Grain Staining

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

6. The disks in the 0.00% solution were transferred to an agar plate held next to the blue flame using the sterilized tweezers. Excess disinfectant was removed from the disks by wiping on the side of the well of the spotting tile. When the 5 disks were positioned (refer to Figure 1 below) the lid was replaced and sticky taped down. A label was added indicating the concentration of disinfectant.

To perform this test, a small drop of water is placed on a clean microscope slide. A metal loop that has been properly sterilized in the blue flame and allowed time to cool is used to

The next step of the project included preparing a Gram stain to discover the cell shape, arrangement, and if the bacteria is gram positive or

1) Apply the stain to your first unknown slide and examine it under the microscope.

Procedure: Filled each test tube with substances provided and subjected them to various conditions. These conditions included, heat, cold water, hot water, acid and basic additions and tested on litmus paper. The reactions were observed and documented at each step.

Then we carefully submerged the nitrocellulose sheet on the transfer buffer for about 5 minutes, and set up the transfer apparatus in the method shown in the lab outline week 2 step 3-6. Afterwards we took apart the transfer apparatus and detached the nitrocellulose sheet. Then we put the nitrocellulose sheet for 5 minutes into the blocking buffer, and we left nitrocellulose sheet in a container till week 3. The second part of week 2 was staining half our PAGE gel from week 1. Which consisted of pouring deionized water and use the shaker machine for 5 minute intervals, and repeating the the process three times. Then the Fresh Coomassie stain was added to the gel and was rinsed (UFV BIO 202 Lab,

The drops of crystal violets, approximately 15 drops, were flooded until the smear were all covered and then allowing resting for one to two minutes. After two minutes, the slide was titled over the sink and washed off, with the distilled water bottle, by aiming the stream of water above the smear. The specimen appeared blue-violet when observed with the naked eye. The drops of Gram’s iodine were applied on the slide until covered and then allowed to react for one minute or more. After the time elapsed, the slide was rinsed again with distilled water following immediate drops of Gram stain decolorizer added one drop at a time.

The purpose of this lab was to identify an unknown microorganism using lab techniques. The importance of identifying microorganisms is essential to the survival of humans, expansion of modern day medicine and improvement of quality of life. In 1884, Hans Christian Gram designed a differential staining technique to identify bacteria that would change the future of microbiology. He give rise to a staining process, known as the Gram stain to differentiate microorganisms into two groups between positive and negative gram staining microorganisms. The Gram stain is essential in a lab technique as it distinguishes the cells based on the physical properties of the individual cell walls, and is almost always the first test preformed to differentiate a microorganism. The identification of weather a microorganism is gram positive or negative can revel the bacteria’s virulence, cell wall structure, resistance to antibiotics, resistance to physical disruptions and so much more. In order to identify the unknown provided, unknown #27, the Gram stain was the first test preformed. After discovering that the unknown bacterium was indefinitely a gram positive microorganism, the vast possibilities were narrowed down. However, In order to more definitively identify the unknown, the next step was to preform biochemical tests. A biochemical test identifies metabolic

Figure #1 This picture show the gram stain of unknown #3. The image is under 1000x magnification using oil immersion technique. Gram (-)

Apparatus: Spectrophotometer (UV-1201), cuvettes, water bath (set at 37°C), 200µl and 1000µl micropipettes and test tube

On a thin chromatography plate, five spots were placed ( as shown in table 2) and the plate was developed using chloroform/methanol. This was later visualized with dragendorff’s reagent under the UV light. All separated components were observed, identified and recorded.

Spores can be stained using specific dyes, such as malachite green, that are absorbed by spores in the presence of heat (Noonan).

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

This report represents my individual effort. I did not receive or offer aid to anyone when performing this assignment, nor did I plagiarize any material.