Chapter 4

Media and
Dilution Water Preparation
G. L. Davis and P. J. Hickey
(M. Bulthaus, Tech. Comm.)

4.010 Introduction
Accuracy of microbial data obtained from testing dairy foods depends on
which culture media are used and how these media are used in the labora-
tory. Media, reagents, and dilution water must be prepared and handled
properly. Refer to chapter 2 for laboratory quality assurance procedures.
It is the responsibility of the individual analysts to know the properties
of, and safety precautions for, the chemicals they use. For details, refer to
manufacturers’ material safety data sheets (MSDS).
Commercial dehydrated or prepared media are readily available for
almost all methods and procedures given in this edition of SMEDP and
should be used when available. For this reason, media formula or composi-
tional information is not presented in this book. In those few exceptions
where commercial media are not available, the formula of the media is
given with the method. For commercial media, compositional information
and directions for their preparation are on the label. If needed, information
on standard methods agar (SMA) composition may be found in the 16th edi-
tion of SMEDP.13
4.020 Suitability of Water
Only water that has been treated to free it of traces of dissolved metals and
bactericidal and inhibitory compounds should be used to prepare culture
media, reagents, and dilution blanks. Inhibitor-free water is referred to as
microbiologically suitable (MS) water. For routine use in microbiological
analyses of dairy products it is the growth medium or dilution system pre-
pared with the water that is of concern, and not the water itself. While cul-
ture media provide considerable protection from toxic agents present in the
water,16 it is still important that water used as a dilution fluid be free from
substances toxic to microorganisms. A procedure to determine if dilution
water may have a toxic effect is given in section 4.041, and water that pass-
es the toxicity test is considered to be microbiologically suitable.
93
 Guidelines for reagent-quality water have been published in Standard
Methods for the Examination of Water and Wastewater,4,5 the Annual Book of
ASTM Standards,1 Preparation and Testing of Reagent Water in the Clinical
Laboratory,14 and the United States Pharmacopeia2 (USP). All of these publi-
cations recommend treatment of water to remove chemical and biological
substances that may interfere with tests. Treatment should include one or
more of the following: distillation, deionization, reverse osmosis, carbon
adsorption, filtration, ultrafiltration, and ultraviolet oxidation. In general,
MS water must be free of chemical contaminants such as chlorine and met-
als, exhibit low conductivity (high resistivity), and have low levels of het-
erotrophic organisms. Bottled water must meet USP requirements for
purified water to be considered MS water. In addition, source water for
treatment by filtration, ultrafiltration, UV oxidation, and/or reverse osmo-
sis must be closely monitored because these types of treatments are inca-
pable of removing all contaminants. For best results, water treatment
should include distillation, mixed-bed deionization combined with acti-
vated carbon treatment and pre- and postfiltration, or reverse osmosis
treatment with prefiltration and either distillation or mixed-bed deioniza-
tion.
MS water should not be stored for long periods of time. Distilled water
storage reservoirs must be drained and cleaned regularly. Open containers
of USP-certified purified water should be protected from contamination.
Point-of-use water purifiers must be maintained and tested according to the
manufacturer’s recommendations. Lengthy tubing and plumbing connec-
tions that are likely to permit the accumulation of bacteria must be avoided.
MS water that is prepared from chlorinated source water must be tested reg-
ularly for residual chlorine. MS water must also be tested regularly for con-
ductivity (or resistivity), metals, and heterotrophic bacteria.

4.030 Preparation of Dilution Water

Dilution blank preparations vary considerably in dairy and food laborato-
ries around the world.3,4,6,10,12,15,16 Janus8 summarized the results of an
IDF/ISO study involving 8 different diluents. The study group recom-
mended quarter-strength Ringers solution as a reference standard, with
peptone-salt diluent, peptone solution, and phosphate water as alternatives.
The phosphate solution is retained in SMEDP because of its equivalence to
other diluents8 and the lack of sufficient evidence to dictate a change.7,15
Distilled water performed as well as phosphated water (without magne-
sium chloride)8,9 and would be usable where dilutions of milk and milk
products are low. Use of a single diluent is essential, however, to ensure
standardization.

