Introduction to Diagnostic Media

You can identify bacteria by their differing abilities to use carbohydrates, withstand salt, produce gas and acid, produce various substances, etc.

Using diagnostic media is the most interesting part of microbiology. Some media are very colorful. All diagnostic media begin with a basal medium such as E-broth which you can make at home. The composition of the basal medium will depend upon the objective of your diagnostic medium. Read this entire page. You will find reading books and the professional diagnostic pages of this site useful, but these professional pages have not yet been written.

Don't overlook the simple catalase assay and the ferrous iron assay for hydrogen sulfide H2S.

Remember the Basic Requirements for Media

Most beginners use media containing meat or milk products for nitrogen source, yeast extract for vitamins and nucleic acid components and some sugars, and glucose for carbon and energy source. Such media do not require minerals because the above ingredients contain minerals. Media made from the above are good for many bacteria isolated from milk, foods, gut, and many other sources. This page is devoted to the study of such bacteria. Bacteria which use sulfur, carbon dioxide, etc will be studied elsewhere.

Steps in Preparation and Use of Diagnostic Media

Design of the Basal Medium

If you want to test the ability of a bacterium to grow on various carbohydates, design your basal medium to contain everything except a carbon source. Such a medium would contain a suitable nitrogen source and vitamins if needed. If vitamins are needed and you are supplying them by adding yeast extract (YE), do not add any more YE than needed, because YE contains several carbohydrates. E-broth can be used as the basal medium.

If you are testing the ability of a bacterium to use various nitrogen sources such as ammonium chloride, sodium nitrate, urea, whole protein, or amino acids, you would not want to have a rich nitrogen source such as tryptone in the basal medium.

Some bacteria require specific vitamins because they can't make them. Since vitamins are required only in very tiny amounts and the tryptone and other ingredients often contain enough vitamin, the bacterium may grow quite well without you adding vitamin. Therefore, this page for beginners will not discuss tests for vitamins or other biochemicals which are needed only in trace amounts. Scientists running vitamin assays use expensive, fresh, highly purified media ingredients.

Give some suggested Basal media. E-broth, tryptone base.

While minerals only base could be used, the high concentration of mineral will carmelize sugars and produce substances toxic to growth of many bacteria. High minerals media also may split agar so that the medium will not gel.

Add Carbohydate as Desired

Some bacteria such as Thiobacillus make everything they need from minerals and carbon dioxide and these bacteria can grow fine without any carbohydrates in the medium. Such species are some times inhibited by the presence of carbohydrate.

This section is about testing common bacteria for their ability to use various carbohydrates. All of them can use glucose as the sole carbon and energy source. Many of them can use other sugars because they have the enzymes needed to split (hydrolysis) certain compound sugars and starches. Some examples are the disaccharides sucrose (glucose-fructose), lactose (glucose-galactose). Some bacteria have the enzymes to split and use long sugar polymers such as starch, inulin (from dahlia), and cellulose. About 30 carbohydrates have been used by bacteriologists. Sucrose (table sugar), and lactose (milk sugar) are the sugars used most commonly.

Starch is useful because many bacteria can't digest it but many species of Bacillus and other genera can. The assay for starch digestion is very simple. Prepare petri plate containing starch and nitrogen source. Streak 10 to 20 different bacteria on the plate and incubate, usually about 25 to 30 C for 2 to 4 days. Record the amount of growth for each bacterum, then flood the plate with dilute iodine solution for a few minutes. Observe a cleared area (no black color) around the bacteria that digested the starch. If your medium contained glucose the bacterium may use that and leave the starch.

Carbohydrates Used to Differentate Bacteria

Some carbohydrates used are adoditol, arabinose, dulcitol, esculin, inostol, lactose, maltose, mannitol, salicin, sorbitol, sucrose, trehalose, amd xylose.

Amount and Carbohydrates to use.

You may use these as broth tubes containing an inverted small tube to catch the gas, or you may use agar as slants with a generous butt (let slant go only 1/2 way to bottom). You should use a pH indicator: phenol red, Bromcresol purple, and bromthymol blue are often used. Will the dye from red cabbage work (bacteria might eat it)?

needs more detail of basal and quant of test sugar

Add pH Indicators, Oxygen Indicators, Iron as Desired

You may add a pH indicator to the medium to detect acid formation. One is of the most popular is Phenol Red. I usually make up a stock solution of 1.5 mg of Phenol Red per mL and use 1 ml of this per 100 ml of medium. Thus I am using 15 mg phenol red per liter. This colors the medium red to orange-red. If acid is produced during growth of the bacterium, the medium turns yellow. If alkaline pH is produced the medium turn a beautiful red color. Other pH indicators used are _________ _____

Methylene blue is blue in presence of oxygen and colorless when reduced. I make a stock solution of 0.5 mg per mL and use 10 ml of this per liter of medium. Methylene blue can be used in any medium but it has long been used in skim milk. Mix 100 g of dry skim milk with water to make 1 liter and add 10 ml of the stock solution. do not shake during incubation.

