Science Buddies: "Ask an Expert"

Does anyone know how to make serial dilutions and regular dilutions on Staphylococcus Aureas, E. Coli, and Gram positive Bacillus? If you know please and thank you!!! P.S. Please also insert how do you count them after the dilutions are made.

eternal_corrector:

To make serial dilutions you will need the following -
sterile test tubes
sterile water
sterile pipetes (1 and 10 ml)
stock cultures of the bacteria
Set up 6 sterile tubes each containing 9 ml sterile water for each bacterial isolate
label each tube as follows - 10^1, 10^2, 10^3, 10^4, 10^5 and 10^6 (this represents 10 to the minus 1 or a 1/10th dilution)
Transfer 1 ml of bacterial stock solution to the tube labeled 10^1 and shake the mixture genetly, now transfer 1 ml of 10^1 to the tube labeled 10^2. Continue the process until you have finished 10^6.

You now have a series of 1/10 dilutions of the original bacterial culture.

You now plate 1 ml of each dilution on a separate agar plate (use a clean pipete for each transfer) and label dilution and date. After 12 and 24 hours you count the number of colonies that are growing on each plate. More than likely you will not be able to count individual colonies on dilutions 10^1, 10^2, 10^3. There will be too many bacterial to get separation of individual cells.

To determine the bacterial concentration record the number of bacterial colonies on each plate and multiply that number by the dilution. The multiplier for each dilution is as follows -
10^1 = 10
10^2 = 100
10^3 = 1,000
10^4 = 10,000
10^5 = 100,00
10^6 = 1,000,000.

Matt Mulanax

Matt Mulanax:



Um I was just wondering if you can take a look at my new post in the same forum because it seems you're the only one that can answer my last question about the project i'm resaerching on.....Ok thanks!


Eternal_Corrector

eternal_corrector:

Could you give me a little background on your project.

What is your objective/hypothesis and what approach are you going to take to answer the research question(s)?

Thanks

Matthew Mulanax

The purpose of this project is to determine if bacterial resistance (after repeated exposure to a disinfectant) depends on the concentration of the disinfectant. The independent variable is the different concentrations of the disinfectant (percentage). The dependent variable is bacterial inhibition (percentage). The hypothesis is that if the bacteria are exposed to lower concentrations of the disinfectant, then more resistance develops.

When you already understand the background information for my project please go to this URL : https://www.sciencebuddies.org/mentorin ... =1471#1471

But if you have any questions please ask me...Thanks

eternal_corrector:

I am workin on your question and will have a simplified answer this afternoon.

Matthew Mulanax

eternal_corrector:

To make a 10% solution - 10 ml of original or stock solution + 90 ml sterile distilled water
20% solution - 20 ml stock + 80 ml water
30% solution - 30 ml stock + 70 ml water
40% solution - 40 ml stock + 60 ml water
50% solution - 50 ml stock + 50 ml water. Remember these will be dilutions of the original stock. That stock is probably not 100%. Check the label because you will be making % dilutions of what ever is in the container. For example Clorox or Purex household bleach is only 5.25% sodium hypochlorite.

Do you have access to laboratory supplies and equipment that will allow you to handle bacteria and work in a clean environment?

If not your best approach will be to demonstrate an increase in resistance or tolerance to disinfectants by the bacteria. You can do that by following the procedure from the project you based your original question on (web sete below).

https://www.sciencebuddies.org/mentorin ... ?from=Home

This will get you started.

Please ask any questions that you have.

Matthew Mulanax

And if I was to test the disinfectants on a certain bacterium how would I do it? Like do I place the bacteria on a agar petri dish then pour it on the surface or mix it when the bacteria is inside a test tube in liquid form? Lastly, why is it necessary to dilute the bacteria by 1/10? I'm just asking becuase the title only meant (to me) that its only showing to see if bacterial resistance is affected by the diffrent concentrations of disinfetants.... Ok this is the last one... Sorry if there is so many questions... P.S. If it was necessary to test the diffrent concentrations on the diluted bacteria of 1/10, how would I test it...Like which percentages of disinfectants would test the 1/10 dilutions?

Don't try to answer them all at one time if you can't....

Thanks.....

eternal_corrector:

You can ask as many questions as you need to to clarify the subject.

I will have an answer for your questions later today (Sunday). I will explain the 1/10 dilution also.

Matthew Mulanax

eternal_corrector:

I suggested a 1/10 dilution initially to see how active the culture is. It is not necessary to do this for your project.

I suggest you mix bacteria and disinfectant in a test tube, let it incubate for 1 hour then spread 2-3 ml of the solution on a nutrient agar plate. You need to follow this procuedure for each dilution of each disinfectant tested.

The procedure is as follows -
.1% dilution = .1 ml of disinfectant (stock solution) + 9.9 ml bacterial suspension
.5% dilution = .05 ml of disinfectant + 9.5 ml bacterial suspension
1 % dilution = .01 ml disinfectant + 9.9 ml bacterial suspension
5% dilution = .05 ml disinfectant + 9.5 ml bacterial solution
10% dilution = 1 ml disinfectant + 9 ml bacterial suspension
20% dilution = 2 ml disinfectant + 8 ml bacterial suspension
30% dilution = 3 ml disinfectant + 7 ml bacterial suspension
40 % dilution = 4 ml disinfectant + 6 ml bacterial suspension
50% dilution = 5 ml disinfectant + 5 ml bacterial suspension.

