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A1 Methods to Identify Bacteria

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   A1 Methods to identify bacteria -   2 methods: o   Bacteriologic: staining and culturing the organism o   Immunologic: detecting antibodies against organism in the patient’s serum -   3 methods of bacteriologic lab work: o   Observing under microscope after staining o   Obtaining pure culture via inoculation onto bacteriologic medium o   Identifying organism using biochemical reactions, growth culture, dna probes…  -   Blood agar  –  RBCs with no nucleus  –  obligate intracellular bacteria and viruses cant grow  –  e.g chlamydia and rickettsia -   Steps in bacterial diagnosis 1.   Obtain specimen from infection site 2.   Stain specimen  –  gram stain or acid fast stain  –  genus may be found 3.   Culture the specimen on media such as blood agar  –  obtaining isolated colonies (pure culture is important)/ incubate plates with presence or absence of oxygen 4.   Identify organism  –  special tests  –  dna probes, dna fermentation, antibody 5.   Perform antibiotic susceptibility tests -   Neisseria and haemophilus  are grown on chocolate agar (heated blood agar) because the blood contains inhibitors which must be inactivated through heating -   Blood cultures: o   Organisms frequently identified through this are: two gram-positive cocci, Staphylococcus aureus and Streptococcus pneumoniae, and three gramnegative rods, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa  -   Throat cultures: o   primarily to detect the presence of group A  -hemolytic streptococci ( Streptococcus pyogenes ), an important and treatable cause of pharyngitis.   o   also used when diphtheria, gonococcal pharyngitis, or thrush ( Candida ) is suspected   o   Gram stain is typically not done on a throat swab because it is impossible to distinguish between the appearance of the normal flora streptococci and S. pyogenes  -   Sputum culture:   o   Pneumonia or tuberculosis are suspected o   most frequent cause of community-acquired pneumonia is S. pneumoniae, whereas S. aureus and gram-negative rods, such as K. pneumoniae and P. aeruginosa, are common causes of hospital-acquired pneumonias   o   If Legionella pneumonia is suspected, the organism can be cultured on charcoal-yeast agar, which contains the high concentrations of iron and sulfur required for growth   o   If tuberculosis  is suspected, an acid-fast stain should be done  -   spinal fluid culture: o   performed primarily when meningitis is suspected.    o   important causes of acute bacterial meningitis are three encapsulated organisms: Neisseria meningitidis, S. pneumoniae, and Haemophilus influenzae   o   the quellung test or immunofluorescence with specific antisera can identify the organism rapidly   o   subacute meningitis, Mycobacterium tuberculosis and the fungus Cryptococcus neoformans are the most common organisms isolated   Acid-fast stains   o   C. neoformans,    india ink   -   Stool cultures: o   primarily for cases of enterocolitis.   o   Shigella, Salmonella, and Campylobacter. E. coli    common causes of diarrhoea   o   Gram stain may reveal large numbers of certain organisms, such as staphylococci, clostridia, or campylobacters. Gram stain of the stool is not usually done because the large numbers of bacteria  in the normal flora of the colon make the interpretation difficult    o   Salmonella and Shigella, a selective, differential medium such as MacConkey or Eosin – Methylene Blue (EMB) agar is used   -   Urine cultures: o   pyelonephritis or cystitis is suspected   o   most frequent cause of urinary tract infections is E. coli. Other common agents are Enterobacter, Proteus, and Enterococcus faecalis   -   genital tract cultures: o   from individuals with an abnormal discharge or on specimens from asymptomatic contacts of a person with a sexually transmitted disease   o   pathogens in the genital tract is Neisseria gonorrhoeae   o   swabbing the urethral canal (for men), the cervix (for women), or the anal canal   o   Chlamydia trachomatis,   -   Bacteriologic agar types: o   Blood: detects hemolysis o   Charcoal yeast extract : legionella pneumophilia  –  increased conc of iron and cysteine o   Chocolate: Neisseria meningitidis and gonorrhoea  –  heated blood inactivates inhibitors of growth o   Eosin methylene blue: enteric gram negative rods  –  against gram positive bacteria, differentiates bwteen lactose fermenters/ non fermenters o   Macconkey: same as eosin meth. Blue   -   5 laboratory techniques: 1.   