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Dr.T.V.Rao MD

Dr.T.V.Rao MD

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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

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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

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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

Dr.T.V.Rao MD

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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

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Microbiology LaboratorySkills 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

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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

Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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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.

Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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Other Pathogens which are always Incorporated in Evaluations

  • The Enterobacteriaceae

  • Vibrio's,

Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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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.

Dr.T.V.Rao MD

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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

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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

Dr.T.V.Rao MD

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Hanging Drop Procedure:

Dr.T.V.Rao MD

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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.

Dr.T.V.Rao MD

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Hanging Drop Negative Bacteria

  • 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.

Dr.T.V.Rao MD

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Hanging Drop Negative Bacteria

  • 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.

Dr.T.V.Rao MD

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Hanging Drop Negative Bacteria

  • 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.

Dr.T.V.Rao MD

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What you choose for Plating and Isolation Negative Bacteria

  • 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,

Dr.T.V.Rao MD

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Caution in Choosing Media Negative Bacteria

  • 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

Dr.T.V.Rao MD

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Streaking will expose your Skills Negative Bacteria

  • 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.

Dr.T.V.Rao MD

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Importance of Streaking Negative Bacteria

  • 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.

Dr.T.V.Rao MD

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Streaking the plates is Negative Bacteriaa ARTPerfect It

Dr.T.V.Rao MD

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Begin with Skills Negative BacteriaTest 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 -

Dr.T.V.Rao MD

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Crucial Reporting Negative Bacteria

  • 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 )

Dr.T.V.Rao MD

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Day 2 Negative Bacteria

  • 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

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Mannitol Salt Agar helps in Gram Positive cocci Negative Bacteria

  • 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

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Mannitol Salt Agar Negative Bacteria

  • 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

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Gram positive – Mannitol Salt Agar Negative Bacteria

  • 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

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Use of Tryptic Soya Agar Negative Bacteria

  • 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

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Tryptic Soya agar Negative Bacteria

  • 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

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Choose the Selective Medium with caution ( TCBS ) Negative Bacteria

  • TCBS is a selective isolation medium for culture of pathogenic Vibriospp primarily 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

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Salmonella Shigella Agar Negative Bacteria

  • 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

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Picking the isolates after separation Negative Bacteria

  • 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

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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

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A crucial step Gram Negative Bacteria

  • 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

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Answer Sheet and Flow Chart Gram Negative Bacteria

  • 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.

Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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Option 2 following

  • 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

Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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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 BIOCHEMICALFINGERPRINT. 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

Dr.T.V.Rao MD

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Biochemical Reaction Bacterial isolates

Use 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

Dr.T.V.Rao MD

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Biochemical Reaction Bacterial isolates(cont.)

c- Methyl red reaction (MR):

Fermentation of glucose with production of huge amount of acid

Lowering pH is detected by methyl red indicator

d- Voges proskaur’s reaction (VP):

Production of acetyl methyl carbinol from glucose fermentation

Acetyl methyl carbinol is detected by addition KOH

Color of medium turns pink (positive)


Dr.T.V.Rao MD

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Biochemical Reaction Bacterial isolates(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

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Common Tests Bacterial isolatesTo identify Bacterial isolates

  • Indole

  • Methyl Red/Voges Proskauer

  • Citrate

  • H2S production

  • Urea hydrolysis

  • Motility

  • Lactose fermentation

  • Sucrose fermentation

  • Glucose fermentation & gas production

Dr.T.V.Rao MD

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I Bacterial isolatesM 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.

Dr.T.V.Rao MD

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Triple Sugar Iron Agar Bacterial isolates

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.

Dr.T.V.Rao MD

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On 2 pathogensnd 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

Dr.T.V.Rao MD

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Testing for Antibiotic sensitivity pathogens

  • 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;

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Testing Antibiotic Susceptibility pathogens

  • Antibiotic sensitivity test: the in vitro testing of bacterial cultures with antibiotics to determine susceptibility of bacteria to antibiotic therapy. Bauer-Kirby test.

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How results to be Reported after Sensitivity Testing pathogens

  • 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”

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Streaking the Inoculum pathogens

  • 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).

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Making proper inoculum pathogens

  • 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.

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Bacteria are inoculated as lawn culture pathogens

  • 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.

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Disk Diffusion Method pathogens

  • 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.

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Placing the Antibiotic disks pathogens

  • 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

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Zone sizes differ on sensitivity pattern pathogens

  • 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

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Look at the Charts for establishing the zones of Sensitivity pathogens

  • 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.

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Do not do more than necessary pathogens


  • 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

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More Media – Less in Grading pathogens

  • 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

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You are Better Graded in skills pathogens

  • 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

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Keep Your Working Table pathogensClean and Precise to the Needs

Dr.T.V.Rao MD

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Think Beyond Your Placement pathogens

  • 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

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Challenges in Bacterial Identification pathogens

  • 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.

Dr.T.V.Rao MD

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Our Vision to Future pathogens

  • 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

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RT PCR pathogensis 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

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The Future of Microbial Identification pathogens

  • 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

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We Identify with pathogens

  • 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

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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

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Wish to be A Better Microbiologist Decisions

  • 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.

Dr.T.V.Rao MD

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Created by Dr.T.V.Rao MD for benefit of Post Graduate Students in Medical Microbiology


Dr.T.V.Rao MD