Day 4 - Bacteriophages and Gram Staining

First thing first, we checked out previous cultures. We looked at the experiment we did with the bacteriophage on the previous day. For one of the bacteria, the bacteria grew on the rest of the plate but not where the bacteriophage was spread in the shape of the letters FUS (Franciscan University of Steubenville). On the other plate, there was bacteria growing everywhere, even where we put the bacteriophage, meaning that the bacteriophage had no affect against that bacteria. The bacteriophage was host specific to the first bacteria. The broth culture prepared with the first bacteria and with the bacteriophage produced less bacteria then the straight bacteria. This confirms that the bacteriophages attack the bacteria and allows less to grow. 

FUS Bacteriophage Test

Part One

We learned how to prepare a gram stain. This experiment will be done on two bacteria cultures that we have gathered in the past couple of days: the streak plate taken from the bacteria on Grace's throat and the streak plate made from the given bacteria incubated in the broth. 

Left: Grace's throat sample, Right: Broth culture sample

For the first part of this experiment we had to prepare a bacterial fixed smear. This was found on p. 87 of our lab manual. First, we wrote the indicators on the edges, so that we would not confuse the two bacteria. Next, we added one small drop of water to each. In this water, using aseptic technique, we added the bacteria, each sliding having one sample from the two being tested. After letting this dry, we allowed it to set by passing it over the bunsen burner flame three times, so the bacteria would not be washed away with any of the liquids being used. The bacteria was then smeared and ready to be stained.

The whole set up for bacteria staining. :)

The process for the Gram staining is as follows: (1) We placed the slide over the smear rack. (2) Next we covered the sample with the crystal violet for twenty seconds. (3) After rinsing this off, (4) we covered the sample in Gram's idonine for 1 minute. (5) When this was rinsed off, (6) the next step was to put drop by drop of 95% ethanol until no color came off with it. (7) We rinsed this and then (8) added safranin, which we left for one minute. Lastly, (9) we rinsed this off and (10) blotted with bibulous paper. The bacteria is now properly stained!

Smear rack used for staining.

Now we will take a minute to explain what each step did. The crystal violet is the blue die. This would have remained on the stain the most adhesively if it was the gram positive. Gram positive has a thicker cell wall, more peptidogylcan and will hold the darker stain because of the crystals it forms with Iodine in the peptidoglycan. The ethanol was to wash off any excess die that was not taken up into the cell walls. Lastly, the safranin, the pink dye, would have attached to anything that did not have the crystal violet stain. This meant that if it was not gram positive that held the crystal violent die, but held the safranin pink dye, then the bacteria would be gram negative.

For the sample given to us and put into the brother culture, the end color was pink, meaning that the crystal violet dye didn't take and that the safranin did. This is an indicator that it is gram negative. The shape on this was found to be bacillus- long chains of rod shaped bacteria.

Broth culture bacteria - Gram Negative, Boccilli 

Our results for Grace's original throat smear showed that the bacteria was a gram positive but we heated the sample too long, and we will go back Monday and retry this stain. Until then, have a good weekend!

*App of the Day - This type of staining is the regular staining that is used in hospitals to determine the what type of bacteria are in samples that they take. They will regularly check their hospitals for Staphylococcus, especially the MRSA strand, because it can frequently pop up and needs to be strictly monitored.