MEDICAL MICROBIOLOGY I

Lesson 4
Automated Identification and
Staining of Bacteria

Automated Identification of Bacteria

Automation has developed more slowly in
clinical microbiology than it has in other
medical laboratory services in the 1970s
The minimum requirement is automated
result entry and identification of
microorganisms. Systems requiring manual
result entry are not discussed

Automated Identification of Bacteria

The instrument must have a data base for the
identification of a large variety of different
microorganisms
Instruments such as automated enzyme
immunoassay systems that identify a relatively
small number of microorganisms are not
described

Automated Identification of Bacteria

Autobac Series II (formerly called the Autobac;
Organon-Teknika, Durham, N.C.) and the
Avantage Microbiology Center (formerly called
the Abbott MS-2) and Quantum II
Microbiology System (Abbott Laboratories
Diagnostic Division, Irving, Texas)
These systems are no longer manufactured
but are still in service in some laboratories

BD Phoenix Automated Microbiology

System
The BD Phoenix Susceptibility System
incorporates the use of an oxidation-reduction
indicator, turbidometric growth detection fullon panel antimicrobial concentrations and the
BDXpert System

BD Phoenix ESBL Resistance Marker Detection

Confirmatory extended spectrum -lactamase
(ESBL)testing for Escherichia coli, Klebsiella
pneumoniae and Klebsiella oxytoca
Definitive -lactamase detection in Staphylococcus
sp.
Methicillin resistance in Staphylococcus aureus
Predictive mecA detection utilising Cefoxitin for
Staphylococcus aureus
Vancomycin resistance in enterococci
High-level aminoglycoside resistance in enterococci
Vancomycin resistance in Staphylococcus aureus

BD Phoenix AP Workflow Efficiency

Provide the laboratory workflow efficiency and
standardised isolate inoculum
The BD Phoenix AP is the first system to incorporate
automated nephelometry in ID/AST testing
The Phoenix AP incorporates the BD EpiCenter
Barcode Printing Software into the workflow
Each ID broth may be identified with an EpiCenter
generated barcode label that may include patient
name, patient location, sample accession number,
specimen type, Phoenix panel type, and isolate
number

BD Phoenix AP Workflow Efficiency

EpiCenter provides the real-time data access
and analysis tools professionals such as
microbiologists, infection control officers,
physicians and pharmacists can use to
improve patient care

AutoMicrobic System (Vitek Systems)

A fully automated, computerised instrument
Identify members of the family Enterobacteriaceae
with the use of a sealed, disposable accessory card
(the Enterobacteriaceae Biochemical card)
containing 26 biochemical tests
Consists of modules:
The diluent-dispenser
The filling module
The reader-incubator module
Computer control module with data terminal

AutoMicrobic System (Vitek Systems)

Plastic disposable cards consisting of
microcuvettes containing dehydrated media
are inoculated and incubated in the AMS
An optical system monitors light transmission
changes in the microcuvettes of each card and
transmits the optical measurements to a
computer which analyses the results and
provides a final report

AutoMicrobic System (Vitek Systems)

The first card developed for use in the AMS,
the Urine Identification Card, by which certain
microorganisms in urine are detected,
enumerated, and identified, has been
evaluated
No additional reagent to perform tests, and in
most cases it requires no additional
biochemical tests to make an identification

AutoMicrobic System (Vitek Systems)

The Enterobacteriaceae Biochemical Card (EBC)
identify 30 species of Enterobacteriaceae from
pure culture
The EBC consists of 30 microcuvettes containing
26 biochemicals, one positive broth control,
and three empty wells for future application
Although preliminary results may be obtained
after 4 hours, final identification is determined
after 8 hours

Micro-ID (General Diagnostics)

Provides for identification of microorganisms
in the family Enterobacteriaceae within 4
hours
Only one additional reagent has to be added
to the wells
The Micro-ID system uses the principle of the
Pathotec strips, i.e. detection of enzyme
activity by using substrates and reagents
impregnated into filter paper strips

Micro-ID (General Diagnostics)

In general, the reactions were very easy to read
With both the decarboxylase and the fermentation
tests the colours should be either purple or yellow
Micro-ID panels consists of plastic trays with filter
paper disks set into individual compartments
A limitation of the Micro-ID system is that it is only
suited to identification of Enterobacteriaceae
An oxidase test must be performed first, and only
oxidase-negative organisms are to be inoculated

