Materials and method Bacteria strains and DNA extraction A collection of standard bacterial strains containing E. amylovora strains and several species of bacteria confirmed by Biochemical, Carbohydrates and Virulence tests for identification of E. amylovora isolates (data not shown) were exploited to estimate the specificity test (table 1). Furthermore, in order to assess the performance of two PCR methods and LAMP assay, about 208 symptomatic plant samples, were used. This collection was obtained from various plant tissues (e.g., flowers, shoots, leaves, fruits, and limbs) belonging to apple, pear and quince cultivars of different regions of Iran, during spring and summer of 2009 and 2010. For preparing samples, the same method of (Gorris et al. 1996) was used: 100 micro liters of the each dilution and other standard bacterial routinely cultivated on Luria-Bertani agar (LB) or LB agar medium and incubated at 28 °C for 48h. In the following, total genomic DNA of each strain was isolated by lysis of bacterial pellets from 1 ml of broth culture, incubated overnight in DNA extraction buffer, purified with phenol–chloroform–isoamyl alcohol (25:24:1) and precipitated with isopropyl alcohol (Llop et al. 1999; Schaad et al. 2001). At last, each strain DNA was eluted in 100 µl of elution buffer and stored at −20 ◦C prior to further assessment. whenever pure bacterial cultures were used, the optical density of bacterial culture was measured by a spectrophotometer at 600 nm (2×107 CFU/ml), and one μl of each dilution of bacterial suspension was directly added to the LAMP and PCR reaction mix. In contrast, for infected plant samples, were used Plant Mini Kit (Qiagen). DNA isolation was performed according to the manufacturer’s protocol fo... ... middle of paper ... ... clearly specific-specific for E. amylovora. Comparison of LAMP with Conventional and Nested PCR assays To assess the ability of the LAMP and other PCR methods for detecting E. amylovora in naturally infected plant material (described previously), first of all LAMP method was tested with infected plant material. Among three methods, LAMP assay showed the highest power of detecting the pathogen in all symptomatic samples (Table 3). Unlike single PCR and nested PCR, in LAMP method there is no need to electrophoresis and time detection is reduced to 45 min. The existence of the pathogen in the positive samples was confirmed by its isolation in culture medium. These remarkable results demonstrated that, in one hand, the method has higher specificity and sensitivity than single PCR and on the other, it is slightly better than nested-PCR in a single closed tube.
The isolate possesses some enzymes required for hydrolytic reactions. Hydrolytic enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Hydrolytic enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been
After 5 days of growth each slant was tested using the gram staining technique to confirm the complete isolation of the bacteria. Both isolations were completely successful. Then each sample of bacteria was subjected to a series of tests for identification.
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.
ABSTRACT: Water samples from local ponds and lakes and snow runoff were collected and tested for coliform as well as Escherichia coli. Humans as well as animals come into contact with these areas, some are used for recreational activities such as swimming and some are a source of drinking water for both animals and humans The main goal of this experiment was to see which lakes, snow run off and ponds tested positive for coliform or Escherichia coli and to come up with some reasoning as to why. It was found that the more remote pond with less contact contained the most Escherichia coli. However, another lake that many swim in and use as their drinking water indeed tested positive for a small amount of Escherichia coli. The two samples from the snow showed negative results for both coliform and Escherichia coli and the two more public ponds that aren’t as commonly used as a source of human drinking water but animal drinking water tested in the higher range for coliforms but in the little to no Escherichia coli range. It was concluded that the remote pond should be avoided as it’s not a safe source of drinking water for humans or animals. Other than that, the the other ponds are likely to be safe from Escherichia coli, but coliforms are a risk factor.
The purpose of this laboratory is to learn about cultural, morphological, and biochemical characteristics that are used in identifying bacterial isolates. Besides identifying the unknown culture, students also gain an understanding of the process of identification and the techniques and theory behind the process. Experiments such as gram stain, negative stain, endospore and other important tests in identifying unknown bacteria are performed. Various chemical tests were done and the results were carefully determined to identify the unknown bacteria. First session of lab started of by the selection of an unknown bacterium then inoculations of 2 tryptic soy gar (TSA) slants, 1 nutrient broth (TSB), 1 nutrient gelatin deep, 1 motility
electrophoresis. The way the PCR method works is by first mixing a solution containing the
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
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
Talaro , K., & Chess, B. (2012). Foundations in microbiology. (8th ed., pp. 563-564). New York, NY:
Schumann, Gail L., and Cleora J. D'Arcy. Hungry Planet: Stories of Plant Diseases. St. Paul: American Phytopathological Society, 2012. Print.
In the last decade, the number of prescriptions for antibiotics has increases. Even though, antibiotics are helpful, an excess amount of antibiotics can be dangerous. Quite often antibiotics are wrongly prescribed to cure viruses when they are meant to target bacteria. Antibiotics are a type of medicine that is prone to kill microorganisms, or bacteria. By examining the PBS documentary Hunting the Nightmare Bacteria and the article “U.S. government taps GlaxoSmithKline for New Antibiotics” by Ben Hirschler as well as a few other articles can help depict the problem that is of doctors prescribing antibiotics wrongly or excessively, which can led to becoming harmful to the body.
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.
They’re out there! You can’t see them but they can see you. Right at this very moment they are living on and in your body, and there is nothing you can do about it!
Knowledge is power when discussing the classes you have taken in college and how it affects your everyday life. Microbiology is one of those important classes where although for a majority of majors you do not have to take it and therefore will not learn the information discussed in it, other majors such as biology and nursing require you to take it. They will require you to take it because you will encounter some of the information being taught in it in the work field. As a nurse practitioner I have worked in the field with many patients who were diagnosed with Leprosy, Escherichia coli, Salmonella and other bacterial diseases. All of these diseases that I have encountered in patients were ones I was familiar with due to learning about them as a student in microbiology at CSUB. My children plan on