Lecture 03

27/07/2018

Methods of microbial investigation

 Methods of Microbial Investigation

• Biologists can use their senses of sight, smell, hearing, and even touch
to detect and evaluate identifying characteristics of animals and
plants.

• Microbiologists cannot rely on senses other than sight, because they

are confronted by some unique problems.

• First, most habitats (such as soil, water and the human mouth) harbor
microbes in complex associations. It is often necessary to separate the
organisms from one another so they can be identified and studied.
• Second, to maintain and keep track of such small
research subjects, microbiologists usually need to grow
them under artificial conditions.
• Third, they are not visible to the naked eye.
• They have wide distribution, and undesirable ones can
be unknowingly introduced into an experiment, where
they may cause misleading results.

• Microbiologists have developed several types of

procedures for investigation and characterization of
microorganisms. These techniques are known as the six
“I’s”.
• The Six “I’s”: Inoculation, incubation, isolation, inspection,
information gathering, and identification.
 Specimen Collection
• Microbiologists begin by sampling the object of
their interest. It could be nearly any thing or
place on earth (or even Mars). Very common
sources are body fluids, foods, water, soil,
plants, and animals, but even places like
icebergs, volcanoes, and rocks can be sampled.
 Inoculation
• The sample is placed into a container of
medium that will support its growth. The
medium may be in solid or liquid form, and
held in tubes, plates, flasks, and even eggs.
The delivery tool is usually a loop, needle,
swap or syringe.
 Incubation
• Inoculated media are placed in a controlled
environment (incubator) to promote growth.
During the hours or days of this process, a
culture develops as the visible growth of the
microbes in the container of medium.
 Isolation
• Some inoculation techniques can separate
microbes and spread them apart to create
isolated colonies that each contain a single type
of microbe. This is invaluable for identifying the
exact species of microbes in the sample, and it
paves the way for making pure cultures.
 Inspection
• Cultures are observed for the macroscopic
appearance of growth characteristics. Cultures
are examined under the microscope for basic
details such as cell type and shape. This may
be enhanced through staining and use of
special microscopes.
 Information Gathering
• Additional tests for microbial function and
characteristics are usually required. This may
include inoculations into specialized media that
determine biochemical traits, immunological
testing, and genetic typing. Such tests will
provide specific information unique to a certain
microbe.
 Identification
• One goal of these procedures is to attach a name
or identity to the microbe, usually to the level of
species. Any information gathered from inspection
and investigation can be useful.
Identification is accomplished through the use of
keys, charts and computer programs that analyze
the data and arrive at a final conclusion.
• Depending on the purposes of the particular
investigator, these may be performed in several
combinations and orders, but together, they serve as
the major or basic tools of the microbiologist’s trade.
• Many professional microbiologists use more
advanced investigation and identification techniques
that may not even require growth or absolute
isolation of the microbe in culture.
• Viable but non-culturable (VBNC)
 The Microscope

• An optical instrument used for viewing very

small objects, such as animal, plant cells or
microorganisms, typically magnified several
hundred times.
• The two key characteristics of a reliable
microscope are magnification, the ability to
make objects appear enlarged, and resolving
power, the ability to show detail.
Effects of magnification. Demonstration of the magnification
and image-forming capacity of clear glass “lenses.” Given a
proper source of illumination, a magnifying glass can deliver 2
to 10 times magnification.
The pathway of light and the two
stages in magnification of a
compound microscope
• Workings of an oil immersion lens. To maximize its resolving power, an oil immersion
lens (the one with highest magnification) must have a drop of oil placed at its tip.
This transmits a continuous cone of light from the condenser to the
objective, thereby increasing the amount of light.
Preparing Specimens for Optical Microscopes

• A specimen is generally prepared by mounting a sample on a

suitable glass slide that sits on the stage between the
condenser and the objective lens.
The manner in which a slide specimen, or mount, is prepared
depends upon:
(1) the condition of the specimen, either in a living or preserved
state

