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26. Thermophilic Flat Sour Sporeformers

Karl E. Olson and Kent M. Sorrells
Published Online: December 17, 2013 © American Public Health Association

Citation Full Text PDF PDF Plus

26.1 INTRODUCTION

In 1810 a French confectioner and distiller, Nicholas Appert, invented the method of preserving foods for extended periods of time.
In this method, foods, such as soups, fruits, vegetables, juices, milk products, marmalades, jellies, and syrups, were enclosed in
hermetically sealed glass bottles or jars, then placed into a boiling waterbath for several hours. Most of the foods preserved by this
method did not spoil. Appert is credited with being the father of canning, although he could never explain why his preservation
method worked. He assumed, as in wines, air exposure spoiled the food; therefore during the process he had to exclude all air and
to ensure a tight closure of the glass container, using corks, wire, and sealing wax. A half century later, Louis Pasteur demonstrated
the relationship between microorganisms and food spoilage, validating Appert’s basic processes. However, there were many
incidents of spoilage in which the root cause remained unknown, even after Louis Pasteur’s experiments. Not until 1895, from
studies conducted at the Massachusetts Institute of Technology, was it nally shown that spoilage was due to the growth of some
microorganisms that were not killed due to insu cient heat being applied to the canned product. When classi ed by their growth
temperature range, the abovementioned organisms were placed into the thermophilic group, or so-called thermophiles.
Thermophiles can grow between 40°C and 75°C.

Canned “commercially sterile” products (e.g., tomato products, vegetables, and evaporated milk) can undergo thermophilic at sour
spoilage, if held at high ambient temperatures. In canned low-acid foods, particularly those having a pH no lower than about 5.3,
thermophilic at sour spoilage seldom occurs, if holding temperatures are maintained below approximately 43°C. If these foods are
held at a temperature above 43°C long enough and if the food contains viable spores capable of germinating and growing out in
the product, then the product may undergo at sour spoilage.4

Typical thermophilic at sour spoilage of low-acid canned foods is caused by the growth of sporeforming, thermophilic facultative
aerobes in the genus Bacillus or Geobacillus.13,19

Geobacillus stearothermophilus (formerly classi ed as Bacillus stearothermophilus) and Bacillus coagulans are the typical species
responsible for this type of spoilage.3,7,8,9,10,16,21 These organisms characteristically ferment carbohydrates with the production of
short-chain fatty acids (e.g., lactic, acetic, and propionic) that “sour” the product. They do not produce enough, if any, gas to change
the usual “ at” appearance of the ends of the containers. Although the at sour bacteria are considered obligate thermophiles, in
fact they may grow at temperatures as low as 40°C, especially if the incubated organisms are in the vegetative state and if proper
environmental conditions are imposed. The group’s upper temperature limit for growth is 65°C to 75°C.

Spores of G. stearothermophilus have exceptionally high thermal resistance. A D121°C of 4.0 to 5.0 min, with a z value between 7.8°C
and 12.2°C was reported for G. stearothermophilus.6,20 Thus, their presence in some containers of any given lot of commercially
sterile low-acid canned foods may be considered normal. Since at sour spoilage does not develop unless the product is held at
temperatures above 43°C, proper cooling after thermal processing and avoiding high temperatures during warehouse storage or
distribution are essential. A severe thermal treatment of about 20 min at 121°C is required for foods distributed in warm climates
to achieve a 5 log reduction of thermophilic sporeformers, including G. stearothermophilus. Such severe treatments decrease
nutrient and sensory qualities of the product but insure a shelf-stable food.12

Because of their exceptional resistance, spores of G. stearothermophilus are often used as a biological indicator to validate moist
heat sterilization.1 B. coagulans has much lower heat resistance (i.e., D121°C = 0.07 min) in comparison to G. stearothermophilus.
The recommended sterilization value for canned tomatoes in juice (pH 4.5) against B. coagulans is the equivalent of 0.7 min at
121°C with a z-value of 10°C.11 Okazaki et al.15 reported the isolation of three remarkably heat resistant strains of B. coagulans from
spoiled retort pouch products; one of these strains had a D110°C of 73.6 min, which is considerably higher than previously published
D-values for this organism.17 Bacterial spores enter food processing plants with soil, on raw foods, and in ingredients (e.g., spices,
sugar, starch, or our).6,14,18,20 Populations may increase at any point where a proper growth environment exists. For example, food
handling equipment in a processing line that is operated within the thermophilic growth range (~ 43°C–75°C) may serve as a focal
point for the buildup of an excessive at sour spore population.

