1. Of course the first thing you must do is figure out which types of bacteria you will be using. Most of the time you will be using the bacteria as a tool for the study of some basic biological function. The best selection would (a) not pose any threat to health or the environment, and (b) exhibit the function that you wish to test in an easily detectible way. Of the Gram-negative bacteria, usually E. coli is the organism of choice. Of Gram-positive bacteria, usually Bacillus subtilis or one of the types found in cheese are used. (Grammar: in English: one bacterium and three bacteria; en español: una bacteria; tres bacterias.)

   If you have ordered bacteria from a commercial source, it will arrive and you must then handle it properly, or else you will mess it up and have wasted your time and money. Click this for instructions, which MUST be done!

   Once you have selected and subcultured your test organisms, you can then select the growth medium. This page will call for the use of "solid" media in petri plates, but you may wish to use liquid media that do not contain agar. (Grammar: in English: one medium and five media; en español: una media; cinco medias.) While commercially prepared dehydrate media are a great convenience, do not feel that you cannot work without it. You can make it yourself - and even design your own petri plates. Click this if you need to really start from the very beginning by making your own petri plates and media "from scratch."

   There are a number of agar media that are of general purpose on which most Gram(+) and Gram(-) bacteria will grow, and they can be bought from supply companies as dried powders. Nutrient agar (NA), tryptic soya agar (TSA) are two of the most common types. In essence they are little more than beef or chicken consumé to which agar powder has been added. Most of these powders are extremely hygroscopic (dictionary, anyone?), and so it is VERY important that you both tightly cap the bottles between uses and clean up any spills. Very few media contain components that will deleteriously affect your health. Such additives might be antibiotics or metals such as selenium. So read the labels!

   Two very forgiving media for growing E. coli are EMB agar and MacConkey agar. This web page will recommend MacConkey agar (MA) because the dyes in it are not as potent stains of clothing or lab furniture as is the crystal violet in EMB agar.

   Now for a little about MA: Fermentation is metabolism in the absence of oxygen.Yeast is an alcoholic fermenter of sugars (it converts sugar to ethanol in the absence of oxygen). E. coli is a "mixed acid fermenter of sugars." That means that E. coli will convert sugars to a wide variety of organic acids in the absence of oxygen (anaerobic growth). Those acids are acetic, formic, succinic, etc., etc. Many other Gram(-) bacteria are mixed acid fermenters - BUT they do now make formic acid (HCOOH), which is a rather strong organic acid.

   So Dr. MacConkey, in the early 1900's, concocted his medium so that it contained the sugar lactose, which few other Gram(-) bacteria can use, a pH indicator that turned red under acidic conditions (left side of the plate), a lot of phosphate buffer that had to be overcome by all the "mixed acids" including the formic acid in order for the pH to drop into the "red" zone of the indicator, and a lot of nitrogenous material (amino acids, for example) that could sustain the growth of non-E. coli, if they happened to be present. But those non-E. coli forms would ferment the amino acids into cell mass and NH₃, which would make the pH go basic rather than acidic. Thus the color would not be red (right side of plate). One final thing the inventor thought of was that the dye, when acidic must be insoluble so that it stays right there wherever the bacteria happen to be growing. E. coli would make red colonies, while other Gram(-) bacteria would not be red and actually be a very light pink. (MA usually comes with lactose in it so that you can see that E. coli ferments lactose to make red. However, you can also buy "MA without lactose", and then add your own types of sugar to it to test whether they can use those sugars and thus detect what kinds of sugar mutations might be incorporated into those strains that you have.

   THUS you will be using both NA and MA.
   

2. You next need to hydrate these media. First you should determine how many petri plates you will be needing of the various agar media. Roughly you will need about 30 ml per each plate. Now look at the labels on the bottles you have in front of you. They will tell you how much of the powder you must add make a liter of medium. It is assumed that you have the mathematical knowhow to determine how much per 100 ml.

   Now comes one of the tricky parts to all of this - keeping messes under control. If you try to shake these media out directly from their bottles it will go fluff-woof and lots will go all over the balance, and up into the air. If it is MA, your nose will soon be dripping red. So USE A SPOON to laddle out what you need. Of course, you have set up a wide-mouth powder funnel in a receiving bottle or flask that is about 2 to 3 times larger in volume than you will put into it. Add the requisite amount of water, tightly cap the container and shake it so that ALL of the powder become suspended in the water. Loosen screw caps a little, and REMOVE any rubber stoppers. For flasks, a double layer of aluminum foil over the openings is good. Make sure that the foil apron hangs down a couple of inches all around the top.