(2) the aims of the examiner, whether to observe overall

structure, identify the microorganisms, or see movement

(3) the type of microscopy available, whether it is bright-field,

dark-field, phase-contrast, or fluorescence.
 Fresh, Living Preparations

• Live samples of microorganisms are used to prepare wet

mounts so that they can be observed as near to their natural
state as possible.
• The cells are suspended in a suitable fluid (water, broth,
saline) that temporarily maintains viability and provides space
and a medium for locomotion.
• A wet mount consists of a drop or two of the culture placed on
a slide and overlaid with a cover glass.
• Although this preparation is quick and easy to make, it has
certain disadvantages. The cover glass can damage larger cells,
and the slide is very susceptible to drying and can contaminate
the handler’s fingers.
 Hanging drop slide
• It is made with a special concave (depression) slide, an
adhesive or sealant, and a coverslip from which a tiny drop of
sample is suspended.
• These types of short-term mounts provide a true assessment
of the size, shape, arrangement, color, and motility of cells.
 Fixed, Stained Smears

• A more permanent mount for long-term study can be obtained

by preparing fixed, stained specimens.
• The smear technique, consists of spreading a thin film made
from a liquid suspension of cells on a slide and
air-drying it.
• Next, the air-dried smear is usually heated gently by
a process called heat fixation that simultaneously kills the
specimen and secures it to the slide.
• Fixation also preserves various cellular components in a natural
state with minimal distortion.
• Fixation of some microbial cells is performed with chemicals
such as alcohol and formalin.
 Staining
• Staining is any procedure that applies colored
chemicals called dyes to specimens.
• Dyes impart a color to cells or cell parts by
becoming affixed to them through a chemical
reaction.
In general, they are classified as basic (cationic)
dyes, which have a positive charge, or acidic
(anionic) dyes, which have a negative charge.
• Dyes are related to or derived from the common
organic solvent benzene. When certain double-
bonded groups (C=O, C=N, N=N) are attached to
complex molecules, the resultant compound gives
off a specific color.
• Most dyes form ions when dissolved in a compatible
solvent. The color-bearing ion, termed a
chromophore, has a charge that attracts it to certain
cell parts that have the opposite charge.
 Basic dyes
• Basic dyes carry a positively charged chromophore and are
attracted to negatively charged cell components (nucleic acids
and proteins). These are used for positive-type staining.
• Because bacteria contain large amounts of negatively charged
substances, they stain readily with basic dyes such as
methylene blue, crystal violet, fuchsin, and safranin.
 Acidic dyes
• Acidic dyes with a negatively charged chromophore bind to positively
charged molecules in some parts of cells.
• One example is eosin, a red dye used in staining blood cells.
• Because bacterial cells have numerous acidic substances and carry a
slightly negative charge on their surface, they tend to repel acidic dyes.
• Acidic dyes such as nigrosin and India ink can still be used successfully on
bacteria using the negative stain method. With this technique, the dye
settles around the cell and creates an outline of it.
 Comparison of positive and negative staining

• Positive stain sticks to

cells and gives them
color.
• Negative stain used to
view cellular size, shape
and arrangement.
• Also to make the capsule
prominent around certain
bacteria and yeasts.
 Positive staining methods
• Positive staining methods are classified as simple, differential, or
structural.
• Simple stains require only a single dye and an uncomplicated procedure.
• In most simple staining techniques bacterial cells ready
binding to dyes like malachite green, crystal violet, basic fuchsin, and
safranin.
• Simple stains cause all cells in a smear to appear more or less the same
color, regardless of type, but they can still reveal bacterial characteristics
such as shape, size, and arrangement.
 Types of Differential Stains
• Differential stains use two different-colored dyes, called the primary dye
and the counterstain, to distinguish between cell types or parts.
• dyes of contrasting color to clearly emphasize differences between two
cell types or cell parts. Common combinations are red and purple, red and
green, or pink and blue.
• Differential stains can also pinpoint other characteristics, such as the size,
shape, and arrangement of cells. Typical examples include Gram, acid-fast,
and endospore stains.
 Gram stain

