Lab tests show vinegar-sprayed safflower seed is the ultimate in clean cat litter

Dr. Rafael Garduño

Dr. Rafael Garduño

The Cat's Ask put its product to a scientific test. Read the results in our Q&A with Dr. Rafael Garduño, former Dalhousie University microbiology professor and Canada Research Chair in Food Safety. Dr. Garduño is currently an adjunct professor at Dalhousie University.

General view of the lab where the experiment was conducted

General view of the lab where the experiment was conducted

Plastic Petri dishes containing the Sabouraud agar (solid medium) used to grow fungi colonies

Plastic Petri dishes containing the Sabouraud agar (solid medium) used to grow fungi colonies

Colonies on the agar are manually counted

Colonies on the agar are manually counted

Q: What was the experiment involving The Cat’s Ask safflower seeds designed to test?
A: The experiment was designed to test whether the safflower seeds used as a litter bed were, or were not, supporting the growth of bacteria. The inventor of The Cat’s Ask, Mark Fenwick, knew that the safflower seeds they were using as cat litter in their litterbox prototype “seemed” clean, even after weeks of use by multiple cats. But through the website of the Department of Microbiology and Immunology at Dalhousie University, he approached me in early 2015 for advice on how to determine (from a microbiological standpoint) if the seeds were really as clean as they seemed. That is, were the seeds actually kept clean on a microscopic level by spraying them with a solution of half vinegar, half tap water? You can’t tell something like that by looking at it. The seeds would have to be tested. 

“According to the results, a solution of regular vinegar and water effectively cleans safflower seeds that are used as cat litter extremely well, even after they are used by multiple cats for many weeks.”
— Dr. Rafael Garduño, former Dalhousie University microbiology professor and Canada Research Chair in Food Safety
Light microscope used to observe the stained glass microscopy slides

Light microscope used to observe the stained glass microscopy slides

Q: Do inventors normally approach university microbiology departments to ask for advice on testing?
A: Not normally, no. But it’s not unprecedented for us (that is, university professors) to partner with someone in the community. Besides, it was an intriguing idea and, for a microbiologist, a relatively straightforward thing to test.  

Q: How did you design the experiment?
A: For this experiment, Mark set up three litterboxes in their home. The first one was a “control” litterbox that was kept in a closet, and never used by cats nor sprayed with vinegar and water. So it was basically the seeds exactly as they came out of the bag. The other two litterboxes were used by their four indoor cats. The seed bed in one of the litterboxes was thoroughly sprayed with equal parts vinegar and water, twice a day. The other was sprayed in the same way with equal parts vinegar and water, mixed with one teaspoon of table salt per 750 ml of liquid, also twice a day. Then, twice a week, for six weeks, Mark would gather two small samples out of each of the three litterboxes, so six samples in total, label them with a series of numbers and put them into separate ziplock bags, and then take the samples to a lab for blind testing. That means a total of 72 samples were examined over a six week period.

Q: You just mentioned "blind testing". So was it a blind study?
A: Yes, it was a single-blind experiment in that all 72 samples received by the lab were labelled using a number key and not otherwise identifiable. At the very end of the experiment — that is to say, after the last samples were delivered and analyzed — the key was revealed by Mark. Only then could I know what samples corresponded to each treatment, and results be graphed and interpreted.

Q: What exactly were you testing the seeds for?
A: We conducted three standard microbiological tests that looked at total aerobic bacteria, general coliforms (reported as bacteria capable of using the sugar lactose in a specialized culture medium called MacConkey agar) and total fungi. That is, we tried to cover all the major types of bacteria and fungi you would expect to find in what is essentially a cat toilet, and especially what is known as gram negative coliform bacteria, which are widely used as indicators of fecal contamination. We cultivated the total aerobic bacteria using a nutrient-rich, non-selective medium called Tryptic Soy agar (TSA agar medium). Fungi were grown on a medium called Sabouraud agar. I will not go into too much detail on the microbiological method used, but briefly it involves a dilution process in which microorganisms washed off the surface of the seed samples are suspended in a liquid medium and serially diluted to achieve enough separation between microorganisms. Then the microorganisms (which one cannot see with the naked eye) are grown on the agars mentioned, until each produces enough progeny to form what we call a colony. Microbial colonies are visible to the naked eye and can be counted. So the results of our experiments are reported as colony-forming units (CFU) per gram of sample seeds. That is, a microorganism that can grow to large numbers and form a colony is called a colony-forming unit. The results, therefore, are a direct indication of how many aerobic bacteria, coliforms or fungi (depending on the agar used to culture them) were present on the samples.