   Clean up your work area of any of the hygroscopic powder that has gotten "loose." Tightly recap the supply bottles of powder. (Disposable plastic picnic spoons or knives make good tools for the transfer of powder.)

   At this point you MUST go to the next step. Hydrated, but unsterilized medium, becomes unusable in three hours as any spurious microbes in the powder begin growing and "use up" the nutrients in it. Once sterilize, it can usually sit for weeks or months. It has been "canned."

3. Let us consider MA first. Another property of MA is that Gram(+) bacteria cannot usually grow in it. It also contains a detergent and Gram(+) bacteria don't like detergents. Another thing about Gram(+) bacteria is that in their realm exist almost all of the spore formers. Bacterial spores are often able to withstand boiling - that's why the NA must be sterilized. Anyway, among the Gram(-) bacteria few can tolerate even 80c let alone boiling. Thus, if you are in a hurry you need merely bring MA to a boil with lots of swirling to keep the particulate agar from burning on the bottom of the contain, and then pour it into well dried petri plates. No stray Gram(-) bacteria can be there, and no stray Gram(+) bacteria will be able to grow anyway. For more on MacConkey agar, and especially about the quick, non-sterilizing way to handle it, click this (Agar is an anionic polysaccharide isolated from kelp (a type of huge marine algae. Agar powder becomes soluble in water over 98c. So you must heat NA and MA to boiling to get the particulate agar to go into solution. Then, upon cooling, it will not gel until the temperature is about 45c. Probably nowhere else in life will you ever come upon something that has a melting point that is different from its freezing point!)

   Now let's turn to NA (and MA for those who have the time). Your particulate suspensions of NA and MA in loosely closed containers are set into an autoclave (an automatic sort of pressure cooker) or into your home pressure cooker, and the heat is turned on. (Click this for instructions on using a home pressure cooker.)

   Once the pressure gets to 15 psi (1 atm), you start timing. Twenty minutes is usually long enough to sterilize almost anything. "Sterilize" means to kill every single living thing within that container. You cannot tolerate even one single cell from surviving! Why? Once the 20 minutes has elapsed, turn off the heat and just let the pressure cooker cool by itself. (DO NOT REMOVE THE WEIGHT ON TOP unless you want a medium guyser and sticky stuff all over your kitchen ceiling!) If you are using an autoclave, just make sure that at the beginning you set it for "liquids" or "slow exhaust"

   Once the pressure is down to ambient (dictionary, anyone?), you may remove the weight and open the sterilizer. Be cautious about lifting the weight. It is hot, and if the pressure is not down, it will hiss if you lift it a millimeter. It if does hiss, let the weight remain and wait awhile longer.

4. The first thing you must do, is swirl each agar container. The particulate agar will have settled to the bottoms of the bottles and flasks. It MUST be swirled up into solution if you have hopes that your petri plates will properly gel. Not swirling is one of the most common errors made by newcomers to this trade! Do not shake, but swirl. Shaking may result in a superheated liquid's suddenly becoming a sprinkler of boiling water.

   Your plates are arrayed out on a freshly damp-wiped countertop. Their covers are all on.

   Do all of the following steps in rapid order. DO NOT TALK (spit splatter spores).

   With one mitted hand hold the agar container. Remove the cap or foil. Immediately lift the cover of the first plate and pour the hot solution in so that the plate becomes about 2/3 full. Replace the plate's top. Go the the next plate and do the same - always opening only one plate at a time. You do not want any environmental spores to blow in to contaminate your plates.
   

   If you have a little extra medium you don't need, dump it into a plastic bag lined waste basket. It will solidify in the trash.

   Next rinse your contain with hot water. It's easier to to this than wait until the agar has solidified forming a solid film that is harder to remove during cleaning.

   Turn your attention back to your plates. Perhaps during pouring, bubbles or foam has collected on the molten surfaces of the agar. Doing one plate at a time and replacing the covers, waft a flame across the agar surfaces and the bubbles will disappear. It is best to set your desktop burner so that it has a blow-torch type of flame, which is a good "wafter." (A plumber's torch works very well also.) BUT if you waft too slowly, you will melt the rims of your plastic petri plates, just that when you replace their covers, they will weld shut for good! In other words, waft your big flame quickly!