4.031 Stock Phosphate Solution

Dissolve 34 g of potassium phosphate (monobasic) in 500 mL of MS water,
adjust to pH 7.2 with sodium hydroxide, and make up to 1 L with MS
water. Preferably, place in small vials. Sterilize at 121°C for 15 minutes.
After sterilization, tightly seal the vials and store at 0° to 4.4°C.

94 Standard Methods for the Examination of Dairy Products

4.032 Stock Magnesium Chloride Solution
Measure 38 g of anhydrous magnesium chloride into a 1-L volumetric
flask and add MS water to make 1 L of solution. Dispense into suitable
containers, sterilize, and store as described above [4.031].

4.033 Phosphate Dilution Water (Class O)

Add 1.25 mL of stock phosphate solution to MS water and make up to 1 L.
Dispense in 99 ± 2-mL [2.045] or 9 ± 0.2-mL quantities, or in other volumes
as required, and autoclave at 121°C for 15 minutes [4.091]. Bacteria should
not be suspended in any dilution for more than 30 minutes at room tem-
perature to minimize death or multiplication of the bacteria.

4.034 Phosphate and Magnesium Chloride Dilution Water (Class O)

Add 1.25 mL of stock phosphate solution and 5 mL of stock magnesium
chloride solution to MS water and make up to 1 L. Dispense in 99 ± 2-mL
[2.045], 9 ± 0.2-mL, or other quantities as required, and autoclave at 121°C
for 15 minutes [4.091]. The addition of magnesium chloride may improve
the recovery of microorganisms with metabolic injury induced by toxic sub-
stances in the dilution water.6,11

4.040 Water Toxicity Tests

4.041 Dilution Water Toxicity Test (Class O)
A screening test for dilution water toxicity can be conducted by preparing a
dilution of the appropriate dairy food to provide 100 to 250 colonies per
plate when 1 mL is plated. The diluted sample is then plated in duplicate
after 0, 15, 30, and 45 minutes by the standard plate count (SPC) or equiva-
lent procedure [chapter 6]. Distinct trends toward lower counts among suc-
cessively plated samples suggest toxicity. Decreases of more than 20% in 45
minutes are indicative of dilution water toxicity.

4.042 Water Quality Test (Class O)

A detailed method is described in section 9020 B.3.C. of Standard Methods for
the Examination of Water and Wastewater.5

4.050 Cleaning Glassware and Testing for Detergent Residues

Modern detergents are effective for cleaning laboratory glassware. Most of
these detergents are of the anionic type, usually with alkaline substances
such as phosphates, carbonates, or silicates. Some detergents, especially the
cationic type with quaternary ammonium compounds, are highly inhibito-
ry and great care must be exercised to ensure their removal. Detergents and
soaps have an affinity for surfaces. They displace dirt and allow it to be
washed away, but they themselves are difficult to remove completely.

4.051 Cleaning Glassware

Deposits of milk stone or calcium salts, which are resistant to ordinary
detergents, are occasionally encountered. Remove salts by exposing glass-

Chapter 4 ■ Media and Dilution Water Preparation 95

 ware for several minutes to acid solutions and then rinsing thoroughly.
Commercial dairy-acid cleaners, formulated to remove milk stone from
milk-processing surfaces, can be used on laboratory glassware. Usage levels
should be obtained from the label.
The detergent wash is best done with hot water after a preliminary rins-
ing with warm water to remove most of the soil. Soaking aids in the removal
of stubborn residues. Six to 12 rinses with running tap water, followed by
several rinses with MS water, are sometimes necessary to remove a deter-
gent completely.

4.052 Detergent Residues

If glass petri dishes are used, laboratories should check for inhibitory
residues whenever changing brands or lots of detergents. This procedure is
described in chapter 2 [2.0412].

