Welcome back to Microbiology Lab. My name is Kevin Tokoff. Please make sure to LIKE this video and SUBSCRIBE to my channel for future videos and notifications. In this video, we're going to discuss nitrate broth, sometimes referred to as the nitrate reductase test. This is a complicated test where you actually have to follow a flowchart.
Unfortunately on an exam or a quiz you would not be given this flowchart. You actually have to learn it. But what I hope to do in this video is go over the possible results and then also at the end explain the mechanism as to why we get those results. And I'll include a timestamp in the first comment where if you want to look at just the theory behind the results you can skip to that. But first of all let's discuss the reactions that are going on here.
So this is the first Time in the semester we're actually going to see two reactions simultaneously. The first reaction is catalyzed by an enzyme, which is actually what this test is named from, and that is nitrate reductase. Recall that whenever we have a reaction arrow and I write something above it in blue, that means I'm indicating an enzyme.
Nitrate reductase converts NO3 minus or nitrate into NO2 minus nitrite. And notice the difference between these two words. We have nitrate ending in A-T-E whereas nitrite ends in I-T-E. One way that you can learn that nitrate comes before nitrite is that A comes before I in the alphabet.
So alphabetically speaking nitrate comes before nitrite. Okay but that's the first reaction that's going on here. Nitrate is being technically reduced into nitrite. It's a redox reaction.
And we have a second reaction here which is a second enzyme. This enzyme is nitrite. reductase.
Again notice the I here instead of the A that we saw in the first enzyme. Nitrite reductase converts NO2-nitrite into a couple of products. We have ammonia which is NH3 and then nitrogen gas.
This is just diatomic nitrogen or N2. You can just refer to it as nitrogen and ammonia. And this test is actually going to allow us to determine what set of enzymes we have. Do we have only the first one?
That's one case we can have, so only nitrate reductase. We can have both of them, nitrate reductase and nitrite reductase, or we can have neither of them. So neither of these two enzymes are actually present in the bacteria. And the way we determine that is by following this flow chart on the results day. So recall that we did inoculations for the nitrate reductase test or nitrate broth, and then we put it in the incubator, and now we're going to take it out, and we're going to start running results.
The first thing we're going to do is we're going to add sulfonylic acid and alpha-naphthalamine. Now, depending on where you're taking this, sometimes sulfonylic acid is called solution A, and alpha-naphthalamine is called solution B. But in any case, we're going to add these to the broth.
That's the first thing we do always. And technically, your manual says to add 16 drops of each of these. So 16 drops of sulfonylic acid and 16 drops of alpha-naphthalamine. Now... The easiest case is going left.
So what can happen is when you add those 16 drops of each of these two reagents, immediately the solution will turn red. Okay, it could remain colorless. We'll talk about that in a minute.
But one option is that after you add these it will turn red immediately. So after we add these two reagents, if we get a color change to red immediately, that means that we have our simple result positive for only nitrate reductase, meaning we have the first enzyme here but we lack the second nitrite reductase. Okay so that's for a red color change immediately after you add these reagents. That's the easy result but what will happen for some species is you'll add the 16 drops of sulfonylic acid and 16 drops of alpha-naphthalamine and you'll see no change. It'll just stay the same color as the broth.
Okay In this case, when there's no color change, you may or may not have nitrite reductase. Notice this is the second enzyme. What that means, in other words, is when you have no color change after these first two reagents, you either have both enzymes, both of these two, or you have neither of them.
And so what you have to do is you have to add what's called zinc dust. We have these little bottles that have a zinc powder in them, and what you're going to do is you're going to add a match head size amount of zinc to the broth. Okay and what will happen is when you add the zinc pretty quickly either there's going to be still no color change or there's going to be a red color. But notice this red color is only after there was initially no color change and after you added the zinc dust. Okay, now after you add the zinc dust, assuming no color change at first, if there's still no color change, then what that result indicates is that you have both enzymes present, both nitrate reductase and nitrite reductase, both of these two.
And that's what happens if you add zinc dust and there's still no color change. However, if you add the zinc dust, assuming initially no color change, you add the zinc dust and it turns red, That indicates that you have neither enzyme. So you do not have nitrate reductase and you do not have nitrite reductase.
Neither of these two enzymes are present. And so basically what this amounts to is you need to learn this flow chart. And given a set of information, you need to be able to predict what the result is. So for example, let's suppose you had a question that read something like, so in a nitrate broth, We added 16 drops of sulfonylic acid and alpha-naphthalamine, and we had an immediate red color change. What's the result?
Well, considering that you had an immediate color change after adding these two reagents, that would indicate that you have only the first enzyme, nitrate reductase. And so that would be the only enzyme you would list. You could have another question where it said, first sulfonylic acid and alpha-naphthalamine were added. after no color change, zinc dust was added and then we got a red color. What's the result?
Well, if you added these two reagents and there was no color change, then you added zinc dust and got a red color change, that means you have neither of these two enzymes present. You have none of them. Neither nitrate reductase nor nitrite reductase.
