You’ll need the following items to complete this lab session. (The standard kit for this book, available from www.thehomescientist.com, includes the items listed in the first group.)

All living things require a suitable “life support” environment if they are to grow and flourish. Just as people need air, water, food, and protection from temperature extremes, bacteria and other microorganisms grow only in environments that supply their essential requirements.

The process of encouraging growth of particular organisms by providing conditions optimal for those organisms is called culturing. For example, growing plants and livestock is called agriculture. Fish farming is pisciculture, a form of aquaculture. In each case, human activity is directed at modifying the environment to suit the needs of the organism.

Culturing microorganisms is no different. We provide everything the microorganism needs to grow and flourish, including nutrients, suitable temperatures and other environmental factors, and a place to grow undisturbed and unthreatened. That place to grow is a gel or a liquid, and is called a culturing medium.

Gels are usually based on agar, which when dissolved in hot water and allowed to cool forms a semisolid gel much like dessert gelatin. (Gelatin is seldom used for general bacterial culturing because many bacteria species eat gelatin, turning it into a runny mess. With the minor exceptions of a few marine bacteria, no common microorganisms eat agar.)

Gel media have the advantage of providing a firm, flat surface for bacterial growth. If a mixture of two or more bacteria species is placed on the gel (a process called inoculation), those species form separate and distinct colonies, the form, color, and other characteristics of which are useful in identifying the particular species, or at least narrowing down the possibilities. Also, because colonies each contain only one species of bacteria, it’s easy to obtain a specimen of that single species and use it to inoculate another culturing vessel, thereby producing a pure culture of that single species.

Liquid culturing media do not separate growing bacteria into colonies. If multiple bacteria species are introduced into a liquid culturing medium, they grow willy-nilly throughout the medium. But liquid media have the advantage of allowing bacteria to grow much faster than gel media, because bacteria grow throughout the entire volume of the liquid, rather than being limited to just the surface of a gel. For that reason, liquid culturing media are used almost exclusively for producing pure cultures.

The primary characteristic of a culturing medium is the mix of nutrients included. For bacteria, which are what we’ll be culturing, a typical nutrient mixture contains some form of carbohydrate (usually a saccharide such as glucose or sucrose, but sometimes glycerol or another carbon source) and nitrogen in the form of proteins or inorganic nitrates. Other components may be added to encourage or discourage the growth of particular classes (or even species) of bacteria.

Such media are called selective media, because they favor the growth of one type or species of bacteria at the expense of others. For example, eosin-methylene-blue (EMB) agar contains methylene blue, which is toxic to most Gram-positive bacteria and therefore favors the growth of only Gram-negative bacteria. Conversely, mannitol-sugar agar (MSA) favors the growth of Gram-positive bacteria.

Some media, called differential media, provide a visual indication of the types of bacteria growing on the medium, usually by including a dye that changes color when exposed to the waste products of particular types of bacteria. For example, McConkey (MCK) agar is differential for lactose fermentation. It includes the dye neutral red, which is yellow at pH 8.0 or higher and red at pH 6.8 or lower. When it is prepared, McConkey agar is slightly basic, and therefore has a pale yellow color. If McConkey agar is inoculated with lactose-fermenting bacteria, as those bacteria grow and produce acidic waste products, the agar turns red. If the bacteria growing on the McConkey agar are not lactose fermenters, the agar remains a pale yellow color.

Another important distinction in culturing media is whether the identities and quantities of nutrients are known:

The most important advantage of defined media is that they can be tailored to provide optimal growing conditions for specific bacteria or other microorganisms. The most important disadvantage of defined media is that they cannot be used for most microorganisms because those microorganisms require resources that cannot be provided by defined media. (For example, viruses can be cultured only in the presence of the cells that host them.) Even most bacteria cannot be cultured on defined media, simply because we don’t know all of their requirements. Conversely, most microorganisms can be cultured on undefined media, if they can be cultured at all, but at the cost of not knowing exactly what we’re giving them to eat.

Culturing media are used in vessels that protect the growing cultures from contamination. Liquid media are used in test tubes (culture tubes) or flasks, which are simply filled, stoppered (usually with sterile cotton balls, but sometimes with solid rubber stoppers or screw-on caps), and sterilized. The most common vessels for gel media are the Petri dish (also called a Petri plate) and the slant tube.

Autoclaving is an important preparation step, because it kills every living thing in the culturing vessel. You might think that boiling the medium during preparation would accomplish that, but it doesn’t. The problem is that the boiling point of water—100 °C at standard conditions—is sufficient to kill only bacteria, protozoa, viruses, and fungi. So what’s left? Spores, which some microorganisms form when exposed to adverse conditions, such as high temperatures or extreme dryness.

Spores are not killed quickly at 100 °C, a temperature they can survive for anything from several hours to days or more. Fortunately, temperatures just a bit higher are sufficient to kill them quickly, which is where autoclaving comes in. In an autoclave (or pressure cooker), water is boiled under pressure higher than atmospheric pressure, which increases the boiling point. In a standard pressure cooker, water boils at 121 °C, which is sufficient to kill spores after only 15 minutes’ exposure, called the 15-15 rule.