4.053 Glassware pH Check

Acid and alkaline residues can be detected on cleaned laboratory ware by
adding and dispersing a few drops of aqueous 0.04% bromthymol blue (BTB)
indicator. Localized blue (alkaline) or yellow (acid) zones in the pH indicator
solution demonstrate the incomplete removal of cleaning solutions. Prepare
the BTB solution by dissolving 0.1 g of BTB in 16 mL of 0.01N sodium
hydroxide and diluting to 250 mL with MS water; the solution must be green.

4.060 Media
4.061 Dehydrated Media
Dehydrated media should be stored in tightly closed containers below 30°C
and protected from light. If the environment is hot and humid, media may
be stored in a refrigerator or freezer. Properly stored, most media remain
stable until the manufacturer’s expiration date; however, purchases should
be planned to facilitate complete turnover within 1 year. Media should be
dated upon receipt and when first opened.
After the seal has been broken, permitting entrance of air and moisture,
the quality of a medium may depend on the storage environment. Use of a
desiccator for storage of opened media will extend shelf life. If possible,
media should be used or discarded within 6 months after breaking the seal.
Suitable package sizes should be purchased to minimize the repeated use of
a material from unsealed containers. Any medium that has absorbed mois-
ture and become caked must be discarded. Contamination may take place if
unclean spatulas are used to transfer material from containers.

4.062 Prepared Media

The life of any prepared medium, whether in tubes, bottles, or plates,
depends on the storage conditions and type of medium. Prepared media
should not be stored unless protected against water loss. This may be
accomplished by using screw-capped tubes and bottles (with caps tightened
after sterilization) instead of cotton-plugged or loosely covered containers.
Prepared plates should be used within 1 week unless stored in moisture-
proof containers such as plastic bags. Media in screw-capped tubes should
be used within approximately 6 months.

96 Standard Methods for the Examination of Dairy Products

 Commercially prepared media should be used prior to the expiration
date. If dehydration has occurred, media should be discarded.

4.070 Basic Steps in Media Preparation

Weigh carefully the proper amount of dehydrated medium.
Place the requisite amount of MS water into a suitable container (e.g.,
borosilicate glass; stainless steel or heat-resistant plastic such as polypropy-
lene, polycarbonate, or polysulfone).
Add the weighed, dehydrated medium to part of the water. Mix with a
stirring rod or magnetic stirrer. If necessary, adjust the medium pH with a
suitable acid or alkali solution. Add the remaining water and mix again.
[Note: Reconstituted media prepared from commercial dehydrates seldom
require pH adjustment if made according to the manufacturer’s directions.
Errors in weighing dehydrated media and overheating may cause pH prob-
lems. Recheck medium preparation procedures if problems occur. If signifi-
cant pH problems continue and medium preparation procedures are con-
sidered satisfactory, the commercial dehydrate should be replaced. See sec-
tion 4.080 for information concerning pH adjustment.]
If necessary to effect complete solution, heat to boiling on wire gauze
over a free flame or on a hot plate with a built-in magnetic stirrer. Stir often
to prevent burning of the medium. A nonpressurized, free-flowing steam
unit may be used. Media containing agar should be boiled 1 minute to
ensure solution of the agar. Prolonged boiling may cause undesirable foam-
ing. This can be reduced by holding the flask in cold water for a few seconds
after the initial boiling has been accomplished. If necessary, restore water
lost by evaporation. For example, weigh the container before and after heat-
ing. Add sufficient water to restore the original weight before distributing
and sterilizing the rehydrated medium. A microwave oven may be used to
dissolve dried medium if it is compatible with the container in use.
Distribute the medium into a suitable container. This can be most easily
accomplished for a tubed liquid medium by using automatic pipeters. A
hand-held adjustable pipeter is most suitable for small numbers of test tubes
while an electrically operated dispensing apparatus is more satisfactory for
large numbers [2.046]. In each instance, before use, the machine must be
flushed with MS water followed by the medium until the water has been
completely removed from the system. After use, the apparatus must be
flushed with warm water, detergent solution, tap water, and MS water. If
any residues accumulate, the apparatus should be disassembled and
scrubbed. A small amount of water should be left in the syringe portion to
prevent drying out and subsequent “freezing” of the plunger and barrel.
The amount of medium per container should be limited so that no part
of the medium is more than 2.5 cm from the container wall or the surface of
the agar. (This is to ensure rapid equilibration of temperature when the con-
tainer is placed in a water bath.)
Sterilize at 121°C for 15 minutes [4.091] or according to recommended
procedures for each medium. Checking of pH before use is recommended.
Melt and hold solid media at 44° to 46°C until ready for use, but not
exceeding 3 hours. Hold inhibitory-substances media at 64°C [12.021,
12.022]. Solid media may be melted in boiling water, by flowing steam not

