Clinical Engineering Merge into Medical Laboratory Science

Innovative Practices in Microbiological Laboratory Assessments

Continuous Professional Development Program

for Medical Laboratory Professionals

Published by
College of Medical Laboratory Science, Sri Lanka
No 25/2, Norris Avenue, Colombo 08
www.cmls.org / cmlssrilanka@gmail.com

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 Academic Panel

Dr. Lakshmi Kiranmayi

Medical Microbiologist
Training and Scientific Support expert

Dr. Lilani Karunarathne

Consultant Clinical Microbiologist
Medical Research Institute, Colombo

Dr. Darshana Wickramasinghe

Consultant Clinical Microbiologist
District General Hospital, Ampara

Dr. Muditha Abeykoon

Consultant Clinical Microbiologist
Teaching Hospital, Kagalle

Mr. N. M. Shafeek
Medical Laboratory Technologist
Teaching Hospital, Kandy

Mr. Gayantha Vidyarathne

Lecturer
School of Medical Laboratory Technology, MRI

Ms. Prabhani Pushpamalie

Medical Laboratory Technologist
National Hospital, Colombo

Mr. Dhanushka Lalinda

Medical Laboratory Technologist
North Colombo Teaching Hospital, Ragama

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 “College of Medical Laboratory Science, Sri Lanka
will lead the profession toward the expected advancement of
Sri Lankan Laboratory Service”

Dr. Anil Jasinge

Director General of Health Services
Ministry of Health and Indigenous Medicine

Today I’m happy to see the College of Medical Laboratory Science, Sri Lanka has
identified the future of medical laboratory science and come forward with the responsibility
of preparing Sri Lankan Medical Laboratory Service for tomorrow. Automation and
innovative practices in Microbiological laboratory set up is one important area to be
improved in Sri Lankan Medical Laboratory Service.

College of Medical Laboratory Science has identified this requirement and this approach
gives a good sign of moving Sri Lankan Medical Laboratory Service with automation for
the betterment of patient care.

It is believed the College of Medical Laboratory Science, Sri Lanka will lead the profession
toward the expected advancement of Sri Lankan Laboratory Service. I congratulate the
effort of the College of Medical Laboratory Science taking the battle of developing Sri
Lankan laboratory service with parallel to the innovations of world’s medical laboratory
service in such disease diagnosis and management.

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 “Patient’s time, convenience and satisfaction are highly significant
factors beyond the “cost”. There is nothing which cannot be
changed on this consumer’s influence”

Ravi Kumudesh
President
College of Medical Laboratory Science, Sri Lanka

Is not Microbiology automation a waste of money?

Cost of test is compared significantly when automation is come in to discussion. It becomes

a major reason for refusing automation. The people who fear toward automation are being
used cost per test as a reason for their reluctance.

Some people believe that automation of microbiology is limited and it will not move
beyond culture, ABST and identification. Some people may refuse automation criticizing
and highlighting drawbacks in laboratory trials.

In final analysis cost includes time, convenience, quality and satisfaction of the consumer.
Science and Technology fulfills this consumer’s need. Patient’s time, convenience and
satisfaction are highly significant factors beyond the “cost”. There is nothing which cannot
be changed on this consumer’s influence. Therefore the pressure towards changing the
existing traditional Microbiology laboratory set up is potentially high.

There are several trials and experiments are being conducted beyond our sense. The
traditional Microbiology laboratory set up which we well trained will be changed swiftly
near future. Our Medical Laboratory Professionals should be responsible to develop that
“instinct” or sense among them.

I wish to develop the sense of future laboratory innovations among medical laboratory
professionals.

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 Program

Clinical Engineering Merge into Medical Laboratory Science

Innovative Practices in Microbiological Laboratory Assessments

08.00 am - 09.00 am Registration and Refreshment

09.00 am - 09.45 am Opening Ceremony
09.45 am - 10.30 am Overview on Automation in Microbiology Assessments
Dr. Lakshmi Kiranmayi
(Microbiologist, Training & Scientific Support expert)
10.30 am - 11.15 am Importance Microbiological Analysis for Clinician’s
Decision towards better Patient Care
Dr. Lilani Karunarathne
(Clinical Microbiologist, Medical Research Institute)
11.15am - 12.00 N Place of Medical Laboratory Technologist in Clinical
Microbiology
Dr. Darshana Wickramasinghe
(Clinical Microbiologist, District General Hospital, Ampara)
12.00 N - 01.00 pm Lunch
01.00 pm - 01.45pm Procalcitonin: Advancing Decision Making in Sepsis
Dr. Muditha Abeykoon
(Clinical Microbiologist, Teaching Hospital, Kegalle)
01.45 pm - 02.15 pm Evolution in Culture Media towards Rapid Diagnosis
Mr. Vidyarathne
(Tutor, School of Medical Laboratory Technology, MRI)
02.15 pm - 03.15 pm Automaton in Microbiological Assessments
Dr. Lakshmi Kiranmayi
(Microbiologist, Training & Scientific Support expert)
03.15 pm - 03.45 pm Consequences of Existing Sri Lankan Microbiology
Laboratory Practices
Mr. N. M. Shafeek
(Medical Laboratory Technologist, Teaching Hospital,Kandy)
03.45 pm - 04.00 pm Panel Discussion
04.00 pm Onward Closing Ceremony

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 Preface

Today Medical Laboratory plays a major role in health care delivery.

