Medical Microbiology I - Lecture4

MEDICAL MICROBIOLOGY I

Lesson 4Lesson 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 The minimum requirement is automated

result entry and identification of

microorganisms. Systems requiring manual

result entry are not discussed


The instrument must have a data base for the

identification of a large variety of different

microorganisms

Instruments such as automated enzyme Instruments such as automated enzyme

immunoassay systems that identify a relatively

small number of microorganisms are not

described


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 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 full-

on panel antimicrobial concentrations and the on 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 Staphylococcussp.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 testingautomated 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 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 testscontaining 26 biochemical tests

Consists of modules:

The diluent-dispenser

The filling module

The reader-incubator module

Computer control module with data terminal


Plastic disposable cards consisting of

microcuvettes containing dehydrated media

are inoculated and incubated in the AMS

An optical system monitors light transmission 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


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 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


The Enterobacteriaceae Biochemical Card (EBC)

identify 30 species of Enterobacteriaceae from

pure culture

The EBC consists of 30 microcuvettes containing 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 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 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 controlgrowth 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


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 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


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 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


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 agentsantimicrobial 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


Each analysis module provides positions for

eight cartridges and contains individual

electro-optical systems for monitoring

turbidity within every cuvette in each 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


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 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


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 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


Organism-antimicrobial agent combinations

exhibiting growth rate either similar to or

exceeding that of the untreated control are

designated as resistantdesignated 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 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


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 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


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 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


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 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


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 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 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 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 handlebacteria 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 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 negativeand 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 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)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 -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

cellcell

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-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 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 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


In general, the size and shape of

microorganisms is often less distorted with

indirect staining procedures, especially when

sampled from a broth culturesampled 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


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 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


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 cellstain 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 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


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


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 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 non-

acid fast cells become clear


Acid fast cells retain the carbol fuchsin and

remain pink / red

Non-acid fast cells take up the methylene blue

and become blue-greenand 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 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


One is produced within each vegetative

bacterium

Once the endospore is formed, the vegetative

portion of the bacterium is degraded and the 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


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 usedspore 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


The bacteria are decolourised and cooled with

water

Malachite green is soluble in water unless it is

bound to the spore coatsbound 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


When describing the spore-forming bacteria,

the location of the endospore is usually stated

as central, terminal, or subterminal

Medical Microbiology I - Lecture4

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