In fact, this “rule” is not a rule at all, because the actual time needed for sterilization varies with the size of the container being sterilized, its composition, the degree of contamination of the material being sterilized, and other factors. For example, a 250 mL flask takes much longer to sterilize than a test tube because it takes longer for the liquid in the flask to reach the maximum temperature in the pressure cooker. Similarly, a metal tube sterilizes faster than a glass tube because the metal conducts the heat faster, and a polypropylene tube takes longer still because the plastic insulates the liquid inside the vessel. In short, there is no hard and fast rule about how long sterilization requires.

You might think the easy answer would be simply to autoclave or pressure-cook culturing vessels for a long time to ensure they’re in fact sterile. The problem with that is that heating the medium too long may destroy nutrients. So, we shoot for a happy medium, so to speak, by autoclaving long enough to kill all or nearly all of the spores present while not cooking the nutrients to death.

We’ve written this lab session on the assumption that you have (or can borrow) a pressure cooker. Even an inexpensive model from Walmart is fine. Although ordinarily we strictly segregate lab equipment from food-preparation equipment, there is, by definition, no risk to using a pressure cooker both for ordinary cooking and for sterilizing media. The pressure cooker kills anything present inside it, whether you’re pressure-cooking a roast or a bunch of culture tubes.

With a pressure cooker, there’s no need to follow aseptic procedures while making up the media, filling glass culturing vessels, and so on, because sterilization is the final step. If you don’t have a pressure cooker, or if you’re using plastic culturing vessels that cannot be autoclaved (as we are in this lab session), aseptic procedure becomes much more important.

Use the following guidelines to keep your culturing media as uncontaminated as possible:

If you do not have a pressure cooker to sterilize the culture media, culture plates and tubes should be used as soon as possible after you make them up, and certainly within 24 hours. If you use a pressure cooker, agar plates and tubes remain usable for several days and broth tubes for a week or more. In any event, store unused culture vessels in the refrigerator until you are ready to use them, and then allow them to come to room temperature before inoculating them.

There are also times when you want to preserve a live bacteria culture but not encourage growth. For example, in the following lab sessions we use a purchased mixed culture of three species of bacteria. We would prefer to purchase that culture only once, so we’ll preserve the culture by transferring a small amount of it to a tube of sterile isotonic saline (also called normal saline) solution, in which many species of bacteria can survive for extended periods. This solution is essentially a dilute solution of ordinary table salt. It provides no nutrients, so the bacteria we inoculate into it will not grow (reproduce). Some, however, usually survive by going dormant, essentially putting themselves into hibernation until a food source is again available.

In this lab session, we’ll prepare a nutrient broth based on chicken stock (as a nitrogen source) and table sugar (for carbohydrates). We’ll use that nutrient broth to prepare broth culturing tubes as well as agar culturing plates and agar culturing slant tubes. We’ll also prepare and sterilize isotonic saline tubes that we’ll use in the next lab session to preserve our purchased bacteria culture for later use. We’ll need about 10 mL of nutrient medium for each broth culture tube, about 5 mL for each agar slant tube, and about 35 mL for each 90 mL Petri dish.

Again, if you do not have a pressure cooker, make up only enough medium for your immediate needs. If you do have a pressure cooker, you can save time by making up additional medium for later use. That’s what we’ll do in these instructions. Modify them as necessary if you do not have a pressure cooker.

For immediate use, we decided to make up enough nutrient broth to fill four broth tubes (4 X 10 mL = 40 mL), and enough nutrient agar to fill two slant tubes (2 X 5 mL = 10 mL), and two Petri dishes (2 X 35 mL = 70 mL). That requires a total of 120 mL of nutrient broth. To save time later, we also decided to make up an extra 125 mL of nutrient agar, which we’ll store in the 125 mL polypropylene bottle and autoclave. That medium will remain usable for at least several weeks if refrigerated, and is sufficient to fill four 90 mm Petri dishes, which we’ll use in a later lab session. Accordingly, we’ll make up 250 mL of nutrient broth.

Use the 125 mL polypropylene bottle from the kit that contains the nitrogen-free fertilizer concentrate A. If you haven’t used all of that solution by the time you start this lab session, simply transfer the fertilizer concentrate to another container and label it. Peel off the original label from the 125 mL polypropylene bottle and relabel the bottle with its current contents.

We’ll use these Petri dishes, slant tubes, and broth tubes in the next lab session. Store them in the refrigerator until then. Store the Petri dishes inverted (with the gel side up). Store the slant tubes and broth tubes upright, either in the test tube rack or in a beaker or similar vessel. Remove the Petri dishes and tubes from the refrigerator and allow them to warm to room temperature before you use them.

Q1: Is the nutrient media we made defined or undefined? Why?

Q2: Why do we store unused culturing media in the refrigerator?

Q3: How do unselective and selective media differ? Why might you choose an unselective medium?

Q4: What is the defining characteristic of a differential medium?

Q5: Why might you choose a broth medium rather than a gel medium, and vice versa?

Q6: Why is agar so widely used for making up gel culturing media?