MS-2 (Abbott Diagnostics)

An automated system designed to perform
antimicrobial susceptibility testing by
comparing growth kinetics of an organism in
the presence of an antimicrobial agent with
growth kinetics of an untreated control
Components of the system include the cuvette
cartridge, the disk loader-sealer, a control
module (control), and one or more analysis
modules

MS-2 (Abbott Diagnostics)

A test organism is inoculated into a cartridge with
11 cuvettes; 1 serves as an untreated growth control
and the other 10 contain antimicrobial agents
Growth is monitored turbidimetrically at 5-min
intervals, and susceptibility is determined
automatically by computer comparison of growth
kinetics in the presence of an antimicrobial agent
with kinetics in the untreated control, i.e. a
comparison of growth curves

MS-2 (Abbott Diagnostics)

Antimicrobial agents for the MS-2 are supplied
as filter paper elution discs which are
automatically dispensed and sealed into the
bottom of the appropriate cuvette chambers
by using the loader-sealer unit
The cuvette cartridge has 11 lower chambers
(cuvettes), one of which serves as a growth
control, with the other 10 each containing
individual antimicrobial agents

MS-2 (Abbott Diagnostics)

The upper part of the cartridge is a chamber in
which the culture grows into logarithmic phase
before being automatically distributed into each of
the 11 test cuvettes, i.e. before contact with the
antimicrobial agents
The computerised control module stores the
information entered by the operator concerning the
test organism (source, culture number, etc.), controls
the sequence of events leading to analysis, accesses
and stores the data, computes the evaluation of
susceptibility, and prints out the final report

MS-2 (Abbott Diagnostics)

Each analysis module provides positions for
eight cartridges and contains individual
electro-optical systems for monitoring
turbidity within every cuvette in each
cartridge at 5-min intervals
Each module also provides a constant
temperature (35C) and continual agitation
throughout the test

MS-2 (Abbott Diagnostics)

After the cartridge is inserted into the analysis
module, growth in the upper chamber is
monitored by the instrument at 5-min
intervals, and when the culture has shown a
clear-cut increase in optical density (change of
at least 0.008), indicating entry into
logarithmic phase growth, it is automatically
transferred into each of the 11 lower cuvettes
by the introduction of pulses of air pressure
into the upper growth chamber

MS-2 (Abbott Diagnostics)

After the culture is transferred into the lower
cuvettes, the antibiotic is eluted into the
broth, and the antibiotic action, if any, occurs
The turbidity in each cuvette is read every 5
min, and all values are stored in the computer
memory
Any antimicrobial agent causing a reduction of
growth rate less than 10% of the control value
is considered to be effective, i.e. the organism
is susceptible

MS-2 (Abbott Diagnostics)

Organism-antimicrobial agent combinations
exhibiting growth rate either similar to or
exceeding that of the untreated control are
designated as resistant

Analytab Products Inc. (API) System

Provides one such kit, API 20E, which permits
the determination of 20 different biochemical
characteristics on one strip
With the API 20E, most enteric bacilli can be
identified to the species level within 18 to 24
hr after primary isolation
A few isolates require additional tests which
can be completed after 1 or 2 additional days

Analytab Products Inc. (API) System

For identification of enteric Gram -ve rods
The technique is basically a modification of
one of the many little tube methods
A plastic strip holding 20 miniaturised
compartments, or cupules, each containing a
dehydrated substrate for a different test
Some of the tubes are to be completely filled
whereas others are topped off with mineral oil
so that anaerobic reactions can be carried out

Analytab Products Inc. (API) System

1. Incubating the API 20E
The strip is then incubated in a small, plastic
humidity chamber for 18 - 24 hours at 37C
Living bacteria produce metabolites and wastes
as part of the business of being a functioning cell
The reagents in the cupules are specifically
designed to test for the presence of products of
bacterial metabolism specific to certain kinds of
bacteria

Analytab Products Inc. (API) System

2. Reading API 20E results
After incubation, each tube (an individual test)
is assessed for a specific colour change
indicating the presence of a metabolic reaction
that sheds light on the microbes identity
Some of the cupule contents change colour due
to pH differences, others contain end products
that have to be identified using additional
reagents

