bacterial identification

• Light Microscopy


  Analytical tool used for magnifying specimens
• Light Microscopy


  • Has potential to view objects at 1000 times their original size
• Basic Principles of Light Microscopy

  1. Light Source - produces light; can be focused by a condenser lens

  2. The light that illuminates the specimen reaches a lens known as the objective lens

  3. Objective Lens - creates a magnified image that is inverted

  4. Eyepiece / Ocular Lens - further magnifies the image which the eye receives
• Compound Microscope


  Utilizes multiple lenses
• Magnification and Scaling


  1. Total magnification is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens

  2. Eyepiece Reticle - Help estimate the size of objects under the microscope, Scale that is projected over the image, At higher magnification, the tick marks in the eyepiece reticle will represent smaller distances than when viewed at lower magnification
• Resolution


  Shortest resolvable distance between two objects under the scope, As the objects become clearer, the resolution increases and the shortest observable distance between them decreases
• Bacteria


  • Living microorganisms, Characteristics that can be visualized by staining and aid in identification and classification of different bacterial species: Varied shapes, Cell arrangement, Does it form capsules?, Does it form spores?

  
• Gram Staining


  Crystal Violet is applied to bacteria which was heat fixed on the slide -> Mordant (Gram's Iodine) is applied -> Decolorizer is applied -> Counterstain (Safranin) is applied, Bacteria with thick peptidoglycan layer will stain purple, Gram Positive - purple, Gram Negative - reddish pink / red
• Capsule Staining


  Uses Negative Staining Method, Stain background with an acidic colorant (ex. Congo Red), Stain bacterial cells with crystal violet, Bacterial cells with capsule: Capsule will appear as a clear halo around the cells
• Endospore Staining


  Apply malachite green stain to bacteria fixed to a slide, Wash the slide with water, Apply counterstain (Safranin), Vegetative Cells - will appear pinkish green, Bacterial cells w/ Endospores - retain the green color of the stain; appear bluish-green in a colony under the microscope
• Serial Dilutions


  A process through which the concentration of an organism is systematically reduced by successive resuspension in fixed volumes of liquid diluent
• Streak Plating


  Streaking is accompanied by introducing a diluted sample to one section of the solid medium supplemented with nutrient which is divided into thirds, The inoculum is then spread over each third of the plate in a zigzag pattern, As different sections of the plate are streaked, crossing from the previous sample only once, the sample is spread more thinly
• Spread Plating


  An aliquot of a single sample is spread evenly over the entire surface of solid medium, Typically, since the bacterial numbers in the mix sample are unknown, a spread plate is made for each of the dilutions, After incubation, enumeration can be performed using these spread plates
• Colony Forming Units (CFUs)


  Formula: CFUs = Colony counts of each plate x Dilution factor / Volume plated (mL)
• Clonal Colony


  When a bacterium in an agar plate undergoes multiple rounds of asexual reproduction

  
• Streak Plating Procedure


  The agar plate is visually divided into 5 parts with the first 4 fragments coming from the circumference of the plate and the 5th being the plate center

  2. A loopful is then streaked onto the 1st segment in a zigzag pattern

  3. Then, either a new disposable loop is used or the used wire loop is sterilized by flaming it until it is red hot along the length of the wire

  4. It is then cooled in the air for a few seconds before being dragged through the 1st section to create 3-4 separate lines, each carrying only a fraction of bacteria onto the 2nd section

  5. The remaining sections are streaking in the same manner using a sterile loop each time and a single pass through the previous streak

  6. The CFUs are then harvested and restreaked to ensure that subsequent work involves only a single bacterial type referred to as pure culture
• Purity of Streak Plates


  The initial streak plate may contain colonies originating from cells of different bacterial species or different genetic make-up, Through subsequent isolation of a single colony where all units are derived from a common mother cell, the 2nd streaking procedure generates a relatively clonal bacterial population that is suitable for further characterization or inoculation into broth

  
• Culturability of Microorganisms


  • Bacteria are able to inhabit almost every environment on earth from desert, tundra, to tropical rainforests, This ability to colonize vastly different niches is due to their adaptability and vast metabolic diversity, which allows them to utilize a wide variety of molecules for energy generation, The massive array of diversity leads to the phenomenon that <1% of the bacterial species on the planet are considered culturable
• Culturing Microorganisms


  Performing manipulations of media and environment in the laboratory allows: 1. Researches to experiment to find optimal conditions for culturing a species of interest, 2. Enables enrichment - the process of changing conditions to select for specific species from a mixed culture
• Types of Species According to the Nutrient and Environmental Requirements

  • Generalists - Able to tolerate a wide variety of states or environment, Fastidious Organisms - Can be culturable but only when specific conditions are met, Extremophiles - Named for their preference for extreme conditions
• Indicator Media


  To further enrich an organism of interest, some media types contain indicators which give insight into the metabolism of the organism
• Mannitol Salt Agar (MSA)


  • Inhibits the growth of organisms sensitive to high salt, Gram-negative bacteria typically can't survive on MSA because it is toxic to most gram-negative species, Gram-positive Staphylococcus genus
• Fastidious organisms


  Can be culturable but only when specific conditions are met, e.g. Neisseria sp. or Haemophilus sp. require media containing partially broken down red blood cells and high CO2 concentration which may also discourage the growth of other species
• Extremophiles


