Laboratory identification-of-clinically-significant-bacteria
•Download as PPTX, PDF•
3 likes•82 views
Presentation designed for healthcare documentation specialists to explain clinical terms.
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Laboratory identification-of-clinically-significant-bacteria
Similar to Laboratory identification-of-clinically-significant-bacteria (9)
Recently uploaded
Recently uploaded (20)
Laboratory identification-of-clinically-significant-bacteria
- 3. identificationof bacteria cell morphology(microscopy) cocci strepto- staphylo- diplo- tetrads sarcinae
- 5. streptococci
- 8. identificationof bacteria cell morphology (microscopy) cocci strepto- staphylo- diplo- tetrads sarcinae bacilli diplobacilli streptobacilli vibrios spirilla spirochetes
- 9. bacilli
- 10. vibrios
- 11. spirilla
- 12. spirochetes
- 13. identificationof bacteria cell morphology(microscopy) colonymorphology
- 14. identificationof bacteria colonymorphology diameter outlineshape elevation translucency color coloring/decoloringofmedium
- 17. identificationof bacteria cell morphology(microscopy) colonymorphology differential staining
- 18. identificationof bacteria differential staining Gram stain HansChristianGram
- 20. Gramstain
- 21. Gramstain
- 23. identificationof bacteria differential staining Gram stain acid-faststain
- 24. acid-faststain
- 25. acid-faststain
- 26. identificationof bacteria cell morphology(microscopy) colonymorphology differential staining metabolic characteristics (waste products andgrowthconditions)
- 27. identificationof bacteria metabolic characteristics temperature requirement oxygenrequirement pHrequirement nutritionalrequirement
- 28. identificationof bacteria cell morphology(microscopy) colonymorphology differential staining metabolic characteristics serologic (immunologic)tests
- 29. identificationof bacteria serologic (immunologic)tests agglutination particlecoupled to antibody orantigen resultingparticlemixedwith specimen iftargetantigenor antibody ispresent,agglutinationoccurs
- 32. identificationof bacteria serologic (immunologic)tests agglutination Coombstest RobinCoombs specifictypeof agglutinationtest
- 34. identificationof bacteria serologic (immunologic)tests agglutination ELISA(enzyme-linkedimmunosorbentassay)
- 37. identificationof bacteria serologic (immunologic)tests agglutination ELISA(enzyme-linkedimmunosorbentassay) “blot”tests Southern blot namedforEdwinSouthern DNA Northernblot RNA Western blot proteins Easternblot post-translationalmodificationofproteins
- 38. Western blot
- 39. Western blot
- 40. identificationof bacteria cell morphology(microscopy) colonymorphology differential staining metabolic characteristics serologic (immunologic)tests selective/differential media
- 41. identificationof bacteria selective/differential media selectivemedia “select”for certain species,strain(s),etc. only desiredorganism(s)willgrow mediacontentswillinhibit growthof, or kill,other organisms example antibioticmedia –only resistantorganismswill grow differentialmedia differentiateby changing appearancein presenceof certain organisms example bloodagar–containsmammalianblood,changes appearancein presenceof hemolyticorganisms
- 44. identificationof bacteria cell morphology(microscopy) colonymorphology differential staining metabolic characteristics immunologictests selective/differential media polymerase chain reaction
- 45. polymerasechain reaction(PCR)applications medicine tissue typing (organ transplants) infectious diseases HIV detection and viral load determination Mycobacterium tuberculosis and otherbacteria that do not growreadily in the lab forensics genetic fingerprinting parental testing
- 49. identificationof bacteria cell morphology(microscopy) colonymorphology differential staining metabolic characteristics immunologictests selective/differential media polymerase chain reaction
- 50. identificationof bacteria cell morphology(microscopy) colonymorphology differential staining metabolic characteristics immunologictests selective/differential media polymerase chain reaction
Editor's Notes
- How many times have you been transcribing or editing and heard a term like “acid-fast,” “PCR,” or “Coombs test,” and wondered, “What does that mean? What exactly is the background behind this term? or How is this particular test performed?” The main purpose of this presentation is give you a little background information on different methods of identifying of bacteria. Bacteria can be identified several ways, some far more specific than others, and many of these methods are relevant to clinical medicine.
