SKILLS IN MEDICAL MICROBIOLOY

Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

Why Microbiologists need Skills

• Successful microbiology work demands

reliable precision in identifying microbes,

observing their characteristics and behavior,

applying biological theory to observations

and drawing accurate conclusions based on

biological data, known facts and concepts,

original research and existing research.

Dr.T.V.Rao MD 2

SKILLS for MD, MSc in MicrobiologyAt the end of the course the student shall be able

to

• 1. Plan the laboratory investigations for diagnosis of infectious diseases

• 2. Perform laboratory procedures to arrive at the etiological diagnosis of diseases caused bacteria, fungi, viruses and parasites.

• 3. Perform and interpret immunological and serological tests.

• 4. Operate routine and sophisticated instruments in the laboratory

Dr.T.V.Rao MD 3

Bacterial Identification a Part of MD,

M.Sc. ( Microbiology ) Examination

• The Indian Medical Curriculum for Post Graduates

in Microbiology emphasizes with Identification of

Bacteria from Pure and a Mixture of Bacterial

agents. A student must have minimum skills to

Identify, Isolate, Characterize , prove the

pathogenic nature and test its Antibiograms

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Why We Should Identify a Bacteria

• Accurate and definitive microorganism

identification, including bacterial identification, is

essential for correct disease diagnosis, treatment

of infection and trace-back of disease outbreaks

associated with microbial infections. Bacterial

identification is used in a wide variety of

applications including microbial forensics, criminal

investigations, bio-terrorism threats and

environmental studies, including Epidemiological

studies.Dr.T.V.Rao MD 5

Microbiology Laboratory

Skills Test: Bacterial Unknown ?

• This skills test will examine your ability to

i) isolate two different bacterial, or even three

from a mixed culture and ii)

identify each bacterium using pertinent diagnostic

characteristics. Many times you have already

performed or are presenting performing these

diagnostic tests. Therefore, you should be familiar

with how they are conducted.

Dr.T.V.Rao MD 6

How to Evaluate

• The Teachers should try to evaluate students with a Mixture of One commensal, One truly pathogen, and other can be a opportunistic pathogen, as more understanding can be created in the learners, the Importance of Emerging opportustic bacterial pathogens

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A student is given a Mixture of

Bacterial agents• The student will receive a

mixed culture unknown containing two or three different bacteria. They can be of any combination from among those bacteria listed by your Laboratory when you were trained as student when practicing

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Read the Question and create Mind

Map for all Procedures

• Carefully read the Clinical question provided to you, it

guides you what is a probable microbes to be

identified.

• To begin, read the instructions given by Examiner

carefully. Begin working immediately. The “Mixed

Culture Unknown Answer Sheet and Flow Chart” and

the “Skills Test Log Sheet” must be submitted as a

stapled unit to your Examiner by the stated deadline.

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All post graduates should be familiar with

Isolation, Description and Identification of

Bacteria

Staphylococcus and Micrococcus; Anaerobic Gram positive cocci.

Streptococcus and Lactobacillus.

Neisseria, Branhamnella and Moraxella

Corynebacterium and other coryneform organisms.

Bacillus: the aerobic spore bearing bacilli

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Other Pathogens which are always

Incorporated in Evaluations

•The Enterobacteriaceae

•Vibrio's,

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Record the findings in the Log Book

• You are to keep a

log of your

activities and

observations in

this test

• Start working with

Skill test log

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Skills on Day 1Selecting the Specimen for Evaluation

• The Examiner will provide a rack of

mixed culture unknowns that have been

randomly labeled with a code number.

Select one tube and immediately record

the number of that unknown on the

report sheet that will also be provided

by your Examiner.

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Gram staining – from Mixture you are

Provided• Prepare a heat-fixed

smear from your mixed

culture unknown on a

glass slide. Gram stain

your smear. Observe and

record your results. These

observations may give you

an idea of what to expect

upon isolating your

unknown bacteria.