Its quality is decided by the consumer of medical laboratory service.
Automation and innovations of medical laboratory set up are
developed in order to fulfill the consumer satisfaction.

Medical Laboratory professionals should be able to move with

laboratory innovations and it is their responsibility to adapt with
automation and innovative practices highlighting their
professionalism in healthcare service.

The strategy of continuous education and strengthening with new

knowledge fulfills the knowledge requirement in enhancing
professionalism

The College of Medical Laboratory Science, Sri Lanka attempts to open

eyes of medical laboratory professionals and show the future trends
in all laboratory disciplines holding the responsibility of guiding
medical laboratory profession of Sri Lanka.

Automation and innovative practices in Microbiology is one key area

to be developed among Sri Lankan Medical Laboratory Professionals
as it is developed rapidly in the world. Even though we are still
practicing traditional microbiological laboratory process confidently,
it will be replaced by innovations near future.

This booklet provides knowledge on applications of automation in

microbiological laboratory assessments and innovative practices
towards better patient care.

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Table of Contents

1.0 History of Microbiology ............................................................................. 8

1.1 Development of Microbiology ................................................................ 9

2.0 Microbiological Laboratory Assessments ................................................ 10

2.1 Types of specimens in bacteriological diagnosis ................................... 10

2.2 Pathogenic Bacteria Identification ........................................................ 11

2.2.1. Isolation of bacteria ....................................................................... 11

2.2.1.1 Culture media .............................................................................. 11

2.2.1.2 Colony morphology ...................................................................... 14

2.3 Chromogenic culture media .................................................................. 15

2.3.1Principle of Chromogenic ................................................................. 15

2.5 Motility test .......................................................................................... 17

2.6 Biochemical Tests.................................................................................. 17

Advantages of API test kit: .......................................................................... 19

2.7 Immunological techniques – for time- and cost-efficient detection ...... 20

2.8 Molecular methods ............................................................................... 21

2.10 MALDI-TOF mass spectrometry........................................................... 22

3.0 Automation in Microbiology ................................................................... 23

3.1 Automated Media Dispensing System................................................... 23

3.2 Automation in gram staining................................................................. 23

4.0 Procalctonin............................................................................................ 25

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 1.0 History of Microbiology

Microbiology is the study of microorganisms, which are unicellular or multi cellular microscopic
organisms. This includes eukaryotes such as fungi and protists and prokaryotes such as bacteria
and certain algae etc. History of Microbiology was initially centered in the causes of infectious
diseases but now including practical applications of the science. Many individuals have made
significant contributions to the development of microbiology.

Historians are unable to describe the first observation of microorganisms, but the microscope
was available during the mid-1600s, and an English scientist named Robert Hooke had made key
observations on strands of fungi among the specimens of he viewed. In the 1670s and the
decades thereafter, a Dutch merchant named Anton van Leeuwenhoek made careful observations
of microscopic organisms, which he called animalcules and provided accurate descriptions of
protozoa, fungi, and bacteria.

After van Leeuwenhoek died, the study of microbiology did not develop rapidly because
microscopes were rare and the interest in microorganisms was not high. In those years, scientists
debated the theory of spontaneous generation, which stated that microorganisms arise from
lifeless matter such as beef broth. This theory was disputed by Francesco Redi, showing that fly
maggots do not arise from decaying meat (as others believed) if the meat is covered to prevent
the entry of flies. Lazzaro Spallanzani disputed the spontaneous generation theory by showing
that boiled broth would not give rise to microscopic forms of life.

Louis Pasteur worked in the middle and late 1800s. He performed numerous experiments related
to microorganisms. Pasteur had to disprove spontaneous generation to sustain his theory and
Pasteur's experiments put to rest the notion of spontaneous generation. His work also encouraged
the belief that microorganisms were in the air and could cause disease. Pasteur postulated
the germ theory of disease, which states that microorganisms are the causes of infectious disease.

The German scientist Robert Koch provided the proof for the germ theory. The procedures used
by Koch came to be known as Koch's postulates. They provided a set of principles whereby other
microorganisms could be related to other diseases.

