Lactic Acid Bacteria Lab Notebook Assignment

Camille Terry

MCB 301

Section B

9-19-16

Lactic Acid Bacteria Lab Notebook Assignment

Purpose/ Relevance:

        The purpose of this experiment is to isolate a pure culture of bacteria from a natural environment that contains multiple organisms. This is done by creating an enrichment used to adjust culture conditions so that the growth of a particular bacterium is enhanced. This enhancement allows an increase in the percentage of bacteria desired. Our first isolation is using cabbage to isolate lactic acid bacteria Leuconostoc. When constructing an enrichment to isolate a particular growth of bacteria there are many factors that come into play. The first thing you look at is the growth conditions this particular bacteria needs to be surrounded by in order to thrive. Some growth conditions consist of salt concentration, pH, temperature. These factors are adjusted to either inhibit the growth of other bacteria for isolation or used to stimulate the growth of bacteria. Leuconostoc  is the organism in which we are trying to isolate so it’s important to identify the type of environment it grows in to make the experiment successful. Lactic acid bacteria are a gram positive bacteria and are found in nutrient rich environments and require multiple growth factors. We will then isolate leuconostoc which produces lactic acid bacteria and observe them on three different media which are: tomato juice agar, heart infusion agar with glucose and lastly heart infusion agar with sucrose. All three plates will be used to identify the growth of a capsule if grown under the right conditions. Overall this experiment will allow us to isolate lactic acid bacteria using an enrichment and observing them on media for growth.

Data/Results:

Bubble observation:  There were not any bubble present when I retrieved the bottle of cabbage. The contents of the bottle had simmered down to the bottom of the bottle

Measured pH: The pH was measured at 3.5

Table 1: Plate observation

Table 2: Gram stain observations

Wet Mount:

We didn’t get to the wet mount but the results should have been the same as the gram stain minus the stain with clusters. There should be no motility.

In comparison to the previous slides we have looked at the growth is much smaller in cell size.

Discussion:

In the Lactic Acid Bacteria lab, we were able to isolate and examine the different growth observed on each growth media and identify whether we were able to increase the frequency of bacteria desired and inhibit the other bacteria present in the sample. In each portion of the experiment we observed different results that contributed to the overall success of the experiment. We isolated Leuconostoc from an enrichment culture. We learned that the enrichment culture is used to meet the growth requirements of the desired bacteria which will be isolated. Leuconostoc thrives on an anaerobic nutrient based environment. Our enrichment consisted of cabbage, fermentable sugar and salt as well as air being excluded by filling the bottle to the top with water. Each component plays an important role in the isolation process. First we added nutrients to the medium that stimulated the growth of the desired organism, then we excluded nutrients required by the unwanted organism, then added chemical agents that inhibit the growth of unwanted organisms, and lastly choose physical conditions to stimulate the growth of the desired organism. I observed that there were no bubbles in the cabbage jar. We were also instructed to get the pH of the enrichment bottle which was 3.5. If lactic were present, then the pH would be between 4 to 5. Does this mean lactic acid isn’t present? Under the wet mount there was a shape of a circle with no motility.  After the cabbage bottle was incubated we then had to isolate the culture onto three different types of growth media. The first growth media we used was tomato juice agar which is used to isolate bacteria that can thrive under low pH levels. According to our results in table 1 the tomato juice agar yielded positive results. We can see that lactic acid tolerates low pH levels when most others can’t. This makes it selective. The next media we will look at is the HIA-glucose agar. This plate also yielded positive results because we see growth. One thing that stood out was the color of the colonies that grew. I identified some colonies that were yellowish. I assume that is some sort of yeast living on the media. This showed that the medium is non-differential and non-selective. The last growth media observed is the HIA- Sucrose/NaN3. This plate also yielded positive results. 1/3 of the plate was filled with a slimy growth that indicates a dextran capsule. The selective NaN3 binds heme and inhibits cytochrome oxidase. The production of a capsule makes it differential. The capsule protects the cell from dehydration. Under the gram stain we were able to clarify that leuconostoc is gram positive with both the tomato juice agar and the HIA-glucose agar. We didn’t get to the wet mount procedure but I presume that the results should be the same as the gram stain without the stain. There should be clustering and no motility. Overall the results collected were the expected results and we can conclude that we isolated leuconostoc.