Microbiology - Laboratory Procedures
4. LABORATORY PROCEDURES IN BACTERIOLOGY
Microbial cells can be observed with an optical microscope. As most cells
are clear and colorless, there has to be a means of making them visible.
The most common means is staining. There are many staining procedures. One
very common one is the Gram stain (named for its discoverer, Christian Gram).
A small amount of the culture is spread thinly on a microscope slide. The
slide is heated briefly to fix the cells (to make them adhere
to the glass). Then a solution of a dye, Crystal Violet, is applied briefly
and the excess is washed off. A solution of potassium iodide is applied,
followed by alcohol. Some kinds of bacteria (Gram positive) remain stained
(blue) while the alcohol washes the stain out of others. The bacteria which
have lost the blue dye are then counterstained with a red dye
such as safranin, so that Gram negative cells appear red. (See a
note below about dyes.) The staining procedure usually kills the bacteria,
but some stains (vital stains) allow survival. A negative stain
uses India ink, or a solution of an intensely black dye (nigrosin), which
is not absorbed by the cell. The ink or nigrosin is not washed off, and
the unstained cells are seen against a black background.
An optical technique, phase contrast microscopy, can be used to
see bacteria without staining. That technique uses the fact that the cell
contents are more concentrated than the liquid medium, so that they have
a higher refractive index and deflect some of the light. (The inventor of
this technique won a Nobel prize for it; obviously, I will not try to explain
it here.) An older technique, dark field microscopy, illuminates
the cells from the side so that only the light they deflect enters the objective
Microscopy is tedious, and can only be used to see small numbers of cells,
which are often dead. Culture techniques have been very useful for studying
metabolism and growth. Bacteria are grown in or on a nutrient medium
(plural: media). Because making these media from their components
is a time-consuming job, several companies supply dehydrated prepared
media. Components of the media are selected so as to nourish most bacteria,
or only specific ones (selective media). Development of solid media
made it possible to grow individual colonies from which pure cultures can
be produced. Media are most often solidified with agar, a polysaccharide
from seaweed. The ingredients, in water, are mixed with 1.5% to 2% agar
and brought to boiling temperature to dissolve the agar, which solidifies
again on cooling to about 35-40 °C. Gelatin and silica have limited use
to solidify media.
A liquid medium is commonly called a broth (German/French: bouillion),
while a solid one is an agar. Nutrient broth and nutrient
agar are very common growth media. There are many such media, typically
with names such as MacConkey Broth, Azide Blood Agar, Levine
EMG Agar, Brilliant Green Bile Broth, etc. These names combine proper
names, chemical names, and dye names; and translation requires some care.
Dyes in stains and media: Dye names are more nearly proper names than chemical
names or descriptions, but one dye may have several names. For instance,
Brilliant Green is used in selective media to inhibit growth of most bacteria
other than certain sewage organisms (coliforms). It has at least ten other
names in English, and my references show two slightly different chemical
structures for the same name. The most common German name appears to be
Brilliantgrün, but that can have other meanings (e. g., a chromium oxide
pigment). Crystal Violet is also known as Gentian Violet (Enzianviolett).
Be very careful! Laboratory supply catalogs usually list the more common
growth media and stains, and it might be a good idea to see if your proposed
translation is on such a list. Some manufacturers of prepared media publish
extensive information; e. g., the Difco Manual (Difco Laboratories, Detroit,
MI, 10th Ed., 1984).
In the laboratory, bacteria are grown in culture tubes, which look like
chemical test tubes except that they usually have straight rather than rolled
lips. Larger volumes are grown in Erlenmeyer (conical) flasks. Some conical
flasks are very wide in comparison to their height, and are called Fernbach
flasks. Some other flasks look like flat bottles lying on their sides (Roux,
Blake, Povitsky, Coli, or tissue culture flasks).
The necks of the tubes or flasks are plugged with nonabsorbent cotton or
are covered with caps made of plastic or stainless steel) to keep airborne
bacteria out of the culture while allowing air to enter. (Most laboratory
cultures are aerobic).
