Culturing Bacteria in the Clinical Lab

Pure Culture

Obtaining a pure culture is mandatory to establish the identity and susceptibility.  Everybody knows what a pure culture is, right? It’s a culture consisting of a single type of microorganism derived from a single cell. When the specimen arrives in the lab, you have to isolate the bacterias, then take your inoculum and you put it on a sterile.  If there’s multiple bacteria, like e.coli and staphylococcus, you have to get separate cultures of each before doing a pure culture.  Only then can you do the C&S report.

Let’s say someone goes to the doctor because they have a sore throat.  The doctor takes a specimen.  There’s several types of bacteria it could be (E. coli, S. aureus, pseudomonas, etc). For it to be identified, you can’t do the test with all three present. The bacteria have characteristics that are unique to themselves.  So we give them what they want (food) to make them happy.  We put them in selective media to do that.

The Mannitol Salt Agar (MSA) favors the growth of S. aureus because the salt inhibits most bacteria.  Remember S. aureus is salt tolerant and grows up to 10% salt concentrations but minimally at 15%.  It then ferments mannitol salt which ferments large yellow colonies, so you could ensure that’s s. aureus. So that selective media works only with specific types of bacteria.

The Eosin Methylene Blue (EMB) favors the growth of fecal coliforms such as E. coli.  In EMB it becomes a blue/black color.

The MacConkey agar is used for growing gram negative bacteria because it inhibits gram positive bacteria. In MacConkey’s, the agar changes to a pink color.  If you ever mix gram + and gram – and you put that mix onto MacConkey agar, which bacteria are you going to get growing?  Only the gram negative version.

Candle jars must be provided for microaerophiles.  Microaerophilic bacteria require oxygen for growth but at lower concentration than is present in the atmosphere.

The point of this selective media is that the bacteria don’t grow if the medium and incubation conditions are inappropriate.

Side note: When you have an organism that’s difficult to grow, you use a DNA probe test.  For example, chlamydia is very difficult to grow, so they will just use DNA.  In fact in UCLA and other labs, the screening for gonorrhea is based on genomics.  You can’t do a pure culture when you have a 1,000 people coming in to a hospital everyday.

Sterilization (Autoclaving)

Sterilization usually involves autoclaving which involves heating (121°C/250°F) under high pressure (15psi) in an autoclave.  An autoclave is like a big pressure cooker. This allows things to reach higher temperatures than otherwise would be possible because it forces a higher vapor pressure to build up so water will boil at a higher temperature to kill things (conversely, water boils at lower temperatures at high elevations where the air pressure, aka the vapor pressure, is lower. If you’re camping up at 12k ft elevation, you can’t even get the water to be hot enough to cook food when it boils because it will boil at 70 degrees! ::the more you know star across the screen goes here::).

You can’t use autoclaving on heat sensitive materials such as proteins. You therefore use filtration to sterilize them. There are other methods which we’ll look at but for our purposes right now, the most common process is the autoclave.

Isolation techniques

The most common procedure used for pure culture isolation is the quadrant streak isolation. When you have cross contamination and multiple appearing colonies, that’s not a pure culture.

You need to get a loop of the specimen, streak it on one section, flame and cool, turn it 45 degrees and streak a second time, overlapping the first streak.  Then repeat it a third and fourth time.  If done properly, you’re going to get isolated regions in the third region typically and if there were three different organisms, you’ll get separate colonies or all three.  Then you could take each of these to a separate plate and then you do a streak and then you get pure culture on a separate plate.

Why does the flaming and cooling separate them?  It dilutes the cultures over the plate.  If you don’t flame/cool you’ll never get isolation.  You have the organisms together and you separate them, so you spread them out.

Pour plate and spread plate methods are not used for isolation and pure culture clinically.

Other procedures are tedious and counter productive.  When you do the pour plate, it’s used for literally counting the bacteria in a clinical specimen.  That would be impossible because you need a gigantic room just to do one organism.  You would put it on different media and multiply the number of bacteria on the plate, by the dilution.  The only results that are countable are the ones where you could count the dilution with 30-300 CFU/mL.  If you have to do this for every single specimen, it would be counterproductive because you MAY get isolated colonies.  The pour plate and the spread plate are more used to count the number of specimen in juice, water, milk, anything, etc.  The only time you use that to diagnose for a clinical reason is for UTI.  How is UTI defined?  The isolation of a >100,000 (10^5) CFU/mL.  We learned this during the case history chart analysis.