Chapter 4 ■ Media and Dilution Water Preparation 97

 under pressure, or in a microwave oven. The time to ensure complete solu-
tion will vary with the method and the volume in the container. Do not per-
mit media to boil over in the microwave oven. If a precipitate forms, discard
the media; do not remelt.
If it becomes necessary to formulate a dehydrated medium that is not
commercially available, use only reagent-grade chemicals and ingredients
approved by the USP and other components prepared by culture media
suppliers. Follow the manufacturer’s instructions for storing stock materi-
als. Discontinue use of materials that show any evidence of contamination,
decomposition, or dehydration.

4.080 Adjustment of pH
Prior to use, determine the hydrogen-ion concentration (pH) of culture
media at 25°C electrometrically by using a potentiometer (pH meter).
Determinations made at 45°C to take advantage of the fluid state of agar
may be low! For example, SMA shows a pH of 7.04 at 25°C and a pH of 6.8
at 45°C.17 Meters measure pH accurately only if samples and buffers are at
the same temperature. Commercially prepared reference buffers should be
used.
Prepare the pH meter as described in 2.0413. Macerate solid medium
thoroughly with a glass rod before inserting electrodes. Be sure electrodes
and sample are at 25°C. Alternatively, use flat surface electrodes that can test
agar surfaces without prior mixing and breaking of the medium.

4.090 Sterilization
Before sterilization, bring the medium to boiling temperature while stirring
frequently. Restore lost water if necessary [4.070] and autoclave. Because the
pH of the medium may change during sterilization and because there may
be potential browning reactions, it is important not to exceed the recom-
mended temperature and time. Sugar broths should not be subjected to a
sterilization cycle longer than 45 minutes. Reduce pressure with reasonable
promptness to prevent boiling or undue changes in the nutritional proper-
ties of the medium. Remove from the autoclave when atmospheric pressure
is attained. It is preferable to use flasks or bottles from which the melted
medium may be poured into plates; however, if desired, test tubes contain-
ing 15- to 20-mL amounts may be used for pouring agar into plates.

4.091 Steam Sterilization

Sterilize media, water, and materials (such as rubber, cork, cotton, paper,
and heat-labile plastic tubes and closures) that are likely to be charred in the
dry-air sterilizer by autoclaving at 121°C for 15 minutes. Start timing after
the temperature has reached 121°C. Autoclave media and dilution blanks
within an hour of preparation. Slightly loosen stoppers to allow the passage
of steam and air out of closed containers in the autoclave. Make certain the
load is loosely packed. For efficient sterilization, separate containers by at
least 1 cm in all directions. Before allowing steam pressure to rise, automat-
ically or manually expel all air from the sterilizer through an exhaust valve.
If manual means are used, make sure that all air has been exhausted and
that only steam is being exhausted before pressurization begins. (If air

