Isolation of bacteria includes several techniques by which different bacterial colonies from a mixed culture can be separated. This isolation is important as it helps in studying the particular organism with its distinguished traits. Bacteria are in habit of living in an association with other organism/bacteria as this association will help in the better survival of an organism. These microbial populations will cooperate together and achieve better nutrients for each other because the waste of one may serve as a nutrient for the other. Similarly the waste from the metabolism of the one may provide the favourable condition to the other for growth.
These isolation techniques were developed by Robert Koch in 1883 in which he added agar to the liquid nutrient broth. With the addition of agar the liquid media was transformed into the solid media and helped in isolating different bacterial colonies. In this media the nutrient broth supplemented the nutrientinal requirements of the bacteria whereas agar provides the solid substrate for bacterial growth.
The pure culture contains a similar kind of bacteria that has risen from a single parent colony having similar cells. There are many methods by which pure cultures can be obtained a few are discussed below.
SERIAL DILUTION.
Serial dilution is a technique in which we find out the overall count of colonies present in the given sample. Serial dilution is followed by spread plate technique or the pour plate technique for the further isolation of microbes.
PROCEDURE FOR SERIAL DILUTION.
This procedure is applicable for finding out the total number of colonies from water and soil samples. The procedure includes the following steps.
• A set of 6 test tubes is taken which are then labelled as 1...
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...and will increase the number of the required selected colonies. The new cells which will form as a result of the reproduction of the old cells are called sub culture. This technique is of great importance as it is used to expand the number of cells as well increase the life expectancy of the selected microbes.
ENRICHMENT AND SELECTIVE MEDIA.
The bacteria can also be isolated by providing the selective media to a bacterial culture. Since the selective media can only support a specific group of microbes therefore by application of it the required group can be isolated from a non-requiring one. Specific materials are added to the culture plates which will prove as poison for non-requiring group ultimately inhibiting the further growth of those microbes. When these microbes will be eliminated then only required group will grow and survive which can then be isolated.
After 48 hours of incubation the agar plates were viewed. Individual colonies were tested for successful isolation by gram staining and then viewing the stained bacteria under a microscope. Isolation was successful. One colony of each unknown bacteria was transferred to an agar slant for growth. The agar slants were stored at room temperature over the weekend so that they would not grow too much.
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
I also inoculated a tryptic soy broth (TSB), a nutrient gelatin deep, a motility agar deep, a fluid thioglycollate medium (FTM) tube, and a TSA plate with my unknown culture. All of these inoculated media were incubated until the next class period (about 48 hours). Then when I came to class most of my inoculated tubes and my streak plate appeared to have growth. The next step I took was making a gram stain to determine the gram reaction and cellular morphology of my unknown. I used my working slant to do this, after careful examination of the gram stain, I learned that my unknown was a gram-positive bacterium. I then preceded by making a negative stain to see the size of the cells of my unknown bacteria. The cell shape was cocci and the cells occurred in clusters of tetrads. After discovering that my unknown bacteria was gram-positive cocci, I turned to page 207 of the lab manual to narrow down my options, there was only four out of the gram-positive list that were
Phenotypic methods of classifying microorganisms describe the diversity of bacterial species by naming and grouping organisms based on similarities. The differences between Bacteria, Archaea and Eukaryotes are basic. Bacteria can function and reproduce as single cells but often combine into multicellular colonies. Bacteria are also surrounded by a cell wall. Archaea differ from bacteria in their genetics and biochemistry. Their cell membranes are made with different material than bacteria. Just like bacteria, archaea are also single cell and are surrounded by a cell wall. Eukaryotes, unlike bacteria and archaea, contain a nucleus. And like bacteria and archaea, eukaryotes have a cell wall. The Gram stain is a system used to characterize bacteria based on the structural characteristics of their cell walls. A Gram-positive cell will stain purple if cell walls are thick and a Gram-negative cell wall appears pink. Most bacteria can be classified as belonging to one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli) (Phenotypic analysis. (n.d.).
The purpose of this project was to identify unknown bacteria species from a mixed culture. The two unknown species were initially plated onto Tryptic Soy Agar (TSA), Eosin Methylene Blue (EMB), Mannitol Salt Agar (MSA), and blood agar plates to distinguish between the two different bacteria using colony size, color, shape, and growth characteristics. By identifying and inoculating the differing types of colonies, the two unknown bacteria were purified and able to be tested
I identified the genus and species of an unknown bacterial culture, #16, and I applied the following knowledge of morphologic, cultural and metabolic characteristics of the unknown microorganism according to the laboratory manual as well as my class notes and power point print outs. I was given an incubated agar slant labeled #16 and a rack of different tests to either examine or perform myself; the tests are as follows: Gram Stain; Nutrient Gelatin Test; Carbohydrate Fermentation; Dextrose, Lactose and Sucrose; IMVIC tests; Citrate, Indole, Mythel-Red and Vogues Proskauer test; as well as a Urease and TSI Test. Materials and Methods/Results Upon receiving the Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed a sterile drop of water on the slide and used an inoculating loop, flame sterilized, I took a small sample of the unknown growth in my agar slant and smeared it onto the slide in a dime sized circle and then heat fixed it for ten minutes.
