CONVENTIONAL MICROBIOLOGICAL

TECHNIQUES DR.T.V.RAO MD

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My tribute to those who made life safe

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Robert Koch Perfected Culturing the Common Pathogens

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A great quotes by Ronald Ross

• I have failed in finding parasites in mosquitoes fed on malaria patients, but perhaps I am not using the proper kind of mosquito

• “The screws of my microscope were rusted with sweat from my forehead and hands, and its last remaining eye-piece was cracked

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Beginning of Diagnostic Microbiology

•In the late 1800s, the realization that identifiable microbes caused specific diseases led to pathogens Specific medical diagnosis. Although the time honoured techniques of growing bacteria in broth or solid cultures and staining and examining them under microscopes are still important today20-02-2016 Dr.T.V.Rao MD 5

A GREAT QUESTION TODAY HOW KEEN WE ARE WITH THE WORK

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The Real Good of the Past in Diagnostic Microbiology

• In the good old days, the microbiology laboratory used to be a labour intensive place equipped with incubators and microscopes. Microbiologists were patient scientists waiting at least 24 hours before their isolated cultures were grown enough for identification

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Where we stand Today• Most neglected and least

invested specialty• Reasons can be many

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CONVENTIONAL METHOD MEANS

•Based on or in accordance with what is generally done or believed:

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What is a Technique

A way of carrying out a particular task, especially the execution or performance of an artistic work or a scientific procedure20-02-2016 Dr.T.V.Rao MD 11

American Society for Microbiology encourages Artistic and Conventional Techniques on Streaking and Culturing

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A MATTER OF CONFLICTWHO WILL COLLECT THE SPECIMENS

• Proper specimen collection, container labelling, and culture requests are the responsibility of the ordering physician. Technologists in the Clinical Microbiology Laboratory will be familiar with specimens of choice and proper collection techniques

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SPECIMEN COLLECTIONwhose duty it is ?

The specimen is the beginning. Alldiagnostic information from the laboratorydepends upon the knowledge by whichspecimens are chosen and the care withwhich they are collected and transported.

—Cynthia A. Needham

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If you accept the Truth many Suboptimal Specimens are Processed

• I should share my experience the ideal collecting of a sample remain with greater challenges, we many times receive suboptimal sample for processing, few posses courage to reject, it just goes

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LABORATORY HANDLES THE SPECIMENS

• The technologist in the laboratory will directly handle specimens of clinical and environmental source which are received from the Postal Service or hand carried to the laboratory.

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Are we Collecting the Right specimens

• Select an appropriate sites or organs for sampling.

•Use sterile equipment for sampling.

• Labelling the sample

•Keep and transfer the sample inappropriate medium and condition.20-02-2016 Dr.T.V.Rao MD 17

Microbiological techniques• Bacteria will grow on

practically any source of organic food which provides carbon compounds to be respired for energy, and nitrogen compounds to be incorporated into proteins for growth. These substances are normally provided dissolved in water.

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General View on the Parameters Used in the Process of MicroorganismIdentification

•Before one can proceed to identify a microorganism, the characteristics of that organism have to be determined in details. The major characteristics which are observed

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Gram Staining continues to be Most rapid method to diagnose

• The rapid Gram stain evaluation is reliable, easy to perform, and well suited for the routine clinical laboratory

• Eg – CSF examination in emergencies

• Sputum in case of acute pneumonias

• Bacterial Vaginitis • Any other transudates • Pleural fluid• Pericardial fluids

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Molecular Biology in Infancy

• Molecular biology techniques promise to revolutionize the diagnosis of infectious disease—to date a promise still in its infancy

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Laboratory Medicine Under threatMicrobiology is No exception

•Great questions to many Microbiologists

How much Time we are spending in the Laboratory

Who are doing the Bench work Are laboratories equipped with minimal

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The young Microbiologists should know

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Use a bright field light microscope

