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Lecture 1.
Subject of Microbiology.
SHORT HISTORICAL OUTLINE
PRINCIPLES OF THE CLASSIFICATION OF MICROORGANISMS.
BACTERIAL CELL STRUCTURE.
What’s microbiology?
Term itself –- mikros – small, bios – life, logos – science.
It is the science, which studies a smallest organisms, invisible to naked eye,
named microbe.
Figure 1.1
Microbiology studies microorganisms from various point of views.
There are different subtypes of microbiology according to the subject of
investigation: general, technical, agricultural, veterinary, medical, sanitary, and
also such sciences as immunology and virology.
Medical microbiology:
Obtain, isolate and describe the infectious agent.
Define and distinguish methods of treatment.
Elaborate new methods of diagnostic.
Elaborate new methods of prophylactic.
The breef history of microbiology.
320 years ago the first microscope was developed by Antony Van
Leeuwenhoek, Dutch microscopist.
He was the first person, who sow and described microbes. He himself made
simple lenses, which magnified 160 – 300 fold.
In 1695 he published his work – “ The Secrets of nature discovered by A. V.
Leeuwenhoek”, where he discribed all living particles, which he observed in
water, soil, feces.
Figure 1.2 The microscope of A. V. Leeuwenhoek
The discovery of A.V. Leeuwenhuek stimulated the great interest among
scientist to invisible world. However, more then 150 years scientists were unable
to use these wonderful investigation for learning nature of infectious diseases.
But during these times many scientists made the attempts to apply science
dates to practical problems being faced in the battle against epidemic diseases.
One of them was Russian scientist D. Samoijlovich.
He made the conclussion that plaque is transmited by spesial infectious
particles. To prove this he carried out a dangerous experiment. He inoculated
himself with infectious material taken from a person with bubonic plaque ( 1771).
In 1798 the English physician E. Jener published his results of vaccinations
against smallpox. He proved that vaccination of humans with cowpox protects
them from infections with smallpox. Those discoveries played important role for
further development theoretical and practical problems of prophylaxis and struggle
against infectious diseases.
Figure 1.3. E. Jener
Louis Pasteur: the name of great French scientist.
Had two Ph.D. deegres – in chemistry and physics, concerning the
polarization of liquids;
He studied the crystals of wine acid in the microscope
Proved microbiological nature of alcoholic, lactic and buturic fermentation
Demonstrated a new type of respiration – anaerobic – in some of microbes
Then he started to investigate the wine diseases – french wine became sour.
He told that when in the wine are presented yeast cells – the wine is good,
lactobacteria – bad. He suggested to heat at 560C. It is a method of pasteurization.
Figure 1.4 Louis Pasteur
Then he presented and proved his famous theory that there is no self-origin of
life (microbes). He proved that spontaneous generation of living substases does
not exist.
First of all he investigated the chicken’ diseases, and namely, anthrax.
Also he discovered the nature of rabies and developed the method of
producing of antirabies vaccines and began to use this vaccine to treatment
(prophylaxis).
German school. Robert Koch (1843) made a great progress of medical
microbiology. He discovered a solid nutrient media ( gelatine, coagulated serum,
meat peptone agar (MPA) and applied them to isolating of pure cultures of
microbes.
He also introduced aniline dyes and immersion system in practice of
microscopy.
Proved the bacterial nature (ethiology) of anthrax.
Discovered choleric agent and tuberculosis agent. And obtained tuberculin
from tuberculoid bacterium.
Figure 1.5 Robert Koch
English school of microbiology represent Lister, who opened the new way
for surgery. He discovered and introduced in surgery practise the methods of
antiseptic – cleance with carbolic acid.
That is why microbiology opened the new way for surgery.
Now – aseptic.
Figure.1.6 Lister
Also a great role microbiology play in therapy.
examples: role of immunology for treatment of rheumatism. Gastric and
duodenal ulcer – Helicobacter pilory – decreased number of cancer.
Russian Scool.
Great role of understanding of inflamation nature was made by
I.E. Mechnikov – all basic ideas of immunology: immune status, immune
resistance, specific and non-specific factors of defense.
