Plating failure - procedure error? - (Jan/09/2008 )

I'm trying a simple experiment in which I want to count colony forming units of any bacteria, but nothing has grown in my nutrient agar. Here is what I did, and I'm wondering where I went wrong before I try it again:

1. Warmed up the bottle of nutrient agar in the microwave until it started to boil.
2. Poured liquified agar in a thin layer in petri dishes. Let them cool and solidify (not sure how long, probably less than 30 minutes)
3. Source of bacteria:
a. cutting board smeared with food, including raw pork juice
b. left at room temp exposed to atmosphere for about 3 hours
c. rinsed for a few seconds with warm tap water
d. swiped surface with regular cotton swab
e. put cotton swab in 20 ml of previously boiled water and stirred
f. serial dilution (transferred 2 ml into 18 ml of previously boiled water, stirred) several times
4. transferred 1 ml from last 3 dilutions into separate agar plates
5. tilted plates until liquid covered entire surface, immediately replaced cover
6. stored them upside down in homemade incubator consisting of styrofoam cooler heated with 22 watt light bulb (~100 degree F environment)
7. waited patiently for colonies to form

It has been over a week and absolutely nothing visible has formed on the agar! Separate sections of the cutting board were also cleaned with various soaps, but none of these produced any bacteria either.

I did not follow aseptic techniques during the dilution, so if anything I should get more bacteria rather than less. Here are my ideas on what might have happened:

1. There was no bacteria in the first place
2. swiping with a cotton swab didn't collect any bacteria
3. dipping the swab into the water didn't transfer any bacteria
4. the previously boiled water was too hot and killed the bacteria
5. the agar was too hot and killed the bacteria
6. the incubator was too hot or too bright and killed the bacteria
7. the nutrient agar was boiled for too long or in some other way became an ineffective growth medium (it was shipped by air and stored in the refrigerator for only 1 day before use)

Should I use a different type of agar when I try again, or...?

The media could be a problem if it is in any way selective. It appears to me that your ideas #4 & #6 are the more likely suspects; Given the nonaseptic conditions, #6 is probably the problem since you've not produced any 'ambient' colonies either.

I will try again with the same nutrient agar, but wait longer for it to cool down (as well as the boiled water.) I also asked the customer service rep when I ordered more agar and dishes, and he thinks the bright light in the incubator may have killed the bacteria. The 22 watt bulb I used was a compact fluorescent, which gives off light equivalent to a 100 watt incandescent bulb, so it was definitely plenty bright inside the white styrofoam cooler. He suggested covering the plates with an opaque cloth. What do you think of this approach?

compact fluorescent, throws off a fair bit of UV light. So, that would be a problem.

The other problem is the nutrient medium. What might you be using? BHI medium? Chocolate medium? Blood?

And strangely enough, if your cutting board was fairly clean (with detergent) and used fresh meat, you might not have had that many bacteria and fungi spores to begin with. Compounded by the serial dilution, you may not actually have that many bacteria actually on your plate.

I guess I can't say for sure what the agar was, other than "nutrient agar", which I believe is not selective to a specific bacterium. I did get a nice spot of mold growing on an extra plate that was not used in the experiment, and therefore not put in the incubator, so I concluded from that evidence that at least the agar was good.

I re-ran the experiement with a fresh set of agar plates. This time, we poured the plates and left them out at room temperature over night before we used them. The source of bacteria was even worse than the first time: the packaging from raw chicken left out for several days (it reeked.) Also, we poured the boiled water and let it sit for quite a while to ensure it was at room temperature before we put the bacteria in and did the serial dilution. And lastly, in the incubator we covered the plates with several layers of towels that blocked the light from the bulb. Unfortunately this also significantly reduced the temperature in that area, from 97 to about 76 F, but we decided to proceed anyway, given that the higher temperature is just an accelerant anyway.

We also plated a few undiluted samples, just in case the serial dilution wasn't done correctly.

This time we successfully grew colonies! We also did not put the plates upside down in the incubator, which turned out to be slightly not level, causing the 1 ml of sample to pool slightly to one side in the dish. The results showed most of the colonies along the edge of the pool, and this is where we counted. The results showed that soap significantly reduced the concentration of bacteria compared to just rinsing the cutting board, as expected. The only surprising result was that "Dial Complete" had more bacteria than "Dial Gold", even though it has 4X the amount of Triclosan, but of course we have no way of testing for statistical significance as we didn't do any replicates anyway.

In the end, it was a fun project, and I appreciate your helpful comments very much.