9050 PREPARATION OF CULTURE MEDIA*
9050 A. General Procedures
1. Storage of Culture Media
Store dehydrated media (powders) in tightly closed bottles in the
dark at ⬍30°C in a low-humidity atmosphere (e.g., a desiccator).
Do not use them if they are discolored, caked, or no longer a
free-flowing powder. Use stocks of dehydrated media before their
expiration date or within a year of purchase if they contain selective
agents (sodium azide, bile salts, antibiotics, sulfur-containing amino
acids, etc.) in order to maintain optimum selectivity.
Use commercially prepared media wherever available. Avoid
preparing media from essential ingredients unless necessary. See
also Section 9020B.5j.
For media prepared onsite, review manufacturer’s instructions,
product material safety data sheets (MSDSs), and analytical meth-
ods. Prepare culture media in batches that will be used within
2 weeks unless the method specifies otherwise. If media are con-
tained in screw-capped tubes, however, they may be stored for up to
3 months at ⬍30°C. (See Table 9020:V for specific details on
storage time and temperature.) Store media out of direct sunlight
and avoid excessive evaporation. Place prepared Petri dishes in
air-tight containers or plastic bags, close with twist-ties, and store
under refrigerated conditions. Invert Petri dishes to prevent moisture
condensation on agar. Do not use plates with condensation drops
because this will cause colonies to spread. If necessary, dry plates
by placing them with lids slightly ajar in a laminar-flow hood.
If refrigerated, liquid media in fermentation tubes may dis-
solve enough air to produce an air bubble in the inner Durham
tube when later incubated at 35°C. Bring all media (especially
fermentation or carbohydrate broth) to room temperature before
use and discard tubes containing any air bubbles.
After prolonged incubation or storage, selective agents may
break down and evaporation may change media ingredients’
concentrations. Discard tubes with growth due to contamination
or an evaporation loss of more than 1 mL. A loss of 10% or more
(e.g., 1 mL or more from an initial 10 mL) can affect most-
probable-number (MPN) calculations.
2. pH Adjustment
After sterilization, determine and record medium’s pH. The
directions for preparing each medium typically specify the final
pH; if so, adjust pH as needed. If a specific pH is not prescribed,
adjustment is unnecessary. During sterilization, the pH usually
will drop 0.1 to 0.2 but occasionally as much as 0.3 in double-
strength media. If the media contain buffers, the decrease in pH
will be negligible. The initial pH required to obtain the correct
final reaction will have to be determined.
To determine final pH, cool medium to 44 – 46°C, aseptically
remove a small quantity, set meter for the higher temperature (if
* Approved by Standard Methods Committee, 2015.
Joint Task Group: Margo E. Hunt (chair), Gil Dichter, Nancy H. Hall, Robin K.
Oshiro.
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not done automatically), and measure pH. Alternatively, com-
pletely cool a sample of the medium to room temperature before
determining final pH. If pH adjustment is necessary, use a sterile
stir bar and pipet a sufficient quantity of filter-sterilized 0.1N
sodium hydroxide (NaOH) or 0.1N hydrochloric acid (HCl) into
the bulk medium to reach the proper pH.
If dehydrated media are reconstituted according to directions,
their pHs seldom require adjustment. However, the final pH may
be unacceptable if dehydrated medium was weighed improperly,
reconstituted medium was overheated, or there was a problem
with the medium itself. If the prepared media’s pH values are
consistently outside the allowed range, determine the cause. The
pH may need to be adjusted before sterilization.
3. Sterilization
After rehydrating a medium, dispense promptly into clean
culture vessels without cracks or chips and sterilize within 2 h.
Do not store nonsterile media.
Sterilize media in an autoclave at 121°C. Review method and
manufacturer’s requirements. The required exposure time will
vary with form and type of material, medium, presence of
carbohydrates, and volume. Sterilize most carbohydrate broths at
121°C for 12 to 15 min; however, there are exceptions. For
example, A-1 media must be autoclaved for 10 min at 121°C.
When the pressure reaches zero, remove medium from autoclave
and cool quickly to avoid decomposition of sugars due to pro-
longed heat exposure. To permit uniform heating and rapid
cooling, loosely pack materials in small containers. The maxi-
mum heat exposure for most carbohydrate broths (from closing
loaded autoclave to unloading) is ⬍45 min. The maximum heat
exposure for A-1 medium is ⬍30 min. Preferably use a double-
walled autoclave to permit preheating before loading to keep
total heating time within the limit. Adjust autoclave times as
volumes/loads increase. Presterilized media may be available
commercially. Do not re-autoclave media.
After sterilization, examine media to determine whether any
unanticipated color or clarity variations occurred or media compo-
nents precipitated. Mark media containers with preparation date and
record all pertinent information in appropriate logbooks.
4. Quality Control
See Section 9020B.5j.
5. Bibliography
BUNKER, G.C. & H. SCHUBER. 1922. The reaction of culture media.
J. Amer. Water Works Assoc. 9:63.
RICHARDSON, G.H., ed. 2004. Standard Methods for the Examination of Dairy
Products, 17th ed. American Public Health Assoc., Washington, D.C.
