Lab 4: Using The Spectrophotometer To Measure The Rate Of Bacterial Cell Division: A Bacterial Population Growth Curve

Lab 4: Using The Spectrophotometer To Measure The Rate Of Bacterial Cell Division: A Bacterial Population Growth Curve

Introduction

            This was a wrap-up of the past lab reports submitted early. This is the chance for everyone to see how well we actually did at growing bacteria. So because bacteria multiply so fast due to binary fission. We are able to grow our own cultures and use them to study a growth pattern. The point of this lab was to follow the growth of a population of E. coli bacteria cells through each and every individual stage of growth they undergo. We measure this by using a spectrophotometer. This machine measures light or absorbance, this can be done by shining light through and measuring the turbidity as the number of cells increase. The point of this is to determine the stages of growth. Starting with the lag phase then reaching the log phase, stationary phase and last is the death phase however the death phase can’t be measured using a spectrophotometer.

            

This is how the graph is suppose to look.

Methods & Materials

            Materials were the usual

·      Lab coat

·      Gloves

·      Quvet

·      Samples that were assigned to you

·      Ethanol

·      Patience

Methods for this lab were pretty straight forward for our end however we also had a helper (Casey) that came in every hour to create the sample we needed for 8 hours straight. For us, we were assigned 3 different time periods for each group that we needed to calculate the light penetration to know the growth rate. We were given mostly the beginning hours therefor we could take samples straight out of the tube and test them. However, for the hours around 6 or so and after, you have to dilute the solution in order to get a more accurate reading. We needed to find the abundance (light penetration) for each test. The work shown below is the results of the groups combined work.

Time (hr)	Absorbance (nm)			Average	Standard Deviation
1	0.007	0.054	0.01	0.023666667	0.021483844
2	0.134	0.117		0.1255	0.0085
3	0.132	0.132	0.378	0.214	0.115965512
4	0.82	0.462	0.775	0.685666667	0.159219625
5	0.456	0.488	0.476	0.473333333	0.013199327
6	0.598	0.386		0.492	0.106
7	0.718	0.744		0.731	0.013
8	0.72	0.86		0.79	0.07
9	1.006	1.126		1.066	0.06
18	1.054	1.154		1.104	0.05

Results:

            

The graph above shows a time vs abundance curve, This graph is to be compared to the graph in the introduction. Our Lag Phase is basically from zero to about the first point on the graph because our bacteria were taken out of a LB broth to be put back into it therefor there was no need for an adjustment period for the. Our Log phase was all the way from the first point to the point at 9 hours. Then of course the hour 9 to hour 18 is our stationary phase. We are unable to show the death stage with a spectrophotometer because even if the bacteria are dead, they still block light.

            Discussion

Due to Binary fission, Cells divide very fast and multiply, thus if you capture light as it passes through a clear quvet as the bacteria grow over time then the water gets cloudy. The water gets darker due to the over populating cells multiplying in the liquid thus blocking the light from passing through. This is why A increases over time.

Like stated above we can look at our graph and see the separate stages that include Log phase was all the way from the first point to the point at 9 hours. Then of course the hour 9 to hour 18 is our stationary phase. We are unable to show the death stage with a spectrophotometer because even if the bacteria are dead, they still block light.

Now using equations for growth rate during the lag phase I got y = 1(1+1)^1 = 4

Then for the log phase we used y = 4(1+1)^8 =1024

And finally we can find the stationary phase which tends to be 0 because its stationary (no food, no growth)

Now if we add antibiotics to the bacteria, the growth will be stunned and decrease however mutations from the bacteria and then overcome the bacteria which in return looks something like

If we added it around 180 min into the medium the absorbance would temporarily stun the growth, then basically starting another curve stacked on top of it. This would look something like

Lab #3: Streak Plate, Culture Transfer Instruments and Techniques, Isolation and Maintenance of Pure Culture

Lab #3: Streak Plate, Culture Transfer Instruments and Techniques,

Isolation and Maintenance of Pure Culture

Introduction

                  Microorganisms are basically everywhere in the environment. Like us, they are generally living as a giant mix of population or communities. However, in our case we want to single out the bacteria on focus on one at a time. In order to do this, one has to isolate each bacteria into pure cultures. Another words we have to differentiate each type, we can do this by using a selective or differential growth media. This will differentiate the bacteria into selective areas or colors for you to single out. Now the fun part… Once you have isolated the selected bacteria you want to study then you can literally pick it up with a toothpick and incubate it in some LB broth. This multiplies the bacteria exponentially allowing you to harvest if you feel like being evil and freezing them for later use then that’s also an option.

Methods and Materials

                  Before working with your microbes, your work space must be nice and sanitary. Mainly for not contaminating your culture but also because your partner may be dirty and smell bad. Who knows! But anyway, you have to slap on your rubbers like your local doctor and sanitize your lab table with college kids best friend (Alcohol). This allows you to not contaminate your culture and keeping it that pure culture you spent so much time growing. So grab some paper towels and at least 70 percent ethanol (not recommended in drinking) and wipe down your table as well as your gloves.