The classic works of Mechnikov on the biological theory of immunity opened a
new stage in the development of medicine. He discovered and studied the process
of intracellular digestion as a mechanism of defence against pathogenic microbes
which have penetrated into the body.
He discovered that some cells of mesoderma, leycocytes.
Those cells was named phagocytes. That is why we now Mechnicoff as a founder
of cell- theory of immunity, in 1908 he was awarded Nobel prize.
Figure.1.7. I.E. Mechnikov
Year
1674
1796
1859
Event
Leeuwenhoek discovers microorganisms.
Jenner creates a vaccine for smallpox.
Pasteur disproves spontaneous generation of microorganisms.
1865
1876
Lister introduces antiseptic techniques.
Koch proves that specific microorganisms cause specific
diseases.
Koch uses agar to obtain a pure culture.
Metchnikov discovers phagocytic theory of immunity.
lwanowski discovers viruses.
Ehriich articulates the principle of selective toxicity.
Fleming discovers penicillin.
1881
1883
1892
1894
1929
Subject of microbiology. Microbiology deal with:
All what’s invisible (inside the world of living mater).
All belong to Protista biologic kingdom: eucariota, procariota (eubacteria and
archebacteria), viruses, viriods, plasmids and prions.
Figure 1.8.
The modern classification:
There are 2 Upper kingdom of living mater : procariotes and eucariotes.
Group of Procariotes contain:
Cianibacterium
Arhebacterium
Eubacterium (true bacteria)
Eucariotes contain: animals; plants; fungi (micota)
In this group for microbiology are important:
From animals: class Protozoa
From plants –- unicellular Water-plants- Algae.
From fungi – all representatives.
Figure.1.9.
That is why Microbiology deal with:
Protozoa
eucariota
have nucleus, chromosomes
free living, conditionally pathogenic and human parasites
representatives: malaria, trypanosomosys, leishmania etc
Fungi
eucariotic cells
dimorphism (yeast and mold forms)
sexual and asexual reproduction
growth cycle – reproductive and vegetative phases
Bacteria
procariota
science – bacteriology
conditionally pathogenic and pathogenic;
normal microflora, antibiotic producents,
smallest free-living forms
no sexual reproduction, could create spores
archebacteria: no peptidoglycan in cell wall;
no pathogenic species for human;
eubacteria: bacteria, ricketssia, mycoplasma, chlamydia
Main classification of bacteria is Bergey’s Manual, which include Two
division:
1.cyanobacteria (cyanophyta) Water-plants
2.true bacteria which include 19 parts.
Main of them represent:
Bacteria ( rod-like, cocci) (aerobic and anaerobic), (endospore forming or no)
Spirochetes and spiriles
Vibrions
Actinomycetes ( – important as producents of antibiotics)
Obligate intracellular parasites (Ricketsia and Chlamidia)
Viruses (are grouped in an independent kingdom) Viriods, Prions
are genetic parasites
no cell structures and protein synthesis systems
on animals, insects, plants, bacteria (phages) and human
contain DNA or RNA
are not visible with the light microscope
viriods cause plant diseases
prions are infectious particles associated with scrapie, a degenerative central
nervous system of sheep
Principles of classification All bacteria has binary nomenclature:
Genus and:
Species – pool of microorganisms with common origin, similar genotype
(>60% of DNA homology) and maximum adjacent phenotypic signs and
properties.
Morphological, tinctorial, cultivation, mobility, spores production,
physiology, biochemical, phages sensitivity, antigenic, cell wall structure etc.
Aditional terms:
Clone – a population of microorganisms descended from a single individual
by asexual reproduction.
Strain – clones that are presumed or known to be genetically different. ( It is
specimen of microbic culture the same species, which was isolated from different
places, or from one place in different times.
Figure 1.10.Main Bacterial Morphology.
Cocci: monococci, diplococci (gonococci, meningococci, pneumococci),
streptococci, staphilococci, tetracocci, sarcines (8 – 16 cells)
Rod-like: mono, strepto, diplo; with or without spores
Spirals forms: vibrio, spirillae.
Figure 1.11.Structures of bacterial cell.
Intracellular structures.
What are bacterial cell consist from?
– Proyoplast, nucleoid, endospores, plasmides, volutine granuls.