Some bacteria produce hydrogen sulfide (rotten egg odor) from the sulfur containing amino acids. Add __ ferrous sulfide per liter of medium. If sulfide is produced, it will react with the iron to forming a black deposit. Use 0.2 g of ferrous sulfate per liter of medium. This can be added to any medium.

Heat the Medium to Dissolve

If the medium contains agar, heat to slow boil for 2 to 5 minutes to melt the agar. If this is not done, the agar will settle and top part of tube will not gel. If there is no agar and everything is dissolved, there is no need to heat the medium

Dispense into tubes for Stabs, Deeps, or Slants

If dispensing into 13 x 100 mm culture tubes, use 2.5 ml for slants, 3 ml for stabs, and 5 ml for deeps. Use about 4 ml for broths.

Add a small inverted test tube to capture gas

If desired, add a small inverted test tube to capture gas released during bacterial growth. This is done only if the medium is a broth. Broth means liquid, it is not neccesairy a meat broth.

If the medium is agar and a gas is generated, the agar will be fractured or bubbles may be seen. Often the agar is moved high in the tube by gas forming in the bottom of the agar. These things only happen when incubation is at 37C. At room temperature, gas production is so slow that it diffuses out of the agar without forming bubbles.

Plug Tubes or Apply Tube Cap

Before autoclaving, install KimKaps or stopper tubes with cotton plugs. These devices are to exclude dust which carries microbes.

Autoclave for 15 Minutes at 15 Pounds Pressure

Place tubed media in wire baskets. Jars autoclaved in plastic pans were not sterilized. If you use soft drink cans to hold your tubes, punch holes in the bottom of cans to permit steam to flow up through the cans. All air must be driven out of the pressure cooker before applying the pressure regulator. Begin timer when guage reaches 15 pounds.

15 minutes at 15 pounds is adequate for 8 oz glass bottles. If you set bottles directly on the bottom of the cooker, the bottoms of the bottles will break off due to expansion. A piece of metal, drilled full of 3/8 inch holes will prevent breakage, but it is better to use the support that came with the cooker. When I want to pour agar into plastic plates, I autoclave the agar in 8 oz boston rounds, but any glass bottle or jar will do. You can make caps for the bottles and jars out of aluminum foil if you do not have autoclaveable lids. Polypropylene lids (found on pop bottles) do not melt, but they may shrink. If in doubt test the caps on a piece of paper in a pre test for autoclaveability. Glass pop bottles with the original caps do fine, but may be too tall for your cooker.

Six quart pressure cookers are large enough to sterilize 0.1 disposeable pipents, but pressure canners are convenient when available. Your first job is to find glassware and containers which will fit into your 4 qt or other size pressure cooker. Common 150 mm tubes are too tall to fit 4 and 6 quart cookers, but they can be autoclaved at an angle. Autoclaving pipets is the big problem, there you can consider cutting glass tubing into length short enough to fit inside 150 mm culture tubes with a cap. You can count drops instead of using volume marked pipets.

Never tighten caps during autoclaving. Tubes with tight caps may explode in your face.

Cooling stabs, deeps, and liquid media overnight in the autoclave reduces contamination.

If slants are wanted, cool to about 50C and then remove tubes and lay on a stick to slant.

Store Sterile Media in a Dust-free Location

You will have much less contaminated media if you store your sterile media in capped milk cartons and other dust-free containers.

Use Media Before Expiration Date

Students need not be concerned about expiration dates. However, professsional labs must adhere to expiration dates. I have used decade old tubed media with no obvious problems when the medium as in screw-cap tubes.

Inoculate media properly

You may use a loop for all inoculations. Touch the loop to a colony or a pure culture. Unless the culture is very old, just touching the loop to the bacteria will pick up enough bacteria. Insert the loop to the bottom of the assay tube. If a slant, also move the loop along the surface of the slant in a wavy line. Sterile applicator sticks, about 1 mm by 150 mm are satisfactory for one time use.

Incubate at correct temperature

Incubate at a temperature which gives good growth of the bacterium. At room temperature, growth and gas production is slower than 37C an the gas will difusse out of the agar an may not form any bubbles or cause fracturing of the agar giving a false indication of "no gas".

Record Results in a Readable Form

When possible record results in a table format for easy understanding by others.

Autoclave discarded used tubes.

Autoclave used glassware before washing. A used tube contains thousands of millions bacteria and might be capable of infecting hundreds or thousands of people. Autoclaving used glassware for 15 minutes at 15 pounds of pressure insures the items are harmless.

Wash Tubes with Detergent

Wash autoclaved tubes with detergent and warm water, rinse, invert, dry, and store in dust-free boxes.

Additional Information

1. This page gave guidelines for designing your own assay media using supplies you have at hand. Even if your media are far different that commercial media, you can learn the principles of common diagnostic media.

2. E-broth, Potato media such as PDA, and many other can be useful to the beginner when he adds Ulrich Indicator and/or iron.

3. Page b042.htm gives the formula for many commercial diagnostic media. Study of those pages will help you design your own media from what you have available. Also examine page b030.htm to learn the composition of simple media which support many common genera of bacteria.

