Fermentation in the

Food Industry:
An Introduction to Biotechnology
Lynn Dokken, Biology Instructor, Montgomery, MN
Barry Schmidt, Agriculture Instructor, Montgomery, MN
Biotechnology Workshop, July 14, 1992
University of Wisconsin - River Falls
UPDATED JULY, 1995
Tim Buttles, Eau Claire North High School, Eau Claire, WI
Brenda Gilliland, Rapid City High School, Rapid City, SD
Rhett Hanson, Platteville High School, Platteville, WI
Cathee Pearson, Rutland School District, Rutland, SD

TABLE OF CONTENTS
I.

Introduction

Page 2

II.

Activity Description

Page 2

III.

Objectives

Page 2

IV.

Timeline

Page 3

V.

Teacher Background

Page 5

VI.

Description of Labs Included in the Unit

Page 10

VII.

Evaluation Guidelines

Page 14

VIII.

Full Laboratory Exercises

Page 23

--Root Beer Production

--Cheese Making
--Kimchee: Korean Delight
--Make Your Own Sauerkraut
--Fermenting ("Dough-Raising") Power of Bread Yeasts
--Making Yogurt
--Yeast Fermentation Lab

Page 23
Page 25
Page 27
Page 28
Page 29
Page 30
Page 32

IX.

National Standards

Page 34

X.

References

Page 36

Page 1

I. INTRODUCTION
In designing this project, one of the primary objectives is the creation of an activity that will
have application in both Biology and Agriculture curricula. The activity can be incorporated into a
Cell Biology Unit in Biology or Advanced Biology, into a Food Processing Unit in Agriculture, or as a
Fermentation Unit in Biotechnology. The intent is to use this activity as an introduction to
fermentation and will be followed by the introduction of some modern techniques and applications of
biotechnology.
This unit is designed so that the individual teacher can easily make modifications according
to:
limitations of time and space
budget
equipment
instructor comfort level
student experience or ability
teacher and student interest
local industries
Before beginning this unit, students are expected to have experience in basic lab procedures, lab
safety procedures, library research skills, oral presentation skills, and class journal expectations.

II. ACTIVITY DESCRIPTION

This activity will introduce the students to fermentation as well as provide hands on
experience with a variety of food fermentation processes. In this exercise, students will study
fermentation as a natural biological process and also its implications in the food and agricultural
industries. Included in this exercise will be laboratory activities designed to explore the production of
everyday foods which are produced by fermentation processes.
Students will be choosing a group fermentation project which will include an inquiry based lab,
a personal journal, and a presentation based on their lab results and research. In addition to the
project students will be exposed to the history, chemistry, economics, culture, and careers
associated with fermentation. Because of its importance in the food industry, food safety information
and activities will be shared with the students.

III. OBJECTIVES
A. TEACHER'S OBJECTIVES
In the presentation of this unit the teacher will:
1. determine the level of instruction appropriate for student assimilation
2. determine the amount of information appropriate for student assimilation
3. instruct students in laboratory protocol relevant to this exercise
4. evaluate student objectives

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5. integrate the activity with appropriate curricula

6. facilitate discussion and student involvement
7. learn and follow all food safety guidelines in the classroom
8. meet National Science Education Standards for content, assessment, and program,
Benchmarks for Science Education, National Resource Council recommendations for
agricultural education, and the School-to-Work Initiative

B. STUDENT OBJECTIVES
At the completion of this activity the students will be able to:
1. explain fermentation in its general form
2. define respiration in words
3. write the formula for respiration
4. contrast aerobic and anaerobic respiration
5. identify 3 types of microorganisms which are used in food production
6. list and describe the processes used to produce several fermented foods
7. identify ways that biotechnology may be used to improve our food and food supply
8. maintain a journal comprised of daily observations, reactions, activities, self observations
and peer observations.
9. demonstrate proper food safety techniques.
10. observe, record, and analyze student generated data.
11. coordinate lab and research data into a coherent presentation.

IV. TIMELINE
The following outline is a sample designed for classes that are approximately fifty minutes in
length. If your class period is shorter or longer than this, you may need to change this schedule.
The assumption is that the students have already worked in the laboratory setting and are familiar
with procedure and aseptic techniques. The students in the class will be divided into groups of four
or five. This grouping can be based on grade average, technique, appropriate behavior in the lab,
and any other criteria the teacher is comfortable with. Each group will be responsible for one lab
project (found at the end of the packet, or use your own). The fermentation of yeast lab (Day 6) can
be done as a class laboratory experiment or a teacher demonstration. Suggestions have been made
concerning videos, outside speakers, and field trips. The students will be expected to check the
Page 3

progress on their lab each day and record this information in their daily journal (discussed later in the
packet.)

Day 1
Unit expectations explained
Journal requirements explained
Students put into groups
Topic selection and distribution of lab materials
Proposal sheet
Day 2
Food safety guest speaker
--county home extension agent
-- public health inspector
Collect and discuss proposal sheets
Day 3
Lab setup
--Each group will set up and begin their experiment
--Remind the students of lab procedure and aseptic technique
Day 4
Anaerobic fermentation lecture or video
--video-The History of Cheese Making (Wisconsin Milk Marketing Board)
Day 5
Lab work and research
--Use the library to find information for your project
Day 6
Fermentation of yeast lab/demo
--Teacher choice of demo or lab experiment
Day 7
Research
Day 8
Research
Day 9
History of fermentation lecture/discussion
--discussion of history
--student discussion on how cheese making began
--student discussion of expectations from their lab

Page 4

Day 10
Field trip or speaker on careers in biotechnology
--local hospital
--local laboratory
--local veterinarian
--area tech school with a bioscience technician program
--area college with a biotechnology or food science degree
--local fermentation industry (cheese plant, kraut plant, bakery)
Day 11
Food safety case studies
*Please consult Safe handling Beyond the Retail and Wholesale Shelf Unit 11 pages 1-15
from Food Science, Safety and Nutrition by the National Council for Agricultural Education
*Case studies are included in this reference which can be read and discussed in class or
written about in student journals. These case studies discuss the following topics:
Staph outbreak at a convention
Botulism in chili peppers killing twelve
Salmonella poisoning from restaurant
Clostridium poisoning meals on wheels
Listeriosis from coleslaw in Canada
*Class discussion or lecture on consumer responsibilities such as food purchasing safety,
storage, preparation and serving, proper cooking temperatures, leftovers safety and how
fermentation relates to these safety issues.
Day 12
Work on presentations
Day 13
Presentations
Day 14
Presentations
Day 15
Evaluation/closing
--Students will be asked to write a response to several questions. The questions will ask the
students to synthesize the information from their lab with other groups and teacher presented
information. The students will also have to evaluate their lab project and propose future
research.

