Science Buddies: "Ask an Expert"

How can you test for fungal growth and bacterial growth on a hard surface? It would need to be tested for repeated days on a small plastic test surface. Vice

Hi,

There are two basic methods for microbial testing on sold surfaces. One method involves swabbing a sterile swab using a defined surface area, and the second method is by contact plates that are pressed onto the surface. The basic methods including the advantages and disadvantages are described in the following website:

http://faculty.weber.edu/coberg/3853%20 ... edures.htm

Here is information from the Science Buddies website on microbial testing:

https://www.sciencebuddies.org/science- ... ques.shtml

Be sure to check out the Science Buddies safety guide on microbial testing.

https://www.sciencebuddies.org/science- ... fety.shtml

If you are interested in both bacteria and fungi, you will need two different types of culture media. Do you know what media you will be using for the two groups of microbes? What is the purpose of your project?

Donna Hardy

Actually I will be testing a cleaning solution over many days and not actually the plastic like I originally thought. I want to test for both fungus and bacteria. Would I dip a swab in the solution and then swab an agar like plate? Is there one for fungus? Would I keep adding to the same plate over the days? or do different plates for different days? Vice

Hi Vice,
Both bacteria and fungi will grow on agar plates (wikipedia has a good overview: http://en.wikipedia.org/wiki/Agar_plate) -- actually, mold that sometimes contaminates bacterial plates in labs are all fungi.

From what you have posted, it appears that you want to test the efficacy of some type of cleaning solution designed for plastic surface-- is this correct?

Before you begin, however, you should consider a few factors, such as:
1) What kind of mold and/or bacteria am I interested in growing? Where may I find sources of these bacteria/mold?
2) What chemicals are in my cleaning solution, and what effects are they predicted to have on certain types of bacteria and mold?
3) What factors will I be holding constant (controls), and what factors will I be changing (variables)?

Try to elaborate more on what it is exactly you want to investigate first.

Lily

Hi,

To grow fungi, you can use potato dextrose agar. For bacteria you can use nutrient agar. Here is a homemade recipe for both media:

http://en.wikipedia.org/wiki/Potato_dextrose_agar

This recipe does not include a source of nitrogen, so I would recommend adding 2 grams of yeast extract, available from a health food store, to ensure growth of a wide variety of fungi.

For bacteria, you can use the following recipe for nutrient agar:

http://www.science-projects.com/PlatesSelfMade.htm


Since it sounds like sterile swabs would be more convenient for you to use, you can use a sterile swab to collect the microorganisms from a specific surface area for each sample and then transfer the swab to a tube containing 1 ml of sterile water. The microorganisms could then be resuspended into the water by mixing well. You would then transfer 0.1 ml of the water to the surface of each plate and use another sterile swab to spread over the surface of the plate evenly. I recommend that you do a trial run and test this procedure before you do a major experiment. You want to practice your sterile technique and adapt the method to the materials you have available to use. If you will be comparing your results over time, you want to make sure that your technique and growth media will give consistent results.

What will you use for a control in this experiment?

Donna Hardy

Vice,
In addition to the great ideas others have posted, something else to think about is how you will count the fungi or bacteria you find on your plates. Will you try to count individual colonies or try to estimate the numbers? If you have too many to count you could also think about diluting your samples before plating. This sounds like a great project!

Matt Badtke

To elaborate more on what Matt was saying in the previous post, a common way of counting bacteria colonies is using a microscope and a grid. If the bacterial colonies (or fungal colonies) appear fairly evenly distributed on a plate, you may divide up your plate into, for instance ten sections, and only count one or two of the sections, and multiply appropriately to get a good estimate of the number of colonies you have on your plate.

Hi,

I just realized I didn’t completely answer your question. An agar plate is used just one time for one sample. Once the surface has been swabbed and the sample transferred to the surface of the agar plate, the plate is turned upside down and incubated for a few days to allow the bacteria and fungi to grow. A new agar plates will be needed for each sample.

How are your going to tell the difference between bacteria and fungi?