It is always necessary to transfer small amounts of the culture from a mature
culture to fresh medium or to microscope slides. That requires care to prevent
contamination. In a typical transfer, the bacteriologist will:
- heat a wire inoculating loop (mounted in a handle) to red
heat in a flame or electric heater
- while holding the loop, remove the plug or cap from the culture
- while holding the loop, plug or cap, and the culture vessel,
flame the mouth of the vessel by rotating it above a gas flame
- insert the loop into the culture and remove it with a drop of
- flame the mouth of the vessel again, while still holding the
loop and the plug or cap
- replace the plug or cap in or on the vessel
- remove the plug/cap from a fresh vessel of medium
- flame the mouth of that vessel
- insert the loop to make the actual transfer
- remove the loop and flame the vessel mouth again
- replace the plug or cap in or on the new vessel
- and, finally, flame the wire loop again.
It is rather regrettable that bacteriologists did not evolve from arachnids,
as the extra arms would have been useful.
Cultures can also be grown in solid media in tubes. After the melted agar
medium is poured into the tubes, the tubes can be tilted at about 45°
to cool, forming slants, or held vertically to produce butts. The
bacteriologist can pick up some inoculum on an inoculating needle (rather
than a loop) and make a stab culture by plunging the needle deeply into
the solid medium. In a stab culture, aerobic organisms grow at the top,
anaerobes at the bottom, and microaerophiles in between. Slants are also
used for surface culture. Culture are also grown in Petri plates (see
Work requiring transfers (inoculation) is best done in a laminar flow
bench in which air is forced through a large HEPA (high efficiency particulate
air) filter to remove particles of about 0.5 mm and larger. Some such
benches made for clean room use blow filtered air toward the operator;
microbiologists are better off with laminar flow benches which blow downward,
with enough inward air flow to prevent escape of microorganisms. Work
with disease-producing organisms (pathogens) is often done in glove
boxes or in laboratories in which the microbiologists are enclosed in
impermeable garments with filtered air supplies.
It is often necessary to determine how many living (viable) bacterial
cells are present. As the concentration may easily be as high as 1,000,000,000
(109) cells/milliliter, a sample taken with a sterile pipet is diluted
in several steps by pipetting 1 ml into 99 ml diluent for a 1:100 dilution,
or into 9 ml diluent for a 1:10 dilution. The aim is to get a concentration
in the range of 30 - 300 colony-forming units (CFU; German Koloniebildendeeinheiten,
KBE) per milliliter. The CFU terminology is relatively recent, and is
used because several cells may stick together and eventually produce a
single colony. The bacteriologist, never sure of the actual concentration,
will usually test 3 to 5 different dilution stages.
The final counting is done by plating the cells C growing them
in or on a solid medium in a Petri plate. The Petri plate is a dish about
75 or 100 mm in diameter, with vertical sides about 10 mm high. (There
are also square Petri plates.) The bacteriologist can make either:
- a pour plate: the sample is pipetted into the plate, and then a tube
of about 10 ml of melted agar medium is poured onto it. The sample and
liquid agar are mixed by swirling, covered with a lid (same shape but
slightly greater diameter), cooled to solidify, and incubated.
- a spread plate: the solid medium is already in the plate, and the
sample is pipetted in, then spread with a sterile glass rod bent in
the shape of a hockey stick, and incubated.
After incubation, typically for 18-20 hours, but sometimes longer, the
separate cells (or colony-forming units) will have grown into visible
colonies which can be counted. Plate counts less than 30 are too small
to be useful (statistically) and plates with more than 300 the colonies
may be too crowded to grow well. High counts are often reported as TNTC
(too numerous to count). The count is multiplied by the dilution factor
to determine the population in the original culture. Counting is best
done by placing the culture plate on a colony counter which
provides illumination and magnification. Counts can also be done with
an automatic colony counter using a video camera.
