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Neil Cameron

Electronics Projects with the ESP8266

and ESP32
Building Web Pages, Applications, and WiFi
Enabled Devices
1st ed.
Neil Cameron
Edinburgh, UK

Any source code or other supplementary material referenced by the

author in this book is available to readers on GitHub via the book’s
product page, located at www.​apress.​com/​978-1-4842-6335-8. For
more detailed information, please visit http://​www.​apress.​com/​
source-code.

ISBN 978-1-4842-6335-8 e-ISBN 978-1-4842-6336-5

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© Neil Cameron 2021

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Preface
It’s never been so easy and practical to access information over the
Internet, develop web pages to update sensor information, build mobile
apps to remotely control devices with speech recognition, or
incorporate Google Maps in a GPS route tracking app. The combination
of Wi-Fi functionality, high computing power, and low cost of the
ESP8266 and ESP32 development boards extends the range of
opportunities for microcontrollers. Communicating with devices and
accessing information over the Internet with the ESP8266 and ESP32
microcontrollers is the focus of Electronics Projects with the ESP8266
and ESP32.
The first section (Chapters 1 to 6) of the book demonstrates the
ease of use and the power of the ESP8266 and ESP32 microcontrollers
to access and display information on the Internet. Projects include
building an Internet radio, an Internet-based clock, and an
international weather station and a project with the ESP32-CAM
camera to upload pictures to a web page.
The book’s second section (Chapters 7 to 9) covers web page design
projects for updating your web page with sensor information using
real-time graphics or controlling a remote device through a web page.
You’ll learn about AJAX (Asynchronous JavaScript and XML), which
combines XML (eXtensible Markup Language) HTTP (Hypertext
Transfer Protocol) requests for updating a web page with JavaScript to
manage those requests, JSON (JavaScript Object Notation) to combine
information transmitted by a server to the client, the two-way fast
communication WebSocket protocol, MQTT brokers, and IFTTT (If This,
Then That) for communication between devices on different networks.
The practical projects include uploading information to the Internet
and controlling devices from anywhere in the world with the ESP8266
and ESP32 microcontrollers.
Mobile apps are now ubiquitous, making the app build projects in
the book’s third section (Chapters 10 to 13) very relevant. An app to
control remotely located motors connected to an ESP8266 or ESP32
development board mimics robotics used in the automotive industry; a
speech recognition app controls devices; and a GPS tracking app,
incorporating Google Maps, displays the current position and route
information. Each project with the ESP8266 and ESP32
microcontrollers is fully described, as no previous experience in mobile
app design and build is required.
Communication between ESP8266 and ESP32 microcontrollers is
described in the fourth section (Chapters 14 to 18) of the book. The
built-in ESP-NOW communication system, LoRa (long range), and RF
(Radio Frequency) communication are applied to controlling remotely
located devices with the device information updated on a web page by
the ESP8266 and ESP32 microcontrollers. Communication protocols
are extended to signal generation with the ESP8266 and ESP32
microcontrollers transmitting alphanumeric text or signals to produce
sounds, as used in electronic music. Signal generation without a
microcontroller is illustrated with an electronic piano, a motor control
project, and an alarm system including an MP3 player with a movement
detector. The book’s fourth section spans the built-in communication
protocol of the ESP8266 and ESP32 microcontrollers to communication
with back-to-basics electronics. A chapter on measuring electricity with
an ESP8266 or ESP32 microcontroller, applied to a solar panel project,
continues the electronics theme to understand the methodology behind
sensors.
The ESP32 microcontroller is more powerful than the ESP8266
microcontroller and also includes Bluetooth and Bluetooth Low Energy
(BLE) communication. Chapters on practical differences between the
ESP8266 and ESP32 microcontrollers and on specific features of the
ESP32 microcontroller form the last section (Chapters 21 and 22) of
the book. Throughout the book, all differences in libraries or
instructions for the ESP8266 and ESP32 microcontrollers are
described, as each project is compatible with both microcontrollers.
All sections of the book are stand-alone, so you can delve into a
section of the book rather than having to start from the beginning.
Several chapters build on information from earlier chapters. For
example, Chapter 12 (GPS tracking app with Google Maps) incorporates
mobile app design, Bluetooth communication, sourcing information
from the Internet, and updating a web page. Some programming
experience with the Arduino IDE is assumed, although all sketches are
completely described and comprehensively commented. The book
Arduino Applied: Comprehensive Projects for Everyday Electronics is
recommended as an introduction to microcontrollers ranging from
blinking an LED to building a robot car. Schematic diagrams were
produced with Fritzing software ( www.fritzing.org ), with an
emphasis on maximizing the clarity of component layout and
minimizing overlapping connections. Authors of libraries used in the
book are acknowledged in each chapter, with library details included in
the Appendix. All the Arduino IDE sketches and MIT App Inventor
source code for the apps are available to download at GitHub (
github.com/Apress/ESP8266-and-ESP32 ). The Arduino
programming environment and libraries are constantly being updated,
so information on consequences of the updates is also available on the
GitHub website.
Table of Contents
Chapter 1:​Internet radio
Station display and selection
Minimal Internet radio
Summary
Components List
Chapter 2:​Intranet camera
Save images to the SD card
Load images on a web page
Stream images to a web page
PIR trigger to stream images to a web page
Summary
Components List
Chapter 3:​International weather station
ILI9341 SPI TFT LCD touch screen
Touch screen calibration
Painting on-screen
ESP8266-specific touch screen calibration and paint
Weather data for several cities
Summary
Components List
Chapter 4:​Internet clock
WS2812 RGB LEDs responsive to sound
ESP8266 and multiplexer
LED rings clock
Network Time Protocol
 ESP32 and Internet clock
Summary
Components List
Chapter 5:​MP3 player
Control command for the MP3 player
MP3 player control with a microcontroller
Infrared remote control of an MP3 player
Creating sound tracks and two alarm systems
Movement detection alarm
Speaking clock
Voice recorder
Summary
Components List
Chapter 6:​Bluetooth speaker
Summary
Components List
Chapter 7:​Wireless local area network
HTTP request
HTML code
XML HTTP requests, JavaScript, and AJAX
Summary
Components List
Chapter 8:​Updating a web page
XML HTTP requests, JavaScript, and AJAX
JSON
Accessing WWW data
 MQTT broker and IFTTT
Parsing text
Console log
Wi-Fi connection
Access information file
Summary
Components List
Chapter 9:​WebSocket
Remote control and WebSocket communication
WebSocket and AJAX
Access images, time, and sensor data over the Internet
Summary
Components List
Chapter 10:​Build an app
Control and feedback app
Install the app
Servo-robot control app
Speech recognition app
Summary
Components List
Chapter 11:​App database and Google Maps
MIT App Inventor database
MIT App Inventor and Google Maps
Summary
Components List
Chapter 12:​GPS tracking app with Google Maps
 GPS position transmit
GPS position receive
Validate transmission of GPS position
Improve GPS position signal
Summary
Components List
Chapter 13:​USB OTG communication
App receive
App transmit
App receive and transmit
Summary
Components List
Chapter 14:​ESP-NOW and LoRa communication
ESP-NOW
LoRa communication
Summary
Components
Chapter 15:​Radio frequency communication
Transmitting and receiving text
Decode remote control signals
Control pan-tilt servos with RF communication
Control relay with RF communication
Relays
Solid-state relay
Summary
Components List
Chapter 16:​Signal generation
Signal generation
Digital to analog converter
Generating waves
ESP32 8-bit DAC
12-bit DAC
Summary
Components List
Chapter 17:​Signal generation with 555 timer IC
555 timer IC
Monostable mode
Bistable mode
Astable mode
Variable duty cycle
50 % duty cycle
PWM mode
Function generator
Square wave to sine wave
Bipolar junction transistor as a switch
MP3 player and PIR sensor application
Summary
Components List
Chapter 18:​Measuring electricity
Voltage divider
Analog to digital converter
Voltage meter
 Voltage meter with a load
Resistance meter (ohmmeter)
Capacitance meter
Current meter (ammeter)
Current sensor
Current and voltage sensor
Solar panel and battery meter
Inductance meter
Summary
Components List
Chapter 19:​Rotary encoder control
Debouncing
Interrupts
Square wave states
State switching
Incrementing a value
Summary
Components List
Chapter 20:​OTA and saving data to EEPROM, SPIFFS, and Excel
OTA updating
Saving data
Saving to EEPROM
Saving to SPIFFS
Downloading SPIFFS files
Saving data directly to Excel
Summary
 Components List
Chapter 21:​Microcontrollers​
Arduino Uno
Arduino Nano
Arduino Pro Micro
ESP8266 development board
ESP8266 analog input
ESP8266 interrupts
ESP8266 watchdog timer
ESP32 development board
ESP32 digital input
ESP32 analog input
ESP32 pulse width modulation
ESP32 serial input
Wi-Fi communication and web server
ESP8266 and ESP32 interrupts
ESP8266 and ESP32 and an OLED screen
ESP32 and servo motors
Summary
Components List
Chapter 22:​ESP32 microcontroller features
Microcontroller CPU and memory
ESP32 cores
Bluetooth communication
Bluetooth Low Energy communication
Timers
 Real-time clock and sleep mode
Digital to analog converter
Capacitive touch sensor
Hall effect sensor
Summary
Components List
Appendix:​Libraries
Index
Other documents randomly have
different content
fuchsin. The granule of venom has an affinity for eosin; it is never
excreted in granular form, but after intracellular dissolution.