Donna Hardy

How do you tell the difference between bacteria and fungus? If you grow on different media can you tell the difference? Vice

Hi,

Fungi grow as furry, fuzzy colonies on an agar plate; they start out as white fuzz and usually turn dark green, brown, or black as spores develop after a few days. Fungal spores are ubiquitous, which means that they are floating around in the air always ready to germinate and grow as soon as they land on a suitable growth medium like an agar plate or a damaged piece of fruit. Bacteria appear as a wide variety of colony types and are usually white or translucent, but can have a wide variety of colors. Yeasts are fungi, but the appearance of the colonies is similar to bacteria. The Science Buddies website guide has a useful guide to introduce this topic.

https://www.sciencebuddies.org/science- ... ates.shtml

Identifying the specific fungi and bacteria in your samples will be outside of the scope of your project, but you should plan to use the official terminology to describe the form, elevation, and margin of the colonies that grow from your samples.

So what is the purpose of your project?


Donna Hardy

I am doing 3 solutions 4 ways each. I plan to test for bacteria or fungal growth every 5 days. I am assuming I won't get any growth for 15 days but more like 20+ days. That is at least 36 agar plates for each test. One test is going to be at least 36 if not 48 plates. Or start day 10? The solutions should not get contaminated right away but what if I wait too long? Because of cost, can I do just one set of the 12? Should I do a second set? Vice

Hi,

Your project sounds very ambitious. Are you planning to have separate fungal and bacterial media plates? Do you have time to do a pilot experiment before you invest in so many plates? It sounds like you are planning to do a growth curve, which requires lots of dilution and lots of plates. Can you describe your experimental protocol in a little more detail? I’m not sure why you think that you won’t get growth for 15-20 days. Please include the temperature you will use to incubate the plates. Perhaps I can suggest a protocol that will require fewer plates.


Donna Hardy

I am using a syringe to measure .1ml of each solution. How can I sterilize it between solutions. I am doing this at home. If using cotton swabs to streak, do I make four sections like it shows to streak? Or can I just make big streaks across agar? Do the streaks need to follow the same pattern on each plate? Also, I don't have an incubator so once I plate what do I do with plates? One source said to put in plastic bags and turn upside down for three days. Should I section off plates before I streak to help count in case I get growth? I am seeing which solution shows contamination first Vice

Hi Vice,

To sterilize the syringe in between samples, you could use 70% isopropanol, and then rinse it with sterile water 3-4 times. If there is any residual alcohol, it will inhibit the microorganisms, so you want to make sure it is rinsed thoroughly.

Since you are doing this at home, you should put each Petri dish in a zip-lock plastic bag, and invert it and incubate it at ambient temperature, although try to choose the warmest place available. Use a thermometer to measure the temperature of incubation. You should observe the growth of the bacteria/fungi through the plastic bag. Avoid opening the plates since you will be growing unknown microorganisms.

I think you will be using the 0.1 ml/ sample from the syringe to apply your sample to the surface of the Petri dish. After you do this, use a sterile cotton swab to spread the 0.1 ml/ evenly over the portion of the plate you are using. Label the back of the plate with a Sharpie so you will remember what is in the plate.

What kind of agar are you using? Do you have some sort of positive control to use?

Donna Hardy

I have two samples of each type of solution I am using. Can I take half from each for one agar plate or should I really use one for each. I may be testing several days and I have 12 types but two of each type. It will be expensive to test both from each type. Vice

Hi Vice,

I would go ahead and use one-half of a plate; try to leave at least a 1/2 cm space between the two halves. Let me know about your progress.


Donna Hardy

1st question is should I try to identify what type of growth I have? I will attach my descriptions.
2nd question is that my teacher said I should say how big the growth was. I didn't record that. My hypothesis was that one solution would be the last to show contamination. How should I graph? All I have is when I tested and what showed growth. Thanks. Vice


12/4/2011 9:40am

SNO: is a creamy yellow with a reddish brown spot in the middle. The form of the bacteria is circular with a tail going upward. The elevation of the bacteria is convex, with the margin of entire. The surface is smooth and glistening with a translucent opacity.

ACC: is creamy-yellow and white with an irregular and circular form. (A bunch of circular colonies made into one in the middle. On the outside there is a bunch of individual colonies. The bacteria had an elevation of convex, with a margin of entire. The surface was smooth, glistening, and bumpy with opaque opacity.