V. TEACHER BACKGROUND
History of Fermentation
The use of natural biological processes to obtain useable products is certainly not new. Since
recorded history, microbes have been involved in the preparation and processing of items in man's
daily diet.
Lacking any knowledge of microorganisms, or of ways in which contamination of food by them
could be avoided, man learned to live with microbially infected foods. Usually the actions of these
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microbes ultimately made the food unacceptable, either by altering the appearance or odor of the
food to a point which it was no longer appetizing or by producing poisonous toxins, some of which
were lethal. Occasionally, however, microbial infections of food materials made it appear more
appetizing and the taste enhanced. Ultimately, microbial infections of these foods were exploited, so
the fermented foods and beverages now form a large and important sector of the food industry.
Today the main groups of microbes involved in the industry include the yeasts, molds, and bacteria.
Nobody knows exactly when cheese making began, but legend generally has it that its origin
lies in the Middle East. A Bedouin, preparing for a journey across the desert, filled his skin pouch
with ewe's milk for refreshment along the way. After hours in the hot sun, and weary from the jostling
ride on the camel, the Bedouin opened the pouch made from the dried stomach of a sheep only to
discover that the rich milk was no more. In its place lay a thin watery fluid surrounded by a thick
white mass - whey and curds. Having nothing else to drink, he tried the liquid and found it tasted
good; then he nibbled at the gummy curds and was equally pleased with the discovery. Arriving at
his destination, he shared the remaining curds with his tribesmen, who were no less satisfied then
he. Thus, quite by accident, cheese was introduced into man's diet. Today, the manufacture of
cultured dairy products represents the second leading fermentation industry (next to alcoholic
beverages), accounting for approximately 20% of all fermented foods produced world wide.
While no such legend exists for the discovery of sauerkraut, it undoubtedly was also
discovered by accident and trial and error methods.
In the days before refrigeration facilities became available, a number of techniques were
devised for preserving seasonally produced vegetables. One of the most efficient of these involved
packing vegetables tightly in a vessel with salt or brine. This technique is thought to have originated
in the Orient where, even today, it continues to be used extensively. Only in the last 60 years has it
been shown that this method of preserving vegetables involves a microbiological fermentation.
In cheese making, the mystery surrounding the nomad's discovery can be easily explained.
The four essentials of cheese making were acting together that memorable day in the desert: milk
plus a slight churning motion coupled with heat and rennet (the product of an enzyme produced in
the membrane lining of ruminate animals stomach). The cheese discovered by the Bedouin was
probably what we would call cottage cheese or cheese curds. Similar legends attend the origin of
aging, curing or ripening which has lead to the many various cheese flavors we have today.
We now know that in the production of sauerkraut, lactic acid bacteria proliferate in the brine.
These bacteria produce acids which lower the pH. The combined action of the salt and acid lowers
the activity of enzymes responsible for the breakdown of vegetable tissue. At the same time oxidative
changes in the tissues are inhibited and thus prevent spoilage.
Anaerobic Fermentation
Although respiration and breathing are often thought of as the same, they are in fact two
different processes. Breathing is the exchange of gases between an organism and its external
environment. Respiration occurs within all living cells. Cellular respiration involves breaking the
chemical bonds of organic molecules and releasing energy that can be used by the cells.
enzymes
ATP
C6H12O6 +
6O2
6CO2 +
energy

Most students are not familiar with fermentation which occurs in some of the less complex organisms
such as bacteria and yeasts. Fermentation reactions are anaerobic, proceeding without oxygen
being present. Anaerobic reactions involve cellular food products and/or glucose sugar as their
Page 6