Venogen is never met with in the lumen of the gland-tube.6

Collection of Venom.

Venom can be extracted from the poison-glands of either freshly

killed or living snakes.

In cases in which the venom of dead snakes has to be collected, the

best method of extraction consists in fixing the head of the animal to
a sheet of cork and carefully dissecting out the gland on each side.
The reptile being placed on its back, the lower jaw is removed with a
pair of scissors; two strong pins or two tacks are thrust through the
skull, in the median line, in order to keep the head from moving. The
poison-fangs are next drawn out of their sheaths, and, without
injuring them, the two poison-ducts, which open at their bases, are
isolated and tied with a thread in order to prevent the poison from
running out.

The dissection of the glands is then very easy; they are lifted out
and placed in a saucer. The end of the duct is cut between the gland
and the ligature, and with a pair of fenestrated or polypus forceps
the whole of the glandular mass is gently squeezed from behind
forwards, the liquid which flows out being received in a large watch-
glass.

If pressed for time, a more simple method of operating is to hold the

head of the snake in the left hand, with the mouth open and the
lower jaw directed downwards. A watch-glass, capsule, or receptacle
of some sort, such as a cup or plate, is then introduced by an
assistant between the jaws, and, with the index finger and thumb of
the right hand, the whole of the region occupied by the glands on
each side of the upper jaw is forcibly compressed from behind
forwards; the poison flows out by the fangs.
The extraction of the venom from living snakes is effected in the
same manner. The animal being firmly held by the neck, as close as
possible to the head, so that it cannot turn and bite; it can be made
to eject the greater portion of the liquid contained in its two glands
by compressing the latter with force from behind forwards, as one
would squeeze out the juice from a quarter of an orange (fig. 85).

