Negative Staining is a technique by which organisms
remain unstained against a dark background.
A small quantity of India ink 10% nigrosin is mixed with
the material on a slide. A smear is made by means of
another slide and the preparation is allowed to dry. The
smear is examined and the spirochetes are seen as clear
transparent objects against a dark brown background.
Capsules may also be demonstrated by this method.
This method is used to observe the morphology but also
demonstrates the motility of organisms. A special slide
with a concave center is used or else a ring of plasticine
can be placed on the slide. A drop of the culture of
bacterial suspension is placed on a coverslip. Vaseline is
placed near the concave area of the slide approximately
the corners of the coverslip. The slide is placed over the
coverslip so that the drop of culture is directly under the
concave area and the Vaseline adheres to the coverslip.
The slide is then quickly inverted and placed under
the microscope. Motile organisms will be seen darting
through the medium in which they are suspended. Motility
should be differentiated from Brownian movement which
is caused by bombardment of the molecules of the fluid.
In motility, the organisms move in a definite direction,
whereas in Brownian movement they show no direction.
Four factors are to be taken into account
1. Media providing optimum growth
4. Cultural characteristics, e.g. size, shape and pigmentation of colonies.
Media can be (a) basic (b) enrichment (c) selective, and
These contain the necessary constituents for growth—
meat extract, peptone and salt, and these are nutrient
broth (liquid) or nutrient agar (solid). Many organisms
would grow on these types of media and need no other
These are used for organisms, which need an additional
source of nutrition. This can be done by adding blood
or serum to the nutrient agar or broth. An enrichment
medium used for growth of the Mycobacterium tuberculosis
3. Differential and Selective Media
These media by virtue of their chemical composition
inhibit the growth of some organisms while at the same
dye or an indicator in the decolorized state. Bacteria,
which ferment lactose with the production of acid will
of lactose non-fermenting organisms. Some media, which
are used are also highly selective in their action on other
organisms. Such media as SS agar, deoxycholate citrate
agar and bismuth sulfite agar will inhibit the growth of
the majority of coliform bacilli along with many strains of
proteus and will permit the successful isolation of enteric
pathogens. Tellurite glycerin agar and mannitol salt agar
are selective media for the isolation of coagulase positive
Staphylococcus from material containing other organisms.
Phenyl-ethyl-alcohol agar is a selective medium for the
isolation of gram-positive cocci in specimens or cultures
contaminated with gram-negative organisms particularly
proteus. Infusion agar containing potassium tellurite
and blood/serum inhibits the growth of normal throat
commensals and encourages the growth of C. diphtheriae.
Some medias make use of the selective antimicrobial
activity of some antibiotics and are useful for isolating
cycloheximide and chloramphenicol will support the
growth of dermatophytes and most fungi, while markedly
inhibiting the growth of many saprophytic fungi and
These are largely used for biochemical reactions. The
most common example is sugar media containing various
carbohydrates such as glucose, lactose, maltose, etc.
Christensen’s urea medium is used mainly in the iden-
826 Concise Book of Medical Laboratory Technology: Methods and Interpretations
tification of Proteus, which has the ability to hydrolyze
the urea, and consequently because of the presence of
phenolphthalein in the medium, a change of color is
Most bacteria, pathogenic in humans, give optimum
growth when incubated at body temperature, i.e.
37°C. Some saprophytes, however, grow best at lower
temperatures, even as low as 4oC (cryophilic) and others at
high temperatures. The latter are known as thermophilic
bacteria and are used in testing effectiveness of sterilization
Most organisms need oxygen for growth and are incubated
in normal atmospheric conditions. Some pathogens, e.g.
tetanus bacilli, will grow only in the absence of oxygen. This
is achieved by using McIntosh and Fildes’ jar, a thick metal or
glass jar with a metal lid which can be clamped down tightly
by bolts. On this lid are 2 holes-one an air inlet and the other
an outlet. There are also 2 electric terminals. On the underside
of the lid is a piece of asbestos saturated with palladium and
covered by wire gauze. This is connected to the terminals,
and acts as a catalyst in combining any oxygen still present
after evacuation of the jar with the hydrogen, which is passed
1. Keep the plates upside down in the jar.
2. Place in the jar an indicator—equal parts of 10% NaOH,
6% glucose and 0.5% methylene blue, boiled until the
solution becomes colorless. It should remain colorless
throughout incubation. If it turns to its original blue
color during incubation, complete anaerobiosis
(oxygenless state) has not been achieved.