reactants. And without oxygen they can produce combinations of ethyl alcohol (C2H5OH), carbon
dioxide (CO2), and lactic acid (C2H4OCOOH) as their products.
We have used the products of anaerobic respiration (fermentation) to our advantage,
supplying ourselves with food, drink, and even fuel for automobiles. Yeasts are used as tiny
"fermentation factories" producing carbon dioxide and alcohol. Certain bacteria and molds ferment
milk, producing carbon dioxide and lactic acid.
It has been stated that the fermentations are the result of growth of bacteria, yeasts, molds, or
combinations of these. Stated more precisely, the changes that occur are caused by the enzymes
liberated by these microorganisms. Some foods usually said to be fermented are actually cured by
the enzymes naturally inherent in the foods. Throughout the centuries fermentation has been one
of the most important methods for preserving food; It still remains one of the most important
methods. Relatively few people, however, are aware that the many food products consumed
regularly are prepared and/or preserved by fermentation processes.
It is essential to understand that the lactic acid- bacteria produce acid which in effect inhibits
the growth of many other organisms. Most species convert sugars to acids, alcohol, and carbon
dioxide. The fermentative yeasts produce ethyl alcohol and carbon dioxide from sugars. They
require oxygen for growth but not for fermentation. The molds have the greatest array of enzymes,
are aerobic, and will grow on most foods to produce various types of digestion.
The changes that occur during fermentation of foods are the result of the activity of enzymes.
The enzymes arise from three sources: Those that are produced by the microorganisms that are
involved in the fermentation, those that are native to the food, and those that are produced by the
microbial flora that happen upon the unfermented food. A good fermentation is one in which the
enzymes produced by the fermentative microorganisms play the primary role.
There are relatively few pure culture fermentations. An organism that initiates a fermentation
will develop until its by-products of growth inhibit further growth and fermentation. During this initial
growth period other organisms develop. They in turn are followed by other more tolerant species.
This succession of growth of different species may be referred to as a natural sequence of growth.
The use of starters or inocula should be based upon these facts. In general, growth will be initiated
by bacteria, followed by yeasts and then molds, if conditions are suitable for growth of these
microorganisms.
Now let us try to relate these biological processes to biotechnology. What is biotechnology?
"In the broadest and simplest terms, biotechnology is defined as the collection of industrial
processes that involve the use of biological systems."(Harlander, 1991).
We have been using bacteria, yeasts, and molds for centuries to produce a host of fermented
foods including buttermilk, yogurt, sour cream, butter, cheese (over 700 kinds), pickles, sauerkraut,
sausage, breads, crackers, pretzels, doughnuts, grape nuts (you thought it was a cereal brand
name?), wines, beer, spirits, soy sauce, coffee, cacao, vanilla, tea, citron, ginger, and more.
Biotechnology is also used in some food processing related areas including processing aids,
ingredients, rapid detection systems, and biosensors. Enzymes acting as protein catalysts, are used
extensively in the food processing industry to control texture, appearance, and nutritive value, and
for the generation of desirable flavors and aromas. Because they are isolated from plants, animals,
or microorganisms, their availability is dependent upon the availability of the source material. Using
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genetically engineered microorganisms for the production of enzymes eliminates the need to rely on
source materials while ensuring a continuous supply of enzymes (Harlander, 1991).
The new technologies have allowed researchers to target the genetics of plants, animals, and
microorganisms and to manipulate them to our food production advantage. What might be in store
for tomorrow's food advancement? Predictions include:
1. Environmentally hardy food-producing plants that are naturally resistant to pests and diseases
and capable of growing under extreme conditions of temperature, moisture, and salinity.
2. An array of fresh fruits and vegetables, with excellent flavor, appealing texture, and optimum
nutritional content, that stay fresh for several weeks.
3. Custom designed plants with defined structural and functional properties for specific foodprocessing applications.
4. Cultures of microorganisms that are programmed to express or shut off certain genes at specific
times during fermentation in response to environmental triggers.
5. Strains engineered to serve as delivery systems for digestive enzymes for individuals with
reduced digestive capacity.
6. Cultures capable of implanting and surviving in the human gastrointestinal tract for delivery of
antigens to stimulate the immune response or protect the gut from invasion by pathogenic
organisms.
7. Microbially derived, high-value, "natural" food ingredients with unique functional properties.
8. Microsensors that accurately measure the physiological state of plants; temperature-abuse
indicators for refrigerated foods; and shelf-life monitors built into food packages.
9. On-line sensors that monitor fermentation processes or determine the concentration of nutrients
throughout processing.
10. Biotechnologically designed foods to supply nutritional needs; meat with reduced saturated fat,
eggs with decreased levels of cholesterol, and milk with improved calcium
bioavailability.(Harlander, 1991)
Additional Information on Food Fermentations
Sausage
Semi-dry fermented sausages
Summer sausage: Pediococcus cerevesiae and Lactobacillus plantarum
Dry fermented sausages
Pepperoni: Pediococcus cerevesiae and Lactobacillus plantarum
Genoa salami: Micrococcus spp. mixed with either Pediococcus cerevesiae or
Lactobacillus plantarum
Snack sticks: Pediococcus cerevesiae and Lactobacillus plantarum
Lactic acid is produced, lowering the pH of the sausage to preserve and flavor.

Page 8

Enzymes
Chymosin
Replaces rennet in over 80% of the cheese produced. Rennet is extracted from the
stomachs of milk-fed veal calves. The supply is linked to the veal market, causing wide
shifts in availability and price of the rennet. Recombinant chymosin is produced by an
E. coli. The gene for chymosin was transferred from a calf to E. coli.
Amylase
Amylase is the enzyme that will break starch into its separate glucose components.
Amylase is used in the brewing industry for malting and used in baking.
Glucose isomerase
The enzyme glucose isomerase converts or isomerizes glucose,(an aldehyde), to
fructose, (a ketone), which is a sweeter product. The fructose is 1.8 times sweeter than
glucose, so less is needed for the same taste.
Pectinase
Pectinase is the enzyme that breaks down pectin a polysaccharide found in fruit.
Pectinase is used to remove particulate matter, or clarify, fruit juices.
Glucose oxidase
Glucose oxidase is an enzyme used in the production of dried egg whites. When
drying egg whites the glucose present in the white can react with amines in a reaction
known as Maillard browning. This will cause the dried egg whites to turn brown. The
addition of glucose oxidase will breakdown the glucose and prevent the glucose from
reacting and causing the off color of the egg whites.
To produce these recombinant enzymes, the gene is first transferred into bacteria. The
bacteria are then grown in fermenters. The enzyme is then purified and sold.
Other additives
Commercial Gums: (Dextran, Gellan, Rhansan, and Welan)
thickeners and stabilizers
Xanthan gum:
Xanthan gum is a polysaccharide produced by the bacterium Xanthomonas campestris
on the cell wall. Xanthomonas campestris occurs naturally on the leaves of plants in
the cabbage family. Commercially xanthan gum is produced by aerobic submerged
fermentation. The bacteria are mixed with sugar, a nitrogen source, trace elements,
and other growth factors in a large stainless steel tank. During fermentation aeration,
agitation, pH, and temperature are precisely controlled. After fermentation the solution
is pasteurized to kill all bacteria. The gum is separated using alcohol to precipitate the
gum. Xanthan gum is used in many products including cakes, muffins, ice cream,
sherbert, sour cream, salad dressings, sauces, gravies, syrups, and toppings. Taken
from "Xanthan Gum, 5th Edition", by Kelco, 500 W. Madison, Suite 3180, Chicago, IL
60661, phone 1-800-535-2687

Page 9

Diagram of respiration and fermentation

CELLULAR
RESPIRATION

ALCOHOLIC
FERMENTATION

LACTIC ACID
FERMENTATION

GLUCOSE

PYRIVIC ACID

CO2
+
WATER
+
38 ATP

GLUCOSE

PYRUVIC ACID

CO2
+
ALCOHOL
+
2 ATP

GLUCOSE

PYRUVIC ACID

LACTIC ACID
+
2 ATP

VI. LABS INCLUDED FOR USE IN THIS UNIT

Root Beer
demonstrate the action of yeast on a mixture of sugar, water and flavorings through aerobic and
anaerobic respiration

materials:
Bottles--washed and sterilized
Wine corks or caps and crowns
Stirring spoon
Large (20 liter/5 gallon) enamel kettle or pot--DO NOT USE ALUMINUM!!
*59 ml Schillings root beer concentrate
*2.27 kg Sucrose (table sugar)
*19 l Chlorine-free water
*Containers to measure out needed volumes of materials--your choice--see procedures
*9.5 g Yeast dissolved in 236 ml warm water
*Use proportions to suit needs

possible variations:
Flavorings other than Schillings Root Beer Extract
Amount of sucrose
Amount of root beer extract
Quick rise yeast vs. normal
Amount of yeast
Different sources of sugar (ie.: dextrose)
(Nancy Heitel, et. al., 1988)

