Microbiology lab

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    • Gram stain
    • Procedure
      • Step: 1
        • A  clinical specimen on a microscope slide is treated with a solution of crystal violet and then iodine, the bacterial cells will stain purple
      • Step: 2
        • If the stained cells are then treated with a solvent, such as alcohol or acetone
          • gram- positive organisms retain the stain,
          • gram-negative species lose the stain, becoming colorless
      • Step: 3
        • Addition of the counterstain safranin
          • stains the clear, gram-negative bacteria pink or red.(MCQ)
    • Mycoplasma, that lack cell walls ,cannot be identified using the Gram stain. .(MCQ)
    • Gram stain applications:
      • gram-negative intracellular diplococci in urethral pus provide a presumptive diagnosis of gonorrhea. .(MCQ)
      • Clinical problem-solved due to gram staining
        • a specimen may show organisms under the microscope but appear sterile in culture media
        • Interpretation
          • This discrepancy may suggest the presence of either
            • fastidious organisms
              • bacteria with complex nutrient requirements that are unable to grow on the culture media employed
            • fragile organisms, such as gonococcus or anaerobic organisms
              • they may not survive transport.
        • Gram staining offers a solution
          • In these cases, direct visualization with the Gram stain may provide the only clue to the nature, variety, and relative number of infecting organisms.
    • Gram stain limitations:
      • Visualization with the Gram stain requires greater than 104 organisms/mL. Liquid samples with low numbers of microorganisms (for example, in CSF), require centrifugation to concentrate the pathogens.
    • Acid-fast stain
      • Ziehl-Neelsen  is the classic acid-fast stain .(MCQ)
      • used to identify organisms that have waxy material (mycolic acids) in their cell walls. .(MCQ)
      • Principle :
        • Most bacteria that have been stained with carbol- fuchsin can be decolorized by washing with acidic alcohol.
        • Certain acid-fast bacteria retain the carbolfuchsin stain after being washed with an acidic solution .(MCQ)
      • Mycobacterium tuberculosis
        • acid-fast bacterium
        • appears pink, often beaded, and slightly curved
      • Nocardia are partially acid-fast.(MCQ)
        • stains show red and blue clusters of the same bacterium on the same slide or vary from slide to slide.
      • Parasitic oocysts of Cryptosporidium,Cyclospora,and Isopora are acid-fast. .(MCQ)
    • India ink preparation
      • useful in detecting Cryptococcus neoformans in CSF .(MCQ)
      • Principle :
        • One drop of centrifuged CSF is mixed with one drop of India ink on a micro- scope slide beneath a glass cover slip.
        • Cryptococci are identified by their large, transparent capsules that displace the India ink particles.
    • Potassium hydroxide preparation
      • Treatment with potassium hydroxide (KOH) dissolves host cells and bacteria, sparing fungi .(MCQ)
      • One drop of sputum or skin scraping is treated with 10 percent KOH, and the specimen is examined for fungal forms.
    • Wet mounts are used for specific specimens such as unspun urine or for motility.
    • Growing bacteria in culture
      • Certain pathogens are very slow growing .(MCQ)
        • M. tuberculosis
      • Certain pathogens are cultured only with difficulty
        • Bartonella henselae
    • Specimen collection
      • gonococci and pneumococci are very sensitive to heating and drying. .(MCQ)
        • Transport media must be used to extend the viability
      • When anaerobic organisms are suspected, the patient’s specimen must be protected from the toxic effect of oxygen
        • It is important to
 use an airless syringe for liquid specimens, such as pus,
rather than a swab in a tube
    • Growth requirement
    • What are fastidious organism
      • Organisms that require either a large number of growth factors or must be supplied with very specific ones are referred to as fastidious.
      • Media commonly used to grow fastidious bacteria
        • Chocolate agar (agar with lysed red blood cells). .(MCQ)
          • used for both Haemophilus spp. and Neisseria spp.