• Use a bright field light microscope to view and interpret slides, including

• 1. correctly setting up and focusing the microscope

• 2. proper handling, cleaning, and storage of the microscope

• 3. correct use of all lenses

• 4. recording microscopic observations

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Properly prepare slides• Properly prepare slides for

microbiological examination, including

• A. cleaning and disposing of slides

• B. preparing smears from solid and liquid cultures

• C. performing wet mount and/or hanging drop preparations

• D. performing Gram stains

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Properly use aseptic techniques• Properly use aseptic techniques

for the transfer and handling of microorganisms and instruments, including

• A. sterilizing and maintaining sterility of transfer instruments

• B. performing aseptic transfer

• C. obtaining microbial samples

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Use appropriate microbiological Media and Test Systems

•A. Isolating colonies and/or plaques

•B. Maintaining pure cultures

•C. Using biochemical test media

•C. Accurately recording macroscopic observations20-02-2016 Dr.T.V.Rao MD 28

Use standard microbiology laboratory equipment correctly

•A. using the standard metric system for weights, lengths, diameters, and volumes

•B. lighting and adjusting a laboratory burner

•C. using an incubator right Temperature to be monitored

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Streaking is the Beginning of learning Alphabets in Microbiology

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Streaking the Urine specimens is the best way to learn the matters with precision

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Streaking is Key to success in Separation of Pathogens from Commensals

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KNOW THE MICROBES BEFORE YOU PROCEED

• The cultural characteristics of a microorganism usually vary depending on the media used and many other factors. Some experienced microbiologists could have a good guess about the identity of a microorganism just by its cultural characteristics, but this was proven to be a bad technique. Students as well as microbiologists are advised to follow strict procedures for the identification of isolates from clinical specimens.

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Macroscopic (colony) morphology

•Size

•Shape

•Colour ( Pigment )

•Surface appearance

•Haemolysis

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General View on the Parameters Used in the Process of MicroorganismIdentification

• Before one can proceed to identify a microorganism, the characteristics of that organism have to be determined in details In clinical terms, it is the shape, size, colour, elevation and other characteristics of the colony formed on the culture plate. In taxonomy, it includes the nutrient requirements for the growth of the organism and the physical factors such as temperature, pH and the incubation period. These factors are used to identify certain pathogenic species but less commonly used in routine procedures

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Is our experience Matches Scientific Approach

• Some experienced microbiologists could have a good guess about the identity of a microorganism just by its cultural characteristics, but this was proven to be a bad technique. Students as well as microbiologists are advised to follow strict procedures for the identification of isolates from clinical specimens.

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BIOCHEMICAL CHARACTERISTICS

• Frequently, the identity of a species requires detailed knowledge of its biochemical activities, since other characteristics are not sufficiently distinctive or differential. For example, the bacterium Escherichia coli, a normal inhabitant of our intestinal tract, is indistinguishable microscopically from Salmonella typhi, the bacterium that causes typhoid fever. However, if these two bacteria are examined for their metabolic (or biochemical) characteristics, they are found to be very different and distinguishable on this basis.

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Morphology and Staining

• This includes the microscopic appearance of a stained preparation of the organism. Useful information to be taken into account, are the size of the individual cells, cell shape and arrangement and staining reaction if differential staining procedures is used.20-02-2016 Dr.T.V.Rao MD 38

TILL TO DATE THERE IS NO FASTER METHOD AS GRAM’S STAINING

• Some laboratories which have a little facility could give the report of a microbiological examination of a clinical specimen just by stating their morphological characteristics and the sensitivity testing results

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LABORATORY HANDLES THE SPECIMENS

• The technologist in the laboratory will directly handle specimens of clinical and environmental source which are received from the Postal Service or hand carried to the laboratory.

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Gram staining most Rapid method as the Situation warrants

• Look up reference images. If you are not certain what a bacteria is, look through a collection of reference images, sorted by shape and result of the gram stain. You can find databases online at the National Microbial Pathogen Database

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Disadvantages of Microscopic methods

•Microscopy may suggest an etiologic agent, but it rarely provides definitive evidence of infection by a particular species.

• Microscopic findings regarding bacterial morphology may be misleading, because many species can be pleomorphic and conclusions can be influenced by subjective interpretation of the investigator.

