Welcome to the Clinical
Microbiology Lab
Is this evidence of an
Which colony is the
“important” one?
Why does the bacteria
causing disease need
to be identified to
treat the infection?
Why not just give
These are questions asked and
answered by the microbiology lab.
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Investigation of disease in the Clinical
Microbiology Lab
In a clinical microbiology lab, experienced clinical
microbiologists begin their investigation of disease by
using microscopy to examine smears of original
samples to obtain important early information.
The arrows are
pointing to
bacteria…but the
identification is
near impossible at
this point.
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General Sequence of Events in Identifying Bacteria
List of steps toward identifying the cause of infection:
1) Microscopic examination of specimen to determine
if there’s bacteria present
2) Gram stain to determine type of bacteria
3) Growth of bacteria in broth
4) Plate broth bacteria on media plate (culture)
5) Select isolated pathogen
6) Perform biochemical testing
7) Perform antibiotic susceptibility
Thoughts on Identification of Bacteria
 Maybe we need to go back a bit. The entire reason to
identify a bacteria that we suspect is causing disease
is to be able to treat it. Bacteria is almost always
treated with antibiotics – but they must be specific to
the bacteria. One antibiotic won’t work for all
 When a specimen arrives in the micro lab, it’s looked
at microscopically and gram stained, but then it
needs to grow outside the body so that we can work
with it.
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Thoughts along with Videos…
 Video on Gram staining (the lady in this video refers to their
lecture and lab manual – don’t be confused, these are theirs, not
 Gram Stain Video
 Inoculating a broth with the bacteria is almost
always the first step in growing it.
 Video on isolating bacteria
Isolating Bacteria
 Once there’s sufficient growth (8-12 hours) the
bacteria present is put on a petri dish of media.
There is likely a number of bacteria in the broth, the
pathogenic ones need to be isolated out.
 This process sometimes requires that steps be
repeated because the bacteria to be tested needs to
be absolutely pure.
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Gram Stain
 After looking at the specimen under the microscope,
a Gram stain is done.
 The Gram stain is a technique for staining and
detecting bacteria and yeasts. It is the most
commonly performed procedure in the clinical
microbiology laboratory.
 Four reagents are used to perform a Gram stain:
crystal violet, Gram's
iodine, acetone-alcohol,
and safranin.
Gram Stain
 Grams stains are used to differentiate types of
bacteria. Gram-positive and Gram-negative bacteria
stain differently because of the structure of their cell
Gram negative = red or pink
Gram positive = purple
Gram Stain
 Gram stain results can have a dramatic effect on
patient care. Hospitalization may be required when
the Gram stain indicates bacteria are present in a
normally sterile body fluid.
Sterile body fluids include blood and CSF. It’s very dangerous
to have bacteria in these fluids.
 Remember, the point of all this work is to identify
the pathogenic bacteria so that it can be treated. The
initial choice of antibiotic therapy can be guided by
the Gram stain results.
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Gram Stain
 Once a gram stain identification is made a culture
plate (or petri dish) is set up so that the bacteria can
grow. Gram Stain Video
Gram Stained Bacteria
 Another use of Gram stain is observing the
arrangement of its colonies as it grows.
 Both of these characteristics are helpful when
identifying bacteria.
Gram Positive Cocci
 The Gram-positive coccus (singular) is a spherical
bacterium. Gram-positive cocci may appear in four
clinically significant groupings: dipplo (two cells growing
very close to one another), chain, tetra (4 cells close),
 Gram positive dipplococci for example is frequently
Streptococcus pneumoniae.
See how these cells
are growing in pairs?
That’s what dipplococci
look like.
 Another example of the importance of knowing the
shape and arrangement of bacteria would be the
differences seen between Strep. anginosus which
grows in chains and Staph. epidermidis.
While both are gram positive cocci, S. anginosus is often the
cause of oral infections and very pathogenic. Staph.
epidermidis is found in great numbers on our skin and is not
pathogenic at all.
See how the
of cells look
Gram Positive Bacilli
 The Gram-positive bacilli is a rod shaped bacteria.
Gram-positive bacilli may also appear in four
clinically significant groupings...
Long, wide
Long, narrow,
often chaining
Gram Negative Cocci
 Gram-negative cocci may appear in two clinically
significant groupings...
Gram Negative Bacilli
 The Gram-negative rod or bacillus is a rectangular
shaped bacterium. Rods are variable in length,
width, and staining characteristics. There are five
clinically significant shapes of Gram-negative rods...