Dr.T.V.Rao MD 14

Limitations of Gram Staining

• However, it is conceivable that you may isolate two bacteria having the same Gram-stain reaction and morphology, in which case it may be difficult to distinguish between the different isolates.

• Eg Two gram negative bacteria, E.coli and Salmonella spp

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Hanging Drop Procedure:

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Hanging Drop give clues to Basic

Identification in Gram Negative Bacteria

• 1. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick. If too much Vaseline is used, it will be squeezed toward the center and mix with the drop or squeeze out the edges and get on the objective lens of the microscope.

• 2. Place a loopful of the culture to be tested in the center of the prepared coverslip.

• 3. Turn the clean concavity slide upside down (concavity down) over the drop on the coverslip so that the Vaseline seals the coverslip to the slide around the concavity.

• 4. Turn the slide over so the coverslip is on top and the drop can be observed banging from the coverslip over the concavity.

• 5. Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives.

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Hanging Drop

• 6 Turn the objective to its lowest position using the coarse

adjustment and CLOSE THE DIAPHRAGM.

• 7. Look through the eyepiece and raise the objective slowly using

the coarse adjustment knob until the edge of the drop is observed

as an irregular line crossing the field.

• Move the slide to make that line (the edge of the drop) pass

through the center of the field.

• 9. Without raising or lowering the tube, swing the high dry

objective into position (Be sure the high dry objective is clean).

• 10. Observe the slide through the eyepiece and adjust the fine

adjustment until the edge of the drop can be seen as a thick,

usually dark line.

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Hanging Drop

• 11. Focus the edge of the drop carefully and look at each

side of that line for very small objects that are the

bacteria. The cells will look either like dark or slightly

greenish, very small rods or spheres. Remember the high

dry objective magnifies a little less than half as much as

the oil immersion objective.

• 12. Adjust the light using the diaphragm lever to maximize

the visibility of the cells.

• 13. Observe the cells noting their morphology and grouping and

determine whether true motility can be observed.

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Hanging Drop

• 14. Brownian movement should be visible on slides of all the organisms, but two should also show true motility.

• 15. Wash the depression slide and after soaking in Lysol or common bleach buckets.

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What you choose for Plating and

Isolation• Obtain one (1) of each of the following agar plate

media:

• MacConkey Agar (MAC) Blood Agar

• = Mannitol Salts Agar (MSA)

• = Tryptic Soy Agar (TSA) [Nutrient Agar (NA) may be substituted for TSA]

• Your choice can be wide, but you should be appropriate and optimal choosing other media available in the Laboratory,

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Caution in Choosing Media

• Too many media will confuse the situation, Leads to unnecessary questioning.

• If you choose inappropriate Medium you loose the path of Identification.

• Optimal media are few for identification all basic pathogens

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Streaking will expose your Skills

• Using your very best technique, separately streak a loopful of the mixed culture

• Unknown onto the MAC, MSA, and TSA plates, blood agar. Or any other of your choice. Incubate these plates at 35°C- 37°C overnight.

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Importance of Streaking

• Use your best skills in streaking the culture plates.

• The streaked plate indicates your skills,

• Examiners can identify you past experience with laboratory bench work.

• Best Instructions are well represented in Koneman’s Diagnostic Microbiology.

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Streaking the plates is a ART

Perfect It

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Begin with Skills Test Log Sheet

• Mixed Culture Unknown Number xxx

• Date: xx/xx/xxxx

• Description of Gram Stain Results of Mixed Culture

Unknown: (Optional)

To create a systematic reporting Log on to

URL - http://www.as.ysu.edu/~crcooper/STLS.pdf

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Crucial Reporting

• Your crucial reporting

of identification starts

from 2nd and by 3rd

day you will need to

submit by the stated

deadline (as listed in the

laboratory syllabus or as

provided by your examiner )

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Day 2• Observe your streak plates for

isolated colonies and record your

results.