Extracellular structures –
flagelas as a mechanism of motion (monotrichous, lophotrihous,
amphotrihous, peritrichous.)
Figure.1.12. Flagelas
capsuls – polysaccharide outer mucose layer, which defens cell from outer
action.(important espesially for pathogenic microbes inside host organism)
Pili (fimbrias) – filamentous structures which take part in processes of
conjugation (transmission of DNA )and adhesion.
Figure 1.13 Capsuls
Figure 1.14. Pili
cell envelope
So called: Gram+ and Gram– depend from structure of cell-wall
Figure.1.15. Structure of cell envelope.
Comparision of a Procaryotic cell and a Eucaryotic cell
Structure
Appendages
Glycocalyx
Outer membrane
and periplasm
Cell wall
Procaryote
Pili, flagella, axial filaments in
spirochetes
Usually; can be a thin slime layer or
thicker capsule
Gram-negatives only
Eucaryote
Flagella; structurally very different from
procaryotic flagella
In some algae, fungi, protozoa, and human
cells that lack walls
Never
All eubacteria except mycoplasmas;
basic component is peptidoglycan
Algae, fungi, protozoa, plants, but never
with peptidoglycan; no cell walls in
animals, including human cells
All; a phospholipid bilayer
Cytoskeleton and cytoplasmic streaming in
all; pseudopods in amoebae
Always membrane-bound; include nucleus,
endoplasmic reticulum, Golgi apparatus,
80S ribosomes (except 70S in organelles),
mitochondria and/or chloroplasts (algae
and some protozoa have both)
Paired chromosomes; associated with
histones
Mitosis for growth and meiosis for
reproduction
Never
Plasma membrane All; a phospholipid bilayer
Cytoplasm
Undifferentiated
Organelles
Not membrane-bound; they include
nucleoid, 70S ribosomes, various
inclusions, and the periptasm in Gramnegatives
DNA
Usually one circular chromosome
Cell division
Binary fission or budding for both
growth and reproduction
Principally in Gram-positive bacteria,
but in some Gram-negative
. Endospores
Size
Typically one or a few micrometers
Typically 10 micrometers or more
Modern diagnostic methods in microbiology
1.Microscopic
light microscopes: fluorescenes, dark-field, etc.
EM – 1939 – thin structure, DNA etc.
scanning EM – fimbriae and pili
native or stained biological material
define cell form, their arrangement
ability to be stained by different dyes
2.Bacteriological or physiological methods
isolation of pure culture and its identification
grow on special nutrient media – for heterotrophes, not for intracellular
parasites – cultural properties;
3.Biochemical methods
Investigation of fermentative activity of microbes.
4.Serological methods
Identification of antigenic composition of bacteria.
determine specific antibodies or antigenes in patients blood
Investigation of patient serum with serological reactions: agglutination,
precipitation, ELISA, immunofluorescence etc.
Allergic probes
sensitivity of macroorganism to various infectious agents or their metabolits
5.Biological methods
contamination (infecting) of lab animal
Investigation of Diagnostical triade:
Isolation of causative agent from sick human
Microscopy of obtained material
Isolation of pure culture and its investigation
Model of the disease on lab animal
6. Genetics methods
Investigation DNA (RNA) sequence.
PCR.
7. Sensitivity to bacteriophages and antibiotics
Laboratory Equipment and Procedures
The microscopy
Laboratory equipment and
procedures
Light microscopes
Stained bacteria
Ultraviolet microscope
Staining reactions
Fluorescence microscope
Staining techniques
Dark-field microscope
Preparation of a pure culture
Phase-contrast microscope
Pure culture techniques
Electron microscope
Culture media
Transmission electron microscope
Control of pH and temperature
Scanning electron microscope
Oxygen requirements
Techniques for microscopic study of Sterilization methods
bacteria
How bacteria are identified
There are certain basic techniques that the student of microbiology must learn to
use in the laboratory. These techniques are used in growing bacteria, isolating them in
pure culture (containing only one kind of bacterium), observing them, and finally
in identifying the organisms.
THE MICROSCOPE
Light Microscopes
Accounts in seventeenth-century scientific literature convey the excitement
with which the
Royal Society of London awaited letters and descriptions from Leeuwenhoek.