And then we could have another question that's similar to the previous one where we added sulfonylic acid and alpha-naphthalamine. there was no color change and after adding zinc dust there's still no color change. Okay in that case we have both of these enzymes nitrate reductase and nitrite reductase present. And generally a question referring to nitrate broth if it tells you that it added zinc dust that pretty much means that there was no color change initially. So if you added zinc dust it's either going to be both or neither.
The only way you're gonna have a positive result for only the first enzyme nitrate reductase if the color change was immediate in which case there would be no need to add zinc. All you added was the first two reagents right here and you got that color change. Alright so the key here learn the flow chart and be able to predict if it's only the first enzyme both or neither.
I'm kind of being a broken record here. Now at this point in the video some of you may want to quit I'm just going to talk about the theory and the mechanism as to why we get these three results. This is not terribly important for our exams here where I'm giving it, but I want to make sure I at least cover the theory.
All right, so why is it that in this first case, it turned red after we add sulfonylic acid and alpha-naphthalamine? Well, the reason that it turns red immediately is because really that red color change is dependent on nitrite, really the important compound here. nitrate, nitrite, nitrogen, and ammonia, the important one is the nitrite.
So let's think about this. If I only have the first enzyme, okay, so I do not have the second one. Well if I have the first one, nitrate can be converted to nitrite, okay, but since there's no second enzyme, none of the nitrite will be converted to these other products over here.
So my broth, when I pull it out of the incubator, is going to be loaded with nitrite. Reason being is the bacteria already converted all the nitrate in the broth into nitrite. So in this case, going for the first enzyme only, I have plenty of nitrite present.
So when I add the sulfonylic acid and the alpha-naphthalamine, and you by no means need to know this, the nitrate initially reacts with the sulfonylic acid and then this intermediate reacts with the alpha-naphthalamine, to produce something that's basically a red dye. Okay? And so, the key to getting that initial red color with the sulfonylic acid and the alpha-naphthalamine is they react sequentially with the nitrite to produce the red color. And so, if I have that nitrite left over because I only have the first enzyme and I lack the second one, then that nitrite can be transformed into that red dye.
And that's why I get the red color immediately. All right, now let's talk about why we don't get any color change initially when we have... Both enzymes or neither, so initially no color change.
Remember that the initial color change is dependent on nitrite. Now, in the case where I have both enzymes, both nitrate reductase and nitrite reductase, let's think about this. I have the first one, so nitrate is converted to nitrite, but I also have the second enzyme, so any nitrite that's formed is immediately consumed to produce nitrogen gas and ammonia.
So, when I pull a broth that has a species that has both enzymes in it out of the incubator, I'm going to have a bunch of nitrogen and ammonia that are produced in that broth. Now technically the nitrogen will bubble off and you won't have any left, but the key is you won't have any nitrite, none of that stuff. In the case where I have neither enzyme, I have neither, nitrate cannot be converted any further.
So my broth is still going to be loaded with that initial nitrate. which is what gives the broth its name. So again, all nitrate, I have no nitrite in this last case. Okay, now the easiest one to consider first is really the neither case.
So why is it that when I add zinc dust, it turns red? Well, to understand this, zinc dust or the zinc ion, what zinc can actually do without an enzyme, this is a non-enzymatic process, but zinc can actually cause nitrate to convert into nitrite artificially. So let's think about this. If I add the zinc to artificially convert the nitrate into nitrite, then all this sulfonylic acid and alpha-naphthalamine that's left over from the first step and still in the broth can now react with this nitrite that's been produced and produce the red color. So that's why in the case of neither enzyme, it turns red after the zinc because We still have nitrate left because we have neither enzyme.
The zinc converts the nitrate into nitrite. And that's why when you have the neither case, it turns red after the addition of zinc dust. Because the zinc artificially converts nitrate into nitrite.
And then these two reagents that are still in the broth react with the nitrite to produce the red color in the same way as before. So neither turns red after the zinc dust. But what about both?
this case. Well, if I have both enzymes present, I should have a bunch of nitrogen gas and ammonia that are in the broth, basically. Okay, so no nitrite and no nitrate.
What's the function of the zinc dust? The zinc converts nitrate into nitrite. Well, I don't have any nitrate in the broth where there's both enzymes because everything was converted all the way to nitrogen and ammonia. So if there's no nitrate, I can't produce any nitrite.
So there's going to be no nitrite in this broth and therefore no color change to red. And so if it still remains colorless, so no color change, you have both enzymes. All right.
And so I hope I've made some sense with the rationale behind this. Again, in some courses, they may not expect you to have memorized all the mechanisms, all the rationale as to why you have the particular color changes or lack thereof. But the major thing that you need to glean from this.
video is understanding the flow chart, adding what reagents, and you get what result. Okay, so if it turns red immediately, it's only the first enzyme. If you have an initial no color change, you have to add zinc dust. And then if it's still no color change, it's both enzymes.
But after the zinc, if it turns red, it's neither enzyme. All right, so hopefully this video made sense to you. Please make sure to like this video and subscribe to my channel for future videos and notifications. Thank you very much.