Making cheese
prepare cheese from milk and buttermilk
Page 10

materials:
500 ml whole milk
50 ml, 500 ml, &600 ml measuring devices
hot plate
thermometer
fine-mesh cheesecloth
cotton twine
labels
50 ml buttermilk
50 ml, 500 ml, &600 ml containers

variations:
Milk--goat, sheep, 2%, 1%, skim, cream enriched
Rennin/rennet addition at day 1, step 1
Specific bacteria inoculation
Mold inoculation at day 1, step 1
Addition of flavorings/salt/colorings

Kimchee
using 2 liter bottles to study lactic acid fermentation

note to teacher:
Cabbage should be kept covered with its own juices at all times. Gas bubbles will escape each
day as the lid is pressed down onto the cabbage. This gas is produced as bacteria grow on the
sugary contents of the Chinese cabbage juice in the salty solution. As pickling proceeds, there
will be an increase in and change in acidity. Windows to air out classroom would be nice to have
when doing this lab!! Let students know that anaerobic lactobacilli are found almost everywhere
in our environment.

materials:
Chinese cabbage (cut into 5-7 cm chunks)
1 red hot chili pepper, chopped
2 cloves garlic, thinly sliced
3 tsp. non-iodized (pickling) salt
one 2 liter soda bottle
large plastic lid (petri plate lid)
pH indicator paper
small plastic pipette

variations:
different cabbage types
change seasonings or ingredients
place bottles in various temperatures

reference:
1990 Fast Plants & Bottle Biology Projects, Department of Plant Pathology, University of
Wisconsin, 1630 Linden Dr., Madison, WI 53706.

Sauerkraut
lactic acid fermentation of cabbage

Note to Teachers:
Containers used in making kraut should be cleaned and rinsed well. Crocks should have
shiny glaze to the surface, and not be cracked or chipped. Metal containers are definitely not
Page 11

to be used. If plastic is used, only use food-grade plastic. The dyes in a nonfood plastic are
not intended for food use, and should not be used for food.

materials:
Cabbage sliced into thin strips
Non-iodized salt
Container (Consider the fact that cabbage will require anaerobic conditions while fermenting
in this container. If a fermenting crock is your container of choice, be careful that it is not
chipped or cracked. Food-grade sturdy plastic pails are excellent containers. Do not use
metal containers of any type)
variations:
Different types of cabbage can be used.
Cabbage can be cut into different sizes to see how size varies the resulting product.
Spices or seasonings can be added for a variety of kraut flavors.
Sauerkraut can be canned for long-term storage.
references:
Mennes, Mary E., "Make Your Own Sauerkraut" , Food Science, University of WisconsinMadison, Food Management Specialist, UW-Extension.

Fermenting Power of Bread Yeasts

observing the raising quality of different types of bread yeast

materials
50 or 100 ml/graduated cylinder (to measure 30 ml distilled water)
100 ml graduated cylinder, greased with Vaseline
Flour
Two brands, A and B, of active dry yeast. (either A or B should be a yeast cake)
Saccharomyces cerevisiae, young streak culture (solid medium)
Square sheets of brown wrapping paper
Buffered methylene blue stain:
Mix 1 part of 1:5,000 methylene blue & l part of a phosphate buffer solution (99,75 ml of
0.2 M KH2PO4 to 0.25 ml of 0.2 M Na2HPO4) to give pH of 4.6.
microscope
microscope slides & coverslips

variations
Different brands of yeast
Different forms of yeast
Rapid Rise vs. normal
Different temperatures

References and Resources:

Boyd, Robert F. 1988. General Microbiology 2nd ed. Times Mirror/Mosby College
Publishing, St. Louis.
Frazier, W.C., and D.C. Westhoff. 1978. Food Microbiology, Third Edition, McGraw-Hill, Inc.,
New York.
Grula, Dr. Mary M., Oklahoma State University, Dept. of Botany/Microbiology,
Stillwater, OK 74078-0289.

Page 12

Yogurt
Simple lactic acid fermentation

materials:
Food grade containers, washed pH paper or meter
Food grade thermometer
Microscope slides
Hotplate
Bunsen burner
Beaker tongs
Innoculating loop (a toothpick will work)
2 cups milk
Crystal violet stain
1/3 cup nonfat dry milk
Microscope (oil immersion if available)
2 tablespoons plain yogurt (Old Home Black Label works well)

Possible variations:
Type of milk (whole, 2%, skim, chocolate)
Different temperatures for incubation
Different brands of yogurt for starter culture
Different levels of starter
Different levels of non-fat dry milk

Adapted from:
"Bacteria in Yogurt" pages 44-47 of Laboratory Experiments in Biotechnology and Related
Areas Volume III: Experiments with Microorganisms, Dept. of Biological Sciences, Mankato
State University, Mankato MN, 1988.
"Bacteria in Yogurt" pages 5-6 of The BioNet Booklet,Volume 3, Spring 1995, BioNet c/o Rod
Johnson, Eau Claire North High School, 2700 Mercury Ave, Eau Claire, WI 54703.
"Homemade Yogurt, Sour Cream, and Buttermilk" by M. Wagner, R. Bradley, and M. Mennes,
University of Wisconsin Extension publication B2768, available from Agricultural Bulletin, Rm.
245, 30 N. Murray St, Madison, WI, 53715, 608-262-3346.

Yeast Fermentation, Variation in CO2 production by yeast

glucose consumption and CO2 production by yeast

materials:
15 mL plastic centrifuge tubes with caps
7% yeast solution (4 packages yeast and 400 mL water)
5% glucose solution (20 grams glucose in 400 mL water)
TES-TAPE (available at Wal-Mart and pharmacies)
large beakers (250 to 500 mL)
water bath at 40 degrees C
permanent fine point lab markers

possible variations:
regular vs. Rapid-Rise yeast
levels of sugar
type of sugar
other temperatures (ice water, boiling water)

adapted from
Fermentation, Respiration, and Enzyme Specificity: A Simple Device and Key Experiments
with Yeast, by L.Reinking, and K. Miller, The American Biology Teacher, Vol. 56, March
1994, pp. 164-168.
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Alternative labs:
"Dinner Date with a Microbe" by A. Gillen and R. Williams, The American Biology Teacher, May
1993, pages 268-274. Provides kitchen microbiology recipes and health information for yogurt,
sauerkraut, and root beer.