Spores of at sour bacteria show exceptional resistance to destruction by heat and chemicals; hence, they are di cult to destroy in
a product or in the plant. Methods to minimize spore contamination include control of spore population in ingredients and
products entering the plant, as well as the use of sound plant sanitation practices.

Inadequate cooling subsequent to thermal processing is a major contributor to the development of at sour spoilage. Localized
heating of sections of stacked canned foods that are placed too close to heaters is another.

26.2 GENERAL CONSIDERATIONS

26.21 Sampling

26.211 Ingredients
Approximately half-pound samples from each of ve bags, drums, or boxes of a shipment or lot of dry sugar, starch, our, or similar
ingredients should be collected and sealed in cans, jars, plastic bags, or other appropriate containers and transported to the
laboratory.14 Samples of ingredients used in lesser proportions in the nished product (e.g., spices) may be sampled in
appropriately smaller volumes. In any case, samples should be reasonably representative of the entire lot or shipment in question.
For liquid sugar, collect ve separate 6- to 8-oz. (200- to 250-mL) samples from a tank or truck when it is being lled or emptied.2

26.212 Equipment and Product in Process

Only those units held at temperatures within the thermophilic growth range are of direct concern. Scrapings or swab samples of
food contacting surfaces or of wet surfaces positioned directly over food materials, from which drippage may gain access to the
food materials, may be cultured. Collect the samples in sterile tubes or sampling bags. Examination of food samples taken before
and after passage through a particular piece of equipment (e.g., a blancher or a ller) will reveal whether a signi cant buildup of at
sour spores is occurring in that item of equipment. Multiple samples of a volume equivalent to the volume of the container being
packed generally are taken. Unused clean metal cans and covers are convenient sample containers. (Do not use glass containers to
collect samples because of the danger of breakage or of being dropped into the equipment.) Solid materials in the presence of
excess liquid may be collected in a sieve or similar device; this permits draining excess liquid. Chill the samples thoroughly without
delay to prevent the growth of thermophilic bacteria prior to the laboratory examination. Immersion of the sample container in
cold tap water usually is adequate for this purpose.

26.213 Finished Product

The method of sampling depends on the objective of the examination. When spore contamination levels during production are the
concern, obtain processed containers representing conditions (1) at the start of operations when the shift begins, (2) before
midshift shutdown, (3) at start-up after midshift shutdown, and (4) at the end of the shift. Samples of each time period should
consist of at least 10 containers. The probability of nding one positive can in 10 samples if 25% of the production contains viable
at sour spores is 0.95.14 Incubate at 55°C for 5 to 7 days.

When known or suspected insu cient cooling or storage at temperatures above 43°C is suspected, obtain containers at random
from the production lot in question. Record the locations from which each container was obtained, that is, its position on a pallet
and the location of the pallet. The larger the number of samples examined, the greater will be the probability of detecting at sour
spoilage. The probability of detecting at least one spoiled container in the sample when the real spoilage level is 1% is about 95%
with a 300-unit sample; 89% with a 200-unit sample, and 62% with a 100-unit sample11 (see the chapter “Sampling Plans, Sample
Collection, Shipment, and Preparation for Analysis”). Because growth of at sour organisms may cause a slight loss of vacuum or a
loss of consistency of a product, separation of these products sometimes is possible without destruction of normal product cans.11

26.22 Spore Recovery

Much of the work in the area of spore recovery deals with recovery from heat resistance tests, and these results may or may not
apply in the recovery of the spores from ingredients and the recovery of vegetative cells from production equipment. Recovery of
heated spores occurs best at 45°C to 50°C and in neutral media. After spore heating, water is the best diluent for spore recovery,4
and best recovery occurs if heating has been done in distilled water.

For optimum vegetative growth, the cell needs an adequate oxygen supply and a culture medium at pH 7. No growth of G.
stearothermophilus occurs at pH 5.

