BISC209/F13: Culture Media

Background

The composition of medium is an important factor when attempting to culture microorganisms. The components and pH can be manipulated to favor the nutritional preferences of particular bacterial groups if the desired microbes are unlikely to be successfully isolated in a general purpose medium. Keep in mind that the composition of selective or enrichment media (particularly the differences between them and general purpose media) provides valuable information about the metabolic range of the microbes that grow there.

The recipe for each selective or enrichment medium described here includes the composition and concentration of all ingredients (in g/l or % [wt/vol or vol/vol]) and the desired final pH. Deionized (filtered) water is generally used unless a particular medium requires a purer type of water (distilled, salt-free, DNAase or RNAase free for example). Tap water is not used in media preparation because it may contain undesirable compounds such as chlorine, copper, lead, and detergents. Some of the media or reagents described here can be purchased from companies such as BBL Microbiology Systems or Difco Laboratories in a dehydrated, premixed form. If commercially purchased in dehydrated form, the manufacturer provides the instructions for preparation.

Pourite™ is an anti-foaming agent from American Scientific Products that is commonly added to non-commercial medium containing agar. One drop of Pourite™ is added to volumes up to 800 ml and 2 drops to volumes of more than 800 ml to prevent foaming.

General Purpose Media
Nutrient broth (NB) and Nutrient agar (NA):

NB: 0.3% Beef extract, 0.5% Peptone, Deionized water to 1 L at pH 6.6- 7.0 at 25°C

NA: 0.3% Beef extract, 0.5% Peptone, 1.5% Agar; Deionized water to 1 L at pH 6.6- 7.0 at 25°C

NA+Starch: NA (above) plus added 2.5% starch

Dilute NA: NA diluted 1:10 (0.03% beef extract, 0.05% Peptone, 1.5% agar)

Nutrient broth/agar is a moderately rich, general purpose, solid medium that meets the nutritional requirements of many culturable bacteria. It contains beef extract, soy digest, and enzymatically digested gelatin to support the growth of a wide variety of chemoheterotrophic organisms. Fungal growth is reduced in this media. In broth form, the solidifying agent (agar) is not included. Sometimes it is diluted to help slower growing microorganisms not be overshadowed by quicker growing or spreading microorganisms.

Defined vs. Undefined Media
These terms refer to media in which all ingredients and the concentrations of those ingredients are known (defined) or media that contains components that vary in concentration, molecular formula, or may or may not be uniform in different batches or brands of medium (undefined). A wider variety of microorganisms will grow on undefined media than on defined so most general purpose media, such as nutrient agar or tryptocase soy agar (TSA), are undefined. Some common ingredients in undefined media include tryptone (trypsin digested casein, a milk protein) or peptone (an enzymatic digest of varying proteins, often meat scraps). These are not pure substances. They contain left over enzymes, vitamins, salts, amino acids, peptides, and polypeptides. They are important sources of nitrogen. When we are interested in enrichment for particularly bacteria, it is more common to use defined media because we can control the sources of nitrogen or carbon. Many of the selective media are defined because your goal is to grow only one type of microorganism based on some known metabolic characteristic it shares with few others.

Selective / Differential / Enrichment media

Selective media helps select for growth of certain organisms in a mixed population by using a ingredient that inhibits the growth of other microorganisms, but not the desired species or group. Enrichment media can be considered a subgroup of selective media since its composition is usually designed to enhance the growth of certain microorganisms by including nutrients that the desired microorganism or group can use for an essential process while its competitors can not. Sometimes enrichment media also limits alternate sources of nutrition or contains an ingredient that inhibits the growth of competitors. Differential media does not select for any particular group by inhibiting or enhancing the growth of one group over competitors, but this type of medium is able to show a visible difference between or among groups of microorganisms. Media can be any permutation or combination of selective, differential, and/or enrichment, depending on its ingredients and its use.
For more information culture media you can access an e-book of the Difco manual of culture media at: | http://www.archive.org/details/difcomanualofdeh09dige