Technologists in
Micro use these
adjectives to
describe what they
see on the Gram stain.
because these
descriptions are used
universally, the doctor
has a good idea about the
identification of the
Long, narrow
Curved rod
Spiral rods
Colony Morphology
 Once the bacteria is grown in a broth, it is put on a
specific media in a petri dish, or culture plate.
 Cultures are generally incubated at 37◦ C in special
atmospheres to maximize growth of pathogens.
 Along with the arrangement of growing colonies,
colony morphology, or the shape of a colony after
growth on the plate is often diagnostic.
Colony Morphology
 Common morphologies:
Colony morphology
This is an
picture because
it shows the
same bacteria
Gram stained
and on the plate.
Describing Bacteria
 If we were going to describe the isolated bacteria on
the preceding slide we might say something like,
 “Gram positive bacilli, small, round, slightly
mucoid (wet looking), slightly raised colonies.”
Colony morphology
 So, the technologist actually uses these observations
to describe and identify disease causing bacteria.
 Some bacterias hemolyse blood in this media (BAP
media) during growth. The degree of hemolysis can
be used to describe and identify the bacteria.
This is a great illustration of three
types of hemolysis: alpha hemolysis,
beta hemolysis, and gamma
Using these two images and the following
descriptors, describe the bacteria seen here
Gram stain Morphology Colony type Hemolysis -
Mixed culture
Pure culture
Pathogenic bacteria
Often the source of the specimen determines if the
bacteria is pathogenic.
 For example, Streptococcus pneumoniae is a
pathogen in the blood, but considered a normal
inhabitant of the throat. Normal inhabitants are
called normal flora. The gut has thousands of
species of bacteria as normal flora.
 Techs need to know where specific bacterias
should be (therefore normal) or shouldn’t be,
(therefore pathogenic).
Biochemical Identification
One of the most distinguishing features of
bacteria is their biochemical versatility. The
types of biochemical reactions each organism
undergoes acts as a
"thumbprint" for its
This is based on the following chain of logic:
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Biochemical Identification
 Each different species of bacterium has a different
molecule of DNA (i.e., DNA with a unique series of
nucleotide bases).
 Since DNA codes for protein synthesis, then different
species of bacteria must, by way of their unique DNA, be
able to synthesize different protein enzymes.
 Enzymes catalyze all the various chemical reactions of
which the organism is capable. This in turn means that
different species of bacteria must carry out different
and unique sets of biochemical reactions.
Biochemical Identification
 The battery of biochemical tests used is dependent
upon the gram stain of the bacteria. Testing can be
or automated. This panel is read on an instrument using
something very similar to spectrophotometry.
Antibiotic Susceptibility
 Once the pathogenic bacteria is identified, the doctor
will want to know how to treat the patient.
Obviously, certain antibiotics are more effective
against some bacterias than others. They are often
chosen by the degree of their selective toxicity.
 The selective toxicity of antibiotics means that they
must be highly effective against the microbe but have
minimal or no toxicity to humans.
Antibiotic Susceptibility
 Remember that certain bacteria are normal and even
helpful in certain locations. If normal flora is killed,
health is compromised. This makes selecting the
right antibiotic at the right does very important.
Antibiotics That Work
 Antibiotics are chosen depending upon the gram
stain of the bacteria. In other words, an antibiotic
will be effective against a gram positive bacteria, but
not against gram negative bacteria.
 Discs with particular antibiotics are placed on a plate
with a pure culture of the bacteria.
Antibiotic Susceptibility
Discs with antibiotic
The antibiotic works if there is a
zone of inhibited growth. These
zones are measured giving the
doctor a clue to which antibiotic
may be most effective.
Would this antibiotic be
effective against this bacteria?
Antibiotic Susceptibility
Manual testing
Automated susceptibility testing
Microbiology lab
 All of this testing in the micro lab generally takes
only 48 hours.
 Some rare labs, in large hospitals, are using PCR for
screening of disease-causing bacteria that is highly
contagious. This enables the lab to report the
identification of the bacteria in less than 12 hours.
Review Questions
 True or false: The first thing done when a specimen
is received in micro is the Gram stain.
What color is a Gram negative bacteria?
What color is a Gram positive bacteria?
Why does pathogenic bacteria need to be isolated?
Define normal flora.
Briefly explain the benefit of doing biochemical tests
on unknown bacteria.
What is the purpose of performing antibiotic

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