It is necessary to take care that the reptile cannot coil itself round
furniture or other objects in the vicinity of the operator, for if this
should happen there would be the greatest difficulty in making it let
go, especially if dealing with a strong animal such as a Cobra, Rattle-
Snake, or Fer-de-lance.

Snakes of the last-mentioned kind are especially difficult to manage.

In order to avoid the risk of being bitten, it is always wise to begin
by pinning down the head of the animal in a corner of its cage by
means of a stick, and to seize it with a pair of long fenestrated tongs
shaped like forceps. The operator then easily draws the reptile
towards him and grasps it firmly by the neck with his left hand,
always as close to the head as possible, at the same time raising the
body quickly in order to prevent it from taking hold of anything. In
this way the most powerful snake is perfectly under control.
Fig. 85.—Collecting Venom from aLachesis at the Serotherapeutic Institute at São
Paulo (Brazil).
Fig. 86.—Chloroforming aCobra in order to Collect Venom, at the French Settlement
of Pondicherry, in India (Stage I.).

At Pondicherry, where is collected the greater portion of the venom

of Naja tripudians used by me for the vaccination of the horses that
produce antivenomous serum, it is customary to chloroform the
snakes in order to render them easier to manipulate.

The reptile is placed in a large covered jar, containing a pad of

absorbent wool impregnated with chloroform (figs. 86, 87), and in a
few minutes it is stupefied. It is then grasped by the neck with the
hands, and the edge of a plate is slipped between its jaws. On
compressing the two poison-glands with the fingers, the venom
dribbles out on to the plate.

A detailed description of this technique will be found in a note kindly

drawn up for me by my friend Dr. Gouzien, late head of the Medical
Staff of the French Settlements in India, and reproduced further on
in the section of this book devoted to documents. The note in
question was accompanied by figs. 17, 18, 19, 86, 87, and 88, which
are reproduced from photographs, for which I am indebted to the
kindness of M. Geracki, Engineer of the Savanna Spinning Mill at
Pondicherry, Dr. Lhomme, and M. Serph, Assistant Surgeon-
Dispenser.

The collection of the venom having been completed, the snake is put
back into its cage again, the tail and the body being introduced first,
and then the head. The lid or trap-door is half closed with the left
hand, and, with a quick forward thrust, the right hand releases its
grasp of the reptile and is immediately withdrawn; at the same time
the left hand completes the closure of the cage. The snake is
temporarily dazed, as though stunned, and it is only after the lapse
of a moment that it thinks of darting open-mouthed at the walls of
its prison.

When it is desired to procure large quantities of venom, as is

indispensable in laboratories where antivenomous serum is
prepared, the endeavour must be made to keep the snakes alive for
the longest possible time. It then becomes necessary to resort to
artificial feeding in the manner previously described (see p. 17), for
they very often refuse to feed themselves.
 Fig. 87.—Chloroforming a Cobra in order to Collect Venom, at the French Settlement
of Pondicherry, in India (Stage II.).

Except when a snake is moulting, the venom can be extracted from

its glands about every fortnight; and it is better that the extraction
be not performed concurrently with artificial feeding, since, owing to
the fact that the venom serves the animal as digestive juice, the
reptile will soon perish if deprived of the means of digesting the food
that it is obliged to receive. It is best, therefore, to select one day of
the week for artificial feeding, and the corresponding day of the
following week for the extraction of the venom.

When the venom has been collected, it must immediately be placed

in a desiccator over calcium chloride or sulphuric acid, in order to dry
it rapidly. In hot countries, and where no laboratory specially
equipped for the purpose exists, it will suffice to dry the venom in a
current of air, or even in the sun. It then concretes in scales of a
citrin colour, more or less dark, according to the concentration of the
liquid. In this dry condition, placed in well-corked bottles, protected
from damp air, it may be kept almost indefinitely without losing
anything of its original toxic power. On the contrary, if the
desiccation be imperfect it undergoes a somewhat rapid change, and
assumes a disagreeable odour of meat peptone. I have kept samples
of various venoms, dried as described, for fifteen years without any
sensible diminution of their activity.

Fig. 88.—Collecting Cobra Venom at Pondicherry (Stage III.).

 CHAPTER V.
THE CHEMICAL STUDY OF SNAKE-
VENOMS.
In the condition in which they are received on issuing from the
glands, venoms always present the appearance of a thick saliva, of
an oily consistency and more or less tinged with yellow, according to
the species of snake by which the poison has been produced. They
are entirely soluble in water, the addition of which renders them
opalescent. Tested with litmus they exhibit a slightly acid reaction;
this acidity, which is due to the presence of a very small quantity of
an indeterminate volatile acid, disappears on desiccation, so that
solutions of dried venom are neutral. The taste of venoms is very
bitter. Their density, which is slightly greater than that of water,
varies from 1030 to 1050.

Venoms are composed of a mixture, in variable proportions, of

proteid substances, mucus and epithelial débris, fatty matters and
salts (chlorides and phosphates of lime, ammonia and magnesia),
with from 65 to 80 per cent. of water.

The elementary analysis of Cobra-venom made by H. Armstrong7

gave the following results:—

Carbon 43·04 per cent.

Hydrogen 7·00 “
Nitrogen 12·45 “
Sulphur 2·50 “
Residue Small quantities.
Not much is to be learnt from these figures; it would be of far
greater importance to know the exact constitution of the proteid
substances to which venom owes its physiological properties.
Unfortunately, our knowledge of the chemistry of the albuminoid
matters is still too imperfect for it to be possible for us to determine
their nature.