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1.1 Development of Microbiology

In the late 1800s and for the first decade of the 1900s, scientists works towards the germ theory
of disease as articulated by Pasteur and proved by Koch. There emerged a Golden Age of
Microbiology during which many agents of different infectious diseases were identified. Many
of the etiologic agents of microbial disease were discovered during that period, leading to the
ability to halt epidemics by interrupting the spread of microorganisms.

Despite the advances in microbiology, it was rarely possible to render lifesaving therapy to an
infected patient. Then, after World War II, the antibiotics were introduced to medicine. The
incidence of pneumonia, tuberculosis, meningitis, syphilis, and many other diseases declined
with the use of antibiotics.

Modern microbiology reaches into many fields of human endeavor, including the development
of pharmaceutical products, the use of quality control methods in food and dairy product
production, the control of disease causing microorganisms in consumable waters, and the
industrial applications of microorganisms. Microorganisms are used to produce vitamins, amino
acids, enzymes, and growth supplements. They manufacture many foods, including fermented
dairy products (sour cream, yogurt, and buttermilk), as well as other fermented foods such as
pickles, sauerkraut, breads, and alcoholic beverages.

One of the major areas of applied microbiology is biotechnology. In this discipline,

microorganisms are used as living factories to produce pharmaceuticals that otherwise could not
be manufactured. These substances include the human hormone insulin, the antiviral substance
interferon, numerous blood clotting factors and clot dissolving enzymes, and a number of
vaccines. Bacteria can be reengineered to increase plant resistance to insects and frost, and
biotechnology will represent a major application of microorganisms in the next century.

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 2.0 Microbiological Laboratory Assessments

Microbiological Laboratory Assessments consists of wide range of organisms including Fungi

and viruses etc. This section has focused on Bacteriological laboratory assessments.

2.1 Types of specimens in bacteriological diagnosis

Selection of the type of specimen is carried out on the basis of clinical signs and symptoms.

 Blood Cultures
 Blood for Antimicrobial assay
 Chlamydia Examination
 CSF and operative specimens
 Eye Swabs
 Feces
 High Vaginal, Cervical and Urethral Swabs
 Intravascular line tips
 MRSA screening specimens
 Nose Swabs
 Pernasal swabs for pertussis
 Respiratory specimens
 Seminal analysis
 Skin scrapings, hair and nail clippings
 Sputum
 Throat Swabs
 Urine
 Wound swabs and Pus swabs

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2.2 Pathogenic Bacteria Identification

The isolated bacteria are further processed through one or few of the procedures mentioned
below so as to identify the bacteria.

 Isolation of Bacteria
 Staining of the isolated bacteria
 Motility testing
 Biochemical testing
 Immunological methods
 Molecular techniques
 Nucleic acid-based techniques
 MALDI-TOF mass spectrometry

2.2.1. Isolation of bacteria

Many microbes are pathogenic. They cause a number of diseases with a variety of symptoms,
depending on how they interact with the patient. The isolation and growth of suspected microbe
in pure culture is essential for the identification and control the infectious agent.

In a Microbiology laboratory, the various species may be isolated from one another. A culture
which contains just one species of microorganism is called a pure culture. The process of
obtaining a pure culture by separating one species of microbe from a mixture of other species, is
known as isolation of the organisms.

2.2.1.1 Culture media

Culture media contain nutrients and physical growth parameters necessary for microbial growth.
All microorganisms cannot grow in a single culture medium and in fact many can’t grow in any
known culture medium.

Organisms that cannot grow in artificial culture medium are known as obligate microorganisms.
Bacterial culture media can be classified on the basis of composition, consistency and purpose.

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Classification of bacterial culture media on the basis of consistency includes Solid (Agar
concentration 1.5-2.0%), Semisolid (Agar concentrations 0.5%) and liquid (Agar concentration
0%). Composition of culture media classifies as synthetic (prepared from purified ingredients) or
Non-synthetic (contains at least one unpurified component). Synthetic medium may be simple or
complex depending up on the supplement incorporated in it.

Classification of Bacterial Culture media can be done with the purpose. Basic or General-Purpose
media, Enriched media (Added growth factors), Selective (Differential growth suppression) and
enrichment media (extracting fastidious organisms). Selective and enrichment media are designed to
inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a
mixture of bacteria. Enrichment medium is used to increase the relative concentration of certain
microorganisms. Transport media prevent overgrowth of contaminating organisms or commensals,
drying of specimen and maintain the pathogen to commensal ratio and inhibit overgrowth of
unwanted bacteria. Anaerobic media need for growth of organism in low oxygen content, reduced
oxidation-reduction potential and extra nutrients. Assay media are used special purposes such as
assay of vitamins, amino acids and determining antibiotic potency.