We have advanced a long way in the last 350 years with respect to the equipment
available for the microscopic study of microorganisms. The microscope you will use in
the laboratory is no longer the "simple" (single lens) microscope of the type made
and used by Leeuwenhoek but rather what we call a compound microscope because
it has two sets of lenses (see Figure 2.1). One set is next to the object to be studied and,
therefore, is called the objective. The other set, the ocular, is (as the name implies) the
one next to your eye.
Figure 1.16 Modern binocular (two eyepieces) microscope. Note the
mechanical stage, which facilitates the movement of slides.
Both objectives and oculars are designed for different magnifications. The
objectives usually are mounted in a rotating wheel known as a turret or
revolving nosepiece; any one objective may be rotated into place depending on
the magnification desired.
Oculars are made in different magnifications — that is, X5, X 10, and X
15 (X meaning power or times actual size) — but most frequently the X 10 is
used. Now, how do these two lenses work together to give a higher magnification of the specimen than is possible with one lens alone?
Suppose that the objective is X10, which means that it makes an image 10
times as big as the object under study. Here you have, in effect, a simple
magnifying glass. Now, suppose that you view this magnified image through a
second lens (the ocular) that will magnify it five more times. How much larger
than the object is the final image that you see? The answer is 50. The total
power of any microscope, then, can be determined simply by multiplying the
objective power by the ocular power.
The usual laboratory microscope has three objective lenses: low-power,
high-power, and oil-immersion. The latter is the highest-powered of them all
and is used almost exclusively to study bacteria. A drop of clear oil is placed
directly on the cover glass over the specimen, and the oil-immersion objective
is lowered into the oil. The light rays traveling from the specimen through glass
and oil do not bend as much as they would if they were to pass through glass and
air; therefore, the image is clearer. The so-called high-power objective is
actually intermediate in the power range; because it does not require oil, it is
usually referred to as high-dry.
The oil-immersion objective usually magnifies 97 times; with a X10 ocular,
the total magnification of the objective would be X 970. It is possible to obtain
somewhat higher power with different lens combinations, but there is a definite
limit to magnification by this means. The high-dry usually has a X 44 objective;
thus, with a X10 ocular the overall magnification is 440. The low-power is
usually a 10 X 10, or a hundredfold, magnification.
A physical law states that the smallest detail we can see (called the
resolving power) is limited by the wavelength of light being used to illuminate
the specimen. The limit of resolution in ordinary visible light can be calculated
to be approximately 0.2 mkm, and all the lenses in the world will not let you see
something smaller than 0.2 mkm unless a different type of light source is used.
Ultraviolet Microscope
A variation on the ordinary light microscope is the ultraviolet microscope.
Because ultraviolet light has a shorter wavelength than visible light, the use of
ultraviolet light for illumination can increase the resolving power to twice that
of the light microscope. The limit of resolution then becomes 0.1 mkm. Since
ultraviolet light is invisible to the human eye, the image must be recorded on a
photographic plate. These microscopes use quartz lenses, and they are too
intricate and expensive for common use.
Fluorescence Microscope
Bacteria stained with a fluorescent dye will be a different color under the
fluorescence microscope than they are under an ordinary light microscope. The
quality known as fluorescence can be observed in the presence of ultraviolet
light. Mycobacterium tuberculosis stained with auramine (a yellow dye) will
appear bright against a dark background. Because most bacteria do not stain
with this dye, this procedure is useful in identifying the tubercle bacillus.
Fluorescence microscopy may also be used to detect foreign substances or
antigens (such as bacteria, rickettsiae, or viruses) in tissues. In this technique
the specific antibody protein is first separated from the serum in which it
occurs. It is then combined, or conjugated, with a fluorescent dye. Because
antibody-antigen reactions are specific, fluorescence will occur only if the
antigen in question is present and binds to the fluores-cently labeled antibody.
Antibody-antigen reactions are discussed further.
Dark-Field Microscope
The dark-field microscope is used for viewing living bacteria, particularly
those so thin that they approach the limit of resolution of the compound
microscope. Many pathogenic spirochetes fall into this category, particularly
the syphilis spirochete, Treponema pallidum.