As early as 1843 it was pointed out by Lucien Bonaparte that in the

venom of Vipera berus the most important principle is a proteid
substance to which he gave the name of viperin or echidnin, and
which he compared to the digestive ferments. Later on Weir Mitchell
and Reichert, and subsequently Norris Wolfenden, Pedlar, Wall,
Kanthack, C. J. Martin, and MacGarvie Smith, showed that venoms,
like diastases, exhibit a great complexity in composition; that all
their characteristic toxic constituents are precipitable by absolute
alcohol, and that the precipitate, when redissolved in water, recovers
the properties possessed by the venom before precipitation.

According to Armand Gautier,8 venoms contain alkaloids. The latter

may be obtained, in very small amounts, however, by finely
pulverizing dried venom with carbonate of soda, and systematically
exhausting the mixture with alcoholic ether at a temperature of 50°
C. These alkaloids have yielded crystallized chloraurates and
chloroplatinates, and slightly deliquescent crystallized chlorhydrates.
The latter produce Prussian blue when treated with very dilute ferric
salts, and mixed with a little red prussiate. They therefore represent
reductive bodies analogous to ptomaines.

Norris Wolfenden did not succeed in extracting these alkaloids from

Cobra-venom, whence they had nevertheless been isolated by
Armand Gautier. Wolcott Gibbs, and afterwards Weir Mitchell and
Reichert, likewise failed to find them in Crotalus-venom. The toxicity
of these bases is, moreover, but very slight, for the totality of the
alkaloids extracted by A. Gautier from 0·3 gramme of Cobra-venom
did not kill a small bird.
It is therefore to the toxalbumins that the toxic properties of venoms
are essentially due.

All venoms are not equally affected by heat. The venoms of

Colubridæ (Naja, Bungarus, Hoplocephalus, Pseudechis) and those of
the Hydrophiidæ are entirely uninjured by temperatures approaching
100° C., and even boiling for a short time. When the boiling is
prolonged, or when venoms are heated beyond 100° C., their toxic
power at first diminishes, and then disappears altogether. At 120° C.
it is always destroyed.

The venoms of Viperidæ (Lachesis, Crotalus, Vipera) are much less

resistant. By heating to the coagulating point of albumin, i.e., to
about 70° C., their toxic properties become attenuated, and they are
entirely suppressed between 80° and 85° C. Lachesis-venoms are
the most sensitive; their toxicity is lost if they be heated beyond 65°
C.

On separating the coagulable albumins of the venoms of Colubridæ,

by heating to 72° C., followed by filtration, we obtain a perfectly
limpid liquid, which is no longer injured by boiling, and in which the
toxic substance remains wholly in solution. The albuminous
precipitate, when separately collected and washed, is no longer
toxic. The clear liquid, after being filtered, is again precipitated by
absolute alcohol, and the precipitate, redissolved in an equal
quantity of water, is just as toxic as the original filtered liquid.

The venoms of Viperidæ, when coagulated, by heating them to a

temperature of 72° C., and filtered, are almost always inert. The
albuminous coagula, if washed, redissolved in water, and injected
into the most sensitive animals, produce no harmful effect whatever.

The results of dialysis likewise differ when we experiment with the

venoms of Colubridæ and Viperidæ. The former pass slowly through
vegetable membranes, and with greater difficulty through animal
parchment. The latter do not dialyse.

Filtration through porcelain (Chamberland candle F) does not

sensibly modify the toxicity of the venoms of Colubridæ; on the
contrary, it diminishes that of the venom of Viperidæ by nearly one-
half.

By using a special filter at a pressure of 50 atmospheres, C. J. Martin

has succeeded in separating from the venom of an Australian
Pseudechis two substances: a non-diffusible albuminoid, coagulable
at 82° C., and a diffusible, non-coagulable albumose. The former
produces hæmorrhages; the second attacks the nerve-cell of the
respiratory centres.

All venoms exhibit most of the chemical reactions characteristic of

the proteids:—

Millon’s reaction.

Xantho-proteic reaction (heating with nitric acid and subsequent

addition of ammonia = orange coloration).

Biuret reaction (caustic potash and traces of sulphate of copper).

Precipitation by picric acid, disappearing on being heated,

reappearing when cooled.

Precipitation by saturation with chloride of sodium.

Precipitation by saturation with sulphate of magnesium.

Precipitation by saturation with ammonium sulphate.

Precipitation by a 5 per cent. solution of sulphate of copper.

Precipitation by alcohol.

According to C. J. Martin and MacGarvie Smith, the albumoses of the

venoms of Colubridæ are hetero-albumoses, proto-albumoses, and
perhaps deutero-albumoses in small quantities. They can be
separated in the following manner:—

The solution of venom is heated to 90° C., and filtered in order to

separate the albumins coagulable by heat. The filtrate, saturated
with sulphate of magnesium, is shaken for twelve hours. By this
means there is obtained a flocculent precipitate, which is placed
upon a filter and washed with a saturated solution of sulphate of
magnesium. The filtrate is dialysed for twenty-four hours in a stream
of distilled water, and then concentrated, likewise by dialysis, in
absolute alcohol. Thus we obtain a few cubic centimetres of liquid,
which contains a small quantity of proteids in solution. These
proteids can be nothing but a mixture of proto- and deutero-
albumoses with peptones. That there is actually no trace of the latter
can easily be ascertained.

Neumeister9 has shown that it is impossible to precipitate all the

proto-albumoses of a solution by saturation with neutral salts, and,
since the filtrate becomes slightly turbid when a few drops of a 5 per
cent. solution of sulphate of copper are added to it, we must
conclude that it contains a small proportion of these proto-
albumoses.

