Handling Newly Arrived Cultures

Packet of Swabs Handling of Newly Arrived Bacterial Cultures
Flaming an
inoculation loop*

A new arrival appears in your mailbox: a culture of bacteria, which you need to use in your experiments. What do you do with it? Foolish people immediately open the tube, sniff it, and start smearing it on their petri plates. Smart people do it much differently because they recognize that mistakes might be made and they will have to go back to the culture in a retrial. They don't want to go back to a culture that has gotten moldy from not using correct microbiological procedures. And the smart researcher also knows that the culture might suffer accidental breakage - fumbling and dropping it - during repeated use.

Supplies you must have available:

Flame source (either a lab burner or a plumber's torch)
Packets of sterile swabs OR an inoculating loop (see picture)
For each new culture, you should have three uninoculated slants or stabs (for instructions on how to make them, click the picture to the right)
Marking pen to write on the tubes

So what is proper procedure for newly arrived cultures? What follows pertains only to cultures that pose no threat to health or the environment. (Click this link for information on harmless microbes.) What you do with the new arrivals depends on the form in which the culture arrived. Choose from the following to learn how to handle your new culture:

on an agar surface (petri plate [rarely], slant [most common],
as a dry powder or pellet sealed within a glass ampuole, or
on a glycerol moistened piece of blotting paper sealed in plastic film
this form is very convenient to mail as the disks of paper can be taped to a card and slipped into an envelope)

The New Arrival is a Slant or Stab

When you get the culture in a slant, you should do the following steps. BUT do several dry runs before you do it for real! The whole process should become nearly automatic and take about 15 seconds. Practice until you can do it in that time. (This is a bacteriologist's skill that is absolutely indispensible!)

Loosen, but do not remove, the cap of the tube.
Turning now to the instrument you will use to transfer the bacteria:
1. For a inoculating loop:
  Heat the entire length of the wire portion to red hot, and then pass part of the handle through the flame to burn off any dust. In any and all cases, the entire length that will go into the tube must be flamed.
2. For a sterile swab:
  Peel back the paper on the handle end of the packet revealing the handle end of the swab. Do not touch the inner surfaces of the pealed back paper. If there are more than one swab in the packet, separate their handles as far as possible from each other. Pull a swab out of the pocket and hold it only by the END of its handle.
The swab or loop's hand's pinkie (little finger) then encircles the loosened tube cap. You then rotate the tube and unscrew it from the cap. Continue to hold the cap in your pinkie! Always hold the tube horizontally so that dust (germs!) won't fall into the tube.
Waft the threaded end of the tube through a flame to burn off any dust and germs. Do NOT roast the end of the tube - just "waft"!
Insertion of your transfer instrument
1. Loop: Insert the wire end and be sure that it is cool by stabbing it into the agar at the bottom of the tube. Then touch the circular loop to the streak of bacterial growth. You do not need to get great big globs of it. Just mere touching will bring up millions of bacteria. Remove the loop from the tube trying not to touch the insides of the end of the tube (that is where contamination is most likely to be if there is any at all).
2. Swab: insert the swab and twirl it on the streak of bacterial growth. Withdraw the swab trying not to touch the inside of the end of the tube (that is where contamination is most likely to be if there is any at all).
Waft the screw end of the tube in the flame again, and screw it into the cap you have been holding in your pinkie. (The pinkie thing precludes the cap from ever being turned uupside down so that dust won't fall into it. Besides, it allows just one person to do all the steps.)
Since the above process works for removing bacteria from any slant (or "stab"; see figures on this page), we shall now continue by considering only those times for a new arrival
THE NEXT THING you MUST do is make two or three replicate slants or stabs of the culture: do all the pertinent of the above steps with freshly made slants as your transfer the bacteria from your loop or swab to these new tubes. When they grow up, they will be your duplicates AND should be your "working" cultures. These are commonly called "subcultures". Save the original in a dark place (a drawer). Why dark? Even though glass is UV opaque, it is not totally opaque and slowly the UV that comes in from outside windows and from fluorescent lights will kill the bacteria.
You may then use the same loop or swab of bacteria to apply them to petri plates or liquid cultures.
Use the marking pen to label your newly inoculated subcultures and your plates, etc., with the identity of the bacterial strain used.

The New Arrival is an Ampuole Containing Powder or a Pellet

You should have ready a cooled flask of sterile growth medium appropriate for the organism that you have ordered.

Have at the ready a sterilized ampuole file and a beaker of alcohol. While alcohol will not kill everything, it is a good start.
Wearing plastic gloves, dip your fingers into the alcohol to semi-sterilize the outer surfaces of both the gloves.
Allow the alcohol to evaporate and don't touch anything in the meantime.
Using tongs, dip the ampuole into the alcohol and remove and place on a sterile surface.
File the crack line on the ampuole
Hold the top and bottom of the ampuole with sterilized paper towels and crack open the ampuole
Immediate dump its contents into the sterile liquid growth medium.
Once turbidity has appeared, make some streak plates of it.
Once colonies have grown on the streak plate, isolate and test the colonies that grow up for the proper characteristics. Usually, almost all colonies have the same morphologies (look alike), and that is a good sign that you will easily isolate what you hope for. If, however, you get a variety of colonies, then you must test each until you find the one(s) with the proper characteristics.
Make subcultures in slants of the proper isolate. (As always, it is good to test the clone for having the desired characteristics).

The New Arrival is a Disk of Moist Blotting Paper

Non-breakable cultures are often sent through the mail as small Saran-wrapped disks of bacteria that have been absorbed in dilute glycerol into blotting paper.

Have a petri plate with the proper agar growth medium in it.
Momentarily dip the small packet into alcohol to semisterilize it.
Use a sterile sissors to snip open one side of the small, flat packet.
Use a small, sterile tweezers to extract the disk.
Place the disk onto the agar in the petri plate. It is best to place the disk near the edge of the agar.
After 2 or three hours, slip a flame-sterilized inoculating loop between the disk and the agar surface and then make a streak plate of it.
Usually all colonies look alike, and you can make subcultures of one of the colonies. (As always, it is good to test the clone for having the desired characteristics).

How to make slants and stabs:

In a 400 ml beaker, make about 100 ml of Nutrient agar (but use only about half of the powder that the instructions on the label call for. Heat this until it boils (microwaving is the preferred method but you must watch it closely as it will sudden burst into foam). If heating it in a pan on a hotplate, swirl constantly while heating (pretend it is gravy, which you don't want to burn). It seems best to then pour the stuff from the pan into the beaker, and then pour from that into screw-topped culture tubes - fill the tubes about 1/4 full. Put on the caps almost tight and then autoclave to sterilize.
Slants: Take tubes immediately out of the autoclave and find the right place to set them on a rather flat incline so that the proper 'slant' is achieved for them to solidify. Once cool and solid, screw caps on tightly to prevent evaporation. Longevity: cultures in slants last for several years if they are stored in the dark and do not dry out.
Stabs: fill tubes 1/3 full, cap and sterilize. Stand them vertically to solidify. When cool, screw caps on tightly. To inoculate these, stab an inoculated loop down into the agar for about 2/3 way to bottom. Stabs preserve most bacteria for years and years in the dark if the caps are airtight and evaporation doesn't occur.
Always store cultures in the dark as UV damage accrues from fluorescent lights and sunlight.

* Figure used by permission of Morton Publishing Company.

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