Winogradsky Column: Case Study

The purpose of this lab was to identify two unknown bacteria from a mixed culture. The reason for identification of unknown bacteria was to help students recognize different bacteria through different biochemical tests and characteristics. This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

The purpose of this study project was to carefully isolate and identify two unknown bacteria from a mixed culture. The ability to properly evaluate biochemical test results is also necessary for the identification to be successful. The goal was to apply all of the methods and techniques that have been learned in the microbiology laboratory course for the proper identification of unknown bacteria. A certain amount of bacteria that were used throughout the course were possible bacteria that could be found in a mixed culture. The bacteria that were identified in the mixed culture were Staphylococcus Aureus and Kocuria Rhizophila.

In a laboratory setting, it often becomes necessary to identify an unknown organism. In this experiment, researchers classified an unidentified bacterium based on its physical structure, colony morphology, optimal conditions and metabolic properties. A Gram stain using crystal violet, iodine, and safranin and a simple stain using methylene blue characterized the organism’s cell wall. Cultural behavior was classified by inoculating the organism onto nutrient agar and incubating it at 37° C for 48 hours, and observing its behavior, as well as using SIM medium to test for motility. Optimal growth temperature was

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

Two smears of the unknown bacterium #5 were inoculated while the second smear was used for a back up. The unknown bacterium dried for at least forty minutes. After the smears dried, the slides were heat fixed two times to ensure the stability of the organism. The slide was placed on top of the staining rack then over the small sink.

FISH analysis showed that the new extraction method was successful for four out of seven probes used on the sludge samples. The general probe EUB 338 showed a high density of bacteria in the seeding sludge and highly reduced densities after 24 and 28 days of operation, though still some patches with high bacterial density could be detected. In the samples from days 24 and 28 cloudlike structures were observed in all wells including negative controls, which showed high light intensity. These structures coincided with reduced hybridization ability of the EUB 338 probe (cf. figure 14 G-L) Whether the background of these structures was too high, the probe directly bound to the substances, which caused these structures or the hybridizing or fluorescent ability of the

A broth in test tube labeled #2 was used to identify the unknown organisms. A T-soy agar plate was the first media I inoculated. I used the streak isolation technique to isolate a pure strain from colonies, so that organisms can be identified. I inoculated a MacConckey to help me identify my Gram negative organism and observed that the organism was a non-lactose fermenter, then I performed a Citrase test which was negative. The last test I performed was a TSI, the butt was positive for hydrogen sulfide production and fermentation of glucose. All 3 results helped me conclude that my Gram negative organism was Proteus vulgaris. P vulgaris inhabits the human intestinal tract of human and animals. It is also found in soil, polluted water and fecal matter. This bacterium is

The Psychrotrophic counts and Enterobactriaceae counts at the beginning of storage were determined to be 2.21 log10 CFU/ g and 1, 59 log10 CFU/ g, respectively. Significant (P<0.05) increases have been determined in all samples treatment during storage (Figures 5 and 6).

The bacteria that was contained within Unknown tube #12 is believed to be Pseudomonas aeruginosa, Figure 1. The bacteria tested to be Gram Stain negative, producing a pink, red color retained from the staining process. When the species of bacteria was plated on nutrient media, the cells produced an irregular and spreading configuration as shown in Figure 2. This same plating test provided the margins and elevation, lobate and hilly, respectively. The specimen was stabbed in a Fluid Thioglycollate Medium (FTM) tube using an inoculated loop of the bacteria. The results of this experimentation indicate the type of oxygen requirement of the bacteria. The test found the bacteria to be aerobic as colonies of the bacteria began to form along the top of the FTM tube (Manual 2017).

Spores are oval, subterminal swelling the cell. The culture media characteristics of Clostridium difficile bacteria the colonies on blood agar plates are circular, flat, opaque, grayish or whitish. The incubation on Brucella blood agar supplemented with hemin and vitamin K1. Blood is not hemolysed. Cultures in PYG broth are turbid with a smooth sediment and have a ph of 5.0-5.5 after incubation for 5 days. The bacteria Grow at temperature 25 C. The optimum 30-37 Abundant gas is produced in PYG deep agar cultures.

The genus Dehalococcoides was originally discovered in 1997 with the discovery of Dehalococcoides ethenogenes (later renamed Dehalococcoides mccartyi Loffler et al. 2013). It was identified at Cornell University by Maymo-Gatell et al. (1997). The original source for Dehalococcoides spp. was an anaerobic sewage digester. Later studies revealed the original source to be from groundwater sites contaminated with chlorinated solvents (Seshadri et al. 2005). Dehalococcoides mccartyi was originally referred to as strain 195, it has the capability to fully dechlorinate chlorinated solvents like perchloroethylene (PCE) to ethene under anaerobic conditions. At the time, no species had been found that could accomplish the complete transformation to ethene (Maymo-Gatell et al. 1997). The cell wall for Dehalococcoides mccartyi

Each mixed culture that was tested had one gram positive and one gram negative bacterial species. The possible species of bacteria that could have been isolated from the mixtures included the following: Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Enterobacter aerogenes, Salmonella enterica, and Pseudomonas aeruginosa. The identities of the unknown species were determined through comparing the experimental data against data acquired from earlier experimentation.

The purpose of this experiment is to obtain isolation of individual species of particles from the mixed culture. This is completed through the isolation technique of streak plate. The objective of this experiment is to replicate the technique of streak plate but on a much larger scale. Because it is on a larger scale the particles are able to be visually observed as they are isolated using the streaking technique as the experiment is conducted. The benefits of the streaking technique is when a cultures has multiple species they are able to be more easily identified once they have been isolated. This experiment is much like the experiments completed on an agar plate but on one a much larger scale and where techniques

In the experiment, we put calcium carbonate, calcium sulfite and shredded paper. Shredded paper contains cellulose which enables the bacteria (Clostridium) to break it down by fermentation to gain energy. Calcium sulfite was added to make H2S gradient. The bacteria Desulfobrio use sulfite as electron receptor. Low sulfur bacteria and high sulfur bacteria make layers. Calcium carbonate was used as carbon source by sulfur bacteria. These chemical materials eventually makes cycles of them. Typically, column contains O2, H2S, CO2, ethanol, organic acids, H2SO4 and possibly other chemical compounds that consist of carbonate, sulfite and the other in pond mud. We sealed top of column and that makes O2 gradient. Desulfovibrio gains energy using organic molecules which is made by Clostridium (anaerobic bacteria) and

Knowing the conditions agar-degrading bacteria accommodate in situ, it is possible to approximate similar conditions in vitro. Leon et al achieved this by spreading samples of sea water on agar plates containing 0.25% casein hydrolysate, 0.05% yeast extract, 2.5% NaCl, 0.06% NaH2PO4, 0.5% MgSO4, 0.002% FeSO4∙7 H20, 0.01% CaCl2 and 1.5% Agar with a pH of 7.25 and incubating them for 48 hours at 25°C(6). After 48 hours, single colonies that had made small pits in the agar (thus indicating the presence of agarase) were inoculated onto new plates with the same conditions as above. The aim of this selective and differential process is to ensure a pure culture, as non-agarolytic bacteria should not be able to survive the first plate, let alone a second one. This inoculation