The deposit retained upon the filter after washing with sulphate of
magnesium is redissolved in distilled water, and dialysed for three
days. An abundant precipitate then becomes collected in the
dialyser. This is centrifuged. The clear liquid is decanted with a
pipette, then concentrated by dialysis in absolute alcohol, and finally
evaporated at 40° C. until completely desiccated. The solid residue is
washed and centrifuged several times in distilled water, after which it
is dried on chloride of sodium.
This method enables us to separate two albumoses, both
precipitable by saturation with sulphate of magnesium, and
belonging to the class of primary albumoses: one of these, proto-
albumose, is soluble in distilled water, the other, hetero-albumose, is
insoluble; but the latter can be dissolved in dilute solutions of neutral
salts. These bodies are respectively identical with those obtained by
the pepsic digestion of proteids.10

In order to study separately the local and general effects of these

different albumoses, C. J. Martin and MacGarvie Smith performed
the following experiment:—

They introduced beneath the skin of the belly of a guinea-pig,

previously shaved and rendered aseptic, two small pieces of
sterilized sponge, about 2 c.mm., one of which was impregnated
with the solution of proteid, while the other served as control. The
two small incisions, one on either side of the median line, were then
sutured and covered with collodion. In this way the maximum of
local effect and the minimum of general effects was obtained. The
solutions of albumoses introduced by this method into the organism
produced an enormous œdema, which, in from six to eight hours,
extended along the whole side of the abdomen containing the
sponge charged with poison.

To test the general toxic effects, the solutions were injected into a
vein or into the peritoneal cavity. It was thus found that the proto-
and hetero-albumoses killed the animals in a few hours.

It must therefore be concluded from these facts that the active

principles of venom are proto- and hetero-albumoses, the albumins
that it contains being devoid of all toxic power.

Many chemical substances modify or destroy venoms, and we shall

see in another chapter that several of them, by reason of their
properties, may be very usefully employed for the destruction, in the
actual wound resulting from a venomous bite, of the venom that has
not yet been absorbed in the circulation.

Among these substances the most important are:—

A 1 per cent. solution of permanganate of potash (Lacerda).

A 1 per cent. solution of chloride of gold (Calmette).

Chloride of lime or even hypochloride of calcium (Calmette), in a

solution of 1 in 12, which is augmented, at the moment of use, by 5
to 6 volumes of distilled water, so as to bring it to the standard
strength of about 850 cubic centimetres of active chlorine per litre of
solution.

A 1 per cent. solution of chromic acid (Kaufmann).

Saturated bromized water (Calmette).

A 1 per cent. solution of trichloride of iodine (Calmette).

All these chemical bodies also modify or destroy the diastases and
the microbic toxins. The venoms, although more resistant to the
influence of heat, behave, therefore, like these latter, and exhibit the
closest affinity with them. Moreover, like all the normal glandular
juices, they possess very manifest zymotic properties, which
singularly complicate their physiological action, and upon which we
shall dwell later on.

Electricity, employed in the form of continuous electrolytic currents

passing through a solution of venom, destroys the toxicity of the
latter, because under these conditions there is always formed, at the
expense of the salts accompanying the venom, a sufficient quantity
of chlorinated products (hypochlorites, chlorates, &c.), and a small
amount of ozone, the oxidizing action of which is extremely
powerful.
With alternating currents of high frequency, Phisalix, repeating the
experiments that Arsonval and Charrin had performed upon
diphtheria toxin, thought that he had succeeded in attenuating
venom to the point of transforming it into vaccine.11 But it has been
shown by Marmier that this attenuation was simply the result of
thermic actions. When, by means of a suitable arrangement, any rise
of temperature was carefully avoided, no modification of toxicity was
obtained.12

The influence of light, which has no effect upon venom preserved in

a dry state, is, on the contrary, very marked upon venom in solution.
Solutions of venom that are destined for physiological experiments
should therefore not be employed without controls, if they be several
days old. Apart from the fact that, if care be not taken to render
them aseptic, they very soon become contaminated with the germs
of all kinds of microbes, it is found that they gradually lose a large
part of their activity, especially when they remain in contact with the
air. By filtering them through a Chamberland candle and keeping
them in the dark, in a refrigerator, in perfectly closed phials, they
may be kept unimpaired for several months.

The addition of glycerine in equal parts to a concentrated solution of

venom is also an excellent means of preservation.

Phisalix has shown that the emanations from radium attenuate and
then destroy the virulence of Cobra- and also of Viper-venom.

“Dry Viper-venom, dissolved in aqua chloroformi in the proportion of

1 in 1,000, is put up in four tubes, three of which are irradiated, the
first for six hours, the second for twenty hours, and the third for
thirty-six hours. Three guinea-pigs, of equal weight, are inoculated
with equal quantities of the irradiated venom; a control receives the
non-irradiated venom. The latter dies in ten hours; the animal
inoculated from the first tube dies in twelve hours; the one
inoculated from the second tube in twenty hours, and the third
proves resistant without any symptom of poisoning. A second
inoculation produces a transitory lowering of the animal’s
temperature by half a degree. At the end of four days it dies after
inoculation with a lethal dose.”

The nature of the solvent exerts a great influence upon the action of
the emanations from radium: if the same experiment be performed
with venom dissolved in a 50 per cent. mixture of glycerine and
water, the attenuation is merely relative after six hours.

Auguste Lumière and Joseph Nicolas, of Lyons, conceived the idea of

studying the effect upon venom of the prolonged action of the
intense cold produced by the evaporation of liquid air.13 The Cobra-
venom employed by these investigators was in solution at a strength
of 1 in 1,000. It was submitted to the action of liquid air, partly for
twenty-four hours and partly for nine days at -191° C. Its toxicity
was in no way diminished.