          • both of them are non hemolytic but require free hemoglobin .(MCQ)
        • Thayer-Martin or New York City agars .(MCQ)
          • used to grow Neisseria obtained from body areas with competing normal flora (any mucosa). .(MCQ)
          • Both are chocolate agars
          • contain antibiotics to prevent growth of the bacteria and yeasts that are part of the normal mucosal flora.
          • Often genetic probes are used instead of cultures for diagnosis of gonorrhea.
        • Regan-Lowe and Bordet-Gengou agars. .(MCQ)
          • media are used for culture of Bordetella pertussis.(MCQ)
          • Rapid non culture methods are replacing culture because it is difficult to culture B. pertussis either from a
            • vaccinated person
            • after the early paroxysmal stage in both vaccinated and nonvaccinated individuals.
        • Thiosulfate-citrate-bile salts-sucrose
          • an alkaline medium used to grow Vibrio cholerae. .(MCQ)
        • (Buffered) charcoal-yeast extract (BCYE) agar .(MCQ)
          • used to grow Legionella.(MCQ)
          • it contains needed iron and cysteine plus charcoal.
        • Lowenstein-Jensen agar.(MCQ)
          • contains egg yolk that provides the necessary lipids for mycobacteria. .(MCQ)
          • replaced with high lipid broth cultures specifically designed for mycobacteria, allowing faster growth.

    microbiology 1

      • Oxygen requirement
        • Strict aerobes
          • cannot survive in the absence of oxygen
          • produce energy only by oxidative phosphorylation
        • Strict anaerobes
          • generate energy by fermentation or by anaerobic respiration
          • killed in the presence of oxygen
        • Facultative anaerobes
          • can grow in the absence of oxygen but grow better in its presence. Aerotolerant anaerobes
          • have mechanisms to protect themselves from oxygen (therefore, being able to grow in its presence or absence)
          • but they do not use oxygen in their metabolism.
        • Microaerophiles
          • require oxygen for their metabolism
          • cannot survive at atmospheric levels of oxygen.
          • Microaerophiles are found in lakes and wet soil where the oxygen concentration is within an acceptable range.
      • Culture Media
        • Two general strategies are used to isolate pathogenic bacteria
          • use enriched media to promote the nonselective growth of any bacteria that may be present
          • use selective media that only allow growth of specific bacterial species
            • used for specimens that normally contain large numbers of bacteria (for example, stool, genital tract secretions, and sputum).
        • Isolation of a bacterium is usually performed on solid medium.
        • Liquid medium is used to grow larger quantities of a culture of bacteria that have already been isolated as a pure culture.
        • Enriched media:
          • Media fortified with blood, yeast extracts, or brain or heart infusions are useful in growing fastidious organisms.
          • Sheep blood agar
            • contains protein sources, sodium chloride, and 5 percent sheep blood
            • supports the growth of most gram-positive and gram-negative bacteria isolated from human sources
          • Haemophilus influenzae and Neisseria gonorrhoeae are highly fastidious organisms
            • require chocolate agar.(MCQ)
            • chocolate agar contains RBCs that have been lysed
            • releases intracellular nutrients, such as .(MCQ)
              • hemoglobin, hemin (“X” factor)
              • nicotinamide adenine dinucleotide (“V” factor)
          • Enriched media are useful for culturing normally sterile body fluids, such as blood and CSF, in which the finding of any organisms provides reasonable evidence for infection by that organism.
          • Failure to culture an organism may indicate that the
            • culture medium is inadequate
            • that the incubation conditions do not support bacterial growth.
        • Selective media
          • MacConkey agar
            • most commonly used selective medium
            • supports the growth of most gram-negative rods, especially the Enterobacteriaceae .(MCQ)
            • inhibits growth of gram-positive organisms and some fastidious gram-negative bacteria, such as Haemophilus and Neisseria species. .(MCQ)
            • contain bile salts and crystal violet to inhibit the growth of nonenteric organisms and Gram-positive organisms
            • Growth on blood agar and chocolate agar but not MacConkey agar suggests a gram-positive isolate or a fastidious gram-negative species. .(MCQ)
            • In contrast, most gram-negative rods often form distinctive colonies on MacConkey agar.