98 Standard Methods for the Examination of Dairy Products

 remains in the chamber, a 15 pounds-per-square-inch gauge pressure can
mean a temperature as low as 100°C!) Because the temperature obtained at
a constant pressure of saturated steam will vary according to atmospheric
pressure, rely on a properly operating and calibrated thermometer (not a
pressure gauge) to ensure sterilization. Maintain and check autoclave as
described in 2.041.
Carefully review label instructions before heat-treating any medium. For
example, violet red bile agar (VRBA) requires boiling for 2 minutes rather
than autoclaving.9
Avoid overloading autoclaves so that the rate of air exhaust or heating is
not appreciably delayed. The volume of the load generally should not
exceed 30% to 40% of the volume of the autoclave. The autoclave should
reach 121°C slowly but within 15 minutes. The maximum time from apply-
ing the steam to unloading the autoclave should not exceed 45 minutes. A
rapid come-up time does not necessarily result in more efficient autoclav-
ing, since air is not replaced by steam when the steam enters the autoclave
too rapidly. A steam flow that is too slow also results in decreased efficien-
cy because air-steam mixtures form.
When nonliquid materials with slow heat conductance are to be steril-
ized, or where the packaged arrangement or volume of materials otherwise
retards penetration of heat, allow extra time for materials to reach 121°C
before beginning to time the sterilization period, and, if necessary, use
longer sterilization periods to ensure sterility.
After sterilization, gradually reduce pressure within the autoclave (no less
than 15 minutes is recommended) because liquids may be at temperatures
above their boiling point at atmospheric pressure. Liquids can be lost
through boiling when the pressure is lowered too rapidly. When dry mate-
rials such as sampling equipment or empty sample bottles are being steril-
ized, pressure may be released rapidly at the end of the 15-minute holding
period at 121°C. This prevents collection of condensate and speeds up the
drying of paper-wrapped equipment.
Used plates, pipets, tubes, etc., must be routinely decontaminated in
microbiological laboratories. Dairy products, especially raw milk, may har-
bor potential pathogens such as staphylococci and streptococci. The same
principles apply for loading the autoclave and sterilizing materials as for
preparing media. Plastic petri dishes are most conveniently sterilized by
placing them in heat-resistant, autoclavable bags, or other containers. When
large quantities of used materials are autoclaved, the sterilization time shall
be at least 30 minutes. Plastic dishes become amorphous when properly
autoclaved. Sterilization tapes or other indicators can be applied to contain-
ers and placed in autoclaves to indicate that sterilization temperatures have
been reached [2.041].

4.092 Hot Air Sterilization

Sterilize equipment with dry heat in hot-air sterilizers or ovens so that mate-
rials at the center of the load are heated to not less than 170°C for not less
than 1 hour. (This usually requires exposure for about 2 hours at 170°C.)
When the oven is loaded to capacity, use a longer period or a slightly high-
er temperature. Spore packs should be used to check sterilization efficiency.

Chapter 4 ■ Media and Dilution Water Preparation 99

4.100 Quality Control
General principles of quality control are covered in chapter 2. This chapter
presents procedures that will provide minimal but adequate quality control
in media production.
Whenever possible, use commercially dehydrated media.
Date the label of each bottle of dehydrated medium, indicating when it
was received and when it was first opened.
A medium must be mixed completely to form a homogeneous solution
in water before it can be sterilized and dispensed. Stirring that causes foam-
ing should be avoided. Restore water lost by evaporation after ingredients
are fully dissolved.
Autoclave performance should be monitored manually or with a contin-
uous temperature-recording device. The bulb of the thermometer should be
located properly on the exhaust line so as to measure minimum temperature
within the sterilizing chamber. For each autoclave run, heat-indicating auto-
clave tape should be placed on each outer container. Records for sterilization
runs should include the date, a brief listing of item(s) sterilized, tempera-
ture, and sterilization time. In addition, routine sterilization procedures
should be checked periodically with a maximum-registering thermometer
and either spore strips or suspensions.
Limit heating of the medium to the minimum necessary to ensure solu-
tion and sterilization. When the autoclave has reached atmospheric pressure
following the recommended cycle, it should be opened immediately.
Prolonged storage of melted media in a water bath should be avoided.
Check and document the final pH of each lot of medium at 25°C [4.080].
Aseptic technique must be followed strictly during dispensing of steril-
ized material. Hands should not touch any part of the dispensing surfaces
that come in contact with sterile material. During interruptions in the cycle
of a mechanical dispenser, the spout of the dispenser train should be placed
inside a sterile container. A dispensing cycle should not be interrupted,
however, except in an emergency.
Media containing dyes should be protected from light by storing them in
the dark or a dark glass bottle, or by wrapping the container with brown
paper or foil.
Each container of autoclaved medium should be labeled with the name
of the medium and the autoclave run number.
Inclusive dates during which each lot and container of dehydrated
medium were used should be recorded for future reference.
Before use, each lot of prepared medium should be inspected visually for
volume, tightness of closure, clarity, consistency, and completeness of label. If
possible, media should be tested with positive and negative microbial con-
trols.