The dark-field microscope differs from the ordinary compound light
microscope only in having a special condenser which produces a hollow cone of
visible light. The rays of light from this hollow cone do not go directly up into
the objective lens. Instead, they are reflected away at a slight angle from the
top of the slide (see Figure 2.3). However, any light rays touching the specimen
will be reflected directly into the objective, with the result that the specimen
looks completely white against a black background. With no specimen in the
field, the field would look dark, since there would be nothing to cause the light
to be reflected up into the objective.
Phase-Contrast Microscope
The ideal way to observe living matter is in its natural state: unstained and
alive. As a rule, however, a microscopic fragment of living matter (such as
animal tissue or bacteria) is practically transparent, and individual details do not
stand out. This difficulty can be overcome with the use of the phase-contrast
microscope.
The principle of this device is complicated. For example, when an ordinary
microscope is used, the nucleus of an unstained living cell is invisible. Since the
nucleus is nevertheless present in the cell, its presence will alter very slightly
the relationship of the light that passes through the nucleus with the light
passing through the material around the nucleus. This relationship, imperceptible to the human eye, is called a phase difference. However, an
arrangement of filters and diaphragms on the phase-contrast microscope will
translate this phase difference into a difference in brightness — that is, into
areas of light and shade that can be discerned by the eye — rendering the
hitherto invisible nucleus (or any other structure) visible.
Electron Microscope
Transmission Electron Microscope
The transmission electron microscope has been a boon to microbiologists
in the past 35 years (see Figure). This microscope uses a beam of electrons
rather than visible or ultraviolet light. Because the wavelength of the electron
beam is much shorter (approximately 0.005 ran, or nanometers) than that of
even ultraviolet light, the resolving power of the microscope is increased
tremendously. With a resolving power greater than one nm (0.001 /im),
magnifications of as much as 1 million diameters are possible. The image produced by
the electron microscope is visible when projected onto a fluorescent screen.
Figure 1.17. The operation of an electron microscope requires a great deal of
experience and skill to obtain clear results. However, the magnification obtainable is
many times that possible with the light microscope.
There are several major problems involved in using a transmission electron
microscope: (1) it requires a skilled technician for operation; (2) it is a very expensive
piece of equipment; (3) it requires the use of very thin specimens (which may be easily
distorted); (4) the specimen being examined must be contained in a very high vacuum
in order that the electrons may move effectively; (5) live specimens, therefore, cannot
be examined; and (6) objects show no color. This means that preparations must be
dried and fixed with chemicals prior to study, and the drying and fixing process may
also result in distortion of some of the cellular components.
An important development in electron microscopy is the use of phosphotungstic
acid as a negative stain. Phosphotungstic acid is electron- dense, allowing structural
details of the cell to be observed while the background and empty areas remain
opaque.
Shadow casting is a technique for revealing surface details. An electron-dense
metal such as platinum or chromium is vaporized under hifh vacuum and deposited at
an angle on the preparation. The uncoated area on the opposite side acquires a
shadow, and the resulting electron micrograph shows a three-dimensional effect.
Scanning Electron Microscope
The scanning electron microscope employs a beam of electrons, but — instead
of being simultaneously transmitted through the entire field — the electrons are
focused as a very fine probe or spot which is moved back and forth over the
specimen. As the probe electrons strike the surface of the specimen, secondary
electrons are emitted and then collected by a cathode ray tube. The strength of the
signal will be seen as dark or light areas on the collector, providing an image of the
specimen's surface. Photographs taken of the cathode ray tube appear as threedimensional micrographs, as is shown in Figure 1.19.
Figure 1.18 Electron micrograph of the spirochete Spirochaeta stenostrepta
shadowed with platinum (X 12,100).
Figure 1.19 The three-dimensional qualities of scanning electron microscopy
clearly reveal the corkscrew shape of cells of the syphilis-causing spirochete T.
pallidum, attached here to rabbit testicular cells grown in culture (X8000).
TECHNIQUES FOR MICROSCOPIC STUDY OF BACTERIA
Living Bacteria
Living bacteria are difficult to see with the average light microscope because they
appear almost colorless when viewed individually, even though the culture as a whole
may be highly colored. However, it is often necessary and desirable to look at living
bacteria under the microscope, particularly to determine whether they are motile.