Selective and Differential Media for Confirming Gram Stain Results

Selective Media for Gram positive Bacteria
Phenylethyl Alcohol Agar (PEA) PEA selects for the growth of gram positive organisms by inhibiting the growth of gram negative bacilli. The alcohol in the medium dissolves the gram negative lipid outer membrane that Gram positive bacteria lack. The thin layer of peptidoglycan, characteristic of gram negative bacteria allows entry of the phenylethyl alcohol into a gram negative bacterium, but not into a gram positive bacterium (gram positive bacteria have a much thicker, more impermeable layer of peptidoglycan in their cell wall). Alcohol interfers with DNA synthesis and kills bacteria with a gram negative cell wall structure. This medium is particularly useful at inhibiting the overgrowth of Gram negative Proteus species that tend to swarm (they are highly motile) all over an agar plate and, thus, using PEA medium makes isolating Gram positive organisms easier in a mixed population.
Recipe: 1.80% Bacto Agar, 1.50% Tryptone, 0.50% Phytone, 0.50% Sodium Chloride, 0.25% Phenylethyl Alcohol (PEA).

Postive control organism: Staphylococcus epidermidis

Selective and Differential Medium for Gram negative Bacteria
Eosin–Methylene Blue (EMB) Agar is a selective and differential medium used to enhance selection of gram negative bacteria. The medium contains peptone, lactose, sucrose, dipotassium phosphate, eosin and methylene blue dyes. Colonies produced by lactose non-fermentors are not dark blue or black because the dyes are not precipitated. The growth of gram positive bacteria is generally inhibited on EMB agar because of the toxicity of methlyene blue dye. In the low dye concentration found in this medium, the protective lipid outer membrane of gram negative bacteria prevents entry of this toxic water-soluble dye because the gram negative outer membrane is a phospholipid bilayer that repels many water-soluble substances. The cell wall structure of gram positive bacteria lacks the protective lipid based outer membrane and that makes them more sensitive to the toxicity of methyene blue.

Recipe:1% peptone, 1% Lactose, 0.2% dipotassium phosphate, 0.04% eosin Y, 0.0065% methylene blue 1.5% Agar. final pH 6.9-7.3

Table 2. Colonial appearance on EMB Agar after 18-24 hours at 35°C.

EMB is also a differential medium. Eosin and methylene blue act as indicators to differentiate between gram negative organisms that ferment lactose from those that do not ferment lactose. Most bacteria that ferment lactose form colonies on EMB agar that are dark blue to black due to precipitation of the dyes by the acid by-products of lactose fermentation. Lactose fermentors have dark colonies from dye precipitation while non-fermentors have light colored colonies. Additionally, E. coli often gives a characteristic green-metallic sheen on EMB, making this medium somewhat differential for E. coli to help distinguish it from other lactose fermenting gram negative bacteria.
Recipe: 0.04% Eosin Y, 0.0065% methylene blue, 1.0% peptone, 2.0% lactose, 0.2% K2HPO4, 1.5% agar, pH 7.1

Organism	Colonial Appearance
Escherichia coli	purple with black center/ green metallic sheen
Klebsiella pneumoniae	dark centered colonies/ sometimes a metallic sheen
Enterobacter aeorogenes	pink colonies/ no metallic sheen
Proteus mirabilis	colorless colonies
Salmonella typhimurium	colorless colonies

Reference: Dehydrated Culture Media and Reagents for Microbiology. DIFCO Laboratories, Detroit, MI. 1984.

Differential Medium For Assessment of Soil Exoenzymes: Amylase, Cellulase, Phosphatase

Nutrient Agar (NA) General Purpose Medium is used to determine comparative number of total culturable bacteria: and for growth of non fastidious organisms once in pure culture. For plate counts use your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) onto a pre-labeled Nutrient agar plate. Use a sterile, disposable spreader to evenly distribute the diluted soil extract all over the culture plate. Repeat for two other dilutions (one 10fold more and one l0 fold less dilute).
Nutrient Agar General Purpose Medium:
0.3% Beef extract, 0.5% Peptone, 1.5% Agar at pH 6.6- 7.0 at 25°C.

Starch Medium is used to determine the % of amylase producing (starch digesting) culturable microbes when compared to the total number counted on NA: Using your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) into the center of a pre-labeled Nutrient agar plate. Use a sterile, disposable spreader to evenly distribute the diluted soil extract all over the culture plate. Repeat for two other dilutions (one 10 fold more and one l0 fold less dilute).
Starch medium :
2.5% (wt/vol) soluble starch in Nutrient Agar

Reference: Beishir, Lois. 1996. Microbiology in Practice 6th ed. HarperCollins Publishers Inc. New York. Module 33: 301-306.