Analytab Products Inc. (API) System

3. The API 20E secret code
Interpretation of the 20 reactions, in addition to
the oxidase reaction (which is done separately),
is converted to a 7-digit code, a process that
seems much like decoding a message with a
super secret spy decoder ring
The technician can look up the code in a huge
manual that has the names of bacterial species
associated with each 7-digit string of numbers

Bacterial Staining
Bacteria are colourless cells and almost invisible
to the naked eyes
Can be stained with dyes to make them easier to
observe
Identifying and categorising different, isolated
bacterial colonies based upon varied appearance
and morphology (form and structure) will permit
the selection and transfer of a single colony to a
mixed culture, and allow transfer of single colony
to a sterile medium for cultivation of a pure
culture

Bacterial Staining
A number of stains have been developed to
distinguish spores, nuclear bodies, capsules, and
characteristics of the cell wall
The staining methods we will use kill the bacteria,
reducing the risk of infection by pathogenic
organisms
Since the rigid cell walls of bacteria prevent
distortion of morphology upon drying, samples can
be spread onto a glass slide and air-dried, then fixed
to the surface by passing the slide quickly through a
flame, melting the complex carbohydrates of the cell
walls to the glass and killing the cells

Preparing Smear
It causes bacteria to adhere to a slide so that
they can be stained and observed
It also kills them, rendering pathogenic
bacteria safe to handle
An objective in preparing smears is to learn to
recognise the correct density of bacteria to
place on the slide

Grams Staining
Grams staining is discovered by Christian Gram
in 1884 when he was staining bacteria in
tissues
The Gram stain is almost always the first step in
the identification of a bacterial organism
It is very useful in the differentiation of
bacteria, especially those which have the same
shape and size but differ in their ability to
retain a crystal violet-iodine complex when
washed with a decoloriser (acetone)

Grams Staining
Microorganisms which take up the complex
are called Gram positive
Microorganisms which give up the complex
and become colourless are Gram negative
These organisms will take up the second stain
or counterstain (usually safranin)

Grams Staining
This staining is based on the physical
properties of the bacteria cell wall
Gram +ve bacteria have a thick mesh-like cell
wall made of peptidoglycan 50 - 90% of cell
wall)
Gram -ve bacteria have a thinner layer (10% of
cell wall), with an additional outer membrane
which contains lipids, and is separated from
the cell wall by the periplasmic space

Grams Staining
Four basic steps of the Gram stain:
1. Applying a primary stain (crystal violet) to a
heat-fixed smear of a bacterial culture
2. Addition of a trapping agent (Grams iodine)
3. Rapid decolourisation with alcohol or acetone
4. Counterstaining with safranin. Basic fuchsin is
sometimes substituted for safranin since it will
more intensely stain anaerobic bacteria but it is
much less commonly employed as a counterstain

Grams Staining
Crystal violet (CV) dissociates in aqueous
solutions into CV+ and chloride (Cl-) ions
These ions penetrate through the cell wall and
cell membrane of both Gram +ve and Gram ve cells
The CV+ ion interacts with negatively charged
components of bacterial cells and stains the
cells purple

Grams Staining
Iodine (I- or I3-) interacts with CV+ and forms
large complexes of crystal violet and iodine
(CV-I) within the inner and outer layers of the
cell
Iodine is often referred to as a mordant, but is
a trapping agent that prevents the removal of
the CV-I complex and therefore colour the cell
When a decolouriser such as alcohol or
acetone is added, it interacts with the lipids of
the cell membrane

Grams Staining
A Gram -ve cell will lose its outer membrane
and the lipopolysaccharide layer is left exposed
The CV-I complexes are washed from the Gram
-ve cell along with the outer membrane
In contrast, a Gram +ve cell becomes
dehydrated from an ethanol treatment
The large CV-I complexes become trapped
within the Gram +ve cell due to the
multilayered nature of its peptidoglycan

Grams Staining
The decolourisation step is critical and must be
timed correctly, the crystal violet stain will be
removed from both Gram +ve and Gram -ve
cells if the decolourising agent is left on too
long ( a matter of seconds)
Counterstain, which is usually positively
charged safranin or basic fuchsin, is applied
last to give decolorised Gram -ve bacteria a
pink or red colour