Laboratory Equipment and Procedures
One satisfactory means of observing living bacteria is in a hanging drop
preparation. A drop of liquid culture (or organisms suspended in water) is placed in
the center of a coverslip. A special slide with a hollow depression in the center is
used. The depression is ringed with a thin film of petrolatum and then turned upside
down over the cover glass so that the drop of culture is in the center of the
depression. The entire slide with the coverslip then is quickly turned over so that the
drop of culture actually hangs from the coverslip down into the depression. The slide
is placed on the stage of a microscope and the organisms are observed using the highdry or the oil-immersion objective (see One can also use a normal flat slide to
determine motility; however, a thin ridge of petrolatum must be applied under the
edge of the coverslip to prevent convection currents caused by evaporation from the
wet mount.
Keep in mind that a bacterium is considered motile only if it seems to be going
in a definite direction. Even nonmotile bacteria will bounce back and forth rapidly
(brownian movement) due to bombardment from molecules of water.
Figure 1.20. This side view of a hanging-drop preparation shows
the drop of culture hanging from the center of the cover glass above the
depression slide.
Stained Bacteria
Bacteria are far more frequently observed in stained smears than in the living
state. By stained bacteria we mean organisms that have been colored with a
chemical stain so as to make them easier to see and study. In general, stained smears
of bacteria reveal size, shape, and arrangement and the presence of certain internal
structures such as granules and spores. Special stains are used for observing capsules
or flagella as well as certain internal cellular details. Stains that reveal chemical
differences in bacterial structure may also be employed. These are called differential
stains.
To prepare bacteria for staining, a small amount of culture is spread in a drop of
water on a glass slide. This is called a smear. The smear is dried at room temperature,
and the bacteria may be firmly bound (fixed) by passing the slide quickly (smear
side up) two or three times through the flame of a Bunsen burner. When cool, the
smear is ready to be stained. Alternatively, many laboratories use methanol to fix the
bacteria to the slide because heat may cause distortions in the appearance of the stained
bacteria. This is done by placing a few drops of methanol on the air-dried smear and
again allowing the smear to air-dry. The following sections outline a few of the more
common procedures used to stain bacteria.
Staining Reactions
Stains are salts composed of a positive and a negative ion, one of which is
colored. In basic dyes, the color is in the positive ion (i.e., dye+ Cl~), whereas in acid
dyes it is in the negative ion (i.e., Na+ dye").
The marked affinity of bacteria for basic dyes is due primarily to the large amount
of nucleic acid in the cell's protoplasm. Thus, when the bacterium is stained, the
negative charges in the nucleic acid of the bacterium react with the positive ion of a
basic dye. Crystal violet, safranin, and methylene blue are a few of the basic dyes
commonly used.
In contrast, acid dyes are repelled by the overall negative charge of a bacterium.
Thus, staining a bacterial smear with an acid dye has the effect of coloring only the
background area. Since the bacterial cell is colorless against a colored background,
this technique is very valuable for observing the overall shape of extremely small
cells. This process is referred to as negative staining.
Staining Techniques
Simple stain This, as the name implies, is the simplest type of staining. One
merely covers the fixed smear with any one of the following dyes: gentian violet,
crystal violet, safranin, methylene blue, basic fuchsin, and other basic aniline dyes.
After 30 to 60 sec the slide is washed off under the water tap and the smear gently
blotted dry. It is now ready to be looked at under the microscope. After locating the
stained specimen with the high-dry objective, one can observe it under oil immersion
by placing a drop of oil directly on the stained smear and lowering the oilimmersion objective into the oil.