Q: Why did you test vinegar and water also with added salt? Why salt?
A: Vinegar is an acid, and acids can kill or prevent the growth of most microorganisms. Chemically speaking, acids have a low pH and alkalis have a high pH. Most microorganisms cannot tolerate low pH, and prefer to proliferate in favourable conditions at neutral pH. Salt is a well-known preservative that prevents the growth of microorganisms and can sometimes kill fungi, so we were curious if it would make any difference in the results. But we discovered that the salt didn’t markedly improve the results, and were basically the same as vinegar/water without salt.

Q: So how clean were the litterbox seed beds after spraying them with vinegar and water? What did you find out?
A: It was evident from our results that the safflower seeds sprayed with vinegar and water and used as litter did not accumulate bacteria or fungi. The sprayed seeds did not support the growth of microorganisms in general, even after being exposed to the cat biological wastes. Now like so many experiments in biology, there were a couple of aspects of this one that were surprising. It is reasonable to expect that the dirtiest samples would come from the used litterboxes, isn’t it? It is also reasonable to expect that the samples from the two working litterboxes would become dirtier as time went on. But, actually, we discovered that neither of these things were true. The "dirtiest" samples (that is the samples with the highest counts of microorganisms) came from the control samples — the seeds that the cats never used! And the other two, even though they were used by cats, actually became cleaner as time went on until they reached a fairly constant minimum contamination level, especially in terms of gram negative coliform bacteria. As you can see from the graphs, the vinegar and water helped them become cleaner and cleaner until about week 4 when they leveled off. 

Q: But that seems very strange! How it be that seeds that were essentially used as a toilet for multiple cats be CLEANER on average than those coming right out of a bag?
A: Basically, it’s because the control seeds were not cleaned with the vinegar solution, whereas the seeds exposed to cat wastes were. Another way to put it is that the cleaning process (spraying with a vinegar solution twice a day) is an effective treatment to prevent microbial buildup or persistence of fecal contaminants, even up to six weeks of continuous use. It should be noted that the control seeds did not become more dirty over time; actually these control seeds ended up with slightly less microorganisms than they started with. These control seeds served a very useful purpose of telling us what happens to the microorganisms in the seeds when one does nothing to them, except storing them. It is clear that these seeds do not accumulate more microorganisms, but those present do persist for up to six weeks.

Q: So what were some of the conclusions of this study?
A: First, according to the results, a solution of regular vinegar and water effectively cleans safflower seeds that are used as cat litter extremely well, even after they are used by multiple cats for many weeks. Second, safflower seeds cleaned with a vinegar solution do not accumulate microorganisms and therefore are an excellent material for litter beds that will not serve as a source of microbial contamination, at least not more than safflower seeds on their own.

Q: What about toxoplasmosis? Was that studied in this experiment?
A: This experiment was NOT designed to study the identification or persistence of Toxoplasma gondii on safflower seeds sprayed with vinegar and water. To begin to look at toxo you would likely need a greater sample size of cats, plus the detection methods are usually PCR assays (DNA testing), which was beyond the scope of this experiment. 

Q: Does this mean that the seeds are so clean after the cats used them that I can feed them to birds?
A: No, the results don’t mean that. These results measure what’s on the surface of the seeds, or what bugs or germs grow on them, but doesn’t detect what happens on the inside. And this is important because the seed shell can certainly be penetrated. It also deals only in samples — not every seed can be tested. In addition, we only tested the seeds from a microbiological standpoint, and never intended to evaluate any nutritional value, which through use and cleaning with vinegar could have deteriorated. Finally, I would add that anything that is deliberately used as a toilet should never re-enter the food chain. So, no, the used seeds should not be fed to birds.

Q: Should used safflower seeds be composted or should they be thrown into the garbage?
A: Every jurisdiction has its own way of disposing of garbage, so it’s hard to generalize. But the rule of thumb is that you should dispose of used safflower seeds in the same way you dispose of meat. Which means that if you compost your meat, you could compost used seeds (not feces, that is, just the seeds). As an added precaution, if you compost the seeds yourself, you should not later use the compost to grow food for human consumption. So it is recommended that you do not use this compost in your vegetable garden, for example. But in your flower garden it should work beautifully.