Indirect (Negative) Staining

The acidic dye nigrosine will be used to
visualise the capsule or sheath that surrounds
some bacteria
Capsules are composed primarily of
polysaccharides or glycoproteins and are
gelatinous in texture
They are readily destroyed by heating and
hence direct staining methods cannot utilised

Indirect (Negative) Staining

In general, the size and shape of
microorganisms is often less distorted with
indirect staining procedures, especially when
sampled from a broth culture
Negative staining is useful to observe the
morphology of individual bacteria
Morphology can often be determined with
confidence with only the high dry lens
This procedure does not necessarily kill the
organism

Indirect (Negative) Staining

The smear will be most dense where the
nigrosine dye was deposited on the slide
The background should be blue-gray
Nigrosine and sodium eosinate are examples
of acidic dyes
Sodium eosinate ionises into sodium and
eosinate ions, with the colouring power of the
dye in the negatively charged eosinate ion

Indirect (Negative) Staining

Since the colouring power of the dye is in the
negative ion and will not readily combine with
another negative ion, an acidic dye does not
stain the negatively charged bacterial cell
Compare to direct staining, the indirect
staining gives a more accurate view of the
bacterial cell to get a clearer picture of its size
and shape

Ziehl-Neelsen Stain (Acid-Fast Stain)

Described by German doctors; Franz Ziehl
(1859 to 1926), a bacteriologist and Friedrich
Neelsen (1854 to 1894), a pathologist
It is a special bacteriological stain used to
identify acid fast organisms, mainly
Mycobacteria, which possess the high mycolic
acid content of certain bacterial cell walls
The reagents used are Ziehl-Neelsen carbol
fuchsin, acid alcohol and methylene blue

Ziehl-Neelsen Stain (Acid-Fast Stain)

Acid fast bacilli will be bright red after staining
It is helpful in diagnosing Mycobacterium
tuberculosis since its lipid rich cell wall makes it
resistant to Gram stain
It can also be used to stain a few other bacteria
like Nocardia
Carbol fuchsin (basic fuchsin in phenol), with
mild heating, is able to penetrate the waxy layer
of mycolic acids present in the cell walls of acid
fast bacteria

Ziehl-Neelsen Stain (Acid-Fast Stain)

Carbol fuchsin is able to enter both acid fast and
non-acid fast bacteria
Phenol and heat assists dye entry into the cell
Carbol fuchsin remains in acid fast bacteria as
the cell wall is resistant to the acid wash acid
fast cells remains pink / red
Without a resistant layer of mycolic acids in the
cell wall, the acid wash removes the carbol
fuchsin from the non-acid fast bacteria nonacid fast cells become clear

Ziehl-Neelsen Stain (Acid-Fast Stain)

Acid fast cells retain the carbol fuchsin and
remain pink / red
Non-acid fast cells take up the methylene blue
and become blue-green

Schaeffer-Fulton Stain (Endospore)

A few genera of bacteria, such as Bacillus and
Clostridium have the ability to produce
resistant survival forms termed endospores
Different from the reproductive spores of
fungi and plants, these endospores are
resistant to heat, drying, radiation and various
chemical disinfectants
Endospore formation or sporulation occurs
through a complex series of events

Schaeffer-Fulton Stain (Endospore)

One is produced within each vegetative
bacterium
Once the endospore is formed, the vegetative
portion of the bacterium is degraded and the
dormant endospore is released
The impermeability of the spore coat is
thought to be responsible for its resistance to
chemicals

Schaeffer-Fulton Stain (Endospore)

In order to observe the inner contents of
bacterial spores, a specialised staining
technique that can drive a dye through the
spore coat must be used
Subsequently, a counterstain can be applied to
give vegetative cells a contrasting colour
Malachite green is the primary stain
The stain must be heated to penetrate the
spore coat

Schaeffer-Fulton Stain (Endospore)

The bacteria are decolourised and cooled with
water
Malachite green is soluble in water unless it is
bound to the spore coats
Safranin is the counterstain
Endospores retain the malachite green and
remains green
Vegetative cells take up the safranin and
become pink / red

Schaeffer-Fulton Stain (Endospore)

When describing the spore-forming bacteria,
the location of the endospore is usually stated
as central, terminal, or subterminal