Some examples for different kind of Media are given below.

1. Bile esculin agar (BEA): It is used for the differential isolation and presumptive
identification of group D streptococci and enterococci.
2. Bile esculin azide agar with vancomycin: Selective and differential culture media
commonly used for the cultivation of vancomycin-resistant enterococci from clinical and
surveillance specimens.
3. Blood Agar: Blood agar is used for the cultivation of fastidious microorganisms. It is a
differential medium which helps to classify/identify the bacteria on the basis of types
of hemolysis (alpha, beta or no hemolysis) present.
4. Bordet-Gengou agar: It is used for the isolation of Bordetella pertussis, the causative
agent of pertussis or whooping cough.
5. Buffered Charcoal-yeast extract agar (BCYE): Enrichment culture media
for Legionella spp, the causative agent of Legionnaires’ disease, also known as
legionellosis.

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6. Buffered Charcoal-yeast extract agar with antibiotics: This culture media is used for
the enrichment and selection for Legionella spp
7. Campy-blood agar(Campylobacter Blood Agar): Selective culture media
for Campylobacterspp. Campylobacter is a leading cause of bacterial foodborne diarrheal
disease worldwide
8. Campylobacter thioglycollate broth: Selective holding medium for recovery
of Campylobacter spp.
9. Chocolate agar: Cultivation of Haemophilus spp and pathogenic Neisseria spp
10. Columbia colistin-nalidixic acid (CNA) agar: Columbia Agar with colistin and
nalidixic acid (CNA) is a selective medium for gram positive organisms. The
antimicrobials colistin and nalidixic acid inhibits gram negative organisms. CNA agar is
used for the selective isolation of Gram positive cocci such as staphylococci and
streptococci.
11. Cystine-tellurite blood agar: Used for the isolation of Corynebacterium diphtheria.
12. Eosin Methylene Blue (EMB) Agar (Levine): Isolation and differentiation of lactose
fermenting and non-lactose fermenting gram negative enteric bacilli.
13. Gram negative broth (GN): Selective (enrichment) liquid medium for enteric
pathogens.

14. Hektoen enteric (HE) agar: Differential, selective culture medium used for the isolation
and differentiation of Salmonella and Shigella spp from other gram negative enteric
bacilli.
15. MacConkey agar: It is commonly used for the isolation and differentiation of lactose
fermenting and non-lactose fermenting gram negative enteric bacilli
16. MacConkey Sorbitol agar : It is a modification of MacConkey agar in which lactose has
been replaced with d-sorbitol as the primary carbohydrate. It is used for the selection and
differentiation of E.coli O17: H7 from stool specimen
17. Mannitol Salt Agar (MSA): Primary purpose of MSA is selective isolation of
Staphylococci and differentiation of Staphylococcus aureus from coagulase negative
staphylococci (CONS)
18. New York City (NYC) Agar: It is a selective culture media for Neisseria gonorrhoeae

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 19. Phenylethyl alcohol (PEA) agar: Selective isolation of gram positive and anaerobic
gram-negative bacilli

20. Salmonella-Shigella agar: Selective for the isolation of Salmonella and Shigella spp.
21. Selenite broth: Used for the enrichment and isolation of Salmonella spp
22. Tetrathionate broth: Selective for the isolation of Salmonella and Shigella spp.
23. Thayer-Martin Agar: Selective culture medium for Neisseria gonorrhoeae and Neisseria
meningitides
24. Thioglycollate broth: It supports the growth of anaerobes, aerobes, microaerophilic, and
fastidious microorganisms.

25. Thiosulfate Citrate Bile Salt (TCBS) Agar: It is a selective and differential culture
medium for the isolation of Vibrio eg. Vibrio cholerae, the leading cause of cholera
worldwide.
26. Trypticase Soya broth (TSB): It is an enrichment broth used for subculturing various
bacteria from primary agar plates.

27. Xylose Lysine Desoxycholate (XLD) agar: XLD agar is used for the isolation and
differentiation of Salmonella and Shigella spp from other gram negative enteric bacilli.

2.2.1.2 Colony morphology

Although one might not necessarily see the importance of colonial morphology at first, it really
can be important when identifying the bacterium. Features of the colonies may Bacteria grow on
solid media as colonies. A colony is defined as a visible mass of microorganisms all originating
from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.

In the identification of bacteria and fungi much weight is placed on how the organism grows in
or on media. This exercise will help you identify the cultural characteristics of a bacterium on an
agar plate - called colony help to pinpoint the identity of the bacterium. Different species of
bacteria can produce very different colonies. Size of the of the colony, surface of the colony,
consistency or texture of the colony, edge/margin of colony, chromogenesis, opacity of colony,
and elevation of colony are the important features of colony which are observed in bacterial
identification.