Silos and Sauerkraut from The AgriScience Institute and Outreach Program published by the
National Association of Biology Teachers, 1994, pages 5-1 to 5-28. Bottle biology labs for making
mini silos and fermentation chambers.

VII. EVALUATION GUIDELINES FOR STUDENT ACTIVITY

Students will be evaluated on their daily journal writings, their lab techniques, the written
presentation, and the oral presentation.

The daily journal writings will be evaluated on the answer to the daily focus question, and a
detailed description of all lab procedures and observations. The journal must also include self
evaluations, group evaluations, definitions to vocabulary words, and summaries of at least three
recent articles related to their topic.

The oral presentations will be evaluated on the delivery and content. The content must include
procedures, results, and a description of two careers related to the topic area.

The written presentation must include a more detailed description of the information from the oral
presentation plus a section describing the importance of fermentation to the economy of the area.

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UNIT EVALUATION SHEET

NAME_______________________

PROJECT GRADE ________

CRITERIA

PERCENTAGE
GUIDELINE

Laboratory setup

PERCENTAGE
RECEIVED

Daily Journal
focus questions
record of lab procedures and observations
peer grade
response to outside activities

---------10
15
5
5

----------

Glossary

Article summaries

15

Unit summary

15

Presentation
written
oral

---------15
15

----------

Extra credit (point total up to teacher)

multiple variations of experiments
current event articles (3 minimum)
find information on schools or careers in biotech

----------

----------

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UNIT GUIDELINES
Daily Journal:
In your daily journal you are required to include:
the date
response to the day's focus question
a record of all lab procedures and observations
any questions that arise during the period
all unit vocabulary words (on glossary page)
self and group evaluation
response to non-lab activities including guest speakers, case studies, field trips,
summaries of at least three articles related to your topic area or fermentation taken from
recent (within 2 years) publications
Group Presentation:
The 5-10 minute oral portion will include:
an explanation of your lab procedures
a summary of your lab results
a brief overview of two careers associated with your topic area
The 4-8 page written report will include:
an introduction to your topic record of lab activities including materials, procedures followed,
observations, and conclusions descriptions of at least two careers in your topic area
importance of fermentation to the economy of the area, state, or country

Page 16

STUDENT PROPOSAL SHEET FOR FERMENTATION LAB

Name:________________
Who is the leader of your laboratory group?
What will you vary in your experimental project(s)?

What is your hypothesis for the effects your variable will have on each experimental project as
opposed to the control?

What steps will you follow to complete your projects?

What materials will each member of the group be responsible for?

Page 17

STUDENT DAILY JOURNAL INFORMATION SHEET

Every day of this unit you are expected to write at add at least one page to your journal. Each day
you should record what you do and what you see.

You are expected to:

Each day summarize the days activities in complete sentence/paragraph form. Be thorough.
Include procedures and pertinent vocabulary as well as any questions which you come up with.

*answer the days focus question found on the board.

*record physical data such as what you see, what you smell, etc.
*record what your group does with your project each day.
*add the word(s) of the day to your glossary. Use this/these words in your entry for the day.
*individually evaluate yourself/your partners effort for the day on a
Scale from 1-5. (1=no effort, 5=high effort)

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DAILY JOURNAL PAGE

Name: _________________________
Date: ___________________

Response to today's focus question:

Record of today's procedures and observations:

Any questions from today's class:

Group evaluation (1-5). Place your partners names in the blanks.

Your name: ____________________________

Your score: _____

Partners name: _________________________

Partners score: _____

Partners name: _________________________

Partners score: _____

Partners name: _________________________

Partners score: _____

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GLOSSARY SHEET
Write in vocabulary terms associated with this unit and define.
UNIT TITLE_________________________

Page 20

SAMPLE DAILY FOCUS QUESTIONS

Why is it important to lower the pH of fermented foods?
How do fermentation and oxidative respiration differ?
Why is the legend of the Bedouin and his discovery of cheese plausible?
Why might have the development of fermented foods progressed relatively slow until the 20th
century?
What is the best way to avoid a Salmonella infections?
What are some of the industries built on fermentation?
What is the chemical equation for fermentation?
What is the difference between breathing and respiration?
List several foods which that are produced by fermentation?
What factors contribute to food spoilage?
How is food safety monitored?
Can you tell if food is safe just by the smell and appearance? Why or why not?

Page 21

SAMPLE VOCABULARY WORDS

fermentation
respiration
biotechnology
enzyme
lactic acid
alcohol
microbe
bacteria
yeast
anaerobic
aerobic
pathogen

Page 22

VIII. FULL LABORATORY EXERCISES

ROOT BEER PRODUCTION.

Introduction:
In this laboratory you will be demonstrating the action of yeast on a mixture of sugar, water, and
flavorings through both aerobic and anaerobic respiration. Your yeast are microbes that will break
down sugar into water and carbon dioxide--the latter causing the root beer to become carbonated.
The oxygen present will eventually be depleted, causing the yeast to revert to anaerobic respiration.
Be sure to follow aseptic technique and use only food-grade containers for this experiment. You will
be responsible for making a control sample following the formula below and at least one other batch
of root beer in which you have changed one factor. You need to hypothesize what differences your
experimental group will have as compared to your control. You must submit a proposal on what your
experimental group(s) will entail before beginning this laboratory work.
Materials:
Bottles--washed and sterilized
Wine corks or caps and crowns
Stirring spoon
Large (20 liter/5 gallon) enamel kettle or pot--DO NOT USE ALUMINUM!!
*59 ml Schillings root beer concentrate
*2.27 kg Sucrose (table sugar)
*19 l Chlorine-free water
*Containers to measure out needed volumes of materials--your choice--see
procedures
*9.5 g Yeast dissolved in 236 ml warm water
*see Caution 2. below
Cautions:
1. Wash hands thoroughly with antibacterial soap and water before and after completing each step
of the laboratory.
2. Alter the volumes and measures (by using proportions) to best suit your needs.
3. Wear goggles and laboratory apron.
Control Group Procedure:
Day 1
1. Make sure all materials, equipment, and your hands are as clean as possible. Wash hands
before handling any materials.
2. Place sucrose and root beer extract into kettle
3. Gradually add chlorine free water
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4. Add yeast/warm water mixture and stir well.