Viable counts can also be done by filtering a known volume of culture
through a sterile membrane filter which has pores smaller than bacteria
(typically 0.22 mm pores), then laying the filter on a pad of nutrient
medium for incubation to produce colonies.
However colony counts are done, the actual numbers found should not be
taken too seriously. Random variation in the sample alone will cause plus/minus
variation of about the square root of the actual count of 30 - 300 colonies.
In another method, the microbiologist pipets 10, 1, and 0.1 ml portions
of a dilution into three to five tubes of media and then counts the number
of tubes showing growth. Tables in reference manuals show the most
probable number (MPN) for a given result. For example, if growth appears
in 2 of 5 tubes with 10 ml inocula, 3 of 5 with 1 ml inocula and 1 of
5 with 0.1 ml, then the MPN is 18 organisms per 100 ml of that dilution.
Total (not necessarily viable) counts can be done in various ways. One
common instrument is a Coulter counter in which a known
volume of sample is forced through a tiny hole in a glass membrane while
a constant electric current is also passed through the solution flowing
through the hole. When a bacterial cell occupies the hole the resistance
is higher and the voltage across the hole jumps, giving a pulse which
can be counted. These counters work quite well for blood cells, but for
bacteria the hole must be so small that it is easily clogged, and many
particles other than bacteria may be counted. In flow cytometry,
individual cells are illuminated by a laser beam and detected by scattered
or fluorescent light.
Sterile is usually understood to mean absence of any
organism which can grow. For better or worse, the mathematics used to
describe bacterial growth and death imply that the actual viable count
becomes steadily smaller but never actually arrives at zero. That has
led to talk of the probability of a single organism surviving, with requirements
of (for instance) a probability of less than 1 in 1012 of such survival.
All pharmaceutical products to be injected are required to be sterile.
If the product is stable to heat, the best way to sterilize is with an
autoclave. The sample, or product, is held for some required period
in steam under pressure (air having been forced out of the autoclave initially).
Small culture tubes and flasks, for instance, are typically sterilized
for 15 minutes at a steam pressure of 15 pounds per square inch (equivalent
to a temperature of 121 °C). Larger containers, and materials into which
steam penetrates more slowly, must be held for longer periods.
Many products cannot stand steam sterilization. Spacecraft of the Ranger
series, about 1965, were required to be sterilized to make sure that they
did not introduce any earth bacteria to the moon.
Apparently the sterilization damaged the electronic systems so severely
that none of those spacecraft landed successfully. (There may have been
other reasons; rocket technology was young).
When steam cannot be used, ethylene oxide, diluted in carbon dioxide and
with some moisture present, can be used in a gas sterilizer for solid
materials. A substantial outgassing period must be provided afterward
to remove remaining ethylene oxide, and chemical analysis may be required
to show that the remaining ethylene oxide is below specified concentration
limits. Other chemical sterilants, used as liquids, include hydrogen peroxide,
peracetic acid, formaldehyde, glutaraldehyde, and a few others. Liquids
which cannot be autoclaved are probably best sterilized by filtration
through very fine filters (typically with 0.22 mm pores).
After sterilization, materials must be tested to make sure that the sterilization
was successful. At one time the standard test for pharmaceuticals was
to transfer a small volume (e. g., 1 ml) from each of 20 containers of
the product into 20 culture tubes of an aerobic medium and into each
of 20 tubes of an anaerobic medium. The tubes were incubated for 2 weeks,
and if no growth appeared in any tube the product was considered sterile.
More recently, it has become possible to test liquids by pumping them
aseptically through sterile membrane filters, and then adding a culture
medium to the container with the filter. The best-known system appears
to be the Steritest 7 system from the Millipore Corporation.
Some pharmaceuticals and cosmetics do not require sterility tests, but
must be tested for presence of certain species of bacteria, yeasts, and
molds (microbial limits tests).
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Copyright © 2010 Denzel Dyer, all rights reserved.