26.3 EQUIPMENT, MATERIALS, AND REAGENTS

Equipment and supplies are needed in accordance with speci cations in the chapters “Sampling Plans, Sample Collection,
Shipment, and Preparation for Analysis” and “Canned Foods—Tests for Cause of Spoilage.” The following additional media and
apparatus are recommended.

26.31 Culture Media

Dextrose tryptone agar

Dextrose broth
Nutrient broth or dextrose tryptone broth (if needed for nutrient supplementation, see Section 26.52)
2% agar

26.32 Equipment

Autoclave: for heat shocking food samples in addition to normal laboratory sterilization purposes
Sanitary can opener: Bacti-Disc Cutter, see the chapter “Canned Foods—Tests for Cause of Spoilage”; for opening canned food
samples aseptically; may be purchased from Wilkens-Anderson Co., No. 10810-01 (www.wacolab.com)
Flasks: 250-mL and 300-mL Erlenmeyer asks, for analysis of thermophilic spore ingredients

Glassware: dilution bottles, 6 to 8 oz for dilution blanks containing distilled water

Incubator: temperature controlled to ~55°C

Microscope
Petri dishes: sterile, either glass or plastic may be used
pH meter: electrometric, pH color comparator with bromcresol purple and methyl red reagents and standards may be
substituted
Pipettes: sterile, 1-mL and 10-mL Mohr pipettes; sample pipettes made from either straight wall borosilicate tubing (7–8 mm in
diameter × 35–40 cm); disposable pipettes may also be used
Waterbath temperature range of 55°C to 60°C (starch)4
Thermometer
Swabs, sterile cotton or alginate swabs

26.33 Reagent

Crystal violet solution

26.4 PRECAUTIONS
Samples other than nished products must be handled so that there will be no opportunities for spore germination or spore
production between the collection of the samples and the start of examination procedures.

Before making a positive judgment on a sample based on pH or microscopic examination of direct smears, be sure that these
characteristics are known for “normal obtained” control products. Controls should be obtained from the same production code as
the suspect samples. If such controls are not available, use a product from the same manufacturer and bearing the next closest
production code. This is particularly important where formulated products are concerned, although it is not necessarily con ned to
such products. Incubated agar plates should not be allowed to dehydrate during incubation. Placement in oxygen-impermeable
bags will minimize dehydration.

26.5 PROCEDURE

Thermophilic at sour spores possess greater heat resistance than most other organisms encountered in foods. This characteristic
is advantageous to the examination of foods and ingredients because, by controlled heat treatment of samples (heat shock), it is
possible to eliminate all organisms except the spore with which we are concerned. Further, heat shock, or activation, is necessary to
induce germination of the maximum number of spores in a population of many species, including the at sours.4,14 Because the
most heat-resistant spores are generally the ones of concern in food canning operations, a heat shock favoring recovery of such
spores is preferable. Unless otherwise speci ed, that is, in a standard procedure, 30 min at 100°C or 10 min at 110°C, followed by
rapid cooling, should be used.

26.51 Sample Preparation and Examination

Sugar and starch: The National Food Processors Association (NFPA, formerly National Canners Association) has suggested a
method and standard for determining thermophilic at sour spore contamination of sugar and starch to be used in low-acid
canned foods.5,6,14,16 There is also an AOAC o cial method2 for this examination.
Sugar (AOAC)2: Place 20 g of dry sugar in a sterile 250-mL Erlenmeyer ask marked at 100 mL. Add sterile water to the 100-mL
mark. Agitate thoroughly to dissolve the sugar. (Liquid sugar is examined by the same procedure, with this di erence: a
volume of liquid sugar calculated to be equivalent, based on degree Brix, to 20 g of dry sugar is added to the 250-mL ask and
diluted with water to 100 mL.)

Bring the prepared sample rapidly to a boil and continue boiling for 5 min, then water cool immediately. Pipette 2 mL of the heated
sugar solution into each of ve Petri plates. Add dextrose tryptone agar (see the chapter “Microbiological Media, Reagents, and
Stains”), swirl gently to distribute the inoculum, and allow to solidify. Incubate the inverted plates at 50°C to 55°C for 48 to 72 hr.