  Named for their preference for extreme conditions, can be very low temperatures due to absent oxygen conditions or very high temperatures due to presence of high salt, conditions are likely intolerable to most other microbes
• Mannitol Salt Agar (MSA)


  Inhibits the growth of organisms sensitive to high salt, gram-negative bacteria typically can't survive on MSA because it is toxic to most gram-negative species, gram-positive Staphylococcus genus are able to thrive, can indicate any colonies able to ferment mannitol as the acid byproducts of fermentation will turn the methyl red indicator on the media to bright yellow
• Eosin Methylene Blue Agar (EMB)


  Contains eosin and methylene blue dyes which are toxic to gram-positive organisms, contains lactose which can allow lactose fermentation, will produce acids that lower the pH, encouraging dye absorption, these colonies take up large amounts of pigment and appear dark and metallic
• Antibiotic susceptibility testing


  Sensitivity of bacteria to antibiotics, measured using broth dilution and epsilometer test (E-test)
• Broth dilution test


  A standardized number of bacteria are needed to a growth media containing serial antibiotic dilution, the lowest antibiotic concentration at which the bacteria can no longer survive or multiply is referred to as the minimum inhibitory concentration (MIC) value of the antibiotic for the given bacteria
• Epsilometer test (E-test)


  A plastic strip impregnated with a predefined gradient of antibiotic is applied over a freshly spread lawn of bacteria on a Mueller-Hinton agar (MH-A) Petri plate, the antibiotic diffuses out into the agar media, where it is taken up by the bacteria, the bacteria cannot multiply and will die off, forming a clear zone (growth inhibition zone) around the E-strip, at the point where the growth intersects with the E-strip, the corresponding value on the scale gives the MIC value of the antibiotic

  
• Antibiotic synergy


  Antibiotics are used in combination to prevent the emergence of antibiotic-resistant strains of bacteria, often results in synergistic rather than additive effect, where the combined effect of the two antibiotics is greater than the sum of their individual activities
• Fractional Inhibitory Concentration (FIC) Index


  Effect is considered significant only when the MIC value of the antibiotic combination decreases by at least two-fold, evaluated by calculating the FIC index, FIC Index ≤ 0.5 = Synergy
• 1. Test Based Methods to Measure Synergy

  1. Non-Cross Test - The E-strips for two different antibiotics with predetermined MIC values are applied to two separate plates, after the antibiotics have diffused into the medium, the original E-strips are removed and the E-strips for the alternate antibiotics are placed such that their MIC scales lay exactly over the MIC scales of the previous strips

  2. Cross Test - Faster version of the non-cross test, the E-strips of the two antibiotics are placed together in a cross formation, such that the scales of their MIC marks form a 90-degree angle at the intersection, the MIC value of each antibiotic in combination with the other antibiotic is read at the point where the growth inhibition zone intersects with the edge of the E-strip
• Sample calculation for FIC Index
• Methods used in the area of microorganism identification

  • Indirect - Conventional methods, isolation and culture of microorganisms, determination of their various phenotypic characteristics
  • Direct - Culture independent, may be used to identify specific microbes in a mixed population, identify non-culturable microbes (e.g. microscopic techniques)

  
• Genotypic methods


  • The most accurate and reproducible method, the sequencing won't lie if the sequencing has been done in a very good environment with a pure isolate, very expensive, ideally not used for clinical specimens where readily available antibiotics can be used, useful for research purposes
• Methods of microorganism identification

  • Chromogenic media
  • Microscopy techniques (brightfield, dark field, SEM, TEM, CLSM, ATM, inverted)
  • Biochemical techniques (traditional media, Wickerham card, API, BBL Crystal, Vtek, Biolog System-Omnilog)
  • Molecular techniques (mass spectrometry, electrokinetic separation, PCR, real-time qPCR, RAPD PCR, sequencing, RFLP, PFGE, ribotyping, WGS, MALDI-TOF MS)

  
• Analytical Profile Index (API)

  Commercial miniaturized biochemical test panels that cover a significant classification of bacteria based on experiments which allows fast identification, developed for quick identification of clinically relevant bacteria, only known bacteria can be identified, API test strips consist of microtubes with dehydrated substances to detect enzymatic activity or the fermentation of sugars by the inoculated organisms, metabolism produces color changes spontaneously or through addition of reagents

  
• API 20E test procedure

  Confirm the isolate is of an Enterobacteriaceae, pick a single isolated colony and make a suspension in sterile distilled water, take the API 20E Test Strip which contains dehydrated bacterial media/bio-chemical reagents in 20 separate compartments, fill up the compartments with the bacterial suspension, add sterile oil into the compartments having ADH, LDC, ODC, H2S and URE, put some drops of water in the tray and put the API Test strip and close the tray, mark the tray and incubate for 18 to 24 hours at 37°C

  
• BacT/Alert and BACTEC


  Automated systems that detect CO2 changes using colorimetric and fluorimetric sensors respectively, culture bottles are commonly used in the clinical laboratory, almost all tertiary hospitals have either BacT/Alert or BACTEC

  
• Traditional method for bacterial identification

  Mixed culture → isolation → pure culture → phenotypically pure