5. Immediately place mixture in clean/sterile bottles, leaving approximately a 5 cm airspace at the
top.
6. Tightly cork or seal the bottles and place them on their sides in a warm (25-30oC) location.
Day 2

Record observations

Day 3

Record observations

Day 4

Once refrigerated, your root beer should be ready for consumption!!

Adapted from:
Nancy Heitel, et. al., "Production of Home Brewed Root Beer," Mankato State University,
Mankato, MN, 1988

Page 24

CHEESE MAKING
Introduction:
In this activity you will be fermenting milk products. The action of bacteria in milk causes a buildup of
lactic acid. This lactic acid causes the milk to curdle, forming a solid curd, and a liquid whey. The
curd is then separated from the whey and aged to make cheese. The physical characteristics of the
cheese depend on many factors. Below is a basic recipe for making cheese. You will be
responsible for making a control sample following this formula and at least one other batch of cheese
in which you have changed one factor. You need to hypothesize what differences your experimental
group(s) will have as compared to your control. Be sure to follow aseptic technique and use only
food-grade containers for this experiment. You must submit a proposal on what your experimental
group(s) will entail before beginning this laboratory work.
Materials:
500 ml whole milk
hot plate
fine-mesh cheesecloth
labels
50 ml, 500 ml, & 600 ml containers

50 ml buttermilk
thermometer
cotton twine
50 ml, 500 ml, & 600 ml measuring devices

Cautions:
1. Wash hands thoroughly with antibacterial soap and water before and after completing each step
of the laboratory.
2. Be aware of shock and burn hazards when using the hot plate.
3. Wear goggles and laboratory apron.
Control Group Procedure:
Day 1
1. Make sure all materials, equipment, and your hands are as clean as possible. Wash hands
before handling any materials.
2. Pour 500 ml whole milk into your 600 ml container and 50 ml buttermilk into your 50 ml container.
3. Heat the whole milk to 37C.
4. Add the buttermilk to the whole milk and stir well.
5. Cover the container with cloth or paper.
6. Incubate at between 25oC and 35oC for 48 hours or until a firm curd has separated from the
whey.
Day 2
1. Prepare a piece of cheesecloth that will be thick enough and large enough to hold your curd.
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2. Pour your curd into the cloth. Collect the whey in the 500 ml container. Gather the edges of the
cloth to form a bag. Tie the bag with the twine and hang it to continue draining. After it has fully
drained, discard the whey and place the bag in the refrigerator.
Day 3

No work necessary today.

Day 4

Remove the cheese and enjoy!

Adapted from:
Pam DiFilippo, San Juan High School, Citrus Heights, CA, "Making Cheese," Biotechnology
Manual, Addison Wesley.)

Page 26

KIMCHEE: KOREAN DELIGHT

FERMENTATION EXPERIMENTS WITH BOTTLE BIOLOGY
Introduction:
Pickling is one of the most ancient forms of preserving food. You will be using microbes to convert
sugars into lactic acid. The microbe involved is called lactobacilli. As the population of lactobacilli
grows, they eat the natural sugars in plant juices and produce lactic acid as a waste product. As the
lactic acid levels increase, so does the acidity. This highly acidic environment prevents the growth of
other bacteria that would under normal conditions feast on the food causing spoilage. In this lab,
you will be making kimchee and study lactic acid fermentation.
Materials:
Chinese cabbage (cut into 5-7 cm chunks)
1 red hot chili pepper, chopped
2 cloves garlic, thinly sliced
3 tsp. non-iodized (pickling) salt
one 2 liter soda bottle
large plastic lid (petri plate lid)
pH indicator paper
small plastic pipette
Procedure:
1. Cut the bottle top off from base 10-15 cm from the top.
2. Alternate layers of cabbage, garlic, pepper and sprinkling of salt in the soda bottle. Press each
layer down firmly until the bottle is packed full.
3. Place the lid, rim side up, on top of ingredients and press down again.
4. Press down occasionally for the next few hours. When there is space, fit the bottle top inside the
bottle bottom, forming a sliding seal. Air should bubble out around the edge of the petri plate
when you press down on the lid. Press daily on the sliding seal to keep the cabbage covered by
a layer of juice at all times
5. Measure and record the acidity of the fresh juice on top each day with a pH indicator paper.
6. When the pH drops to 3.5, your kimchee will be ready. (About 3-7 days) Enjoy!
Cautions:
When working with chili peppers, take care not to touch eyes of mouth. Wash hands thoroughly
when finished.
Bottle used to ferment kimchee in should be cleaned and rinsed well.

Page 27

MAKE YOUR OWN SAUERKRAUT

Introduction:
Sauerkraut is a naturally fermented cabbage. Natural fermentation is one of the oldest means of
food preservation, and reduces the risk of foodborne illness and food spoilage. The juice extracted
from shredded cabbage by adding salt* contains fermentable sugars, and in the absence of air, the
microorganisms feed on the cabbage leaves and will produce lactic acid. This lactic acid creates
and acidic environment unsuitable for other organisms to survive. In this lab you will make your own
sauerkraut.
*The salt used should be a non-iodized pickling or canning salt. Iodine, which is in table salt,
prevents the bacterial fermentation necessary to change cabbage into sauerkraut.
Materials:
Cabbage sliced into thin strips
Non-iodized salt
Container (Consider the fact that cabbage will require anaerobic conditions while fermenting in this
container. If a fermenting crock is your container of choice, be careful that it is not chipped or
cracked. Food-grade sturdy plastic pails are excellent containers. Do not use metal containers of
any type)
Procedure:
1. Clean off cabbage head to remove residual insecticide spray or dust. There are important
bacteria existing on the cabbage leaves which are necessary in the fermenting process,
therefore, do not overclean the head.
2. Cut, slice, or shred the cabbage.
3. Place cabbage in container and sprinkle with salt. Add 2.25 to 2.5 percent salt by weight.
4. Cover and weight down the cabbage to produce anaerobic conditions for the fermenting process
to take place.
5. Set the cabbage back and allow fermentation process to proceed. Remember the effects
temperature can play on fermentation.
6. Check the container daily for film yeasts or molds which may appear on the surface. This can be
removed by skimming the surface of the cabbage. Kraut should be ready in 3 to 4 weeks.