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Staining is limited Sensitive

• Limited sensitivity is because a relatively large number of microbial cells are required before they are seen under microscopy (e.g. 104 bacterial cells/ml of fluid) (Fredricks & Relman, 1999). Some micro-organisms can even require appropriate stains and/or approaches to become visible. • Limited specificity is because our inability to speciate

micro-organisms based on their morphology and staining patterns.

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Problems With Traditional Methods• Cultivation-based methods insensitive for detecting some

organisms.

• Cultivation-based methods limited to pathogens with known growth requirements.

• Poor discrimination between microbes with common behavioural features.

• Failure to detect infections caused by uncultivated (e.g., novel) organisms, or organisms that fail to elicit a detectable host immune response.

• Visual appearance of microorganisms is nonspecific.

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Problems With Traditional Methods

• Examples of Failures With Traditional Approaches

• Detection and speciation of slow-growing organisms takes weeks

• (e.g., M. tuberculosis).

• A number of visible microorganisms cannot be cultivated (e.g., Whipple bacillus).

• Diseases presumed to be infectious remain ill-defined with not detected microorganism (e.g., abrupt fever after tick bite).

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Culture of fungi• Fungi specimens

obtained from non sterile sites must be inoculated onto media containing antibacterial agents. Specimens should be allowed to grow for 4 wkbefore being discarded.

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Use of Physical Separation ProceduresStreak Plate technique

•As we have seen in previous labs, single colonies may be achieved by using the streak plate technique

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Most of us Miss the Parasites

•Parasites cause hidden epidemics, many practitioners do not even think

•Microbiologists evaluate for Bacteriological examination can miss the Parasites and Fungus

• Still our society there sare several parasitic infections prevails

• Simpler techniques are missed as stool examination and entrusted with most of the times to the technicians without much understanding 20-02-2016 Dr.T.V.Rao MD 49

Can we diagnose the stool parasites by conventional methods with accuracy

•Microsporidia

•Isospora Belli & Cyclospora

•Cryptosporidium

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Parasitology Testing Laboratory Services

• This test evaluates stool for presence of parasites and levels of beneficial flora, imbalanced flora, possible pathogenic bacteria and possible fungal pathogens. This Comprehensive Parasitology Profile uses the most technologically advanced procedures to accurately identify a wide range of protozoal parasites, including amoebae, flagellates, ciliates, coccidia and microsporidia. This stool test can help reveal hidden causes behind acute or chronic conditions that develop from parasitic infection or dysbiosis.

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Empowering conventional techniques with modern technology

• Specimens are carefully analysed by highly trained technicians using computer-enhanced video microscopy, new staining procedures, and advanced immunoassay techniques. These accurate detection methods allow for increased detection rates, increasing the awareness of the important relationship between parasitic infection and a broad spectrum of illnesses and diseases

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IS THE CONVENTIONAL METHODS CONVINCING THE CLINICIANS YES / NO

• I am confident it is certainly no many times tests are ordered without forethought on the existing the clinical diagnosis and it is just fishing in troubled water, 20-02-2016 Dr.T.V.Rao MD 53

Not a one step Diagnosis

•Even Today Many Clinicians think Microbiologist diagnosis is one step diagnosis as in Biochemistry as we many steps to be perform coming to minimal conclusions 20-02-2016 Dr.T.V.Rao MD 54

MOVING TO FEATURE NEEDS

• In the get-it-done-yesterday environment Hospitals and Microbiology laboratories are finding that the traditional microbiological methodologies and especially sending microbiological samples to outside labs cost them time, money and opportunities Internalizing the microbiology testing and especially adopting rapid microbiological methods (RMM) can significantly speed up the time to results from 7-10 days to 24-48 hours.

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Limitations of Conventional Microbiological Testing

• Conventional microbiological methods have well-known inherent limitations. These include small test sample volumes, prolonged incubation periods, incompatibilities with membrane filtration, and ambiguity associated with using turbidity as a detection endpoint

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India can face many challenges • The increase in worldwide travel,

coupled with increasing immigration into the India, contributes to the spread and incidence of parasitic infections. In addition, parasitic infections are commonly transmitted through fecally contaminated food, water, or other materials within this country.