Starch2: Place 20 g of starch in a dry, sterile 250-mL Erlenmeyer ask and add sterile cold water to the 100-mL mark, with
intermittent shaking. Shake well to obtain a uniform suspension of the starch in water. Pipette 10 mL of the suspension into a
300-mL ask containing 100 mL of sterile dextrose tryptone agar at a temperature of 55°C to 60°C. Use large-bore pipettes;
keep the starch suspension under constant agitation during the pipetting operation. After the starch has been added to the
agar, shake the ask in boiling water for 3 min to thicken the starch. Then place the ask in the autoclave and heat at 5-lb
pressure (108.4°C) for 10 min. After autoclaving, the ask should be gently agitated while cooling as rapidly as possible. Violent
agitation will incorporate air bubbles into the medium, which subsequently may interfere with the reading of the plates. When
the agar starch mixture is cooled to the proper point (about 45°C), distribute the entire mixture about equally into ve plates
and allow to solidify. Then stratify with a thin layer of sterile plain 2% agar in water and allow to solidify. This prevents possible
“spreader” interference. Incubate the inverted plates at 50°C to 55°C; count colonies in 48 hr and in 72 hr.
Other ingredients: The procedure for sugar and starch may be applied to other ingredients used in low-acid canned foods.14
Modi cations may be necessary because of physical or chemical characteristics of a particular ingredient, e.g., use of smaller
sample sizes or plating smaller volumes of suspension in more than ve plates because of colony particle size interference
during counting.
Calculating counts: Flat sour colonies are round, are 2 to 5 mm in diameter, show a dark, opaque center, and usually are
surrounded by a yellow halo in a eld of purple. The yellow color (acid) of the indicator may be missing when low-acid-
producing strains are present, or where alkaline reversion has occurred. (Subsequent colonies are compact and biconvex to
pinpoint in shape. If the analyst is unfamiliar with subsurface colonies of at sour bacteria, it is advisable to streak subsurface
colonies on dextrose tryptone agar to con rm surface colony morphology.)
The combined count of typical at sour colonies from the ve plates represents the number of at sour spores in 2 g of the original
sample (20-g sample diluted to 100 mL; 10 mL of this dilution plated). Multiply this count by ve to express results in terms of
number of spores per 10 g of sample.

The total thermophilic spore count is made by counting every colony on each of the ve plates, then calculating in terms of number
of spores per 10 g of sample.

26.52 Equipment and Product in Process

The source of excessive at sour spores in a food processing operation may best be determined by “line samples” (Section 26.21).

Use quantities of sample equivalent to the amount of the material included in a container of nished product; prepare several
replicates (5–20); after closing, warm the containers to the initial temperature of the commercial process, subject them to the
normal commercial thermal process, then incubate at 55°C for 5 to 7 days; open and determine growth of at sour bacteria by pH
measurement, supplemented by microscopic examination of a direct smear if necessary. Many line samples are nutritionally
complete so that water may be added to ll the container; however, some formulated product components may lack essential
nutrients, in which case nutrient broth or dextrose broth (see the chapter “Canned Foods—Tests for Cause of Spoilage”), for
example, should be added instead of water. If in doubt, inoculate a control sample with G. stearothermophilus spores, heat shock,
and incubate at 55°C for 48 to 72 hr to determine whether the sample material will support spore germination and outgrowth.

An alternate procedure is to make agar plate counts on serial dilutions of each heat-shocked line sample. Use dextrose tryptone
agar and incubate at 55°C for 48 to 72 hr.

Swabs or scrapings from equipment surfaces should be shaken in a known volume of diluent, and the suspension heat shocked
and plated on dextrose tryptone agar and incubated at 55°C for 48 to 72 hr. Then calculate the numbers of microorganisms per
unit area sampled.

26.53 Finished Product

Incubate containers of processed product at 55°C for 5 to 7 days, open, and examine for at sour spoilage.11 Comparison of pH of
incubated samples and normal unincubated controls will usually be su cient to show the presence of at sour spoilage. If the
results are not clear, con rm the presence or absence of spoilage ora by direct microscopic examination of smears of the product
from both incubated and unincubated control containers. The bacteriological condition of products whose physical characteristics
provide confusing artifacts when seen in a stained smear can be examined best in a wet mount using phase optics.