            • This agar is also used to detect organisms able to metabolize lactose .(MCQ)
            • Clinical samples are routinely plated on blood agar, chocolate agar, and MacConkey agar
          • Hektoen enteric agar .(MCQ)
            • It is also a selective medium
            • It differentiates .(MCQ)
              • lactose/sucrose fermenters and nonfermenters
              • H2S producers and nonproducers.
            • It is often used to culture Salmonella and Shigella species. .(MCQ)
          • Thayer- Martin agar
            • selective medium composed of chocolate agar supplemented with several antibiotics .(MCQ)
            • suppress the growth of nonpathogenic Neisseria and other normal and abnormal flora.
            • normally used to isolate gonococci. .(MCQ)
        • Growth under specific conditions
          • used to identify certain metabolic features of microbes, such as whether it is an aerobe or anaerobe.
          • Suspected Campylobacter cultures are grown in incubators at 42°C under microaerophilic conditions. .(MCQ)
          • Thioglycollate broth
            • a medium with reducing power that develops an oxygen gradient. .(MCQ)
            • They are carefully stab inoculated the full length.
            • If an organism grows only at the top of the medium, the isolate is an obligate aerobe.
            • If an organism grows throughout the medium but grows more heavily at the top, it is a facultative anaerobe.
            • If an organism grows a little way down but not on the surface, it is a microaerophile. .(MCQ)
            • If an organism only grows on the bottom, it is an obligate anaerobe.
      • Identification of bacteria
        • Single-enzyme tests
          • Catalase test:
            • enzyme catalase catalyzes the degradation of hydrogen peroxide to water and molecular oxygen 
            • Catalase-positive organisms rapidly produce bubbles when exposed to a solution containing hydrogen peroxide
            • catalase test is key in differentiating between many gram-positive organisms.(MCQ)
              • staphylococci are catalase positive
              • streptococci and enterococci are catalase negative.
            • catalase is an important virulence factor
              • H2O2 is antimicrobial
              • degradation of H2O2 decreases the ability of neutrophils to kill invading bacteria.
          • Oxidase test:
            • enzyme cytochrome c oxidase
            • part of electron transport and nitrate metabolism in some bacteria.
            • enzyme can accept electrons from artificial substrates (such as a phenylenediamine derivative), producing a dark, oxidized  product.(MCQ)
            • This test assists in differentiating between groups of gram-negative bacteria. .(MCQ)
            • Pseudomonas aeruginosa is oxidase positive. .(MCQ)
          • Urease:
            • enzyme urease hydrolyzes urea to ammonia and carbon dioxide .(MCQ)
            • ammonia produced can be detected with pH indicators that change color in response to the increased alkalinity .(MCQ)
            • The test helps to identify certain species of Enterobacteriaceae, Corynebacterium urealyticum, and Helicobacter pylori. .(MCQ)
          • Coagulase test:
            • Coagulase is an enzyme that causes a clot to form when bacteria are incubated with plasma (
            • differentiates Staphylococcus aureus (coagulase positive) from coagulase-negative staphylococci. .(MCQ)
          • Nitrate reductase (urine dipstick tests): t
            • this test is performed directly on urine.
            • Escherichia coli and other enterobacteria produce nitrate reductase.(MCQ)
            • this test requires that the bacteria remain in contact with the urine for a sufficient time.
            • Staphylococcus saprophyticus does not produce nitrate reductase.(MCQ)
        • Vitek System
          • Automated system
          • small plastic reagent cards
          • contain microliter quantities of various biochemical test media in 30 wells
          • provide a biochemical profile that allows for organism identification
      • Immunologic detection of microorganisms
        • Detection of microbial antigen with known antiserum
          • This method of identification is often rapid
          • show favorable sensitivity and specificity.