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2.3 Chromogenic culture media

Chromogenic Agar Media are culture medium used to isolate, identify, and differentiate specific
microorganism from a heterogeneous population. The medium contains chromogenic substrate
which is utilized by the microorganism to give colored colonies that is specific for each
microorganism. Depending on the color of the result, the presence or absence of target organism
is determined and also accurately differentiated from others.

Figure 01: Chromogenic Media

2.3.1Principle of Chromogenic Media

Chromogenic media contains soluble colorless molecules called chromogens. Chromogens are
composed of two parts: a substrate ( which is the target of a specific enzymatic activity of
the microorganism) and a chromophore.

When the bond between the substrate and chromophore is split by a specific enzyme produced
by the target microorganism, chromophore is released. In its unconjugated form, the
chromophore shows distinctive colour. Due to reduced solubility, chromophore forms a
precipitate that imparts unique color to the colony.

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 Figure 02: Principle of Chromogenic Media

2.3.2 Composition of Chromogenic Agar Media

Like traditional culture media, chromogenic media also contains nutrients such as peptones,
amino acids, yeast extract, minerals, vitamins and solidifers (agar). Depending on the purpose
chromogenic media may also contain inhibitors. Unlike traditional media, which
contain chromogenic substrates or chromogens. These chromogenic substrates such as ONPG,
X-Gal, or X-Glu, together with a specified selectivity of the medium, is the simple principle
behind chromogenic media.

2.3.3. Advantages of Use of Chromogenic Agar Media:

1. Less Labor intensive and more economic:
Though people perceive chromogenic media as expensive alternative, use of a single
chromogenic medium rather two-three selective ones, reduce the cost of sample
processing. Chromogenic media s may eliminate the need of subculture and further
biochemical test for the identification of the isolates.
2. Less time consuming: As chromogenic media eliminate various steps of sample
processing (e.g. subculturing, biochemical testing) results are available within 24 hours as
compared to 48 hours or more by conventional methods. Timely diagnosis not only
ensures better outcome for the patients but also helps in prevention and spread of
infections.

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 3. Easy identification: On chromogenic agar medium, target colonies of specific
microorganisms can be recognized by their colour at a glance. No specialized equipment
needed.

Examples of Chromogenic Agar Medium

Most of the commercially available chromogenic media utilize substrates like indoxyl and its
halogenated derivatives, metal chelators like Esculin, 8‐Hydroxyquinoline, substrates for
β‐glucosidase enzyme etc. Based on these a number of companies have formulated several
variations depending on need and usage. Following are some of the examples.

2.4 Staining of isolated bacteria

Staining of the bacteria forms the foremost and the most important step in the identification of
bacteria. The isolated bacteria are stained by various methods depending upon the bacteria in
focus. Various staining techniques are used in microbiological laboratory assessments. Gram
stain (Identify gram positive and negative organism), Albert stain Identify Corynebacterium spp)
and Acid-fast stain (Identify mycobacterial spp) are commonly used stains.

2.5 Motility test

Motility testing is performed by preparing a wet mount and is then observed under the
microscope. Motility of bacteria can also be tested by inoculating the bacteria in the semisolid
motility medium.

2.6 Biochemical Tests

The staining is followed by use of various biochemical reagents and tests to get closer to the
identification of bacteria. There are many biochemical tests available for bacterial identification.
Few of them are required to be carried out depending upon the bacteria. The commonly used
biochemical tests are as mentioned below

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  Catalase test
 Coagulase test
 Oxidase test
 Sugar fermentation test
 Indole test
 Citrate test
 Urease test
 MR –VP Test
 PPA test

2.6.1 API Test kit method (Analytical Profile Index) for Biochemical Testing

API identification products are test kits are used for identification of Gram positive/negative
bacteria and yeast. It gives accurate identifications based on extensive databases and are
standardized, easy-to-use test systems. The kits include strips that contain up to 20 miniature
biochemical tests which are all quick, safe and easy to perform.

API (Analytical Profile Index) 20E is a biochemical panel for identification and differentiation
of members of the family Enterobacteriaceae. It is a well-established method for manual
microorganism identification to the species level.

API strip holds twenty mini-test chambers containing dehydrated media having chemically-
defined compositions for each test. They usually detect enzymatic activity, mostly related to
fermentation of carbohydrate or catabolism of proteins or amino acids by the inoculated
organisms.

A bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During
incubation, metabolism produces color changes that are either spontaneous or revealed by the
addition of reagents. All positive and negative test results are compiled to obtain a profile
number, which is then compared with profile numbers in a commercial codebook (or online) to
determine the identification of the bacterial species.

