Effect of Heat on the Sterilization of Artificially
Contaminated Water
Catherine D. Groh, Douglas I
NMucPherson, and David]. Groves
Background: The objective of the study was t o evaluate indirect methods commonly used in the field t o decontaminate
water as follows: boiling for 5-10 minutes and heating until ”too hot t o touch.” Water perceived to be “too hot t o touch“
is defined as water in which a subject is unable t o bear having the right index finger fully immersed for 5 seconds.
Methods: Five flasks of water at 25”C,50°C,60”C,70”C, and 100°C were inoculated with 1.82 x lo6 Escherichia coli bacteria.
At the time of inoculation, and at 1 minute, 5 minutes, and 10 minutes, samples were withdrawn from each flask. The
samples were plated and incubated for 18 t o 24 hours. The numbers of colonies were then counted. Finally, subjects
attempted t o immerse, fully, their right index fingers into water at 50”C,55”C,60”C,and 65°C for 5 seconds. Subjects were
normal, healthy individuals who worked at a microbiology laboratory. The main outcome measures were the number of
colonies and the subjects’ intolerance t o heat.
Results: Results showed that water at 50°C has no effect on the number of bacteria, whereas water maintained at 60°C
for 5 minutes and at 70°C and 100°C for any time period effectively kills E. coli bacteria. The majority of subjects found
60°C or cooler t o be “too hot t o touch.“
Conclusions: For travelers or campers, water is safe to drink if heated to boiling, but heating water until it is “too hot t o
touch” is inadequate for safe drinking purposes.
The most common illness in travelers is diarrhea due
to the consumption of contaminated food or water.’ A
common belief is that, in order for it to be made safe to
drink, contaminated water must be boiled for 5 to 10 minutes. The standard recommendation is to boil water for
10 minutes and then to add 1 minute for every 1000 feet
in elevation.’ However, there is evidence that these practices may be excessive. Neumann suggested that water
that is “too hot to touch” is safe to drink,3 while Bandres and colleagues found that heating water to 75°C
killed all detectable b a ~ t e r i a . ~
This study was designed to determine the ability of
heating to sterilize water artificially contaminated with
Escherichia coli bacteria. E. coli was chosen because it is the
most common cause of traveler’s d~arrhea,~
and it has been
shown to be one of the most heat resistant organisms.‘
In order to determine whether water that is “too hot to
touch” is hot enough to kill E. coli bacteria, the temperature of water that people found “too hot to touch”
was assessed.
Methods
Heat Killing of Bacteria
The E. coli strain used in this experiment was a
clinical isolate recovered in our laboratory. All heat-lull
experiments were conducted in Hamilton, Ontario (elevation of 100 meters) at an ambient pressure of 98.7 kPa.
Escherichia coli was grown in trypticase soy broth for 18
to 24 hours at 37’C.The broth was centrifuged for 20
minutes at 2000 g in a swing bucket centrifuge.The liquid phase was discarded, and the pellet resuspended in
5 mL of phosphate buffered solution (PBS). This suspension was thoroughly mixed, and 1 mL was withdrawn
and added to 4 mL PBSA McFarland standard was used
to approximate the concentration of bacteria to lo8per
mL. One mL. was removed and added to a 9 mL dilution blank. (This 1 : l O dilution is the stock inoculum and
has a concentration of approximately lo7per mL.)
To determine the number of organisms per mL,
0.1 mL of stock was diluted in a 0.9 mL dilution blank.
Six serial 1 in 10 dilutions were made.All subsequent steps
in this experiment were conducted in duplicate. A volume of 0.1 mL was removed from the
to
dilution tubes and plated on McConkey’s agar.The plates were
Catherine D. Groh, B.ArtsSc. (Hon), BSc: Faculty of Health
Sciences, McMaster University, Hamilton, Ontario, Canada;
Douglas W. MacPherson, MD, MSc (CTMI, FRCPC and David
J. Groves, PhD, ScM (CCM), Dip/ (ABMM): Faculty of Health
Sciences, McMaster University, Hamilton, Ontario and
Service of Medical Microbiology, St. Joseph’s Hospital,
Hamilton, Ontario, Canada.
Paper presented at the Fourth International Conference on
Travel Medicine, Acapulco, Mexico, April 23-27, 1995.
Reprint requests: D. W. MacPherson, Regional Parasitology
Laboratory, St. Joseph’s Hospital, 50 Charlton Avenue East,
Hamilton, Ontario, Canada L8N 4A6
J Travel Med 1996; 3:ll-13.
11
J o u r n a l of T r a v e l M e d i c i n e , V o l u m e 3 , N u m b e r 1
12
Table 1 Concentration of Bacteria in Flasks
Time'
Temp ("C)
25
50
60
70
100
(Min)
Flask
A'
B
A
B
A
B
A
B
A
B
TO
4.2 X
4.0 x
1.6 X
3.0 X
1.6 X
>3.0 X
104/mL
104/m~
10"/rnL
10'/mL
IO'/mL
1O3/mL
0
0
0
0
TI
2.4
4.6
2.4
1.1
X 10'/mL
1OJ/mL
10'/rnL
104/mL
0
2.5 X Io'/mL
X
X
X
T5
2.4
1.6
2.2
3.0
X
lO'/mL
x IO'/mL
X
10'/inL
x lO3/mL
0
0
0
0
0
0
0
0
0
0
'Time of inoculation exposure to temperature X; +A and B are the two flasks at each temperature
incubated for 18 to 24 hours at 37"C, and then the
colonies counted.