          • Quellung reaction: .(MCQ)
            • Some bacteria having capsules can be identified directly in clinical specimens by a reaction that occurs when the organisms are treated with serum containing specific antibodies
            • it makes the capsule more refractile and thus more visible, but the capsule does not actually swell.
            • This method can be used for all serotypes of S. pneumoniae, H. influenzae type b, and Neisseria meningitidis groups A and C. .(MCQ)
          • Slide agglutination test: .(MCQ)
            • when a specific antibody directed against the microbial antigen is added to the suspension
            • it causes cross-linking of the bacteria.
            • It causes agglutination (clumping) of a suspension of bacterial cells on a microscopic slide
            • Used to identify Salmonella and Shigella species.(MCQ)
        • Identification of serum antigens and antibodies
          • High yield facts about clinical interpretation of level of serum antibodies
            • antibody may not be detectable early in an infection
            • presence of antibodies in a patient’s serum cannot differentiate between a present and a prior infection
            • a significant rise in antibody titer over a 10 to- 14-day period does distinguish between a present or prior infection.(MCQ)
            • High immunoglobulin M (IgM) titers suggest recent infection or exposure. .(MCQ)
            • High IgG titers usually indicate older infection or exposure.
        • Complement fixation test :
          • Procedure
            • Step : 1 – A patient’s serum is first incubated with antigen specific for the suspected infectious agent, followed by the addition of complement
              • If the patient’s serum does contain immunoglobulin (Ig) G or IgM antibodies that target the specific antigen (indicating past or current infection), then the added complement will be sequestered in an antigen–antibody–complement complex (“complement fixation”).
            • Step – 2 : Next, sensitized (antibody-coated) indicator sheep RBCs are added to the solution.
            • Inference:
              • If complement has already been fixed (because the patient’s serum contained antibodies against the added anti- gen), then little complement will be available to bind to the antibody–RBC complexes, and the cells will not lyse
              • If complement has not been depleted by initial antigen–antibody complexes (because the patient’s serum does not contain antibodies to the specific antigen), the complement will bind to the antibody–RBC complexes, causing the cells to lyse.
            • As hemolyzed RBCs release hemoglobin, the reaction can be monitored with a spectrophotometer.
        • Direct agglutination:
          • ordered when a suspected pathogen is difficult or dangerous to culture in the laboratory.
          • It  measures the ability of a patient’s serum antibody to directly agglutinate specific killed (yet intact) microorganisms.
          • used to diagnose Brucella abortus or Francisella tularensis.(MCQ)
        • Direct hemagglutination:
          • Antibodies directed against RBCs can arise during the course of  infectious mononucleosis caused by Epstein-Barr virus .(MCQ)
          • When uncoated (native) animal or human RBCs are used in agglutination reactions with serum from a patient infected with such an organism, antibodies to RBC antigens can be detected.
          • The patient’s antibodies cause the RBCs to clump
          • This test is, therefore, a direct hemagglutination reaction.
          • What is  “cold agglutinins” test.
            • Seen In the case of pneumonia caused by Mycoplasma pneumonia.(MCQ)
            • IgM autoantibodies may develop that agglutinate human RBCs at 4oC but not at 37oC.
        • Latex agglutination test:
          • Latex and other particles can be readily coated with either
            • antibody (for antigen detection)
              • used to rapidly test CSF for antigens associated with common forms of bacterial or fungal meningitis
            • antigen (for antibody detection).
              • widely used for the identification of b-hemolytic streptococci group A.(MCQ)
        • Enzyme-linked immunosorbent assay:
          • quantitate bound antigenin a patient’s serum
          • quantitate antibody in a patient’s serum
        • Fluorescent antibody (FA) stains
          • specific to a genus or species depending on specificity of the primary antibody
          • Direct fluorescent antibody (DFA)
            • uses fluorescent-labeled antibodies that are specific to various microbes.