Lastly, I must mention the recent researches of Hideyo Noguchi,14

with reference to the photodynamic action of eosin and erythrosin
upon the venoms of the Cobra, Vipera russellii, and Crotalus. It was
found by the scientist in question that the toxicity of these various
venoms is more or less diminished in the presence of these aniline
colours, when the mixtures are insolated. Cobra-venom is the most
resistant, just as it is in regard to the other physical or chemical
agents. That of Crotalus, on the contrary, is the least stable.
 CHAPTER VI.
THE PHYSIOLOGICAL ACTION OF
SNAKE-VENOMS.

A.—Physiology of Poisoning in Man and in Animals Bitten

by the Different Species of Poisonous Snakes.
(Colubridæ; Viperidæ; Hydrophiidæ.)

The bites of poisonous snakes produce very different effects

according to the species of snake, the species to which the animal
bitten belongs, and according to the situation of the bite. It is
therefore necessary to take these various factors into account, in
describing the symptoms of poisoning in different animals.

When the quantity of venom introduced into the tissues by the bite
of the reptile is sufficient to produce fatal results—which is happily
not always the case—the venom manifests its toxic action in two
series of phenomena: the first of these is local and affects only the
seat and surroundings of the bite; the second, or general series, is
seen in the effects produced upon the circulation and nervous
system.

It is remarkable to find how great is the importance of the local

disorders when the venomous reptile belongs to the Solenoglypha
group (Viperidæ), while it is almost nil in the case of the
Proteroglypha (Colubridæ and Hydrophiidæ).
The effects of general intoxication, on the contrary, are much more
intense and more rapid with the venom of Proteroglypha, than with
that of Solenoglypha.

In considering the usual phenomena of snake-poisoning in man, we

must therefore take this essential difference into account, and draw
up separately a clinical description of the symptoms observed after a
bite from a Cobra (Colubridæ), for instance, and another list of those
that accompany a bite from Lachesis or Vipera berus (Viperidæ).

The bite of a Cobra, even of large size, is not very painful; it is

characterized especially by numbness, that supervenes in the bitten
part, rapidly extends throughout the body, and produces attacks of
syncope and fainting. The patient soon experiences a kind of
lassitude and irresistible desire to sleep; his legs scarcely support
him; he breathes with difficulty and his respiration becomes of the
diaphragmatic type.

By degrees the drowsiness and the difficulty of breathing become

greater; the pulse, which at first is more rapid, becomes slower and
gradually weaker, the mouth contracts, and there is profuse
salivation, the tongue appears swollen, the eyelids remain drooping,
and, after a few hiccoughs frequently accompanied by vomiting and
involuntary emissions of urine or fæcal matter, the unfortunate
victim falls into the most profound coma and dies. The pupils react
to luminous impressions up to the last moment, and the heart
continues to beat sometimes for two hours after respiration has
ceased.

All this takes but a few hours, most frequently from two to six or
seven, rarely more.

When the reptile by which the bite is inflicted is one of the

Solenoglypha, such as a Lachesis for example, the seat of the bite
immediately becomes very painful and red, then purple. The
surrounding tissues are soon infiltrated with sanguinolent serosity.
Sharp pains, accompanied by attacks of cramp, extend towards the
base of the limb. The patient complains of intense thirst, and
extreme dryness of the mouth and throat; the mucous membranes
of the eyes, mouth, and genitalia become congested.

These phenomena often continue for a very long period, even for
more than twenty-four hours, and are sometimes accompanied by
hæmorrhages from the eyes, mouth, stomach, intestines, or bladder,
and by more or less violent delirium.

If the quantity of venom absorbed be sufficient to cause death, the

patient exhibits, a few hours after being bitten, stupor, insensibility,
and then somnolence, with increasing difficulty of respiration, which
ends by becoming stertorous. Loss of consciousness seems complete
a good while before coma appears. Asphyxia then ensues, and the
heart continues to beat for nearly a quarter of an hour after
respiratory movements have entirely ceased.

In certain exceptional cases death is very rapid; it may supervene

suddenly in a few minutes, even before the local phenomena have
had time to manifest themselves; in this case the venom, having
penetrated directly into a vein, has produced almost immediate
coagulation of the blood, thus causing the formation of a generalized
embolism.

If the venom be introduced in a highly vascular region, or directly

into a vein, the result is almost invariably fatal. On the contrary, if
the derm be scarcely broken, or if the clothing has acted as a
protection, scarcely any absorption will take place. We are here
confronted with the same factors of gravity as in the case of bites
inflicted upon human beings by animals suffering from rabies.

In experiments we are able to eliminate all these factors, and to

follow in an animal inoculated with a known quantity of venom the
whole series of phenomena of poisoning, the intensity of which can
be graduated. Let us see, then, how the various animals that it is
possible to make use of in laboratories behave with regard to
venoms of different origins.

B.—The Physiology of Experimental Poisoning.

In the monkey, the first apparent sign of the absorption of Cobra-
venom, or of the venom of any other species of Colubridæ, is a sort
of general lassitude; the eyelids next become half closed. The animal
appears to be seeking a suitable spot in which to rest; it gets up
again immediately, and walks with a jerky action; its limbs have a
difficulty in supporting it. It is soon attacked by nausea, vomiting
and dyspnœa; it rests its head upon the ground, raises it, trying to
get breath, and carries its hand to its mouth as if in order to pluck a
foreign body from its throat. It totters upon its limbs, and lies down
upon its side with its face against the ground. Ptosis increases, and
complete asphyxia soon supervenes. The heart continues to beat for
some time after respiration has ceased, and then stops in diastole.