• NOTE 1: MAC medium is a

differential and selective growth

medium. Only Gram-negative

bacteria grow on this medium. In

addition, bacteria that ferment

lactose appear as pink colonies

(occasionally purplish) or as white

colonies with a pink center. Non-

lactose fermenters appear clear,

colorless, or white only.

Dr.T.V.Rao MD 28

Mannitol Salt Agar helps in Gram Positive

cocci• Composed of NaCl, Mannitol and

phenol red as pH indicator

• Both selective and differential

• Selective since it allows only the growth of high salt or saline loving organisms.

• Differential since manitol fermenter organisms change the red pH indicator color (neutral) to yellow (acidic).

• Non-mannitol fermenters do not change the color of the media.

• Usually allows the growth of Staphylococci especially for differentiating Staphylococcus aureus from others.

Dr.T.V.Rao MD 29

Mannitol Salt Agar

• Mannitol fermenters includes: Staphylococcus aureus

• Non-mannitol fermenters includes: Staphylococcus epidermidis

• Positive growth but non-mannitol fermenters includes: Micrococcus luteus

• Negative growth includes: Escherichia coli, Pseudomonas aeruginosa

Dr.T.V.Rao MD 30

Gram positive – Mannitol Salt Agar

• MSA medium is also a differential and selective growth medium. Only Gram-positive bacteria grow on this medium. In addition, those Gram-positive bacteria that ferment Mannitol grow as white colonies and cause the surrounding medium to appear yellow. Gram-positive, non-mannitol fermenters appear clear, colorless, or white only with the exception of Micrococcus luteus. The latter microbe appears naturally as a yellow colon

Dr.T.V.Rao MD 31

Use of Tryptic Soya Agar

• TSA and NA media are not differential or selective growth medium.

• Both Gram-positive and Gram-negative bacteria grow on this medium. Hence, the purpose of this medium is to select bacteria based upon differences in colony morphology.

Dr.T.V.Rao MD 32

Tryptic Soya agar

• Tryptic soy agar (TSA) inoculated with (A)Staphylococcus aureus, (B) Staphylococcus epidermidis, and (C) Escherichia coli demonstrating growth of all three organisms. TSA is a general purpose medium that will allow for the growth of all three organisms.

Dr.T.V.Rao MD 33

Choose the Selective Medium with

caution ( TCBS )• TCBS is a selective isolation

medium for culture of pathogenic Vibrio sppprimarily from clinical samples. On this medium most Enterobacteriaceae in faeces are suppressed for at least 24 hours although slight growth of Proteus spp and Streptococcus faecalis may occur but they are readily distinguished from vibrio colonies

Dr.T.V.Rao MD 34

Salmonella Shigella Agar

• Salmonella Shigella Agar (SS

Agar) is a differentially

selective medium for the

isolation of pathogenic

enteric bacilli, especially

those belonging to the

genus Salmonella. This

medium is not

recommended for the

primary isolation of Shigella.

Dr.T.V.Rao MD 35

Picking the isolates after separation

• From your different media, identify two or even

three different bacteria. On the underside of your

medium, mark the location of two distinct, and

well-isolated colonies. Using a loop, pick a small

portion of these two colonies and inoculate each

onto TSA or NA slants. Incubate the slants at 35°C-

37°C. These slants will serve as your stock cultures

to inoculate your diagnostic media

Dr.T.V.Rao MD 36

With Caution – It is crucial to Identify Gram

positive and Gram Negative Bacteria

• Depending upon the nature of your mixed

culture unknown, two different colony types

may not appear on MAC or MSA plates.