The flasks to be inoculated were prepared in the following manner: each was filled with 100 mL distilled
water and was autoclaved for 15 minutes.The water in
the first flask was allowed to reach room temperature. In
water baths, the water in the second, third, fourth, and
fifth flasks was heated to 50,60,70, and 100"C, respectively.The flasks were maintained at these temperatures
throughout the experiment.
The first flask was inoculated with 0.5 mL of the
stock E. coli culture (approximately 5 X 10" bacteria per
inoculum). Immediately upon inoculation, at 1 minute,
5 minutes, and 10 minutes, 0.001 mL was withdrawn
using a calibrated loop and then plated on McConkey's
agar.The plates were incubated at 37°C and the colonies
counted, by direct visual inspection of the plates, 18 to
24 hours later.
The process of inoculation and withdrawal of samples was repeated for the flasks at 50,60,70,and I 00OC.
A volume of 0.001 mL was withdrawn from the flasks
at 50"C, and 0.1 mL was withdrawn from the flasks at
60°C. For the flasks at 70°C and 100"C, 10 mL was
removed and chilled, then centrifuged at 1500 g for 15
minutes.The pellet was resuspended in 0.1 mL, which
was then plated on McConkey's agar.
Determination of "Too Hot to Touch"
"Too hot to touch" was defined as the temperature
ofwater in which a subject was unable to keep the right
index finger fully immersed for at least 5 seconds.This
definition was chosen because of the necessity to standardize the length of time of immersion, since the ability to tolerate a temperature is proportional to the
amount of exposure to the temperature. Five seconds was
chosen because that length of time was feasible to measure with reasonable accuracy both in the laboratory and
in the field.
Using water baths and hot plates, four beakers were
heated to four different temperatures: 50"C, 55"C, 60"C,
and 65°C. Each subject was asked to fully immerse the
right index finger in the 50°C beaker for 5 seconds. If
the subject was able to do this, he or she then cooled the
finger in cool water, so that the finger quickly returned
to normal or near normal skin teniperature.The subject
then attempted to inimerse the sanie finger in 55OC
water.This process continued until the person was unable
to immerse his or her finger in a beaker for the required
time. The water temperature of the contents of this
beaker was then recorded as "too hot to touch" for that
subject.The subjects were not blinded to the experiment.
Results
Heat Killing of Bacteria
The results of E.coli survival in heated water are
shown in Table 1. Escherichia coli were able to survive at
all times in water at room temperature and at 50°C. At
6OoC,they were able to survive for less than 5 minutes
of exposure.They were unable to survive for any length
of time at 70 or 100°C.
Determination of "Too Hot to Touch"
All 15 ofthe 15 subjects tested were able to immerse
their right index finger in water at 50°C for 5 seconds,
but only five were able to immerse their fingers in water
at 55"C, and three were able to immerse their fingers in
the 60°C water. None was able to ininierse their fingers
in the 65OC water.
Discussion
This study shows that water heated to 50°C has no
significant effect on the survival of E. coli bacteria. However, water heated for 5 minutes at 60°C, and for any
length of time at 70°C or 100°C, kills all E. coli bacteria.
Groh, MacPherson, and Groves, Sterilization o f Artificially Contaminated Water
Furthermore, 67% of the subjects found water at 55OC
to be “too hot to touch.”
The implications of this study are that it is not necessary to boil water in order to kill E . coli bacteria, the
most common cause of traveler’s diarrhea. In the interests of fuel conservation, it would be beneficial, for both
economic and environmental reasons, to heat water to
only 7OoC to make it safe to drink.
Unfortunately, in many wilderness situations or in
developing countries, thermometers are unavailable to
determine whether heated water is at 70OC.Since most
people found water “too hot to touch” at 55”C,which
is a temperature at which E. coli bacteria were able to survive for at least 1 nlinute,“too hot to touch”is not a valid
method for determining sterility of water. However, it
may be true that tap water that is “too hot to touch,” such
as that which may be available in hotels in developing
countries, may be safe to drink since it may have been
held in a hot water heating tank for a time sufficient to
kill any bacteria present. Additional studies need to be
done to test this observation.
Even if hot tap water were safe to drink for this reason, the rule of“too hot to touch” would still not be valid
13
in situations where people are heating water themselves,
such as when camping. In these cases, if a thermometer
is unavailable, simply bringing water to a visible boil is
sufkient to kill the bacteria. Furthermore, since the
boiling point of water, at even the highest elevations, will
not be iess than 7OoC,bringing water to a boil at high
elevations will be sufficient to kill the bacteria.
References
1 . Steffen R,Van der Linde E Gyr K, et al. Epidemioloby of diarrhea in travelers. JAMA 1983; 249:1176-1180.
2. Auerbach 15, Ceehr EC, rds. Management of wilderness and
environmental emergencies. 2nd Ed. St. Louis: C V Mosby,
1989:807-808.
3. Neuniann H.Travellers’ diarrhea. Lancet 1970;1:420.
4. Bandres JC, Mathewson JJ, DuPont HL. Heat susceptibility
of bacterial enteropathogens: implications for the prevention
0ftravelers’diarrhea.Arch Intern Med 1988;148:2261-2263.
5. Tellier R, Keystone JS. Prevention of Traveler’s Diarrhea.
Infect Dis Clin North Am. 1992;2:333-354.
6. Neumann HH. Bacteriological safety of hot tapwater in
developing countries. Public Health Keports 1969;9:812-814.
Clothes washing in the Ganges, Varanasi, Uttar Pradesh, India. Submitted by Danielle Gyurech, MD and Julian Schilling, MD.