3. Tightly clamp down the lid.
4. Open the air outlet valve and close the air inlet valve.
5. Attach the apparatus to an exhaust pump, and slowly
evacuate the jar (If a glass jar is used, it should be
evacuated while enclosed in a padded box to avoid
6. Allow hydrogen obtained from hydrogen cylinders or
Kipp’s apparatus in through the inlet valve after closing
7. Attach the terminals to the main current and leave for
20 minutes. This heats the palladiumized asbestos to
assist the combination of hydrogen with any remaining
8. Allow a little more hydrogen in via the inlet valve.
9. Put the jar in the incubator overnight. The present day
McIntosh-Filde’s jars have room temperature catalysts
and need no electrical charge. They are left at room
temperature for 15–30 minutes before allowing more
hydrogen into the jar. There are other, less complicated
methods of achieving anaerobiosis (i.e. an oxygenless
a. Boil a tube of nutrient broth and layer over it
sterile Vaseline. The boiling removes the oxygen
and the Vaseline prevents more entering as the
broth cools. The tube is inoculated using a sterile
b. A sterile iron nail placed in glucose broth which
has been treated as in method (1), will maintain
anaerobic conditions for some time.
c. Robertson’s cooked meat medium and Brewer’s
thioglycollate broth are frequently used in the
culture of anaerobic organisms.
Some organisms are not anaerobic, but do grow better
when the amount of oxygen has been reduced. One simple
technique is to place the plates in a tin or wide mouthed
bottle with a tight fitting lid. A candle is lit inside the
container and the lid replaced firmly. The candle flame
will use off the oxygen and give an atmosphere of 5–10%
CO2. The container is placed in the incubator.
Bacteria grown artificially (in vitro) on agar plates are
described as colonies. These colonies vary in size, shape,
pigment production, and hemolysis on blood agar
depending on the type of media.
Circular, regular, radiating or rhizard.
Smooth, rough, fine, granular shiny, dull, etc.
Usually colonies are 2–3 mm in diameter, smaller ones
Colonies may be discrete or swarming.
May be mucoid, tenacious dry or adherent to the medium.
Some organisms produce pigmented colonies (Staphylococci, Pseudomonas).
Microbiology and Bacteriology 827
On nutrient agar they may be transparent, translucent or
Colonies may be raised, low convex, umbilicated or dome
Colonial growth may bring about color changes in the
media themselves, e.g. hemolysis on blood agar by
hemolytic streptococci. With Pseudomonas, the green
pigment produced may diffuse into the medium.
Organisms that are alike in microscopic and cultural
characteristic are often differentiated by their reactions in
Specific carbohydrate fermentation is a property of some
organisms when grown in sugar media. Sugars most
frequently employed are glucose, sucrose, lactose, mannite,
maltose and dulcite. Usually, these are incorporated into
peptone water, but for the more delicate organisms, Hiss’s
serum water must be used. Meningococci and gonococci
will only react in solid serum-sugar media. Each sugar
medium has a colored stopper and a set ‘color scheme’
may be established for the following sugars.
Glucose (green), Lactose (red), Sucrose (blue), Mannite
(mauve), Maltose (blue and white), Dulcite (pink).
The organism ferments sugar and produces acid and, in
certain groups, gas. Acid production is indicated by a color
change of the medium, due to inclusion of a pH indicator.
Gas production is shown by placing a small Durham’s tube
upside down in the medium during its production. Before
inoculating the medium the tube should be completely
filled with the medium. If gas is produced, small bubbles
of gas will be seen in the inverted tube.
Organisms may further be identified biochemically by
their production of indole, change in pH (as shown by
the methyl red test), by their utilization of citrate and
by another test called the Voges-Proskauer reaction.
These 4 tests are especially useful in the differentiation of
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