VERSALOVIC, J., ed. in chief. 2011. Manual of Clinical Microbiology,
10th ed. American Soc. Microbiology, Washington, D.C.
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 PREPARATION OF CULTURE MEDIA (9050)/Media Specifications
9050 B. Water
1. Specifications
To prepare culture media and reagents, use only distilled or
demineralized reagent-grade water that has been tested and
found free from traces of dissolved metals and bactericidal or
inhibitory compounds. Toxicity in distilled water may be derived
from fluoridated water high in silica. Other sources of toxicity
are silver, lead, and various unidentified organic complexes.
Where condensate return is used as feed for a still, toxic amines
or other boiler compounds may be present in distilled water.
Residual chlorine or chloramines also may be found in distilled
water prepared from chlorinated water supplies. If chlorine com-
pounds are found in distilled water, neutralize them by adding an
equivalent amount of sodium thiosulfate or sodium sulfite.
Distilled water also should be free of contaminating nutrients.
Such contamination may be derived from flashover of organics
during distillation, continued use of exhausted carbon filter beds,
deionizing columns in need of recharging, solder flux residues in
9050 C. Media Specifications
The need for uniformity dictates the use of dehydrated media.
Never prepare media from basic ingredients when suitable
commercially prepared dehydrated media are available. Follow
manufacturer’s directions for rehydration and sterilization. Com-
mercially prepared media in liquid form (sterile ampule or other)
also may be used if known to give equivalent results. See Section
9020B.5j for quality-control specifications.
The terms used for protein source in most media (e.g., pep-
tone, tryptone, and tryptose) were coined by the media develop-
ers and may reflect commercial products rather than clearly
defined entities. It is not intended to preclude the use of alter-
native materials, provided that they produce equivalent results.
NOTE: In the following directions, the term percent solution
means “grams of solute per 100 mL solution.”
1. Dilution Water1
Various dilution water solutions can be prepared in the labo-
ratory or purchased commercially. Below are two of the most
commonly used solutions in the basic water microbiology labo-
ratory.
a. Buffered water:
1) Stock phosphate buffer solution—Dissolve 34.0 g potas-
sium dihydrogen phosphate (KH2PO4) in 500 mL reagent-grade
water, adjust to pH 7.2 ⫾ 0.5 with 1N NaOH, and dilute to 1 L
with reagent-grade water. Sterilize via filtration or autoclave.
Store stock solution under refrigerated conditions and discard if
turbidity develops.
https://doi.org/10.2105/SMWW.2882.183
new piping, dust and chemical fumes, and storage of water in
unclean bottles.
Store distilled water out of direct sunlight to prevent algae
growth. Aged distilled water may contain toxic volatile organic
compounds absorbed from the atmosphere if stored for pro-
longed periods in unsealed containers. Good housekeeping prac-
tices that minimize the presence of airborne particulates usually
will eliminate nutrient contamination.
See Section 9020B.5f and Table 9020:II.
2. Bibliography
STRAKA, R.P. & J.L. STOKES. 1957. Rapid destruction of bacteria in
commonly used diluents and its elimination. Appl. Microbiol. 5:21.
GELDREICH, E.E. & H.F. CLARK. 1965. Distilled water suitability for
microbiological applications. J. Milk Food Technol. 28:351.
MACLEOD, R.A., S.C. KUO & R. GELINAS. 1967. Metabolic injury to
bacteria. II. Metabolic injury induced by distilled water or Cu⫹⫹ in
the plating diluent. J. Bacteriol. 93:961.
2) Magnesium chloride stock solution—Add magnesium chlo-
ride (38 g/L MgCl2 or 81.1 g MgCl2 䡠 6H2O) to 1 L reagent-grade
water. Sterilize and store stock solution under refrigerated con-
ditions, discarding if solution becomes turbid.
3) Working solution—Add 1.25 mL stock phosphate buffer
solution and 5.0 mL magnesium chloride stock solution to 1 L
reagent-grade water. Dispense in amounts that will provide 99 ⫾
2.0 mL or 9 ⫾ 0.2 mL after autoclaving for 15 min. Final pH
should be 7.2 ⫾ 0.1. NOTE: pH values will change with time.
Store under refrigerated conditions after opening and discard if
turbidity develops. Use within 6 months.
b. Peptone water, 0.1%: Prepare by adding 1 g peptone to 1 L
reagent water. Final pH should be 7.0 ⫾ 0.2 after sterilization.
Dispense in amounts to provide 99 ⫾ 2.0 mL or 9 ⫾ 0.2 mL
after autoclaving for 15 min. Store as above.
Do not suspend a sample in any dilution water for ⬎30 min at
room temperature because injury, death, or growth (in peptone
water) may occur.
2. Culture Media
Specifications for individual media are included in subsequent
sections. Details are provided where use of a medium is first
described.
3. Reference
1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1978. Microbiological
Methods for Monitoring the Environment, Water and Wastes;
EPA-600/8-78-017. Cincinnati, Ohio.
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