Remember, these media are selective. If you

have two Gram-negative bacteria, they will

grow on the MAC plate, but not the MSA

plate,

Dr.T.V.Rao MD 37

A crucial step

• Conversely, if you have two Gram-positive

bacteria, they will grow on the MSA plate, but not

the MAC plate. If you have one Gram positive and

one Gram-negative bacterium in your mixed

culture unknown, then you should see the growth

of only one colony type on the MAC and only one

colony type on the MSA plate. In most cases, two

colony types should appear on the TSA (or NA)

plate.

Dr.T.V.Rao MD 38

Answer Sheet and Flow Chart

• Mixed Culture Unknown Number xxx

• Identity of Unknown Isolate 1 …….

• Identity of Unknown Isolate 2 ……

• Identity of Unknown Isolate 3 …..

• Prepare a flow chart of your work for each isolate..

Be sure to identify which flow chart corresponds

to which isolate.

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You may now Gram stain your isolated bacteria by one of

the following two options:

Option 1

: From the location marked on the plates from which you isolated your unknown bacteria, remove some of the colony with a loop and make a heat-fixed smear on a glass slide. Gram stain your smear. Observe and record your results

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Option 2

• Incubate your stock cultures for 12-18 hours (i.e., until Day 3), then from each tube separately remove some of the growth of the unknown bacterium with a loop and make a heat-fixed smear on a glass slide. Gram stain your smear. Observe and record your results.

• Caution As the Examination is final by 3rd this can be only 2nd

option

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Biochemical Test for Identification for

Identification of Genus and Species

• Students should choose the Biochemical tests which are available, familiar in the past, and Rapdily reactive as the Examination is for 2 or 3 days, and should come to maximum conclusions. However should choose few the tests giving late reaction for academic discussion

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Biochemical tests help in Identification of

several Bacterial isolates

• EVERYTHING that a living organism does is the

result of the activity of an ENZYME, the

SUMMATION of the activities of all an organism's

enzymes equals its BIOCHEMICAL FINGERPRINT.

That is, an organism is the totality of its enzymes,

so by determining which enzymes are present in

an unknown organism one can DESCRIBE &

IDENTIFY that organism

43Dr.T.V.Rao MD

Biochemical ReactionUse of substrates and sugars to identify pathogens:

- Sugar fermentation:

Organisms ferment sugar with production of acid only

Organisms ferment sugar with production of acid and gas

Organisms do not ferment sugar

b- Production of indole:

Depends on production of indole from amino acid tryptophan

Indole is detected by addition of Kovac’s reagent

Appearance of red ring on the surface

- H2S production:

Depends on production H2S from protein or polypeptides

Detection by using a strip of filter paper containing lead acetate

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Biochemical Reaction (cont.)

c- Methyl red reaction (MR): Fermentation of glucose with production of huge amount of

acidLowering pH is detected by methyl red indicator

d- Voges proskaur’s reaction (VP):Production of acetyl methyl carbinol from glucose

fermentationAcetyl methyl carbinol is detected by addition KOHColor of medium turns pink (positive)

e-

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Biochemical Reaction (cont.)

f- Oxidase test:

Some bacteria produce Oxidase enzyme

Detection by adding few drops of colorless oxidase reagent

Colonies turn deep purple in color (positive)

g- Catalase test:

Some bacteria produce catalase enzyme

Addition of H2O2 lead to production of gas bubbles (O2 production)

h- Coagulase test:

Some bacteria produce coagulase enzyme

Coagulase enzyme converts fibrinogen to fibrin (plasma clot)

Detected by slide or test tube method

i- Urease test:

Some bacteria produce urease enzyme

Urease enzyme hydrolyze urea with production of NH3

Alklinity of mediaand change color of indicator from yellow to pink

Dr.T.V.Rao MD 46

Common Tests To identify

Bacterial isolates– Indole

–Methyl Red/Voges Proskauer

– Citrate

– H2S production

– Urea hydrolysis

–Motility

– Lactose fermentation

– Sucrose fermentation

– Glucose fermentation & gas production

47Dr.T.V.Rao MD

I M Vi C Tests

• I M Vi C is an acronym that stands for

indole , methyl red, Voges-Proskauer , and

citrate . To obtain the results of these four

tests, three test tubes are inoculated:

tryptone broth (indole test), methyl red -

Voges Proskauer broth (MR-VP broth), and

citrate test.