• Gram staining is a 130-year-old method named for its

developer, Hans Christian Gram. Even today, it is an important
diagnostic staining technique for bacteria.
• It permits ready differentiation of major categories based
upon the color reaction of the cells: gram-positive , which
stain purple, and gram-negative , which stain red.
• This difference in staining quality is due to structural
variations found in the cell walls of bacteria.
• The Gram stain is the basis of several important bacteriologic
topics, including bacterial taxonomy, cell wall structure,
identification and drug therapy.
 Acid-fast stain
• The acid-fast stain, like the Gram stain, is an important diagnostic stain
that differentiates acid-fast bacteria (pink) from nonacid-fast bacteria
(blue).
• This stain originated as a specific method to detect Mycobacterium
tuberculosis in specimens. These bacterial cells have a particularly
impervious outer wall that holds fast (tightly) to the dye (carbol fuchsin),
even when washed with a solution containing acid or acid alcohol.
• This stain is used for other medically important mycobacteria such as the
Hansen’s disease (leprosy) bacillus and for Nocardia, an agent of lung or
skin infections.
 Endospore stain
• The endospore stain (spore stain) is similar to the acid-fast method in that
a dye is forced by heat into resistant survival cells called spores or
endospores. These are formed in response to adverse conditions and are
not reproductive.
• This stain is designed to distinguish between spores and the vegetative
cells that make them. Of medical importance are the gram-positive,
members of the genus Bacillus (the cause of anthrax) and Clostridium (the
cause of botulism and tetanus).
 Structural stains
• Structural stains are used to emphasize special cell parts such as capsules,
endospores, and flagella that are not revealed by conventional staining
methods.
• Capsule staining is a method of observing the microbial capsule, an
unstructured protective layer surrounding the cells of some bacteria and
fungi.
• Because the capsule does not react with most stains, it is often negatively
stained with India ink.
• The fact that not all microbes exhibit capsules is a useful feature for
identifying pathogens. One example is Cryptococcus, which causes a
serious fungal meningitis in AIDS patients.
• Flagellar staining is a method of revealing flagella, the tiny,
slender filaments used by bacteria for locomotion.
• Because the width of bacterial flagella lies beyond the
resolving power of the light microscope, in order to be seen,
they must be enlarged by depositing a coating on the outside
of the filament and then staining it.
• The presence, number, and arrangement of flagella can be
helpful in identification of bacteria.
 Inoculation, Growth, and Identification of Cultures

• To cultivate, or culture microorganisms, one introduces a tiny sample (the

inoculum) into a container of nutrient medium (pl. media), which provides
an environment in which they multiply. This process is called inoculation.
• The observable growth that later appears in or on the medium is known as
a culture.
• The nature of the sample being cultured depends on the objectives of the
analysis. Clinical specimens from body fluids (blood, cerebrospinal fluid),
discharges (sputum, urine, feces), or diseased tissue.
• Samples subject to microbiological analysis can include nearly any natural
material, like soil, water, sewage, foods, air, and inanimate objects.
• A past assumption has been that most microbes could be teased out of
samples and cultured, given the proper media. But this view has had to be
greatly revised due to VBNC.
 Isolation Techniques

• Certain isolation techniques are based on the concept that if an individual

bacterial cell is separated from other cells and provided
adequate space on a nutrient surface, it will grow into a discrete mound
of cells called a colony.

• Proper isolation requires that a small number of cells be inoculated into a

relatively large volume or over an expansive area of medium.