- One very basic method of identifying (or classifying) bacteria is cell morphology – or cell shape. One of the main cell morphologies is the coccus (or plural, cocci). These are spherical cells and can be further classified by how the cocci arrange themselves in a culture. The three main arrangements are streptococci, staphylococci, and diplococci. (Sarcinae and tetrads are other arrangements that are not used frequently because they are difficult to see under a microscope.) These arrangements are based on the manner in which the cells divide.
- A – streptococci (one plane) B – diplococci (one plane) C – tetrad (two planes) D – sarcina (three planes) E – staphylococci (random planes)
- Example: Streptococcus pneumoniae (pneumonia)
- Example: Staphylococcus aureus (normal flora of skin – MRSA)
- Example: (diplococci) Neisseria gonorrhoeae (syphilis)
- Another important cell structure is the bacillus (plural bacilli). Bacilli can also be further classified by arrangement, but this is far less common than it is for the cocci. There are a few variations to the bacillus structure – vibrios, spirilla, and spirochetes.
- Example: Bacillus anthracis (anthrax)
- Vibrios are “comma” shaped bacilli. You also can see a flagellum in this micrograph. Nearly all bacterial cells contain one or more flagella, or cilia to help them move. However, these are not generally seen under a microscope. Example: Vibrio cholerae (cholera)
- rigid helical structures (as opposed to spirochetes, which are flexible) Example: Campylobacter jejuni (common enteric pathogen causing diarrhea)
- More flexible structure (or “backbone”) than spirilla Example: Treponema pallidum (syphilis) Borrelia burgdorferi (Lyme disease)
- Another means of identifying bacteria is colony morphology – or what does a colony look like when grown on an agar plate. Colony morphology, just like cell morphology, is not a specific means of identification, since many bacteria can appear the same or similar. However, it does provide a rough means of identification.
- When bacteria are grown in the lab, on agar plates, the colonies can look very different from species to species. These are some ways in which colonies can appear different. “Coloring/decoloring of media” is included not because it is a morphology, but because this can be an important identification tool when looking at colonies on a agar plate.
- Another very important tool in bacterial identification is differential staining, and one of the best-known differential stains is the Gram stain…
- … named after and developed by Hans Christian Gram, so it is an eponym. This stain differentiates bacterial species based on cell wall structure.
- Gram stain differentiates bacterial species based on the cell wall structure – specifically the layer of peptidoglycan and the presence of an outer membrane. Decoloring step removes stain from gram-negative organisms.
- heat fix culture on slide flood slide with crystal violet, rinse stains all organisms purple flood slide with Gram’s iodine fixes stain in the cell wall (peptidoglycan layer) rinse with isopropyl alcohol decolorizes gram-negative cells, which have thinner cell wall and an outer lipid membrane flood slide with safranin counter-stains gram-negative cells for contrast under microscope rinse
- The acid-fast stain also differentiates bacteria based on cell wall structure, but based on presence or absence of mycolic acid.
- heat-fix culture on slide flood slide with carbolfuchsin, heat over burner flame until steam arises stains all organisms red carbolfuchsin contains phenol (carbolic acid), which “softens” mycolic acid in acid-fast cell walls allow slide to cool, rinse with water rinse with acid-alcohol solution decolorizes non-acid-fast cells, which do NOT contain mycolic acid in their cell walls flood slide with methylene blue counter-stains non-acid-fast cells for contrast under microscope rinse
- Red - “acid-fast” organisms Blue – non-acid fast An important use of the acid-fast stain is in the diagnosis of tuberculosis. A sputum acid-fast stain can confirm or rule out the presence of Mycobacterium tuberculosis (which is an acid-fast organism). Mycobacterium… leprae (leprosy)
- Just very briefly, because this is a very broad topic that can cover an entire college course, certain metabolic characteristics of bacteria can be helpful in their identification.