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Conventional techniques of Malaria

• Current diagnostic methods for malaria include clinical diagnosis, microscopy, serology, molecular diagnosis, and antibody detection. Giemsa staining of the peripheral blood smear is the gold standard for the diagnosis of malaria, but also has limitations. Using microscopy as the only means of diagnosing malaria can lead to false negatives. Another diagnostic tool is Rapid Diagnostic Tests or RDTs also known as dipstick assays. It

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Gold Standard for diagnosis of Malaria

• The gold standard for the diagnosis of malaria is the Giemsa staining of the peripheral blood smear(Thick and thin smear), but its also has its limitations. However, even under optimal conditions the sensitivity of microscopy is only about twenty parasites/μl of blood, and subjective interpretation and reader errors further reduce the accuracy of diagnosis.

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Human dedication matters • Conventional microscopic

examination of peripheral thick and thin blood smears remains the gold standard for malaria diagnosis. Although this method requires a trained microscopist, and sensitivity and specificity vary compared with recent technical advances, it is inexpensive and reliable.

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Medical Microbiologists losing the working opportunities

• While Rapid Microbiological Methods (RMM) offer high degree of automation, significant reduction in time to results, faster product release, ability to employ non-microbiologists to operate the system, and improved control;

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My Dear Young Microbiologists

• Most people want to do excellent work. Apathy becomes a problem when team members feel there is no solution or they have no voice

• I wish you are the future leaders in Microbiology for next 40 years hope you all wish for change to live in comfort,

• Assess your role

• Purpose of the speciality

• Try Impress others with hard work and sincerity

• If Medical People do not wish to work some body take you place ?

•NEXT WHAT ?

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Are we ignoring Anaerobes ?

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Tele Diagnosis

CDC helps in Digital diagnosis ➲The CDC now offers tele diagnosis to help laboratories diagnose

malaria and other parasitic diseases. When laboratories are not certain about identifying parasites on a slide, they can e-mail to the CDC images of the suspected parasites. Experts then review the images and discuss findings with the submitting lab within only a few hours, allowing near real-time diagnosis as well as an opportunity for training in microscopic diagnosis.

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A long parasite of 15 cm was extracted without damage to the physical structure

•An approximately 15 cm long filamentous macroscopic parasite was extracted sent intact in Normal saline, to Microbiology

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Histopathology sections and Photomicrograph

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As reported from CDC – Atlanta USA

• Based on the images, we agree this is a female Dirofilaria (possibly D. repens in India), as indicated by tall, polymyarian musculature, external cuticular ridges, and paired reproductive tubes.

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World First for Malaria - mobile phone diagnosis now available

• xRapid is a world first in mobile health, providing automatic diagnosis of malaria via an iPhone app. It is the first commercially available mobile app that has the functionality to quickly and accurately diagnose a major disease.

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ARE WE READY FOR CHANGE TO AUTOMATION ?

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THE FUTURE OF DIAGNOSTIC MICROBIOLOGY IS CHANGING

• The physical structure of laboratories, staffing patterns, work flow, and turnaround time have all been profoundly influenced by technical advances. The implementation of nucleic acid amplification-based molecular techniques provides complementary, rapid, and on-demand diagnosis services. These changes will continue, and lead diagnostic microbiology inevitably to a modern discipline, which can face many challenges in the future.

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TODAYS PROBLEM WITH DIAGNOSTIC MICROBIOLOGY

• We Teach More than what we see, and diagnose

• WE Talk More ?

• We discuss more ?

• However we do less ?

• 1Bench work 2 Fail to improve the laboratory skills ?

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Laboratories should progress with scientific Developments

•Modern science is fast-moving, and no laboratory can exist for long with a program

based on old facilities. Innovation and renewal are required to keep a laboratory on the

frontiers of science.

• Burton Richter

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Wish to be A Better Microbiologist

• A microbiologist needs to be both brilliant and methodical. An ability to think critically and analytically is a prerequisite, as is an advanced understanding and knowledge of computers.

• Microbiologists must be experts at working with statistics and must stay abreast of developments in statistical techniques

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How we can improve our Diagnostic Microbiology

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