Cellulose Medium is used to determine the % of cellulolytic microbes (those producing cellulase) when compared to the total number counted in NA : Using your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) onto a pre-labeled plate of Cellulose medium. Use a sterile, disposable spreader to evenly distribute the diluted soil extract all over the culture plate.
Cellulose Congo Red Agar:
0.05% K2HPO4; 0.025% MgSo4; 0.188% ashed, acid washed cellulose powder; 0.02% Congo red, 0.5% Noble Agar, 0.2% gelatin, 10%(vol/vol) sterile soil extract (Soil extract prepared as follows:105 g of air-dried sieved soil and 660 ml of deionized water are placed in a 1 litre bottle and autoclaved once at 15 psi for 15 minutes, then again after 24 hours. The contents of the bottle are left to settle for at least a week and then the supernatant is decanted and filtered. The final pH should be 7.0 - 8.0.)
Reference: Hendricks, Charles W., Doyle, J.D., Hugley, B. (1995) A New Solid Medium for Enumerating Cellulose-Utilizing Bacteria in Soil. Applied and Environmental Microbiology. May: 2016-2010.

Phosphate Medium (Pidovskaya medium) is used to determine the % phosphate solubilizing microbes (those producing phosphatases) in a soil community: Using your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) onto a labeled Pidovskaya medium plate.
Pidovskaya Medium:
1.0% glucose, 0.05% yeast extract, 0.01% Calcium Chloride (CaCl₂), 0.025% Magnesium Sulfate (MgSO⁴.7H₂0), 0.251% Calcium Phosphate [Ca(PO₄)], 2.0% agar.
References: Pikovskaya, R.I. 1948. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17, 362-370, modified by Pranjal Baruah (2007) Isolation of phosphate solubilizing bacteria from soil and its activity. Biotechindia.files.wordpress.com/2007/12/isolation.pdf.

Incubate all cultures at room temp until mature colonies have formed and then refrigerate before the bacteria overgrow.

Count the total number of colonies on the Nutrient Agar plate that has between 30-300 colonies and record the dilution. Assess total culturable CFUs for that dilution.

Find the starch plate with between 30-300 total colonies and note the dilution. Choose the dilution that shows 30-300 colonies. Flood the Starch plates with Grams Iodine. Let the plate sit for at least 10 minutes, longer is fine. The plate should turn dark blue as the iodine binds to the starch; Pour off the excess iodine (into paper towels in the biohazard bag on your bench) and wait until the remaining liquid is absorbed by the plate. Count the colonies that show a clear halo, indicating the starch in the medium was digested by amylase secreted from the bacteria in the colony. Count the number of colonies that are able to digest starch (clear zone around the colony). To calculate the % of culturable microbes able to digest starch/CFU in a gram of wet soil you will use the Average total number of colonies calculated from your NA plate count. Make sure that both counts were made on the same dilution. If not, you will have to do a conversion.
Reference: Beishir, Lois. 1996. Microbiology in Practice 6th ed. HarperCollins Publishers Inc. New York. Module 33: 301-306.

Find the cellulose plate showing between 30-300 total colonies. Count the number of colonies that show cellulose digestion activity by looking for positive digestion as a clear zone or halo around the colony. Record as number of cellulose digesting organisms in that dilution.

Do the same for the assessment of number of phosphate digestion microbes in a particular dilution. The positive colonies will be red that show phosphate solubilizing activity.

Calculating the % of digestion positive microbes in the total culturable population

Use the soil extract dilution on the plates counted to normalize all the calculations to CFUs/gram of soil (wet weight) for each assessment medium. If you divide the number of colonies counted by the volume of inoculum plated, times the dilution factor of that inoculum, you will obtain the number of that type of bacteria per gram of soil.

For example, if you counted 150 colonies on the 10^-3 plate the calculation is:
150/(0.1ml vol. of inoculum*1X10^-3dilution)= 150X10⁴ which in scientific notation is written as 1.5X10⁶ CFU/gram

Once you calculate the total number of aerobically culturable bacteria (cfu/g) on the general purpose media, you can determine the % of the total number able to solubilize phosphate by dividing the number of phosphatase positive colonies by the total number of culturable colonies---if the colonies counted are compared from the same plate dilutions.

This calculation of the % of cultured bacteria that are positive for each tested enzymatic activity: (# positive colonies/total count on nutrient agar X 100) gives you a sense of the prevalence and variety of soil organisms in a community with particular substrate utilizing potential.

Links to Labs

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Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10