Cadaveric rigidity very rapidly sets in, and persists for a long time,
even after putrefaction has commenced. During the last moments of
life the pupil remains very sensitive; the animal appears to retain
unimpaired its sense of hearing and sensibility to pain. The electric
excitability of the muscles of the face persists, but that of those of
the limbs and body almost entirely disappears. The application of
volta-faradic currents from the nape to the diaphragm produces no
respiratory movement when asphyxia begins to manifest itself. The
sphincters of the bladder and anus relax after a few spasms, which,
in case of males, frequently provoke the ejaculation of semen; the
urine and fæces immediately escape.

The autopsy reveals slight hæmorrhagic œdema at the point of

inoculation, and hyperæmia of all the viscera, especially of the liver
and spleen, with, very frequently, small hæmorrhagic patches on the
surface of these organs, and on that of the intestine and kidneys.
The serous membranes, especially the meninges, endocardium,
pleuræ, and peritoneum, exhibit ecchymoses; the lungs are
besprinkled with small infarcts, the more numerous the slower the
intoxication. The blood remains fluid and laccate.

In poisoning by the venoms of Viperidæ, the hæmorrhagic

phenomena appear at the outset, and are more intense. Death is
always preceded by a period of asphyxia, indicating that the bulbar
nuclei of the pneumogastric nerve have become affected. At the
autopsy, however, the blood, instead of remaining fluid, is always
found to be coagulated into a mass in all the vessels; it afterwards
gradually becomes redissolved in six or eight hours, and then
appears laccate, as after poisoning by Cobra-venom, but darker.

All mammals exhibit the same symptoms after inoculation with lethal
doses of venom. The same applies to birds; but in the latter the
period of asphyxia is much longer, probably on account of the
reserves of air accumulated in their air-sacs and pneumatic bones.
They gape like pigeons that are being suffocated, rest the tip of the
beak on the floor of the cage, and frequently have convulsive
spasms of the pharynx, accompanied by flapping of the wings. Small
birds and even pigeons are extremely sensitive to venom; fowls are
more resistant.

Frogs, thanks to their cutaneous respiration, succumb very slowly. I

have seen some survive for thirty hours after being inoculated with a
quantity of venom which, when subcutaneously injected into a
rabbit, causes death in ten minutes.

Lizards and chameleons succumb very rapidly. Grass Snakes and

non-venomous snakes in general withstand doses of venom that in
proportion to their weight are fairly large; nevertheless, as indeed
we shall see in the sequel, they do not possess any real immunity. It
is only poisonous snakes that are unaffected by enormous doses of
their own venom, as has already been shown by Fontana, Weir
Mitchell, and Viaud Grand Marais. They are, however, quite capable
of being poisoned by snakes belonging to altogether different
species; strong doses of Crotalus- or Lachesis-venom are fatal to
Cobras or Kraits, and, when several poisonous snakes are shut up
together in the same cage, they are not infrequently seen to kill
each other as the result of repeated bites.

Fishes, which are particularly sensitive to the venom of Hydrophiidæ,

readily succumb to inoculation with other venoms, such as that of
the Cobra. At Saigon, in 1891, I made experiments upon the action
of this latter venom on two specimens of the fighting fishes, that the
natives of Annam rear in aquariums in order to witness their
combats and make bets on them. The fishes died five hours after
intramuscular inoculation with a dose which kills a pigeon in twenty
minutes.

Many invertebrates, such as leeches, crayfish, and gastropod

molluscs (snails), are killed by inoculation with very small quantities
of venom.

C.—Determination of the Lethal Doses of Venom for

Different Species of Animals.
It is very difficult to specify, even within broad limits, the dose of
venom necessary to kill a human being. The quantity of poison
introduced by the bite of a venomous snake depends, as has already
been stated, upon a large number of factors, and, very fortunately,
this quantity is not always sufficient to cause death. Thus in India,
that is to say in the region in which snakes are most numerous and
most dangerous, the mean mortality seems scarcely to exceed 35 to
40 per cent., so far as it is possible to judge from official statistics.
But, by experimenting upon animals, and commencing with known
doses of venom, which has first been dried and then dissolved again
in always the same quantity of physiological saline solution or sterile
distilled water, we can determine exactly, for each kind of venom,
and for each species of animal, the minimum lethal dose per
kilogramme of animal.

The entire series of data collected by investigators who have

devoted themselves to this study may be summed up as follows:—

Minimal doses lethal in twenty-four hours for a guinea-pig weighing

from 600 to 700 grammes:—

Colubridæ.
Venom of Naja tripudians 0·0002 gramme
“ Bungarus cæruleus 0·0006 “
“ Naja haje 0·003 “

VIPERIDÆ.
Venom of Vipera berus 0·04 gramme
“ Vipera russellii (Daboia) 0·001 “
“ Lachesis lanceolatus 0·02 “
“ Lachesis mutus (Surucucu) 0·02 “
“ Lachesis neuwiedii (Urutù) 0·02 “
“ Lachesis flavoviridis 0·007 “
“ Ancistrodon contortrix 0·015 “

Cobra-venom. Dose lethal in twenty-four hours for different animals:

—

Dog 0·0008 gramme per kilogramme

Rabbit 0·0005 “ “
Guinea-pig 0·0004 “ “
Rat 0·0001 “ 150 grammes
Mouse 0·000003 “ 25 “
 Frog 0·0003 “ 30 “

Venom of Bungarus cæruleus (Common Krait), according to Elliot,

Sillar, and Carmichael.15 Minimal lethal doses for:—

Frog 0·0005 gramme

Rat 0·001 “
Rabbit (by subcutaneous injection) 0·00008 “ per kilogramme
Rabbit (by intravenous injection, 0·00004 “ “
according to G. Lamb)