• It generally requires a medium that has a relatively firm surface contained

in a clear, flat covered plate called a Petri dish, and inoculating tools.
Stages in the formation of an isolated colony, showing the
microscopic events and the macroscopic result
 Plating methods
1. In the streak plate method, a small droplet of
sample is spread with a tool called an inoculating loop
over the surface of the medium according to a pattern
that gradually thins out the sample and separates the
cells spatially over several sections of the plate.
• Because of its ease and effectiveness, the streak
plate is the method of choice for most applications.
Steps in a Streak Plate, this one is a four-part or quadrant streak
2. In the loop dilution, or pour plate, technique, the sample is
inoculated, also with a loop, into a series of cooled but still liquid
agar tubes so as to dilute the number of cells in each successive
tube in the series.
• Inoculated tubes are then plated out (poured) into sterile
Petri dishes and are allowed to solidify (harden).
• The number of cells per volume is so decreased that cells
have ample space to form separate colonies in the second or
third plate.
• One difference between this and the streak plate method is
that in this technique, some of the colonies will develop deep
in the medium itself and not just on the surface.
Steps in Loop Dilution; also called a pour plate or serial dilution
3. With the spread plate technique, a small
volume of liquid, a diluted sample is pipetted
onto the surface of the medium and spread
around evenly by a sterile spreading tool
(sometimes called a “hockey stick”).
• As with the streak plate, cells are spread over
separate areas on the surface so that they can
form individual colonies.
Steps in a Spread Plate
 Incubation
• Once a container of medium has been inoculated, it is incubated in a
temperature-controlled chamber (incubator) to encourage
microbial growth.
• Although microbes have adapted to growth at temperatures ranging
from freezing to boiling, the usual temperatures used
in laboratory propagation fall between 20°C and 40°C.
• Incubators can also control the content of atmospheric gases such as
oxygen and carbon dioxide that may be required for the growth of
certain microbes.
• During the incubation period (ranging from a few hours to several
weeks), the microbe multiplies and produces a culture with
macroscopically observable growth.
 Growth in liquid and solid media
• Microbial growth in a liquid medium materializes as
cloudiness, sediment, a surface mat, or colored pigment.
• Growth on solid media may take the form of a spreading
mat or separate colonies.
• In some ways, culturing microbes is analogous to gardening.
• Cultures are formed by “seeding” tiny plots (media) with
microbial cells.
• Extreme care is taken to exclude weeds (contaminants).
 Pure culture
• A pure culture is a container of medium that grows only a single
known species or type of microorganism.
• This type of culture is most frequently used for laboratory study,
because it allows the precise examination and control of one
microorganism by itself.
• Instead of the term pure culture, some microbiologists prefer the term
axenic, meaning that the culture is free of other living things except
for the one being studied.
• A standard method for preparing a pure culture is to use a subculture
technique to make a second-level culture. A tiny bit of cells from a
well-isolated colony is transferred into a separate container of media
and incubated.
 Mixed Culture
• A mixed culture is a container that holds two or
more easily differentiated species of microorganisms,
like a garden plot containing both carrots and onions.
• A contaminated culture has had contaminants
(unwanted microbes of uncertain identity) introduced
into it, like weeds into a garden.
• Because contaminants have the potential for
disrupting experiments and tests, special procedures
have been developed to control them.
Various states of cultures.
(a) A mixed culture of M. luteus and E. coli readily differentiated by their
colors.
(b) This plate of S. marcescens was overexposed to room air and has
developed a large, white colony. Because this intruder is not desirable and
not identified, the culture is now contaminated.
(c) Tubes containing pure cultures of Escherichia coli (white), Micrococcus
luteus (yellow), and Serratia marcescens (red).
 Identification Techniques

• Microscopy helps in differentiating the smaller, simpler prokaryotic cells from

the larger, more complex eukaryotic cells.
• Eukaryotic microorganisms can be identified to the level of genus or species
because of their more distinctive appearance.
• Different bacterial species may appear quite similar, microscopy may not be
enough.
• Biochemical tests, can determine fundamental chemical characteristics such
as nutrient requirements, products given off during growth, presence of
enzymes, and mechanisms for deriving energy.
• A test system for common gram negative cocci
(Neisseria meningitidis) uses 8 small wells of media to be inoculated
with a pure culture and incubated.
• A certain combination of reactions will be consistent with this
species.
• A sample of a key that uses test data (positive or
negative reactions) to guide identification.
• Several modern diagnostic tools that analyze
genetic characteristics can detect microbes based
on their DNA. These DNA profiles can be extremely
specific, even sufficient by themselves to identify
some microbes.
• Identification can be assisted by testing the isolate
against known antibodies (immunologic testing).