Page 28

FERMENTING ("DOUGH-RAISING") POWER OF BREAD YEASTS

Introduction:
Bread dough is usually leavened by bakers' yeast (actively gas-producing strains of
Saccharomyces cerevisiae ). Yeasts ferment the sugar in the dough, producing ethanol and carbon
dioxide. CO2 is the leavening agent and the alcohol evaporates off during baking. Sometimes other
gas-producing microorganisms are involved in bread leavening--these usually are heterofermenting
lactic acid bacteria (sourdough bread or salt-rising bread)
Commercial yeast is prepared and sold in two forms: yeast cakes and active dry yeasts.
Yeast cakes contain, in addition to yeast cells, small amounts of starch, vegetable oils, and some
lactic acid bacteria. Active dry yeast is made by drying the yeast cells to less than 80% moisture.
These yeast cells are dried carefully at low temperatures so the cells will survive. When these yeast
cells are stored at room temperature, they will retain their "dough-raising" ability for many months.
You will be studying the difference between the active dry yeast and the yeast cakes.
Materials:
50 or 100 ml/graduated cylinder (to measure 30 ml distilled water)
100 ml graduated cylinder, greased with Vaseline
Flour
Two brands, A and B, of active dry yeast. (either A or B should be a yeast cake)
Saccharomyces cerevisiae, young streak culture (solid medium)
Square sheets of brown wrapping paper
Buffered methylene blue stain:
Mix 1 part of 1:5,000 methylene blue & l part of a phosphate buffer solution (99,75 ml of 0.2 M
KH2PO4 to 0.25 ml of 0.2 M Na2HPO4) to give pH of 4.6.
microscope
microscope slides & coverslips
Procedure:
A. Examine yeast cells
prepare wet mounts of yeasts
stain slides using methylene blue stain
microscopically examine yeast cells
record the percent of living cells (will appear white)
record the percent of dead cells (will appear blue )
B. Fermenting ("dough-raising") power of yeasts
combine 50 g flour, 1 g dry yeast A, and 30 ml water on brown wrapping paper knead
vigorously for 5 minutes
--repeat step above using yeast B
--place into separate greased cylinders and record volume
--incubate at room temperature and record volume at intervals of time

Page 29

MAKING YOGURT
Introduction:
Yogurt production demonstrates fermentation by Streptococcus thermophilus and
Lactobaacillus bulgaricus. Heated milk is innoculated and maintained at a given temperature causing
bacteria to grow and ferment lactose, the sugar in milk. The bacteria produce lactic acid which
causes the milk to coagulate and adds a sour flavor.
Be sure to follow aseptic technique and use only food-grade containers for this experiment.
You must submit a proposal on what your experimental group(s) will entail before beginning this
laboratory work.
Materials:
Food grade containers, washed
pH paper or meter
Food grade thermometer
Microscope slides
Hotplate
Bunsen burner
Beaker tongs
Innoculating loop (a toothpick will work)
2 cups milk
Crystal violet stain
1/3 cup nonfat dry milk
Microscope (oil immersion if available)
2 tablespoons plain yogurt (Old Home Black Label works well)
Procedure:
1. Combine milk with nonfat dry milk and heat in a double boiler to 190 F. Hold at that temperature
for 10 to 20 minutes so that the protein in the milk mixture will take up more water and make a
better gel. Cool to 115 F (warm) and record the pH of the mixture.
2. Place the plain yogurt in a jar and gradually blend in the warm milk.
3. Cover. Place in a bowl of warm water (115 F), a slightly warm oven or a styrofoam cooler. The
temperature within the oven or cooler should be about 110 to 120 F to provide optimum
conditions for yogurt culture activity.
4. Allow to stand undisturbed until the mixture is firm when the jar is gently wiggled. This may take
as long as 6 to 8 hours. Note the time so that less care will be needed for the next batch. Record
the pH of the yogurt.
5. Chill yogurt as soon as it is set. It can be stored in the refrigerator for up to 3 weeks.
6. Place a drop of water on the slide. Use the innoculating loop to mix a little yogurt with the water
and spread it around the middle 1/3 of the slide.
7. Let the slide air dry.
8. Quickly pass the slide through the flame of the Bunsen burner 3-4 times.
9. Let the slide cool.
10. Cover the slide with 2-3 drops of crystal violet.
11. After 30 seconds rinse the slide with water.
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12. Examine the slide under the microscope and draw the bacteria.
13. Add 2 tablespoons sugar and 1/4 cup fresh, crushed or frozen fruit .
14. Taste your yogurt. (Do not eat any yogurt that smells or looks bad! If in doubt, throw it out!)

Page 31

YEAST FERMENTATION LAB

Introduction:
During anaerobic conditions high levels of NADH develop, leaving a shortage of NAD+. Low
levels of NAD+ slow the rate of glycolysis. Fermentation restores NAD+ levels while producing alcohol
and CO2.
During aerobic respiration glucose is broken down into water and carbon dioxide.
C6H12O6 + O2 6H2O + 6CO2
Under ideal conditions most eukaryotic cells produce 36 ATP molecules from one molecule of
glucose.
During fermentation baker's yeast breaks glucose into ethyl alcohol and carbon dioxide.
C6H12O6 2C2H5OH + 2CO2
This process only yields 2 ATP per glucose molecule.
Materials:

15-ml plastic centrifuge tubes with caps (Corning #25319)

7% yeast solution (4 packages of yeast and 400 ml of water)
5% glucose solution (5 grams of glucose in 400 ml of water)
TES-TAPE (Lilly, available at Wal-Mart and pharmacies)
large beakers (250 - 500 ml)
Water bath at 40C
permanent fine point lab markers

Prelab preparation:
1. Poke 3-4 small holes in the centrifuge tube caps using a pin or thumbtack
2. Prepare the yeast solution by mixing 4 packages of active dry yeast in 400 ml of tap water
3. Prepare the sugar solution by mixing 20 grams of glucose in 400 ml of tap water
4. Preheat the water bath and the solutions to 40C
5. Fill 12 beakers with water and place in the water bath to preheat
6. Fill 12 beakers with water and leave at room temperature
Procedure:
1. Fill each tube halfway with sugar solution.
2. Fill the rest of each tube with yeast solution, extending the fluid level above the top of the tube.
3. Take a small piece of TES-TAPE and measure the amount of glucose.
4. Screw the cap on the tubes (a few drops will spurt out the holes).
5. Check to make sure there are no bubbles visible in the tube
6. Invert the tubes and mark two tubes L and the other two tubes C
Page 32

7. Place one L tube and one C tube in each beaker

8. Return the 40C beaker to the water bath
9. After 5, 10, 15, and 20 minutes
take the L tubes out of the water,
dry the tops with a paper towel
mark the level of the gas bubbles (include any foam as part of the bubble)
return the L tubes to the beakers
take the C tubes out of the water
turn the tubes upright and remove the caps
take two small pieces of TES-TAPE and measure the amount of glucose
recap the tubes and return to the beakers
10. After 20 min. empty the contents of all tubes into the waste beaker
11. Record the ml of CO2 at each mark
12. Wash and rinse out your tubes
13. Graph the change in glucose and CO2 over time in your journal
14. Answer the following questions in your journal

Why did you have two tubes at each temperature?

