3.1 Biochemical test strips

Okay now, to do any identification it's absolutely vital that you have a pure culture and that means that you don't have a mixture of different species, that you're doing the same test on as a mixture. And one way of doing that is to plate out your sample and pick off the single colonies. A single colony – this is actually a pure culture anyway, so all the colonies are the same, but in a real sample in a hospital, so you might get a mixture of different types of colony and the one that you think is the suspicious one then you want to identify. If you pick off a single colony that's the pure culture. Okay, a single colony is all one species or one type of cell and you're guaranteed that.

So what we have to do when we're setting up an API 20E, a biochemical test strip, is pick off one colony and make an emulsion of it in sterile distilled water. this is 10 mls of sterile distilled water. So I'm going to do that now and turn the Bunsen from the safe setting to the hot type, and it's always useful to have two needles because if you sterilise one by the time you have sterilised the second one the first one is in cool and safe to use. So I'm going to pick off just one colony off this plate and then I'm guaranteed that it's a pure culture. As I say you can't do an identification on a mixture. Take the top off, flame it, shake off the colony into the 10 mils of sterile water, flame again re sterilise the loop. Some people say you should sterilise the loop like that – so you don't burn your hands, but I've always done that for years and I've never burnt my hands yet. But, that's slightly safer. Okay so now I've got an emulsion of one type of bacterium, this is an unknown we don't know what it is. I've just called it ‘One’ It’s completely unknown. I've got a second one there too which we would obviously make that up with a separate bottle. And now I'm going to inoculate an API strip.

Well I'm now going to set up an API strip. They usually come in bags like this. And now for the first time you can see an API – 20E that is – as you can see it's got 20 compartments. And in here, are dehydrated biochemical reagents and the bacteria will react with them and give different colours, different reactions, and that will help us very much. It will give us a complete idea. So it's a wonderful system really, because it will do a full ID, in other words species, to the species level.

So I put that there, prop it up. I then need a Pasteur pipette, this is our suspension, pure suspension of the unknown bacterium number one in this case. And you just fill up most of these compartments, just to the top here, but a few there's an exception where you you fill it right up to the brim. So this is OMPG which is a test for beta-galactosidase, it's really a sort of an analogue of lactose, and it changes colour. Arginine dehydrogenase, lysine decarboxylase, ornithine decarboxylase. Now this next one has a box under it and that means that you have to fill the compartment right up to the very top which gives a bit more surface area for oxygen. This is a citrate test and it's a utilisation test to see whether the bacteria can use citrate as their only carbon source. And then we've got hydrogen sulphide production… and now I'm starting to run out of liquid, so I pick up some more. So we got up to hydrogen sulphide… this is the urease testing for the enzyme urease, tryptophan deaminase, the indole test which tests for the production of a substance called indole, the Voges-Proskauer test which tests for another metabolite, and that one again has a box under it so we fill it right up to the top. And then digestion of gelatin. Most bacteria can't digest gelatin, neither can we, but some can. And that's another one that you fill right up to top. Now the rest of the strip to the right here, is sort of, well not sort of, its carbohydrates and related compounds. A very important one, the first one is glucose, because if the bugs don't break down glucose then they're not Enterobacteriaciae, so you're probably using the wrong bacterium with the wrong strip. So that was glucose, and then some other carbohydrates and related things – mannitol, inositol, sorbitol rhamnose, sucrose, melibiose, amygdalin and lastly arabinose – and then I'm finished with that. Okay.

Now there's a couple of other things we still have to do. Some of the compartments have a line under them, a box meant that you fill it fill it up to top, but the ones with the line you have to add oil, sterile oil, which I've got some here. And that's because sometimes they'll produce ammonia or hydrogen sulphide which will affect the results of the other compartments. So that effectively you’re screening it off and making it sort of semi-anaerobic. So anything with a box underneath you put a drop of sterile oil into. So that's the amino acid ones also the hydrogen sulphide compartment and the urease test as well.

And, we’ve still got one other thing to do. This is a special tray to put the strip into but before we close it up, we put a bit of water into it just to stop the strip dehydrating. And then pop the strip in, and put the lid on. And very importantly, we must mark number one, culture number one and the date, and usually you'd put your initials on. Now that strip then goes in the 37ºC incubator for 18 to 24 hours, and then at the end of that time you take it out you can read some of the colours they will have changed hopefully, and but you also have to add some reagents. So that will be the next stage.