Sediment samples were taken from 14 stations during the cruise program organized by the Persian Gulf and Oman Sea Ecological Research Institute (PGOSERI), Bandar Abbas, Iran in February 2014. The sediment samples were collected by Van Veen Grab sampler (Hydro-bios, Germany) and then were transferred to sterilized 50 ml tubes, and were kept refrigerated until shipboard processing later that day. The position and depth of sampling stations were recorded (Fig. 1).
Selective isolation of actinomycetes
Two physical treatments and four isolation media were implemented. Drying treatment was applied by desiccation of sediment samples in a laminar flow hood for a week. After grinding and fivefold serial dilution, the samples were inoculated on
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The purified isolates were inoculated into 50 ml of the Hickey Tresner broth medium (DSMZ) prepared with filtered seawater. The seeded media were incubated in a shaking incubator (220 rpm) at 28 ˚C for 5 days. After centrifugation, 100 µl of the culture supernatants were added to the punctured wells on the Muller Hinton agar (for bacteria) or Potato Dextrose Agar (for fungi) that previously inoculated with test strains. The test strains included Staphylococcus aureus ATCC 6538, Micrococcus luteus ATCC 10240, Escherichia coli ATCC 25922, Pseudomonas aeroginosa ATCC 27853, Aspergillus niger PTCC 5057 and Candida albicans ATCC 10231. The antimicrobial activity of the culture broths were determined by measurement of the inhibition zone diameters (mm) after the incubation period at 35 ˚C (Madigan et al., 1997).
Production and extraction of antimicrobial secondary metabolites
The selected potent isolates were inoculated to the seeding medium containing 1% malt extract, 1% peptone 2% glycerol and were incubated at 28 ˚C in shaking incubator (220 rpm) for 48 hours. After termination of the incubation, the seeded cultures were inoculated to the optimized production medium. After incubation for 5 days at 28˚C, the aliquots of filtrate-fermented broth were extracted with ethyl acetate twice (1:1 v/v). The ethyl acetate layers were evaporated and the crude extracts were kept for the subsequent experiments (Seidel,
An unknown bacterium was handed out by Dr. Honer. The appropriate tests were prepared and applied. The first procedure that was done was the gram stain. Under a microscope, if the gram stain is purple, the bacterium is gram positive, if the stain is red, it is gram negative. The next test was the fermentation tests for glucose, sucrose and
A mixed culture of two unknown bacteria was provided by the instructor. The methods used for
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 purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible
The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were oxidase, catalase, lactose and sucrose fermentation, Kugler/iron agar, nitrate reduction, gelatin hydrolysis, starch hydrolysis, manitol salt, MR-VP, citrate, bile esculin,
Thus we inoculated lactose broth, glucose broth and tryptophone broth with our organism. At our final lab meeting, we observed that the lactose broth was still phenol red in color, glucose tube had turned yellow with a bubble present in Durham tube, and the oily ring in our Indole test was colorless. Taking all the results into consideration and rechecking all of our steps against the guidelines and steps to ensure all required tests had been performed, we concluded that our organism belonged to Group IX genre, Proteus, Providencia, and Morganella. We then proceed to consult with our instructor.
After the incubation period the bacteria was observed for pure colonies. The colonies were sampled and the three streak plate technique was repeated and this sample was incubated for forty eight hours at 37 degrees Celsius. After the incubation of the colonies, a gram stain was performed which is defined in the lab manual.
The purpose to this lab was to isolate and identify two unknown bacteria from a mixed culture provided to us by our instructor. This study was done by applying all of the methods that have been instructed on thus far in microbiology laboratory class. Each test performed, provided us with some key information about the unknown microbes in question and how the bacteria function.
The purpose of this project was to identify the identities of two unknown bacteria in a mixed broth culture by using several separation methods. To separate the organisms, a four-way streak plate technique was used to isolate the two unknown bacteria into separate visible colonies. Then after each colony were clearly isolated; the two unknowns were processed through Gram staining test to determine the Gram stain and morphology. After Gram staining, a carbohydrate test was performed on each unknown to determine if it had glucose, sucrose, or lactose fermentation. The results of the sugar test help determining which biochemical test should be performed next. The Gram positive organism was tested through a carbohydrate fermentation test, then further tested to confirm its identity through an indole and catalase test. The Gram negative organism was tested through carbohydrate fermentation test, then further tested to confirmed its identity through an indole, and TSIA test. After running four biochemical tests, the results conclude that the Gram positive unknown was Staphylococcus aureus. S. aureus was identified based on the fermentation results of the glucose test, negative indole test, and a positive catalase production. S. aureus is a Gram positive circular shaped bacterium that is very common in the U.S and is normally found in the nose, respiratory tract, and on the skin. This bacterium is usually the most common cause of infections after injury or surgery.
Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB broth.
I want to go for next sampling in summer (September). When water temperature and salinity are almost 33 C and 41ppt, respectivly, in the Persian Gulf.
2. Introduction: Each student was given unknown bacteria and was instructed to perform a variety of experimental tests that would help to identify their bacteria. During the process of identification, the unknown bacteria was added to many different testing medias using aseptic technique. They are as follows: lactose fermentation on eosin methylene blue (EMB), TSI (Triple Sugar Iron agar), Phenol red sucrose, the SIM test, H2S by SIM, IMViC (indole, motility, voges-proskauer, and citrate), Urease (urea broth), PDase (Phenylalanine Deaminase), Lysine Decarboxylase, and Ornithine Decarboxylase. Colonial morphology on EMB was used to
Nutrient Broth and Nutrient Agar were used to inoculate bacteria taken from different surfaces. Nutrient agar plate was inoculated with a sample taken from skin surface. A sterile cotton swab was first immersed on sterile water, then, rubbed against the skin with swirling motion and transferred to an agar plate by rubbing
Introduction: Through the conduction of numerous experiments, the identity of two bacterial isolates was determined. The tested specimen was an unknown sample of a mixed culture of two different species of bacteria. The first step that was taken was obtaining a pure culture of each species of bacteria by isolating one species from the other. Once isolation was complete, the isolated cultures were tested using procedures that had been performed during previous lab sessions. A gram stain was performed on the two isolates. The isolate which had tested gram negative was then tested for the presence of cytochrome C and lactose fermentation. For the gram positive isolate, cell shape was determined and a catalase test was performed.
Broth cultures of E.coli, Micrococcus luteus and Vibrio natriegens were streaked onto the respective sections. The plate was then incubated at 37oC for 48 hours. The process was then repeated on nutrient agar plates with 5, 6.5 and 10% salt.