What was the variable in this experiment?

What was the gas that formed in the tubes?

Describe an experiment that you could use to test if a different type of sugar would give the
same results.

Variations:

Regular vs. Rapid-Rise Yeast

Levels of sugar
Type of sugar
Other temperatures (ice water, boiling water)

Adapted from:
"Fermentation, Respiration, and Enzyme Specificity: A Simple Device and Key Experiments
with Yeast" by L. Reinking, J. Reinking, and K. Miller, The American Biology Teacher, Vol. 56,
March 1994, pp. 164-168.

Page 33

X
X

X
X

Case Studies

Field Trip

History of Fermentation

Anaerobic Respiration

Yeast Lab

Food Safety Speaker

Group Presentation

Daily Journal

Standard:
Benchmarks
3A
10I
12D
NSE Content Standards
A
B
C
F
G
NSE Assessment Standards
A
B
C
NSE Program Standards
B
NRC Ag Ed Recommendations
Scientific content
School-to-Work
Career exploration
High academic content

Group Fermentation Lab

IX. NATIONAL STANDARDS

X
X
X
X

X
X

X
X

X
X

X
X
X

X
X

X
X

X
X

X
X

Descriptions of the individual standards:

Benchmarks for Science Literacy
3A Technology and Science:
Technology usually affects society more directly than science because it solves practical
problems and serves human needs
10I Discovering Germs:
Pasteur wanted to find out what causes milk and wine to spoil
12D Communications skills:
Locate information in reference books, back issues of newspapers and magazines, compact
disks, and computer disks.
Use tables, charts, and graphs in making arguments and claims in oral and written
presentations.
Page 34

National Science Education Standards

Content
A Science as Inquiry:
Abilities of scientific inquiry
B Physical Science:
Chemical Reactions
C Life Science:
Cell biology
F Science in Personal and Social Perspectives:
Science and technology in local, national, and global challenges.
Personal and community health
G History and Nature of Science:
Historical perspectives
Assessment Standards
A Coordination with Intended Purposes:
Deliberately designed
Explicitly stated purposes
B Measuring Student Achievement and Opportunity to Learn:
Achievement data collected focuses on the science content that is most important for students
to learn
C Matching Technical Quality of Data with Consequences:
The feature that is claimed to be measured is actually measured
Assessment tasks are authentic
Program Standards
B Curriculum
Inquiry is emphasized as a tool for learning science
The curriculum connects to other school subjects
National Resource Council recommendations for agricultural education
Ongoing efforts should be expanded and accelerated to upgrade the scientific and technical
content of vocational education courses.
New curriculum components must be developed and made available to teachers addressing the
sciences basic to agriculture, food, and natural resources
School-to-Work Initiative
School Based Learning
Career exploration to help students in the career identification process
High academic content enabling admission, if desired, to post-secondary education
Applied and integrated technical and academic curriculum

Page 35

X. REFERENCES
Biology, Visualizing Life--Teachers Resource Binder. Holt, Rhinehart and Winston, 1994.
Biology, Visualizing Life. Holt, Rinehart and Winston, 1994.
Brock, Thomas D. and Michael T. Madigan (1988) Biology of Microorganisms, 5th Ed. Prentice Hall,
Englewood Cliffs, New Jersey.
Coghlan, Andy. "An Explosive Start To To Fast Maturing Cheese." New Scientist, March 16, 1991
"Fermentation" , Modern Biology Laboratories, Holt, Rinehart and Winston, Investigation 7.2, p 4548.
Frazier, William C, and Dennis C. Westhoff (1988) Food Microbiology, 4th Ed. McGraw-Hill, New
York.
Harlander, Susan K. "Biotechnology - A Means For Improving Our Food Supply." Food Technology,
April, 1991
Harlander, Susan K. "Food Technology: Yesterday, Today, and Tomorrow." Food Technology,
Septermber, 1989
Harley, J. and L. Prescott, Laboratory Exercises in Microbiology. William C. Brown Publishers,
1993.
Heitel, Nancy, et al. "Production of Home Brewed Root Beer." Minnesota; Mankato State University,
1988
Kurmann, Joseph A, Jeremija Lj. Rasic, and Manfred Kroger (1992). Encyclopedia of Fermented
Fresh Milk Products. Van Nostrand Reinhold, New York.
Lindquist, John (1990) Bacteriology 102 Lab Manual. University of Wisconsin-Madison.
May, Ranee (1995) Special Topics in Food Science. University of Wisconsin-River Falls.
Muriel Mandell, Simple Kitchen Experiments. Sterling Publishing Company, 1993
Oklahoma State University, Dr. Mary Grula, Fermenting Power of Bread Yeasts.
Pearl, Anita May , Cuttle, Constance and Deskins, Barbara B. Completely Cheese , Jonathan David
Publishers, Inc. Middle Village, New York , 1978
Pederson, Carl S. Microbilogy of Food Fermentations. Westport, Connecticut; AVI Publishing Co,
Inc., 1987
Potter, Norman N, and Joseph H. Hotchkiss (1995) Food Science, 5th ed. Chapman & Hall, New
York.

Page 36

Robinson, R.K. Ed. (1995) A Colour Guide to Cheese and Fermented Milks. Chapman & Hall,
London.
Rose, A.H. , Fermented foods , Academic Press , New York, New York , 1982
Towle, Albert , Modern Biology Holt, Rinehart and Winston, New York, New York , 1989
Varnam, Alan H. and Jane P. Sutherland (1994) Milk and Milk Products. Chapman & Hall, London.
Wood, Brian J.B. , Microbiology of Fermented Foods Volume 1, Elsevier Applied Science Publishers,
London and New York , 1985

Page 37