Gram stain In 1884 the Danish physician Christian Gram devised a special
stain that is probably the most important one used in bacteriology. It is a differential
stain, so called because it divides all the true bacteria into two physiologic groups,
thereby greatly facilitating the identification of a species. The staining procedure has
four steps: (1) the smear is flooded with gentian or crystal violet; (2) after 60 sec, the
violet dye is washed off and the smear is flooded with a solution of iodine; (3) 60 sec
later, the iodine is washed off and the slide is washed with 95 percent ethyl alcohol
for 15 to 30 sec; and (4) the slide is counterstained for 30 sec with either safranin (a
red dye) or Bismarck brown. (The Bismarck brown usually is used by people who are
color-blind to red.) The length of time that each dye is left on the smear is not
critical, and many laboratories have modified this procedure to allow each dye to
remain in contact with the fixed organisms for only a few seconds. Also, many
people prefer to use a 50:50 mixture of acetone and ethyl alcohol for the
decolorization step because this solution acts faster than the 95 percent alcohol
alone. Either decolorizing agent should give the same result, but in the
discussion to follow we shall refer to 95 percent ethyl alcohol rather than the
acetone-alcohol mixture.
The violet dye and the iodine form a complex compound. Some genera of
bacteria readily give up the stain when washed; in other bacteria the stain
resists even a wash of 95 percent ethyl alcohol. Organisms that do not reta in
the dye complex after the 95 percent alcohol wash are called gram-negative
organisms; those that retain the complex are called gram-positive organisms.
Because gram-negative bacteria are colorless after the alcohol wash, one
always counterstains with a different color dye before looking at the smear
under the microscope. The usual counterstain is the red dye safranin; hence
gram-negative bacteria are red. Because alcohol does not wash out the blue
dye complex from gram-positive cells, safranin counterstain has no effect, and
gram-positive cells appear blue or bluish purple. Table 2.2 summarizes the
appearance of both types of cells after each step of the Gram staining
procedure. Table 2.3 lists the Gram reactions of some selected common bacteria.
Many theories have been proposed to explain why some organisms are
gram-positive and others gram-negative. This staining difference results from
the fact that the cell walls of gram-positive bacteria are different from those of
the gram-negative bacteria; the dye-iodine complex appears to become trapped
between the cell wall
Table Gram Reaction of Selected Common Bacteria
Gram-positive
Gram-negative
Bacillus
Bacteroides
Neisseria
Clostridium
Bordetella
Pasteurella
Corynebacterium
Brucella
Proteus
Lactobacillus
Enterobactc
Pseudomonas
Listeria
Escherichia
Salmonella
r
Micrococcus
Franciscella
Shigella
Staphylococcus
Haemophilu
Vibrio
Streptococcus
Kkbsiella
Yersinia
s
and the cytoplasmic membrane of the gram-positive organisms, whereas
the removal of lipids from the gram-negative cell wall by the alcohol wash
permits the dye-iodine complex to be washed out of the cell.
It is essential to know the Gram reaction (positive or negative) as well as the
overall appearance of the bacterium in order to identify it. In addition, other
general characteristics of an organism are associated with the Gram reaction. For
example, most gram-positive bacteria are easily killed by low concentrations of
penicillin, gramicidin, or gentian violet, whereas gram-negative bacteria are
much more resistant to these compounds but are considerably more sensitive to
streptomycin. We discuss the reasons for these differences in more detail in the
next Chapters .
Acid-fast stain The acid-fast stain (also called the Ziehl-Neelsen stain) is
used specifically to help identify organisms in the genus Mycobacterium. This
genus contains many non-disease-producing organisms as well as some virulent
pathogens, of which tuberculosis and leprosy are the most important. The
mycobacteria are called acid-fast because once they are stained with
carbolfuchsin (a red dye), their unique chemical properties cause the stain to
remain even though the stained smear has been washed with acid alcohol (95
percent ethanol containing 3 percent hydrochloric acid). This treatment removes the dye from all other organisms in a smear. One exception is found in
the pathogenic (disease-producing) moldlike bacteria classified in the genus
Nocardia. These bacteria are not as strongly acid-fast as the pathogenic
mycobacteria are, and prolonged washing decolorizes most nocardia. This
property sets the acid-fast group apart from other bacteria and makes it possible
to stain mixtures of large numbers of bacteria (such as those present in sputum)
and still recognize the acid-fast bacteria. This is a real help in the diagnosis of
tuberculosis.
Other stains A number of additional specialized staining procedures are
used to stain parts of the bacterial cell. They include techniques for staining
capsules, cell walls, nucleic acid, flagella, endospores, and other structures. All
these staining procedures involve the use of two or more special dyes, but none
of these stains is used routinely in the identification of a bacterium.