I suggest testing .1%, .5%, 1%, 5% and 10% disinfectant levels to begin with. After 1 hour incubation, spread 2-3 mls evenly over the surface of a nutrien agar plate. Follow this procedure for each disinfectant and bacterial culture tested. Incubate these plates and count colonies on each plate at 24, 48, 72 and 96 hours.

Any growth on the plates will be bacteria that are resistant to the disinfectant. You can test those that survive by running then through the same disinfectant dilution series again to see if more colonies grow on the plates.

Please ask if something is not clear.

Matthew Mulanax

So what happens with the solutions produced by mixing the disinfectants with sterile water? In the part where you "plus" it with the bacterial suspension, do I have to use the disinfectant directly or do I have to use the solution mentioned above?And what kind of graphs or charts would I be able to produce? Like how do I measure resistance and colonies per day?

eternal_corrector:

You have a bacterial culture - most likely nutrient broth. This is the stock culture. Do not dilute.

To a sterile test tube add the required mls disinfectant + the required mls of bacterial stock culture. Incubate for the required time, plate evenly 2-3 mls bacteria and disinfectant on the surface of a nutrient agar plate.

Record the number of colonies on each plate over aperiod of four days.

You can then make charts for each % disinfectant tested showing the number of bacterial colonies surviving after exposure to the disinfectants.

When you repeat the procedure using bacteria surviving the first disinfectant test you should see an increase in the number of colonies growing at the more concentrated disinfectant rates.

Matt Mulanax

Is it okay if you can explain how to make the chart by giving me an example??? Like just use random numbers but make an example of the (%) of RESISTANCE!!! that can be acceptable.....I think there is a diffrence between a "colonies chart" and a "resistance chart".....

Thanks...

eternal_corrector:

These are important sites for you to visit and read.

http://www.infectioncontroltoday.com/ar ... opics.html

http://www.cmmonline.com/article.asp?IndexID=6633496

http://www.selah.k12.wa.us/SOAR/SciProj ... rleyW.html (this is a good example of a resistance science project for you to use as a model)

To determine the percent resistance you must know the original population size (number of bacterial cells exposed to the disinfectant) and the number of cells that survive exposure to the disinfectant. All surviving cells may not be resistant - they may have escaped exposure by virtue of their place in the mass of cells.

Matt Mulanax

Heres something I made up the science project........


--Do Different Dilutions of Disinfectants Affect the Development of Bacterial Resistance?--

--Purpose--
The purpose of this project is to determine if bacterial resistance (after repeated exposure
to a disinfectant) depends on the concentration of the disinfectant. The independent variable
is the different concentrations of the disinfectant (percentage).
The dependent variable is bacterial inhibition (percentage).

--Materials--

35 ml of Staphylococcus aureas
35 ml of Gram positive bacillus
30 ml of pine oil (new bottle of Pine Sol)
30 ml of sodium chloride (new bottle of Lysol)
30 ml of sodium hypochlorite (new bottle of Clorox)
30 sterile test tubes
30 sterile loops
30 nutrient agar plates
1 pipette
1 incubator
60 stick labels

--Procedure--

1.Set up 10%, 20%, 30%, 40%,and 50% concentrations (out of 10 ml) for each disinfectant which
include pine oil which is found in Pine Sol, sodium chloride which is found in Lysol,and sodium
hypochlorite which is found in Clorox.
2.Set up 5 sterile test tubes: the first one containing 9 ml of bacterial suspension, the
second one with 8 ml, the third with 7 ml, the fourth with 6 ml, and the fifth with 5 ml.
3.Now label each test tube according to the amount of bacterial suspension, the type of
bacteria which is Staphylococcus Aureas ,the name of the disinfectant that will be used
(Pine Sol), and the amount of disinfectant in percentage that will be used to treat the
bacteria which is found by subtracting the amount of bacterial suspension out of 10 ml.
4.Repeat steps 2 and 3 with another 10 test tubes to test two more of the reamaining
disinfectants which are Lysol and Clorox.
5.Repeat steps 1, 2, and 3 for the treatment of the remaining bacteria culture
(Gram POsitive Bacillus).
6.Then mix each disinfectant concentration with each of its corresponding test tube gently.
7.Then insert the test tubes in an incubator and let them incubate in room temperature
(25°C/77°F) for one hour.
8.While the incubation process, label each nutrient agar plate according to the labeling information of the test tubes.
9.After 1 hour incubation, spread 3 milliliters of each mixture evenly onto their corresponding nutrient agar plate.
10.Incubate these plates and count colonies on each plate after 24, 48, 72, and 96 hours.
11.If there are signs of bacteria that are resistant to the disinfectant, test them by running
them through the same disinfectant series again to see if anymore colonies grow on the plates.
12.Zones of inhibition (bacterial colonies) are measured each day.
13.Sterilize all materials used and wash hands.

eternal corrector:

Looks pretty good. Check with your mentor/advisor for final approval.

Matt Mulanax

Can you show me how to make an example percentage graph that would be necessary for the project I'm doing...Like for the colonies.....

eternal_corrector:

Could you post some data over the weekend so I can see what you have.

Percentages will be calculated as follows -

Population after treatment/Population before treatment X 100.

I have attached an example of how to plot such data.







This is an example of how suuch a chart could look





150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0

Treatment 1 100% Treatment 2 50% Treatment 3 150% Treatment 4 0%

Treatment 1 did not influence growth
Treatment 2 reduced growth 50%
Treatment 3 stimulated growth of the test organism
Treatment 4 completely inhibited growth
Use a graphing program - bar chart type - and enter treatment number and corresponding value for each treatment


Matt Mulanax