48Dr.T.V.Rao MD

Triple Sugar Iron Agar

• Bacteria that ferment any of the three sugars in

the medium will produce byproducts These

byproducts are usually acids, which will change

the color of the red pH-sensitive dye (phenol red)

to a yellow color. Position of the color change

distinguishes the acid production associated with

glucose fermentation from the acidic byproducts

of lactose or sucrose fermentation. Many bacteria that

can ferment sugars in the anaerobic butt of the tube are

enterobacteria.

49Dr.T.V.Rao MD

Reading the results on TSI enables to

identify the several pathogens

50Dr.T.V.Rao MD

On 2nd day we have to Express with Caution –

Express all other Possibilities

• Citrobacter freundii – still under consideration; lactose fermenter

• Enterobacter aerogenes - still under consideration; lactose fermenter

• Escherichia coli - still under consideration; lactose fermenter

• Klebsiella pneumoniae - still under consideration; lactose fermenter

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Testing for Antibiotic sensitivity

• The method includes several steps

including obtaining a bacterial sample;

identifying the type of bacteria in the

bacterial sample; selecting a set of

antibiotics based on the identity of the

bacteria in the bacterial sample;

obtaining a control sample from the

bacterial sample;

Testing Antibiotic

Susceptibility

• Antibiotic sensitivity

test: the in vitro

testing of bacterial

cultures with

antibiotics to

determine

susceptibility of

bacteria to antibiotic

therapy.

Bauer-Kirby test.

How results to be Reported after How results to be Reported after How results to be Reported after How results to be Reported after Sensitivity TestingSensitivity TestingSensitivity TestingSensitivity Testing

• One should follow guidelines in reporting the results and make matters simple with clear words as

Organism A isolated and sensitive ( or resistant ) to Antibiotic B

Such a report is relevant to present clinical condition “ that the minimum inhibitory concentration( MIC ) of the antibiotic for it has been measured in some way and that, if the organism is reported as sensitive the MIC is less than a half or quarter of the concentration of antibiotics likely to be found in the infected tissues of a patient given the usual schedule of doses i.e. that the infection is treatable”

Streaking the Inoculum

• Kirby-Bauer (also Bauer-Kirby) disk diffusion antibiotic susceptibility testing applies a defined inoculum (compared to

McFarland 0.5 OD standard streaked as a lawn onto a large Mueller-Hinton agar or Blood agar plate (in 3 directions to ensure confluence).

Making proper inoculum

• Swab a Mueller-Hinton

plate with each of the

bacteria. Dip a sterile

swab into the broth and

express any excess

moisture by pressing the

swab against the side of

the tube.

Bacteria are inoculated as lawn Bacteria are inoculated as lawn Bacteria are inoculated as lawn Bacteria are inoculated as lawn cultureculturecultureculture

• Method of inoculation-

Good results are

obtained by placing a

standard loopful of

inoculum suspension

on the plate and then

spreading it with a dry

sterile swab.

Disk Diffusion Method

• After completely swabbing the plate, turn it 90 degrees and repeat the swabbing process. (It is not necessary to re-moisten the swab.) Run the swab around the circumference of the plate before discarding it in the discard bag.

Placing the Antibiotic disks

• Then, using a dispenser

such as the one pictured,

antibiotic-impregnated

disks are placed onto the

agar surface. As the

bacteria on the lawn

grow, they are inhibited

to varying degrees by the

antibiotic diffusing from

the disk

Zone sizes differ on sensitivity pattern

• It has been determined

that zones of inhibition of

a certain diameter (varies

for antibiotic and to a

lesser extent, bacterial

species) correlate with

sensitivity or resistance to

the antibiotic tested

Look at the Charts for establishing the

zones of Sensitivity

• The zone sizes are looked up

on a standardized chart to

give a result of sensitivie,

resistant, or intermediate.

