do a lab summary of minimum 300 words on strak plate method.

How to streak a plate for isolation - the streak plate method

What is a pure culture? How are pure cultures isolated from mixed cultures? Apply a sample containing many cells to the semi-solid agar surface of a Petri dish (plate). Assuming the medium will support the growth of the microbes in this sample, each cell will begin to divide. Eventually, a colony will arise from each original cell that was placed on the agar surface. We can therefore refer to each of these original cells as a “colony forming unit” or CFU. If we “streak” the sample onto the agar surface in a manner that spreads the CFU’s out, then the colonies that arise will also be spread out. This is to say that the colonies are isolated, a word that can be defined 2 ways:

1) an isolated colony is one that is not touching another colony

2) an isolated colony is one that contains only one species ofmicrobe

Colonies of different species of microorganisms present in the mixed culture can often be distinguished by subtle differences in their appearance, an idea referred to as colony morphology. This will be discussed at length in another lab exercise. Imagine that upon examining the plate you see isolated colonies of 3 different colony types. Using aseptic technique, touch a loop to an isolated colony of one type and streak these cells onto another plate. Do this for all 3 colony types and incubate the plates. You have just generated 3 pure cultures of microorganisms from a mixed culture. There are 3 reasons for attempting to isolate the cells in a sample:

1) generation of a pure culture from a mixed culture

2) determination of colonial morphology

3) enumeration of viable (culturable) cell number.

If we wanted to determine the number of viable cells in a sample, we could apply the sample to a plate of media, incubate the plate, and count the number of resulting colonies. Obviously, this would only work if the colonies were isolated. Confluent VS TNTC: If the colonies all grew together on the plate, we would not be able to tell if there were 1000 small colonies present or only one large colony. In this case, we would say that the colonies on the plate were confluent, and no estimation of cell number in the sample could be made. SEE IMAGE: Confluent. You may want to place a smaller sample on the plate in this case, or dilute the sample before plating. If the colonies are isolated on the plate but there are simply too many to count, we would say that the colonies were “too numerous to count” or TNTC. On a standard Petri dish, a colony number greater than 300 is considered TNTC. Again, one would want to plate a smaller sample or dilute the sample before plating.

Streak plate procedure

EVERY TIME that you streak a plate you should streak for isolation, unless you are specifically instructed otherwise. You may streak a plate from any type of sample or culture (agar surface, broth, or virtually any type of clinical or environmental sample). Follow the rules of aseptic technique at all steps. First of all, inspect your plate. Moisture on the underside of the lid is OK. Water standing on the agar is not. Get another plate. Next, inspect your loop to make sure the wire is straight and the loop is complete and flat with no sharp edges that will cut the agar surface of the plate. Label the bottom of the plate with your name, the media type, the organism name or sample type, and your lab section meeting date and time. Also, until you get the hang of this, use your sharpie to section off quadrants on the bottom of the plate as illustrated in the figure two pages ahead. Now, hold your loop in your writing hand as you would hold a pencil, then roll your hand over ¼ turn so that your thumb is on top. SEE IMAGES: Streak plate procedure 1 & 2. Flame your loop and aseptically collect a sample of cells. Pick up and hold the plate as demonstrated. Unless you have very small hands, you should be able to hold and manipulate the plate with one hand as instructed. If not, lay the plate on the bench. Follow the steps of streaking a plate for isolation using the 3 quadrant method as demonstrated. Refer to the figure on the next page.

IMAGE: Streak plate procedure: 3 quadrants

IMAGE: Streak plate procedure - isolation is good!

Things to remember when steaking plates

Flame your loop between quadrants and always cool your loop after flaming by sticking it in the agar on the edge of a plate.
The loop should travel as flatly as possible over the agar surface.
BE GENTLE - do not cut the agar with your loop if you can help it.
Remember, you are trying to isolate cells on the agar surface. Fight the tendency to drag too many cells from quadrant to quadrant.
Spread the cells out as much as possible by trying to cover the entire surface of each quadrant with your loop. I will show you how to do this.

Incubating plates

Always incubate plates in an inverted position in the incubator.
Multiple plates should be stacked then taped together. It is handy to write your information on the tape as well as on the plate. This makes it easy to find your stack of plates in the incubator.

NOTES

* As agar dries up it will become thin and wrinkled. Organisms need water. Get a better plate.

* Wet agar is no good. Cells will “swim” on the surface of the agar rather than forming colonies.

* Good flat, smooth loops make for good streak plates. You want to avoid cutting your agar if possible.

* Your loop should lay flat on the agar otherwise the edge of the loop will cut like a knife.

* You can usually see “tracks” on the plate made by the loop during the streaking process. View these

to see how you are doing as you learn how to operate this new machinery.

* Avoid making“race tracks”and “figures 8s”as you streak your plate. Remember that it is all in the

fingers, not in the wrist or the arm. You must hold your plate and your loop correctly to streak a good

plate.

* Two reasons why you may not have any growth on a plate: a) there are no cells on the plate or b) the

cells are dead. The latter could be because you cooked them with a hot loop.

* Of course I want for you to do everything perfectly, but the truth is, it is hard to streak a plate so poorly

that it will not serve its purposes.