Samples collected from a warehouse where insu cient cooling or storing at elevated temperatures is known or suspected are
examined as above, but without preliminary incubation at 55°C.

26.54 Isolation of B. coagulans From Tomato Products

See the chapter “Aciduric Flat Sour Sporeformers.”

26.55 Isolation of B. coagulans From Milk Products

See the chapter “Aciduric Flat Sour Sporeformers.”

26.6 INTERPRETATION OF RESULTS

26.61 Ingredients

Grocery Manufacturers Association standards for thermophilic at sour spores in sugar or starch for canners’ use state the
following: “For the ve samples examined there shall be a maximum of not more than 75 spores and an average of not more than
50 spores per 10-g sugar (or starch).”14

The total thermophilic spore count standard is, “For the ve samples examined there shall be a maximum of not more than 150
spores and an average of not more than 125 spores per 10 g of sugar (or starch).”11
The sugar and starch standard may be used as a guide for evaluating other ingredients, keeping in mind the proportion of the other
ingredients in the nished product relative to the quantity of sugar or starch used.4,5,11,15

The presence of thermophilic at sour spores in ingredients for foods other than thermal-processed low-acid foods is probably of
no signi cance, provided those foods are not held within the thermophilic growth range for many hours. The at sour bacteria have
no public health signi cance.13

26.62 Equipment and Product in Process

Canned and processed line samples usually indicate a point or points at which a spore buildup has occurred. A high percentage of
positive samples taken from one point in the line, when a low level or no positive samples were found prior to this point, shows a
spore buildup in this piece of equipment. The time of day yielding positive samples may indicate whether the buildup is due to
operating temperatures within the thermophilic growth range, or whether inadequate cleanup and sanitization procedures were
used prior to sampling. The former condition is suggested when a majority of positive samples occurs in those taken after the line
has been in operations for several hours. The latter condition is suggested when samples at the start-up of the line are
predominantly positive and those taken later are predominantly negative.

Plate count data for equipment line samples may be meaningful, especially when taken over a long period of time. They can show
trends regarding buildups, inadequate cleanups, etc. Because counts are made in a laboratory medium that may be a better spore
germination and growth medium than certain speci c food products themselves, and because the sample does not receive the
equivalent of the commercial thermal process, results may re ect greater than the actual potential surviving spore load in the
nished product. They can, however, indicate buildup situations that are undesirable.

26.63 Finished Products

Dormant thermophilic spores are of no concern in commercially sterile canned foods destined for storage and distribution where
temperatures will not exceed about 43°C. However, some canned foods are destined for exposure to temperatures above 43°C
during part or most of their shelf life, that is, those shipped to tropical areas and those intended for hot-vending service. To be
considered commercially sterile, these specialized foods must not contain thermophilic spores capable of germination and
outgrowth in the product.

Randomly selected warehouse samples of low-acid canned foods, some of which are found to have undergone at sour spoilage,
can reveal information about the condition contributing to the development of spoilage. Spoilage con ned to product situated in
the outer layers or rows of cases on a pallet suggests localized heating (e.g., from close proximity to a space heater or having been
too close to the building roof during hot weather). Spoilage con ned to inner cases on a pallet is indicative of insu cient cooling,
that is, stacking cases on pallets while the product was still in the thermophilic growth temperature range. Inner cases are insulated
by exterior cases and may retain heat for several days.

ACKNOWLEDGMENT
Fourth edition authors: Karl E. Olson and Kent M. Sorrells.

