added. One of them is sulfanilic acid and the other is α-naphthylamine, and if the broth turns a red color, it means that nitrate was reduced to nitrite. How