...standing the nature of relationship between the residing microbes inside human cells and about their function is very important to put an end to this war and to live in peace with the natural organisms that are benefitting human body and their survival has become our primary importance.
To study the variation gene expression, we used Vibrio parahaemolyticus, calcium, centrifuge, spectrophotometer, mini-protean unit, SDS-PAGE, and UV transilluminator. During the first week of the procedure, you must come in around 24 hours before actually starting your procedure so you have adequate time for bacterial growth. The process consists of multiple steps of adding different chemicals to your bacteria and centrifuge it after add a chemical each time for different lengths of time. After all of the steps, I added 209 mL of LSB (Laemelli Sample Buffer) so that all test runs have equal amount of cells. Final step of this week is freezing our samples. While week two consisted of using our sample from previous week to make a gel plate that will go into the mini-protean unit. The mini-protean unit will distribute the vibrio in a way we can read its gene expression. After the gel is created in the mini-protean unit you must stain and de-stain the gel so it can be taken a picture of. To take the picture we use a UV transilluminator. For the standard procedure, reference (Department of Biology.
Bacterial cells, like plant cells, are surrounded by a cell wall. However, bacterial cell walls are made up of polysaccharide chains linked to amino acids, while plant cell walls are made up of cellulose, which contains no amino acids. Many bacteria secrete a slimy capsule around the outside of the cell wall. The capsule provides additional protection for the cell. Many of the bacteria that cause diseases in animals are surrounded by a capsule. The capsule prevents the white blood cells and antibodies from destroying the invading bacterium. Inside the capsule and the cell wall is the cell membrane. In aerobic bacteria, the reactions of cellular respiration take place on fingerlike infoldings of the cell membrane. Ribosomes are scattered throughout the cytoplasm, and the DNA is generally found in the center of the cell. Many bacilli and spirilla have flagella, which are used for locomotion in water. A few types of bacteria that lack flagella move by gliding on a surface. However, the mechanism of this gliding motion is unknown. Most bacteria are aerobic, they require free oxygen to carry on cellular respiration. Some bacteria, called facultatibe anaerobes can live in either the presence or absence of free oxygen. They obtain energy either by aerobic respiration when oxygen is present or by fermentation when oxygen is absent. Still other bacteria cannot live in the presence of oxygen. These are called obligate anaerobes. Such bacteria obtain energy only fermentation. Through fermentation, different groups of bacteria produce a wide variety of organic compounds. Besides ethyl alcohol and lactic acid, bacterial fermentation can produce acetic acid, acetone, butyl alcohol, glycol, butyric acid, propionic acid, and methane, the main component of natural gas. Most bacteria are heterotrophic bacteria are either saprophytes or parasites. Saprophytes feed on the remains of dead plants and animals, and ordinarily do not cause disease. They release digestive enzymes onto the organic matter. The enzymes breakdown the large food molecules into smaller molecules, which are absorbed by the bacterial cells. Parasites live on or in living organisms, and may cause disease. A few types of bacteria are Autotrophic, they can synthesize the organic nutrients they require from inorganic substances. Autotrophic bacteria are either photosynthetic or Chemosynthetic. The photosynthetic bacteria contain chlorophyll that are different from the plant chlorophyll. In bacterial photosynthesis, hydrogen is obtained by the splitting of compounds other than water.
Tissue culture allows for the clonal propagation of plant (production of multiple copies of the same genotype).
Microbes are everywhere in the biosphere, and their presence invariably affects the environment in which they grow. The effects
There were five test solutions used in this experiment, water being the control, which were mixed with a yeast solution to cause fermentation. A 1ml pipetman was used to measure 1 ml of each of the test solutions and placed them in separated test tubes. The 1 ml pipetman was then used to take 1ml of the yeast solution, and placed 1ml of yeast into the five test tubes all containing 1 ml of the test solutions. A 1ml graduated pipette was placed separately in each of the test tubes and extracted 1ml of the solutions into it. Once the mixture was in the pipette, someone from the group placed a piece of parafilm securely on the open end of the pipette and upon completion removed the top part of the graduated pipette.
Suspension culture - Cells are cultured in a liquid medium to form a callus. Subculturing needs to be done every 3-14 days due to their faster rate of growth than callus culture. This is the most used method in large scale production.
Microbes are major key components in both are homes and industrial food preparation. There are number of lactic acid which is a form of bacteria which is a large group of beneficial bacteria used in certain foods while they are getting prepared such as yogurt, cheese, sour cream, butter milk and other type of fermented milk products. Things such as vinegars are produced by bacterial acetic acid fermentation. Yeast is also major use in the making of beer and wine and also for the leaving of breads. This also involves fermentations to convert corn and other vegetable carbohydrates to also make beer, wine or gasohol but also bacteria is the agents of are other foods. Other fermented foods will include things such as soy sauce, olives and cocoa. (Microbes and human life, 2013) Single cell proteins are known as dried cells of microbes which are used in protein supplement shacks. They are also called “novel food” and “minifood”. The production of this requires micro-organisms which then serve as the protein source and then the substrate which is biomass which they grow on them. There are a number of both these sources that we are able to use for the production of single cell protein (SCP). The micro-organisms used belong to the following groups of Algae, Fungi and bacteria. (Slide Share, 2012)