---------- These media from page b030.htm may help you design a basal medium ------------

NA Nutrient Agar

Supports the growth of many common bacteria from the intestines and many other species. Nutrient agar is the most widely used medium in highschools, but not in my laboratory. TGY supports the growth of many more species. If your bacterium requires NaCl just add NaCl to TGY. Nutrient Agar is specified in many texts because it has been used since the 1880's when nothing better was available. Nutrient agar is a very low quality medium.

NB Nutrient Broth

Nutrient Broth is Nutrient Agar without the agar. In my laboratory, I use NB only when I have a special reason not to use a tryptone broth.

WR * Water on Rocks

It is not necessary to autoclave this unless you are making stock cultures. Actually, steaming on 3 successive days may be better as autoclaving 15 minutes @ 15 pounds pressure will melt the sulfur into little balls, but that is not serious as the Thiobacillus can still use it. Adding a chip of limestone or marble to each tube will neutralize the sulfuric acid produced by the bacteria as they oxidize the sulfur. My Thiobacillus survived a year on this medium at room temperature.

TGY ** Tryptone Glucose Yeast

This is called Plate Count Agar by many people. It supports more species of bacteria than any other medium. If in doubt try TGY. Bacterial pigments which are pale on some media are usually much brighter on TGY in air at room temperature. Used as slants and stabs. I often add 1 gram of powdered CaCO3 to counteract the acid generated by many bacteria from the glucose. With the carbonate, many stock cultures last years instead of months. If you don't have calcium carbonate powder try ground agriculural limestone or any limestone or blackboard chalk. To avoid settling of CaCO3, stir while filling tubes.
    This is my most important medium. This is the only medium a teacher or student needs. You can do lots of interesting work using only this medium. Some bacteria do better on a more dilute medium such as PYE. Some bacteria would do better if the glucose is left out or reduced to 1 to 5 grams, I have not tried that and have no experiences to relate.

Notes on Choice of Ingredients

Scientists use high quality distilled water when doing exacting nutrition work, but tap water gives good results in the classroom. However, tapwater may give a cloudy preciptate in some instances. I use spring water, but tapwater is just as good. If it has lots of chlorine or junk, you may let it stand a few days before use--especially if you are growing invertbrates in the water.

--- Some useful info for non-chemists -----

MgSO4.7 H2O is Epson Salt.

NaCl is table salt, but use normal salt not Morton's which contains insoluble aluminum salts which should be harmless but will give cloudy medium. I urge you to use only salt that dissolves to a clear solution. After all, aluminum is the active ingredient in underarm deodorants.

If you don't have all the minerals such as Mg, Ca, Mn, etc. stir soil into spring (or tap) water and let the mud settle a couple days and use the clear supernatant water.

If the chemical you have differs in water of hydrate. Solve a proportion to get the correct amount or just ignore the difference. You can't ignore the difference if you are preparing pH buffers by weight.

You may travel the web to ATCC, NRRL, other media sites. Search for +bacteria +media. For more media formulas. However, the above are more than most students and teachers need.

Catalase Assay

Certain flavoprotein enzymes catalyze substrate dehydrogenations that are coupled directly with the reduction of oxygen producing hydrogen peroxide (H2O2). While H2O2 is highly toxic, nearly all aerobic organisms possess a heme containing enzyme which catalyzes the decomposition of H2O2 to water and oxygen.    2 H2O2 == catalase ==> 2 H2O + O2.

To assay for catalase, place a drop of hydrogen peroxide (H2O2) on a culture or a sample removed in a loop. If you get bubbles of oxygen (O2), it is because the bacterium makes the heme enzyme catalase which speeds the breakdown of hydrogen peroxide into water and oxygen. All bacteria make catalase except the following:

The simple catalase test will tell you much about the bacterium you are studying.

Acetobacter peroxydans solves the toxic H2O2 problem by making a peroxidase enzyme which uses H2O2 as the hydrogen acceptor yielding 2 molecules of water and getting rid of the toxic H2O2 but notice no bubbles are generated.    H2O2 + 2 [H] == peroxidase ==> 2 H2O

Since the Lactic Acid Bacteria do not make cytochromes, they can not make hydrogen peroxide, and, therefore, they have no need for catalase. To read about these reactions, study the Electron Transport Pathway in any biochemistry book. The lactic acid bacteria are all species in these genera:

Cell shape and
arrangement
Fermentation
type
Name of
Genus
Further
remarks
Cocci in chains Homofermentative Streptococcus Milk, fecal, mouth,
respiratory tract
Cocci in chains Heterofermentative Leuconostoc Produce dextran slime
friom sucrose
Rods, usually in chains Homofermentative Lactobacillus Most species are
found in milk
Rods, usually in chains Heterofermentative Lactobacillus Wines, Kefir grains,
inactive in milk


Version 2     Revised: 1999 Feb 15


Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164

Suggestions, corrections, and comments are appreciated: Contact Harold Eddleman (indbio@disknet.com).