          • Indirect fluorescent antibody (IDFA)
            • uses unlabeled, known antibodies that bind to bacterial antigens .
            • A fluorescent antibody that detects the bound antibody is applied, highlighting any antibody bound to the organism
      • Fluorochrome dye methods
        • include auramine-rhodamine dyes that bind nonspecifically to waxy cell wall components of both Mycobacterium and Nocardia. .(MCQ)
        • These stains are easier to read than an acid-fast stain because fluorescent dyes light up the bacteria on the black background without interference from the specimen.
        • The fluorochrome dyes are not specific for mycobacteria because antibodies are not involved in the binding of the dye to the bacteria. .(MCQ)
        • An acid-fast stain must be performed to confirm the presence of mycobacteria.
      • Nagler test for Clostridium perfringens alpha-toxin (a lecithinase) .(MCQ)
        • this test uses a lecithin-containing agar to detect lecithinase activity. .(MCQ)
        • One side of the plate has an antibody to the alpha toxin that neutralizes its activity.
        • The test result is positive if there is change around the growth on the side without antitoxin and no change in the media on the side containing the antitoxin since the antitoxin will inactivate the enzyme.


      MICRO LAB REVIEW Part 1.
      Experiments read and reviewed:
      1.Motility Tall
      2.Catalase
      3.Reduction of nitrate
      4.Hemolysis
      -alpha,
      -beta,
      -gamma
      5.CHO Fermentation
      -mannitol,
      -glucose,
      -lactose,
      -sucrose
      -phenol red pH indicator
      6.Unique source of Nitrogen
      -ammonium phosphate (NH4)
      -Brom cresol purple pH indicator

      Disclaimer: For informational purpose. Please consult your lab notes and textbook for confirmation and for more detailed and complete information.
      A tour of the Microbiology Lab – Section one
      Overview of a medical microbiology laboratory
      A tour of a diagnostic medical microbiology highlighting some of the work done. With thanks to Susan UCLH/RBH.
      Introduction to Microbiology Culture Techniques
      Nicole Gentile, PhD Candidate, provides an overview of basic microbiology and the concepts involved, including the bacterial growth curve and classifying organisms based on morphologies. This lecture describes blood, urine and skin/soft tissue cultures, focusing on the types of media, sample collection processes, culture procedures, as well as speciation and susceptibility testing. Basic staining procedures, such as the simple stain, gram stain, spore stain, negative stain, and acid fast stain are briefly discussed. Included in the staining procedure descriptions are explanations of the organisms that the stains identify. In addition to staining procedures, biochemical tests used for differentiating bacteria are covered. Concluding the lecture are some facts about fungi and viruses, focusing on the current 2009 novel H1N1 influenza pandemic.
      Microbiology Lab Tour & Safety
      Tour of SCC microbiology lab, overview of safety and facility
      Mannitol Salt Agar (MSA) Bacterial Growth Medium: Microbiology Lab Tutorial
      Microbiology lab tutorial on Mannitol Salt Agar (MSA) specialized bacterial growth medium. Examples of sterile MSA as well as plates with growth of halophilic mannitol fermenting bacteria (pathogen Staphylococcus aureus) and halophilic non-mannitol fermenting bacteria (normal flora S. epidermidis).
      Microbiology Lab Final
      Broward College North Campus. I got tired of not seeing anything helpful for the Micro Final, so here it is everyone. Help. 🙂 THE NURSING PROGRAM: ALL OF MY RECORDED WORD FOR WORD LECTURE NOTES TYPED WITH IMAGES. EACH CHAPTER IS ON SALE FOR $20.00. PROCESS 1 AND 2!! THEY CAN BE PURCHASED HERE. ENJOY!! ALMOST IMPOSSIBLE TO SURVIVE