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The test kit enables the following tests:

1. ONPG: test for β-galactosidase enzyme by hydrolysis of the substrate o-nitrophenyl-b-D-

galactopyranoside
2. ADH: decarboxylation of the amino acid arginine by arginine dihydrolase
3. LDC: decarboxylation of the amino acid lysine by lysine decarboxylase
4. ODC: decarboxylation of the amino acid ornithine by ornithine decarboxylase
5. CIT: utilization of citrate as only carbon source
6. H2S: production of hydrogen sulfide
7. URE: test for the enzyme urease
8. TDA (Tryptophan deaminase): detection of the enzyme tryptophan deaminase: Reagent-
Ferric Chloride.
9. IND: Indole Test-production of indole from tryptophan by the enzyme tryptophanase.
Reagent- Indole is detected by addition of Kovac’s reagent.
10. VP: the Voges-Proskauer test for the detection of acetoin (acetyl methylcarbinol)
produced by fermentation of glucose by bacteria utilizing the butylene glycol pathway
11. GEL: test for the production of the enzyme gelatinase which liquefies gelatin
12. GLU: fermentation of glucose (hexose sugar)
13. MAN: fermentation of mannose (hexose sugar)
14. INO: fermentation of inositol (cyclic polyalcohol)
15. SOR: fermentation of sorbitol (alcohol sugar)
16. RHA: fermentation of rhamnose (methyl pentose sugar)
17. SAC: fermentation of sucrose (disaccharide)
18. MEL: fermentation of melibiose (disaccharide)
19. AMY: fermentation of amygdalin (glycoside)
20. ARA: fermentation of arabinose (pentose sugar)

Advantages of API test kit:

 Fast and efficient (18-24hour identification of Enterobacteriaceae and other non-

fastidious gram-negative bacteria)
 Economical to run
 User-friendly

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  Standardized
 API strips have a long shelf life, enabling every laboratory to keep the test kits on hand.
 Allow accurate identifications based on extensive databases

A fast but reliable identification of pathogens causing acute infections is a highly important issue
in microbiological diagnostics. Conventional culture-based methods are well adapted and quite
inexpensive but time-consuming and accompanied by certain limitations. Modern molecular
methods have been increasingly incorporated in laboratories, but can they fully replace the
traditional microbiological diagnostics? This article will focus on bacterial identification and
characterisation methods in microbiological diagnostics including currently used technologies
with their advantages and drawbacks. An overview table of the article will summarize all
important information.

2.7 Immunological techniques – for time- and cost-efficient detection

Immunological methods rely on binding of antibodies to specific antigens of target bacteria.

Commonly used techniques are enzyme-linked immunosorbent assay (ELISA) and serological
assays. These methods improved the microbial diagnosis because of their high-throughput
capacity, time- and cost-efficiency and ease-of-use ELISA uses an enzyme-mediated colour
change reaction to detect the presence of the specific pathogen/antigen. An advantage of this
method is the possibility to quantify the pathogen/antigen. Reduced specificity and sensitivity is
a limitation due to the difficulty to generate selective antibodies and the requirement of large
amounts of antigens for quantification.

Another immunological approach is the serological assay which is used to detect human serum
antibodies that appear specifically in response to acute or past microbial infection. However,
these assays are usually inadequate due to their long time the antibodies need to be produced
within the human body.

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2.8 Molecular methods

Molecular methods substantially revolutionized microbiological diagnosis. These techniques are

more convenient than conventional methods as they are generally faster, more specific, precise
and preceding cultivation of microorganisms is not necessary. In contrast to the conventional
methods, the identification and characterization of uncultivable and slow-growing pathogens is
easier to implement. In addition, they can be performed and results interpreted by staff with no
taxonomical expertise. Moreover, they apply more stable genotypic characteristics for
identification than traditional techniques using phenotypic characteristics.

2.9 Nucleic acid-based techniques

Nucleic acid-based technologies include several variations of hybridisations, polymerase chain

reaction (PCR) and sequencing as well as DNA/RNA microarrays. All these methods base on the
selection of genetic sequences through which pathogens can be specifically identified. For this
purpose, generally ubiquitously conserved genes – called housekeeping genes – are used or
random parts of the genome are screened. However, these methods don’t provide information
about the cellular metabolic state because they just detect intact DNA.

Hybridisation methods turned out to be a promising tool for rapid and reliable detection and
identification of bacteria. The assays are based on a direct hybridisation of labelled (e. g.
isotopes, fluorophores) oligonucleotide probes with species-specific DNA/RNA (1,9). A
disadvantage of this method is the necessity of pre-cultivation of the microorganisms to produce
significant amounts of cells to obtain a detectable signal. In addition, the number of probes that
can be used in one experiment is limited and background noise might be a problem (.

The advent of PCR enabled the detection of even small amounts of genetic material
encompassing DNA and RNA by amplification. PCR is very fast, sensitive and highly specific
for bacteria whose sequence is already known. But they are limited in multiplexing and
discovery of novel species or detection of variant strains of a known species.