   The plates can now stand as is for days (preferably in the refrigerator inside of a ziplok plastic bag. Don't allow ants, roaches or younger siblings don't get into them. Do not go to the next step unless you are within 2 hrs of using the plates.

5. About an hour or two before you are going to use the plates, you should start removing the condensation that has collected on the surfaces of the agar and on the inside of the covers. Click this for the method for doing so.
6. There are a number of ways to inoculate a plate. What will be done here is the simple placement of a swath of bacteria onto the surface of the agar. This can be done with a sterile swab or with an inoculation loop. Click this for the method for doing so with either a swab or inoculating loop.
7. While it may seem simple to just put your plates in a place that is warm, there is an art to incubation. You don't want the bacteria to be overgrown by the time you next visit them. That would happen if you place them in too warm a place. And you don't want them to grow so slowly that you see nothing when you need look at them. That would happen if the place were too cool. Just like Goldie Locks, you want the temperature to be just right. Thus, if you want to look at E. coli colonies tomorrow, you should try to find a place that is between 35c and 40c (90-100F). But if you want to look at them after a weekend, you should go for something cooler. Only with experience will you find the "right" temperature for you. (This laxity in temperature shocks medical microbiologists who have a notion that 37c is THE temperature because that is the body temperature of humans and other warmblooded animals. Alas, they don't know about the normal body temperatures of other animals, which range from 30c (sloths) to 43c (canaries).)
8. E. coli colonies should appear red on MA that has sugar in it, or pale pink if there is no type of sugar in it. If you have a MA + sugar plate that has both red and pinkish colonies, the pinkish ones are probably contaminating Gram(-) bacteria that wafted in from the window because of some improper and non-aseptic technique you did.

   On NA the colonies should appear creamy and gelatinous.

9. You school system probably has a prescribed way to get rid of bacterial cultures, and if you are in school and disposing of them, you MUST follow there rules. However, if you at home, you might do something much different. What is described here is for use ONLY if you know the bacteria are harmless ones: Use a stick to dislodge the agar from inside of the plate and flip it into the toilet (imagine what else goes down the toilet - a lot of fecal bacteria of questionable safety!). You can either toss the empty, dirty plates into the garbage (much less dangerous than tossed diapers!), or clean them out with water, dry them and use them under flower pots or as small frizzbees!

ADDING MORE COMPONENTS TO THE MEDIA

Frequently you will need to add other components to the media. If, for example, you are trying to osmotically stress the bacteria, you will want to add something that is non-toxic, non-ionic and very soluble to the medium. Two such things come to mind: sucrose and polyvinyl alcohol. Thus if you wish to grow your bacteria in plates that have differing osmolarities of sucrose, you will need to make several small containers containing just the right amount of NA powder, the requisite amount of water, PLUS varying amounts of sucrose. At this time it would be good if you knew the terminology of solutions.

There are a number of ways to make a series of different concentrations. Not only are there linear relationships between the concentrations (1, 2, 3, 4, 5,...), but also exponential ones (1, 2, 4, 8, 16, ...). And there are lab tricks for making each of these with the minimum of work and mess. It is suggested that you correspond with the author of this website to discuss your situation and which kind of series would be best for you, and how to make what you need.

USING THE BACTERIA FOR BIOCHEMICAL TESTS

Using the bacteria from agar surfaces has tricks of its own versus using cells from liquid media. Generally you are no longer concerned with aseptic technique by this time. Thus you can use flat toothpicks to scrap up globs of bacteria from plates. Try to have all your globs about the same size (thus the biomasses would be about equal). These cells can then be swizzled and mixed into buffers and become ready for the biochemical tests such as enzyme assays. One such classic assay is that for lactase (β-galactosidase) and uses the chemical analog of lactose called "ONPG" (ortho-nitro-phenyl-β-D-galactopyranoside). This ONPG test is described elsewhere. For those of you who may be growing E.coli on various NA plates, scooping the bacteria off, and then assaying them for ONPG reaction rates, you should be able to adapt this flow diagram to your purposes. Of course, you might not be using gal⁺ and gal^- strains of E.coli, but rather you might be using a lac⁺ strain (which is the way it usually exists) grown under a variety of conditions. For example, cells grown under the "control" (normal) conditions in lactose, might be well-formed cells with a low level of yellow ONPG reactivity (shown at the left of the title at the top of this page), while stressed cells might be shrivelled and have a such a high level of ONPG reactivity that the yellow is so intense it looks orange (shown at the right of the title at the top of this page).

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