Hey everyone! For Thursday breakfast (I’m writing this Friday), we went to a cafe and ordered a thoroughly American breakfast. Su Chen and I got orange juice, and I don’t think I’ve ever been presented with a fancier cup of orange juice than the ones they prepared for us. Orange juice here, by the way, is amazingly sweet and freshly squeezed. It tastes a lot like the HEB homemade juice, except minus the price tag and plus a sketchiness (in a “this might make you sick, but probably wont” sort of way) .

Anyway, I’m writing this post on Friday, so I’m going to include 3 different things.

1. Bacteria counts of each of the sampled households from San Juan Cancuc
2. Cursory experiment of re-contamination elimination steps
3. Bacteria counts from the cursory experiment

Bacteria counts of each of the sampled households from San Juan Cancuc

Given the results of this experiment, I believe that it was pretty clear that we needed a larger sample size. From what we observed, we assumed that that the cleanest cups would be in the following order (from cleanest to dirtiest):

1. H3, because they use bleach to wash their cups and garrafon water to rinse the cups.
2. H1, because both are in the Red and seemed very invested in the program (use tap water to rinse)
3. H2, because they seemed more neutral about the program (used stored tap water to rinse)

However, the results were as follows:

I didn’t individually count the number of total coliforms because the purpose of the tests was to gauge the number of bacteria colonies. Furthermore, the tests were so far from expectation (the cup rinsed with garrafon water had 4 colonies whereas the unrinsed cup had 3 colonies) [The difference between 3 or 4 CFUs is insignificant.  However, I don’t think the colonies in #3 are coliforms–there was no gas formation!  -JL]. Although this in itself is not bad, we believe that there could be variation due to the extremely small sample size. For example in H3, the cup that was rinsed was likely dirtier from the get go than the other cup.

However, I would note that there did not seem to be a large difference in total coliforms between using an unrinsed cup and using a rinsed one (either rinsed in treated water or not).  [I agree, based on this experiment, rinsing made no difference at all!  A good research topic for Cántaro and us-JL]

Cursory experiment of re-contamination elimination steps

Since the petrifilm results from the trip to San Juan Cancuc did not give a strong indication of whether it was necessary to a) rinse with treated water b) rinse with untreated water c) rinse the cup at all, I figured that it may be worthwhile to come up with a small test. Obviously, this would have too small of a sample size to be conclusive, but it might point us in an interesting direction. Therefore, I set up 4 different scenarios with contaminated cups.

1. Control: simulated no rinsing of the cup at all
2. Rinse Untreated: simulated rinsing of the cup in tap water
3. Rinse Treated: simulated rinsing of the cup in garrafon water
4. Chlorine: simulated chlorine contact for 15 seconds, and then rinsing of the cup in garrafon water

My detailed method for this can be found in the read more section.

We then poured 20 ml of water in each of the cups and took 1 ml samples from each. We also took a sample from the garrafon water (which had been poured into a smaller container) to make sure that the water we added was not adding contamination.

Bacteria counts from the cursory experiment

The results from this experiment were also very wonky. For example, the garrafon water, which should have been clean, showed a large amount of total coliforms. This could be because the water I used from the garrafon had been poured into a different cup. But even so, the control should have showed at least that much contamination; but instead, nothing. We assumed that we must have made a mistake somewhere in the process. The test is inconclusive. However, I do think that the method in theory has some value (with modification) to be studied more carefully in the future.  [Hm, I wonder what happened between the garrafon and the control sample. I wonder whether the cups themselves might be a problem…  Or whether contamination comes from somewhere in the process.  You are not working under the right sterile conditions and it is hard to pinpoint contamination in these situations. This would be a good protocol for Cántaro to work on if they don’t have it already. -JL]

Have a fabulous weekend!

-Horatia

Method for Determining Efficacy of Cup Rinsing

1. Take sample of contaminated water (10 drops) in each cup and use a new paper towel to wipe each cup and spread around
2. Take samples
   1. Garrafon water
      1. Take sample
   2. Control
      1. Pour 20 ml of garrafon water into cup, take sample
   3. Rinse Untreated
      1. Rinse with tap water for 5 seconds
      2. Pour 20 ml of garrafon water into cup, take sample
   4. Rinse Treated
      1. Rinse with 100 ml of garrafon water for 5 seconds
      2. Pour 20 ml of garrafon water into cup, take sample
   5. Rinse Chlorine
      1. Pour mixture of 3 mL to 99 mL of garafon water (yeah I messed up)
      2. Swirl for 15 seconds
      3. Rinse with 100 ml of garrafon water for 5 seconds
      4. Pour 20 ml of garrafon water into cup, take sample

3. Add 1 ml of each sample to Petrifilm via a pipette and lowered top film. Place in incubator at 37 C for 24 hours

Note: I would allow the water to evaporate next time to prevent inconsistency in the way the contaminated water is spread around. I would also take water from the sample general location of the cup each time. Care should be taken that the container holding garrafon water is not contaminated itself.