In contrast, sequencing technologies provide the most detailed, unbiased information of all
nucleic-acid based methods and are able to reveal unknown organisms. But depending on the
application, it can be cost-intensive and time-consuming. Multiplex sequencing reduces the costs

21
but decreases the sensitivity of the analysis, which might be an issue when pathogens have low
abundance in the clinical sample.

DNA/RNA-Microarrays – collection of various microscopic DNA/RNA-spots (probe) attached

to a solid surface – are found in the middle range regarding cost, processing time, sensitivity,
specificity and ability to detect novel organisms. The currently available arrays are able to test
for the presence of thousands of different microbes simultaneously (e.g. LLMDA), but generally,
a previous labelling (e.g. fluorophore, silver, chemiluminescence) of the samples is necessary
making it more labour- and cost-intensive. However, the usage of label-free devices might
overcome this issue and turn microarrays to a more attractive technique.

2.10 MALDI-TOF mass spectrometry

Another technique to identify pathogens is the matrix-assisted laser desorption/ionisation time of

flight mass spectrometry (MALDI-TOF MS). This approach identifies species in a microbial
community and even differentiates strains based on molecular signatures (e.g. rRNA). A
characteristic spectrum is recorded representing a specific fingerprint for each species/strain.

This technique has fast, easy and high-throughput characteristics, does not need bacterial
cultivation and generates simple and easily interpretable spectra. However, clinical samples are
usually rich in host proteins and normal flora, which might result in overlapping mass spectra.
Moreover, MALDI-TOF MS is limited in sensitivity since a sufficient number of cells is
necessary to prevent their lost in background noise. Furthermore, the initial investments and
maintenance costs are very high, whereas the overall operating costs are low.

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 3.0 Automation in Microbiology

This section will introduce currently available automated devices and equipment in
microbiological laboratory assessments.

3.1 Automated Media Dispensing System

In culture media preparation, sterile dispensing is essential for successful

downstream applications and to meet quality requirements. Furthermore, as a growing need for
cost saving and performance improvements exists, a reliable work flow is necessary.
These requirements create a strong demand for an automated medium dispensing system that
allows reliable walk-away operation and at the same time fulfils quality requirements.
Automated Media Dispensing System reduces Non-uniformity dose, heavy working intensity,
poor efficiency and easy contamination. It improves efficiency, eliminating source of
contamination, solve the limited condition problem of big batch storage and transportation

3.2 Automation in gram staining

Despite recent advances in automated laboratory systems, most microbiology laboratories

continue to rely on manual Gram stain methods for testing patient samples. Most have accepted
manual Gram staining as a standard operating procedure, with only high-volume microbiology
laboratories adopting automated methods. Manual Gram staining can pose a unique set of
problems for Medical laboratory technologists. It is sometimes cumbersome and time
consuming, includes numerous steps that must be performed in a specific sequence with precise
timing, and exposes laboratory personnel to toxic chemicals. Additionally, the quality of the stain
often is directly related to the technologist’s experience and technique. Manually prepared slides
are less reproducible compared with automated Gram-staining methods that eliminate manual
steps and increase efficiency, standardization, and productivity.

Current automated Gram-staining instruments utilize various methods for staining slides. One
method involves automatically dunking slides into staining baths. This type of system increases
the potential for contamination as slides are in direct contact with each other and are exposed to

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the same aliquot of staining solution. The dunking method also exposes laboratory personnel to
toxic chemicals. A modification of this system uses special adapters that minimize contact
among slides, thus reducing the risk for cross contamination. Other automated Gram-stain
instruments use a closed system for storing chemical reagents and waste products, minimizing
technician exposure to toxic chemicals. These systems apply the stain by spraying the slides as
they spin in a rotor. This method uses only 10% of the volume of reagents typically used in
manual staining procedures. This reduction results in lower reagent costs and less waste.

Automated Gram-stainer systems increase the number of slides that a technologist can stain at
one time. Automated Gram-staining instrument can stain multiple slides simultaneously. Some
instruments can process and stain up to 30 slides in about five minutes using varying rotor heads
with the flexibility to fix samples on the slide or to use an off-line fixation process. Some
systems may also provide cytocentrifuge capabilities to sediment cells from liquid suspensions.

Automated Gram-staining instruments eliminate manual manipulations and processing, and

detect Gram-positive and Gram-negative strains with equal clarity. Some utilize less reagents
and produce less waste while providing consistent and reliable results that reduce the potential
for errors or repeat testing. More importantly, some automated Gram-stain instruments reduce
reagent use, waste volumes, and reduce the time required to stain slides, freeing technologists to
perform other tests. After all, the skilled technologist is the most important asset of all in the
microbiology laboratory.