- These different characteristics can help identify a bacterial species, and they are determined or measured by many different methods, which I will not go into in this presentation.
- One very specific and clinically important type of test is the serologic test. The basic premise is that antibodies are highly selective in terms of the proteins (or other cell structures) to which they bind, to the point that they are able to distinguish the proteins coming from one species among many species, or even one strain among many strains. There are several specific tests that use this premise and that are considered serologic tests.
- One is the agglutination test. e.g. – latex agglutination
- An agglutination test can be qualitative (either positive or negative) or quantitative. The highest dilution to produce agglutination is referred to as “titer”
- Another medically important serologic test is the Coombs test. This test is not used to identify bacteria, but I’ve included it here because it is so important medically, and it IS a serologic test. direct Coombs test red blood cells washed (removing serum) incubated with antihuman globulin (Coombs reagent) if antibodies or complement factors have been fixed to RBCs in vivo, Coombs reagent will agglutinate RBCs agglutination = positive test no agglutination = negative test indirect Coombs test first phase washed red blood cells incubated with test serum containing target antibody if serum contains antibodies to any RBC surface antigens, antibodies will bind to RBCs second phase RBCs washed then incubated with Coombs reagent if antibodies have bound to RBCs in the first phase, agglutination will occur agglutination = positive test no agglutination = negative test
- Direct Coombs test Detects if antibodies or complement system factors have bound to RBC surface antigens in vivo (immune-mediated hemolytic anemia) positive Coombs test indicates that an immune mechanism is attacking the patient's own RBC's - Rh disease - systemic lupus erythematosus - infectious mononucleosis Indirect Coombs test Detects in vitro antibody-antigen reactions. - Used to detect antibodies that could cause hemolytic disease - before blood transfusions (cross-matching) - prenatal screening for antibodies that could cause hemolytic disease of the newborn
- A newer method of bacterial identification that is very important medically (mostly in the identification of viruses – such as HIV) is the ELISA (sometimes abbreviated “EIA.” This test is used to identify the presence of a specific antigen (any molecule that induces an immune response and the production of antibodies) in a sample.
- - antibody to desired antigen is fixed to surface - add serum to be tested - if antigen is present, it will attach to antibodies - add antibody linked to enzyme - if antigen is present, antibody-enzyme complex will attach - rinse excess solution - add substrate that will change color in presence of enzyme
- The Western blot uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
- An important method for identifying bacteria in the lab based on certain metabolic characteristics is the use of selective or differential media.
- selective only organism of interest will grow, all others will be inhibited or killed differential medium contains substance that will somehow change in the presence of the organism of interest
- pyogenes – strep throat agalactiae – important in pregnancy – can cause complications in newborns
- The polymerase chain reaction is a process by which a small amount of DNA is replicated in a short time to create millions of copies. This process is very helpful in forensic science to amplify small amounts of DNA in samples for the purpose of identification. It is also helpful in medicine for tissue typing, or to identify viruses or bacteria that do not grow well in the lab. Primer – around 20 bases PCR in diagnosis of diseases PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods. PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes. Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques.
- amplify specific region of strand of DNA usually around 10 kb (kilo base pairs) “ingredients” DNA sample primer (about 20 bases) DNA polymerase usually Taq polymerase (“Taq pol”) isolated from Thermus aquaticus stable at high temperatures required for PCR deoxynucleoside triphosphates (DNA “bases”) buffer solution Denaturation at 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle). Annealing at 54°C : The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore. Extension at 72°C : This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template). A basic PCR set up requires several components and reagents.[6] These components include: DNA template that contains the DNA region (target) to be amplified. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C. Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7] Monovalent cation potassium ions.
- How many times have you been transcribing or editing and heard a term like “acid-fast,” “PCR,” or “Coombs test,” and wondered, “What does that mean? What exactly is the background behind this term? or How is this particular test performed?” The main purpose of this presentation is give you a little background information on different methods of identifying of bacteria. Bacteria can be identified several ways, some far more specific than others, and many of these methods are relevant to clinical medicine.