Venom of Enhydrina valakadien (according to Elliot and Fraser).16

Minimal lethal doses per kilogramme:—

Rat 0·00009 gramme

Rabbit 0·00006 “
Cat 0·0002 “

Venom of Enhydris curtus:—

Rat 0·0005 to 0·0006 gramme per kilogramme

Venom of Notechis scutatus (syn. Hoplocephalus curtus; the Tiger

Snake of Australia):—

Rabbit (by intravenous injection, 0·00006 gramme per

according to Tidswell) kilogramme

Venom of Vipera russellii (Daboia):—

Rabbit (by intravenous injection, 0·00005 gramme per

according to G. Lamb) kilogramme

Venom of Lachesis gramineus (Green Pit-Viper, India):—

Rabbit (by intravenous injection, 0·002 gramme per
according to G. Lamb) kilogramme

Venom of Crotalus adamanteus (Californian Rattle-Snake):—

Rabbit (by intravenous injection, 0·00025 gramme per kilogramme

according to McFarland, G.
Lamb, and Flexner and
Noguchi)

It will have been seen from the foregoing figures, that the respective
sensitiveness of the dog, cat, rabbit, guinea-pig, rat, mouse, and
frog, with regard to the same venom, is in no way proportional to
the weight of these animals.

The species mentioned are, per unit of weight, more or less resistant
to intoxication; and, on experimenting with other animals, as for
instance the monkey, pig, ass, and horse, we find that the monkey is
much more susceptible to intoxication than the dog, and that the ass
is extremely sensitive (0·010 gramme of Cobra-venom is sufficient to
kill it), while the horse is less so, and the pig is by far the most
resistant.

The same weight of dry Cobra-venom, let us say 1 gramme to be

precise, will enable us to kill 1,250 kilogrammes of dog, 2,000
kilogrammes of rabbit, 2,500 kilogrammes of guinea-pig, 1,430
kilogrammes of rat, or 8,333 kilogrammes of mouse.

The lethal dose for a horse being, as I have ascertained by my own

experiments, about 0·025 gramme, 1 gramme of dry Cobra-venom
will therefore suffice to kill 20,000 kilogrammes of horse.

Assuming that man, in proportion to his weight, possesses a

resistance intermediate between that of the dog and that of the
horse, we may consider that the lethal dose for a human being is
about 0·015 gramme. It follows, therefore, that 1 gramme of venom
would kill 10,000 kilogrammes of man, or, let us say, 165 persons of
an average weight of 60 kilogrammes.

Another extremely important fact, which must not be lost sight of, is
that differences of toxicity, which are often considerable, are
exhibited by the venoms of different specimens of the same species
of snake, or by the venom of the same snake collected at different
times. I have found, for instance, in the case of the specimens of
Naja and Lachesis reared in my laboratory, that, according to the
length of time that the animals had been without food, and to the
nearness or otherwise of the moulting period, the venom was more
or less active, and that on evaporation it left behind a more or less
considerable quantity of dry extract. In certain cases, immediately
after the moult and after a prolonged fast, the venom was ten times
more active than after a plentiful meal or before the moult.

The figures given above must therefore not be regarded as

determining the minimal lethal doses of the different venoms, except
in a purely comparative way, and they must be considered only as
data useful to know when it is desired to experiment upon animals
with these substances.

Variations of this kind are observed in the case of all species of

snakes. Thus Phisalix rightly insists upon the necessity of always
noting, besides the species of snake, the place of origin and the
season; for he has himself seen that, as regards French vipers, those
of the Jura, for example, produce in the spring a venom almost
devoid of local phlogogenic action; while vipers from the vicinity of
Clermont-Ferrand, though less toxic, produce much more serious
local effects.

On the other hand, it has been shown by Th. Madsen and H.

Noguchi, in a very interesting study of venoms and anti-venoms,17
that, when we examine the relation between dose and toxicity, we
find that the interval separating the moment of inoculation from that
of death diminishes only up to a certain point in proportion as the
dose is increased. In the case of the guinea-pig, with 0·0005
gramme of Cobra-venom the interval is 3 hours 75 seconds; but
after this, an increase in the dose produces only a relatively
inconsiderable acceleration of death. There is therefore no strict ratio
between the dose inoculated and the time that elapses until death
supervenes.

D.—Effects of Venom in Non-Lethal Doses.

When the quantity of venom introduced into the organism is
insufficient to cause death, the phenomena that precede and
accompany recovery differ very greatly according as the snake from
which the venom was derived belongs to the Colubridæ or Viperidæ.

After a non-lethal bite from a Cobra or Krait, for example,

convalescence usually takes place very rapidly, and, apart from the
local œdema of the subcutaneous tissue surrounding the wound,
which in very many cases leads to the formation of a suppurating
abscess, no lasting injury to health is observed. The venom is
eliminated by the kidneys, without even causing albuminuria, and
sensation gradually returns, in twenty-four or forty-eight hours, in
the part affected by the original lesion.

If the bite has been inflicted by a Viperine snake, the local lesion,
which is much more extensive, almost always results in the
formation of a patch of gangrene. Hæmorrhages from the mucous
membranes, and sanguineous suffusions into the serous cavities,
such as the pleura or pericardium, may supervene more or less
slowly. Pulmonary infarcts are sometimes produced, as well as
desquamation and hæmorrhage from the kidneys, albuminuria, or
hæmaturia. These lesions, which are more or less severe, last for
several days, and then slowly disappear after a period of true
convalescence. In many cases they leave behind them traces which
last for months and even years, and they then more or less affect