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 4.0 Procalctonin

Antibiotic overuse and misuse represents a significant healthcare burden, in terms of treatment
costs, but also the increased risk of resistant micro-organisms. Emerging antimicrobial resistance
is a serious issue of Clostridium difficile infections calls for more effective efforts to reduce the
unnecessary and prolonged use of antibioticsin self-limiting non-bacterial and resolving bacterial
infections. To help achieve this aim, diagnostic tools and biomarkers are urgently needed which
allow better assessment of a patient’s risk of having an infection, and their response to antibiotic
therapy. One such blood biomarker is procalcitonin (PCT), which is increasingly used in clinical
practice for improved patient management. During bacterial infections, PCT blood levels rise
within 4-6 hours. Its kinetics then mirror the severity of infection. PCT levels drop by about 50%
daily when infection is controlled and responds adequately to antibiotics. Based on this
regulation and kinetics, many studies have documented the clinical utility of PCT for different
clinical settings and infections.

 PCT improves early detection of sepsis and risk assessment

 PCT can aid in decision-making on antibiotic discontinuation for patients with suspected
or confirmed sepsis

 PCT used to monitor therapy for respiratory infections has led to a more tailored use of
antibiotics with a reduction in antibiotic exposure of 30-70% depending on the clinical
setting (3)

 PCT used to monitor therapy for respiratory infections has shown secondary gains such
as lower risk of antibiotic-associated side effects, shorter length of hospital stays, and
lower overall costs due to antibiotic savings.

Procalcitonin (PCT) is the precursor peptide – or prohormone – of the mature hormone

calcitonin. PCT is released in multiple tissues in response to bacterial infections via a direct
stimulation of cytokines. PCT shows an interesting kinetic profile. Cytokines such as interleukin
(IL)-6 and tumor necrosis factor (TNF) show a fast-initial spike upon infection with, however,
levels going back to normal within a few hours. The high variability of these markers has been a
major challenge for their use in clinical practice.

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C-reactive protein (CRP), on the other hand, increases slowly with a peak after 48-72 hours and
a slow decrease thereafter. CRP is usually considered a biomarker for inflammation rather than
infection. In adults, PCT increases promptly within 4-6 hours upon stimulation and decreases
daily by around 50% if the bacterial infection is controlled by the immune system supported by
effective antibiotic therapy. These characteristics make PCT an interesting biomarker for
monitoring patients with systemic infections and sepsis and for more informed decisions on
prescription and duration of antibiotic therapy. As PCT levels do not show a steep decrease in
non-responding infections, monitoring its course also has prognostic implications.

PCT production is induced in response to microbial toxins and to certain bacterial-induced

cytokines, particularly interleukin (IL)-1b, tumor-necrosis factor (TNF)-a and IL-6, and is
released in the bloodstream where it can be measured. Conversely, PCT production is attenuated
by certain cytokines released in response to a viral infection, particularly interferon-g (IFN-g).
This selective cellular mechanism makes PCT a useful diagnostic biomarker, which is more
specific for bacterial infections compared to other inflammatory markers (i.e. C-reactive protein)
and helps to distinguish bacterial infections from other inflammatory reactions or viral
infections.

Diagnostic value of procalcitonin in the early recognition of sepsis Globally, an estimated 20 - 30

million cases of sepsis occur each year, with over 6 million cases of neonatal and early childhood
sepsis, and the rate of sepsis mortality remains unacceptably high (between 30 and 60% of
patients with sepsis die). Furthermore, sepsis has significantly increased by an annual rate of 8-
13% over the past decade, due to the aging population, the development of drug-resistant and
more virulent varieties of pathogens, and, in the developing world, to malnutrition, poor
sanitation, lack of access to vaccines and timely treatments. The cornerstone of today’s sepsis
treatment is early recognition of the condition and early initiation of appropriate antibiotic
therapy, as well as fluid resuscitation. Clinical signs, such as the systemic inflammatory response
syndrome (SIRS) criteria, lack both sensitivity and specificity. Therefore, blood biomarkers
(such as PCT) that mirror the severity of bacterial infections, improve the early diagnosis of
sepsis PCT has been demonstrated to be most clinically useful, and superior to commonly used
clinical variables and laboratory tests in the early diagnosis of Sepsis. Moreover, it has been
shown to correlate with the extent and severity of microbial invasion. Thereby, PCT improves

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the clinical work-up of patients with suspicion of sepsis. Low PCT values (<0.25 ng/mL) in
patients with clinical signs of infection indicate a low probability for blood culture proof of
bacterial infection and sepsis. Usually, PCT levels are found to be >0.5 ng/mL.

Figure 04: Sepsis Diagnosis with PCT

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