REFERENCES

1. Abdul Ghani Al-Baali, A. G., and M. M. Farid. 2006. Pouch product quality In: G. V. Barbarosa-Canovas (Editor). Sterilization of Food
in Retort Pouches. Washington University, Pullman, WA. 119–138. CrossRef
2. AOAC O cial Method 972.45. W Horwitz (Editor). 2005. Chapter 17. O cial Methods of Analysis of AOAC International, 18th ed.
AOAC International, Gaithersburg, MD.
3. Ayres, J. C., J. O. Mundt, and W. E. Sandine. 1980. Microbiology of Foods. W. M. Freeman and Company, San Francisco, CA.
4. Cook, A. M., and R. J. Gilbert. 1968. Factors a ecting the heat resistance of Bacillus stearothermophilus spores, J. Food Technol.
3:285. CrossRef
5. Department of Defense. 1985. Military standard. Bacterial standards for starches, ours, cereals, alimentary pastes, dry milks,
and sugars used in the preparation of thermostabilized foods for the Armed Forces. MIL-STD-900C. U.S. Department of Defense,
Washington, D.C.
 6. GMA Science and Educational Foundation. 2007. Chapter 2. Canned Foods, Principles of Thermal Process Control, Acidi cation
and Container Closure Evaluation, 7th ed. GMA Science and Educational Foundation, Washington, D.C.
7. Gordon, R. E., W. C. Haynes, and C. H.-N. Pang. 1973. The genus Bacillus. Agric. Handbook No. 427. U.S. Department of
Agriculture, Washington, D.C.
8. Hammer, B. W., and F. J. Babel. 1957. Dairy Bacteriology, 4th ed. John Wiley & Sons, Inc. New York, NY.
9. ICMSF. 1980. Microbial Ecology of Foods 1: Factors A ecting Life and Death of Microorganisms. Academic Press, New York, NY.
10. ICMSF. 1998. Microorganisms in Foods 6: Microbial Ecology of Food Commodities. Blackie Academic and Professional, New York,
NY.
11. Lund, B. M., and A. L. Snowdon. 2000. Fresh and processed fruits. In: B. M. Lund, T. C. Baird-Parker, and G. W. Gould (Editors).
Microbiological Safety and Quality of Food. Aspen Publishers, Gaithersburg, MD, 738–758.
12. Montville, T. J., and K. R. Mathews. 2008. Food Microbiology: an Introduction, 2nd ed. American Society for Microbiology,
Washington, D.C.
13. Nazina, T. N. , T. P. Tourova, A. B. Poltaraus, E. V. Novikova, A. A. Grigoryan, A. E. Ivanova, A. M. Lysenko, V. V. Petrunyaka, G. A.
Osipov, S. S. Belyaev, and M. V. Ivanov. 2001. Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus
subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus
stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and
Bacillus thermodenitri cans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G.
thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrifcans. Int. J. Syst. Evol. Microbiol. 51:433–446.
CrossRef, Medline
14. National Canners Association Research Laboratories. 1968. Laboratory Manual for Food Canners and Processors, vol. I. AVI Publ.
Co. Inc., Westport, CT. 88–102.
15. Okazaki, T., K. Kakugawa, and T. Yoneda. 2000. Heat-resistance of spoilage microbes isolated from retort pouch foods. Bulletin
of Hiroshima Prefectural Food Technological Research Center. 22:35–38.
16. Olivier, S. A., M. K. Bull, G. Stone, R. J. van Diepenbeek, F. Kormelink, L. Jacops, and B. Chapman. 2011. Strong and consistently
synergistic inactivation of spores of spoilage-associated Bacillus and Geobacillus spp. by high pressure and heat compared with
inactivation by heat alone. Appl. Environ. Microbiol. 77:2317–2324. CrossRef, Medline
17. Peng, J., J.-H. Mah, R. Somavat, H. Mohamed, S. Sastry, and J. Tang. 2012. Thermal inactivation kinetics of Bacillus coagulans
spores in tomato juice. J. Food Prot. 75:1236–1242. CrossRef, Medline
18. Richmond, B., and M. L. Fields. 1966. Distribution of thermophilic aerobic sporeforming bacteria in food ingredients. Appl.
Microbiol. 14:623. Medline
19. Schmitt, H. P., 1966. Commercial sterility in canned foods, its meaning and determination. Q. Bull. Assoc. Food Drug O . US.
30:141.
20. Stumbo, C. R. 1973. Thermobacteriology in Food Processing, 2nd ed. Academic Press, New York, NY.
21. Walker, P. D., and J. Wolf. 1971. The taxonomy of Bacillus stearothermophilus. In: A. N. Barber, G. W. Gould, and J. Wolf (Editors),
England Spore Research. Academic Press, New York. 247.

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