ANC: is a tannish yellow with an irregular shape. It was a blob with small little colonies dotting away from it (like islands). It has an elevation of flat with a margin of entire/undulate. The surface was smooth, glistening, and under high magnification it looked dotty. The bacteria’s opacity was opaque toward the middle, but along the sides it was translucent.

ANO: is a creamy- yellow and white with some circular and irregular forms. There is a bunch of little colonies. The elevation was convex with a margin of undulated. The surface was shiny and smooth with opacity of opaque, but was translucent towards the edges.

ACO: is yellow and has the form of filamentous. The plate was covered but there was a dot where it was raised with an umbonate elevation. The margin wasn’t able to be seen. The surface was dull and smooth, but the dot was rough. The opacity was an opaque.

12/9/2011 6:20pm
Looked at plates with high magnification (slit lamp).
ACC: Nothing. When disinfecting, bacteria was probably killed.
ACO: Was circular with a yellow flat surface. Its margin was entire. It was dull and smooth, with an opaque opacity. It was translucent towards the outside.
ANC: Was circular with a yellowish-white raised surface. Its margin was entire, and its surface was also smooth and shiny. The opacity was translucent.
ANO: Was circular with a yellowish-white raised surface. Its margin was entire, and its surface was also smooth and shiny. The opacity was translucent.
BCC: Was circular with a white raised surface. Its margin was entire, and its surface was also smooth and shiny. The opacity was translucent.
BCO: Was circular with an entire margin. The color was white with a yellow dot. It was convex and had a smooth and shiny surface. The opacity was translucent.
BNC: Its shape was irregular with an entire margin. The color was white with yellowish dots. It was raised with a smooth and shiny surface. The opacity was opaque.
BNO: Irregular with an entire margin. It was raised with the color of whitish-yellow. Its opacity was opaque with clear edges.
SCC: It was circular with an entire margin. The surface was yellowish-whitish with a raised elevation. The surface was smooth and shiny, with opacity of it being opaque with clear edges.
SCO: Circular with a filamentous. It is yellow in the middle, and has an elevation of raised. The margin is entire with smooth and shiny surface. Its opacity is opaque.
SNC: Its shape is irregular with an entire margin. Its elevation is raised. It surface has a yellow /whitish color, and was smooth and dull. Its opacity was opaque.
SNO: There was none. Could have been killed during sterilization.

Hi Vince,

You have done an excellent job in describing the colonies. Very detailed and thorough. This information should allow you to compare the colonies in the plates. It sounds like you had a variety of different bacteria growing in the plates, and not just one colony type. You cannot do anything to further identity the bacteria at this point, as this would require a complete microbiology lab with a microbiologist to help you.

If you did not count the colonies, or measure the percent of the surface that was covered with growth, and if the plates have been discarded, then you will have to report qualitative yes/no results. So growth was either present or absent on each sample. If you still have the plates available, perhaps you can try to estimate the percent of area of the agar surface that was covered with growth. A bar graph would be a good way to report results, as you could list the various samples along the x axis and each bar would be an estimate of the growth. If you have the date and recorded when growth appeared, then you could also do a bar graph and have each bar represent the time required for growth to appear after each solution was used. If you have a few more days, you could post your data and perhaps I could make an additional suggestion.

Donna Hardy

I will try to attach graph. I tried to add legend but couldn't figure out how to add width to right side to allow space. Not sure attached right?

Hi,

I’ve tried several different times to save the attached file, but I can’t seem to open it. What program are you using for your graph? It would be fine if you wanted to try again, or just attach the raw data.


Donna Hardy

I used excell. I used clustered column. Title was "Efficacy of Different Contact lens Solutions". There are 3 set of bars with the name of each type of test (abbreviations) below each bar.
SNC SNO SCC SCO ANC ANO ACC ACO BNC BNO BCC BCO
SALINE Opti-Free RepleniSH Biotrue



The vertical has 1-20 with the caption " Number of days to show contamination.

i know I need box to show what abbreviations stand for but i couldn't figure out how to add space to side of graph. Can i have in separate box below? It needs to be in same space with graph right?

Thanks for all the help. Live long and prosper! Vice

Hi Vice,

Thanks. I finally opened the graph. Your results are great! You have done a very good job of showing the number of days that the contact lens solutions prevented microbial growth. There’s hardly any room left on the graph to include the legend for the abbreviations, so you could include this in a separate box immediately below the main graph. This would be essential so the science fair judges will know what happened just by looking at your graph.