                  Now you are ready to streak some plates! Use a sterile toothpick and your frozen stock of cultured microbes and give it a dip. Use a spreading technique shown below on a sterile dish provided usually by a nice lab guy. You will need about three to four toothpicks, each time dragging your microbes along the dish with a jelly like substance you set up for them. This is how it should look

Figure 1. Streak Plate Technique Methodology. Source: http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/lab4pureculture.html

                  Now that you plated the little guys, you have to incubate them at a temperature of about 37 degrees C for around 24 hours. Now after time has passed and so has your patience then remove from the incubator. Use another toothpick and pick up a nice lonely isolated colony and swirl it in a sterile flask with some yummy LB broth (liquid). Throw it back it an incubator at 37 degrees C for 24 hours, however add a spin of about 100 rpms to it so it gets a nice mix.

                  Now you can use the incubated microbes in the LB broth as a growth curve. Use a sterile pipette tip and transfer 1 mL of culture to a flask of sterile broth. This allows for the growth curve to begin. If a frozen stock sparks some interest then combine 1 mL of the log phase E.coli with 1 mL of glycerol (stops from killing the cells) in a cryogenic tube at 80 degrees C below (-80).

We are beautiful 

Results

                  This is still a set up lab for a future lab so no results just yet, stay tuned for results.

Discussion

                   As a personal note, do not tear the gel-like substance that was made previously when applying your frozen culture with a toothpick. This is very easy to do so don’t go at it like a mad man like me.

                  Don’t forget to add glycerol to your culture if you want to freeze them for later use. If you forget then this will kill them all and all that time was wasted for nothing.

Lab #2: Media Prep and Autoclave, Plate Pouring

Lab #2: Media Prep and Autoclave, Plate Pouring

Introduction

We had made/ used one of many types of commonly used media. Luria-Bertani (LB) being the most common was my groups choice for this lab. It’s very rich in nutrients and food source for bacteria, but even better is that it’s very easy to create. Since its very rich in food for bacteria, it is used to grow pure cultures quickly.

                  LB broth or medium was a sample in the lab and usually always will be. It usually brings out Ecoli (Escherichia coli) and also many other bacteria. The main recipe used in this lab for the LB broth that we had used was a combination of 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1 liter of distilled water; adjust the pH to 7.0 with 1 N NaOH. After the mixture was put together, you have to autoclave the mixture for 25 minutes at 120 degrees Celsius.

                  In this lab we used two different forms of medium for LB: we made a broth and an agar. The Broth is used for a liquid culture of bacteria. Agar then being for more of a solid culture like a petri-dish. R2A was the second media on the list for this lab. It is used as a media that allows more of a slow and controlled growth for bacteria compared to LB which is a very quick growth pattern. R2A however is much more complex, including yeast extract, peptone, dextrose, magnesium sulfate, starch, sodium pyruvate, and others. LB is used for cultures that are pure like Ecoli, compared to cultures that are mixed like water from a river. This is when R2A is handy. It allows you to see all types of bacteria instead of just the fast growing bacteria like LB would show.

Methods and Materials

In a 1L autoclave bottle we added ×20 g LB broth powder and 500 mL ultrapure (DI) water. We had to cover the top with double-layered aluminum foil. Placing a fresh piece of autoclave tape on the top to show sterilization. Place on mixing plate to mix. Autoclave (121 C, 20 minutes) on a liquid cycle, but being sure to add water to the autoclave basin before starting the cycle!. After cycle is complete, remove and let it cool to about 50 C. This is to allow handling of the flask so you don’t burn yourself, caution this is still hot. Pour out the media into petri dishes. First, remove the lid to a dish and remove the foil cover to the flask. Then pour just enough LB agar into the petri dish to completely cover the bottom of the dish. Quickly put the lid back on and swirl the dish gently enough to make sure the entire bottom covered. Do not get any on the sides of the dish. The agar will harden so try and be a little speedy with this process. To end, store plates back in the plastic sleeve the petri dishes originally came in, should be labeled. Make sure dishes are stored upside down in the cold room.

Pics: 

Results:

                  This was a set up lab for another lab to come so hang around for the results later J

Discussion

Why did we make LB broth and agar?

                  We want a comparison between the results of each and how it would vary. We know that LB broth is made to allow growth in a liquid and agar is made to allow growth in more of a solid.

Why did we make LB and R2A?

                  This was to see how different growth patterns are formed in media. LB is a rapid and allows very fast growing. This then allows only the fast growing bacteria to accumulate on the dish which then generally only shows up as one type of bacteria. R2A on the other hand allows for slow growth thus allowing all different types of bacteria to accumulate.

How do your teams’ plates look? Do they look uniform?

                  We moved pretty quickly at first so the pours were more giving than the later pours therefor we definitely didn’t pour evenly. However, we did always cover the bottom completely and I believe our dishes were better than the rest due to me being the one doing them (Too good of a pourer)

Why might we need to adjust the pH to 7 on a broth media?

                  Most of these bacteria are found in living animals or people’s intestines which generally sits around pH of 7. They also like it here because if a bacterium is in too basic of an environment or even too acidic then they tend to die. Neutral is where they thrive.