Many charts have a

corresponding column that

also gives the MIC (minimal

inhibitory concentration) for

that drug.

Do not do more than necessary

• DO NOT PERFORM MORE DIAGNOSTIC TESTS

THAN IS NECESSARY TO OBTAIN THE CORRECT

IDENTIFICATION!!! EFFICIENCY DOES COUNT!!!

Based

• upon your log sheets and flow charts, the

course/ examiner will be able to determine

if you performed your work in an efficacious

manner

Dr.T.V.Rao MD 62

More Media – Less in Grading

• In addition, should

you be observed using

an abundance media

to perform virtually

every test possible,

then you will loose

better grades.

Dr.T.V.Rao MD 63

You are Better Graded in skills

• Based upon the following criteria

i) submission of your work by the deadline,

ii) isolation of two different unknown bacteria,

iii) correct identification of these unknowns, iv)

use of the fewest possible diagnostic tests to

obtain the correct answer, and v) proper

completion of the log sheet and flow charts

Dr.T.V.Rao MD 64

Keep Your Working Table

Clean and Precise to the Needs

Dr.T.V.Rao MD 65

Think Beyond Your Placement

• Students should think beyond Traditional Bacteriological Identification methods, as Developed world switching to Molecular

Methods, and one should be familiar with Basic Molecular Methods, as future of

Medicine Depends on Molecular Methods

Dr.T.V.Rao MD 66

Challenges in Bacterial Identification

• Traditional methods of bacterial identification rely on phenotypic identification of the causative organism using gram staining, culture and biochemical methods. However, these methods of bacterial identification suffer from two major drawbacks. First, they can be used only for organisms that can be cultivated in vitro. Second, some strains exhibit unique biochemical characteristics that do not fit into patterns that have been used as a characteristic of any known genus and species.

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Our Vision to Future

• In the past decade or so, molecular techniques

have proven beneficial in overcoming some

limitations of traditional phenotypic procedures

for the detection and characterization of bacterial

phylotypes. Several non-culture based methods

have emerged in the past 15 years. Real time PCR

and microarrays are currently the most commonly

employed molecular techniques

Dr.T.V.Rao MD 68

RT PCR is Real Emerging Tool

• Real time PCR is highly sensitive and allows

quantitation of bacteria at a species level.

Microarray based bacterial identification relies on

the hybridization of preamplified bacterial DNA

sequences to arrayed species-specific

oligonucleotides. Each probe is tagged with a

different colored dye which fluoresces upon

hybridization. See how microarray technology

works.

Dr.T.V.Rao MD 69

The Future of Microbial Identification

• Using a DNA based assay, one can easily detect bacterial strains directly from clinical samples or from small amounts of cultured bacterial cells, thus improving the sensitivity and decreasing the time required for bacterial identification. PCR has been particularly useful in this regard, which relies on primer sequences designed to facilitate bacterial identification at any level of specificity: strain, species or genus.

Dr.T.V.Rao MD 70

We Identify with

• Microarrays combines the

potential of simultaneous

bacterial identification

and speciation. This

method is versatile and

makes it possible to

detect and discriminate

different bacterial samples

on a single slide.

Dr.T.V.Rao MD 71

We need more rapid methods for

Identification and Clinical Decisions

• The rapid identification of the

bacteria in clinical samples is

important for patient

management and

antimicrobial therapy. DNA

microarray-based approach is

used for the quick detection

and identification of bacteria

using species-specific

oligonucleotide probes

designed for specific regions of

various targeted genes.

Dr.T.V.Rao MD 72

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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Created by Dr.T.V.Rao MD for benefit of

Post Graduate Students in Medical

Microbiology

Email

doctortvrao@gmail.com

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