4.110 Cited References

1. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 1994 annual book of ASTM
standards, Vol. 11.01, Designation D 1193-91, Standard Specification for Reagent Water.
Philadelphia, PA: American Society for Testing and Materials; 1994.
2. ANONYMOUS. United States pharmacopoeia, 22nd ed. Easton, PA: Mack Printing Co.;
1989.

100 Standard Methods for the Examination of Dairy Products

 3. BUTTERFIELD, C.T. The selection of a dilution water for bacteriological examina-
tions. J. Bacteriol. 23:355-368; 1932.
4. CLESCERI, L.S.; GREENBERG, A.E.; EATON, A.D., EDS. Standard methods for the
examination of water and wastewater, 20th ed. Washington, DC: APHA; 1998.
5. EATON, A.D.; CLESCERI, L.S.; GREENBERG, A.E., EDS. Standard methods for the
examination of water and wastewater, 19th ed. Washington, DC: APHA; 1995.
6. HARTMAN, P.A. Modification of conventional methods for recovery of injured col-
iforms and salmonellae. J. Food Prot. 42:356-361; 1979.
7. HUHTANEN, C.N.; BRAZIS, A.R.; ARLEDGE, W.L.; DONNELLY, C.B.; GINN, R.E.;
RANDOLPH, H.E.; KOCH, E.J. A comparison of phosphate buffered and distilled
water dilution blanks for the standard plate count of raw-milk bacteria. J. Milk Food
Technol. 38:264-268; 1975.
8. JANUS, J.A. Investigations into the bactericidal effect of diluents applied for the enu-
meration of Enterobacteriaceae in food products. Report of Group E 50. The
Netherlands: International Dairy Federation; 1980. ISO/TC 34/SC 9.
9. JENSEN, J.P.; HAUSLER, W.J., JR. Productivity of boiled and autoclaved violet red bile
agar. J. Milk Food Technol. 38:279-280; 1975.
10. KING, W.L.; HURST, A. A note on the survival of some bacteria in different diluents.
J. Appl. Bacteriol. 26:504-506; 1963.
11. MACLEOD, R.A.; KUO, S.C.; GELINAS, R. Metabolic injury to bacteria: II. Metabolic
injury induced by distilled water or Cu++ in the plating diluent. J. Bacteriol. 93:961-969;
1967.
12. MALLETTE, M.F. A pH 7 buffer devoid of nitrogen, sulfur, and phosphorous for use
in bacteriological systems. J. Bacteriol. 94:283-290; 1967.
13. MARSHALL, R.T., ed. Standard methods for the examination of dairy products, 16th ed.
Washington, DC: APHA; 1992.
14. NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS.
Preparation and testing of reagent water in the clinical laboratory, 2nd ed. Approved guide-
line. Villanova, PA: National Committee for Clinical Laboratory Standards; 1991.
NCCLS Document C3-A2.
15. OBLINGER, J.L.; KENNEDY, J.E., JR. Evaluation of diluents used for total counts. J.
Milk Food Technol. 39:114-116; 1976.
16. RONALD, G.W.; MORRIS, R.L. The effect of copper on distilled water quality for use
in milk and water laboratories. J. Milk Food Technol. 30:305-309; 1967.
17. WALTER, W.G.; KUEHN, K.; BOUGHTON, A. Electrometric pH determinations of
different agar media at 25° and 45°C. J. Milk Food Technol. 33:343-345; 1970.

Chapter 4 ■ Media and Dilution Water Preparation 101

102 Standard Methods for the Examination of Dairy Products