Donna Hardy

I completed the graph. I'll try to reattach the graph again. Thanks again. Live Long and Prosper!
-Vice
p.s. When it's printed, it'll be bigger. It's originally printed sideways on a PowerPoint document. I had moved the graph to a word document to see if it would make it easier to open.

Hi Vice,

Excellent! Yes, the word document is easier to open. With the color coding, this is clear and readable and very easy to understand. I like graphs of results that tell the whole story. Make sure you position this graph in a prominent position the middle center of your board to make it the “center of attention.” You did a really good job. In your conclusion, make sure you reference you hypothesis and your results. If there’s room, you can include a statement about what you would do different if you did the project again.


Donna Hardy

Thanks again for all the help you've been. I would like to give you credit in my science project... How would I list you? I hope you have a great week! LIVE LONG AND PROSPER!

Hi Vice,

You are more than welcome. I'm very glad I could provide some helpful advice for your project. In your acknowledgements section, you can refer to me at " Donna Hardy from Science Buddies," or just give credit to the Science Buddies organization. Please do let me know about the results from your science fair.

Donna Hardy

I am writing up the report. On the conclusion I told the results.The saline did better than the Opti-Free in general. Not sure how I should present that? Saline should have been the worse. How much should I say in conclusion? I think some of that may have been me that I might have used the syringe too soon after sterilizing with hot water. Do I put that in conclusion? Or do I put that in the recommendation page? There could have been a chance it was how the Opti-Free was stored? That goes in conclusion, right? Not sure what needs to be said? Thanks. Vice

Hi,

You are on the right track. In your conclusion you can state a summary of the results, that the saline was better than the Opti-Free, so your hypothesis was not confirmed. Then you can continue with the conclusion section and discuss the possible reasons for the unexpected results. You have already thought about two good possibilities, so you should mention those. Can you think of anything else? Some of the best projects are those with unexpected results, because it makes for an interesting conclusion section.

Saline is 0.15 M sodium chloride; Optifree is saline with proprietary additives that are supposed to disinfect the contact lenses and provide moisture. Maybe there’s something in the Opti-Free that actually supports microbial growth after a few days. I wish we knew what was in the product.

http://www.drugstore.com/opti-free-repl ... /qxp150933

You should also think of what you would do differently if you were to do this project again, and include this in the conclusion section.

You’re almost done! Let us know how you do in the science fair.

Donna Hardy

My teacher said I should have stats. I don't know of what? What percentage of each solution showed contamination? My results were when each type showed contamination. Each solution had two cleaned and two not cleaned. What would I do stats on?

Hi,

Here are some ideas for including statistics in your project. If you were in high school, I would have recommended that you use the student’s t-test to calculate whether or not there is a significant different between your saline control and the Opti-Free and also between the saline and the Biotrue. Here is information on the student’s t-test:

http://en.wikipedia.org/wiki/Student's_t-test

I don’t know how many data points you have for each bar in your graph, but if you have two or more points and the values are an average, you can try using the following p-value calculator using the 1-tailed test. With a biological assay like you are doing, if the p-value is less than 0.05, then results would be statistically significant because there would be a less than 5% chance that results would have occurred by chance.

http://studentsttest.com


However, you are in 7th grade, and the p-value calculation uses advanced level math formulas. Unless you are a math genius, I don’t think the information above will make sense to you.

You shouldn’t present something that you don’t understand, so I recommend that you do the following:

1. If you have more than one data point for each group, you can calculate the standard deviation for each group. The standard deviation is a measure of the variability of your data. Here’s a SD calculator:

http://www.mathsisfun.com/data/standard ... lator.html

If you have just one data point, then you can’t do SD, so omit this.

2. You can calculate the percentage difference between your control group and the two independent variables. For example, the Opti-Free ANC results are 12/18 or 66% of the control saline values and the Biotrue BNC results are 100% (18/18) compared to the saline control. You can write in the percentage on top of the Opti-Free and Biotrue graphs.

I recommend checking in with your teacher and asking if this would be acceptable. If she wants you to try doing the p-test, then please post all of your raw data if you have more than one result for each group, and I will explain further. Let me know if you have any questions.

I hope this helps.


Donna Hardy