Microsocpic visulaisation 2.   Cultivation and identification 3.   Detecting microbial agents 4.   Detecting microbial dna or rna 5.   Detecting inflammatory or host response to microorganism -   Take a patiet history and physical examination:   o   Example: cough respiratory tract infection; dysuria urinary tract infection o   Age of patient o   Where they have been o   Where they work o   Example: gram + coccus in spinal fluid of new born likely to be strep. Agalactiae rather than strep. Pneumoniae o   Example: Erythema migrans (skin lesion, bright red) indicates lyme disease -   Direct visualisation of the organism: o   Gram stain, acid-fast stain, india ink preparation, potassium hydroxide, o   Microbes excluding viruses can be visualised microscopically o   Gram stain 1.   Heat fix specimen to slide, add crystal violet wait 1 minute 2.   Rinse slide, add iodine, wait 1 minute, purple organisms indicate gram + 3.   Rinse, decolorise with acetone for 5 seconds 4.   Was slidewith water, gram  –   aren’t visible  5.   Apply safranin counterstain 6.   Wash in water, dry in air, gram  –  are visualised as red    Mycoplasma have no cell wall so cannot be visulaised through gram staining    Visualisation requires > 10 to power of 4 organisms/mL of liquid, low conc. Sample are centrifuged o   Acid fast stain= ziehl neelsen (classic), to identify mycolic acid in organisms cell wall, mycobacterium tuberculosis which appears pink, beaded, curved, for patients with mycobacterial infections o   India ink = to detect cryptococcus neoformans in CSF -   Growing bacteria in culture: o   Used mainly for bctria or fungi not for helminths or protozoa o   Example = streak a throat swab onto blood agar to search for group A beta hemolytic streptococcus o   Use characteristics such as colony, size, shape, colour, gram stain, hemolysis, odor, metabolic properties o   Pure cultrues = used for antimicrobial susceptibility testing o   Specimen collection:    Transport in proper conditions to prevent inadequate samples o   Growth requirememtns:    Most bacteria are heterotrophs = require organic carbon for growth    Fastidious = require large number of growth factors or nutrients and must be specific for them  o   Oxygen requirements:    Aerobes = need oxygen, oxidative phosphorylation    Anaerobes = fermentation, no oxygen    Facultative anaerobes = grow in absence of o2 but grow better in its presence    Aerotolerant anaerobes = protects itself from oxygen but don’t use it in their metabolism    Microaerophiles = need o2, cannot survive at atmospheric levels of oxygen, found in lakes or wet soil o   Media:    2 strategies to isolate bacteria    enriched media = non selecyive growth of any bacteria    Selective media = growth of specific bacteria from specimens with large numbers of bacteria    Enriched media    Media containing: blood, yeast extract, brain/heart infusions for growing fatsiious organisms    Sheep blood agar = protein, sodium chloride, sheep blood, supports most gram +/- bacteria    Chocolate agar = RBCs that have been lysed through heating, releases Hb, hemin (X factor), NAD for organisms such as haemophilus influenzae and Neisseria gonorrhoeae    Useful to culture blood and csf which are normally sterile    Selective media    Macconkey agar = o   Promotes growth of gram  –  rods, such as enterobacteriaecae; o   inhibits growth of gram + organisms and some fastidious gram  –  bacteria (haemophilus and Neisseria) o   used to detect organisms able to metabolise lactose    hektoen enteric agar o   lactose/sucrose fermenters + non fermenters o   h2s producers o   Culture: salmonella and shigella species    Thayar martin agar o   Isolate gonococci o   Identifying bacteria    Single enzyme tests:    Catalase: H202 h20 + o2 ; organisms produce bubbles if catalase +; differentiates between staph. (catalase +) and strept./ enterococci (catalse -) ; it is a virulence factor because h202 is antimicrobial so it shows how well organisms can survive against drugs    Oxidase: differentiaties gram  –  bacteria, pseudomonas aeruginosa is oxidase positive, cytochrome c oxidase (ETC, nitrate metabolism) accepts substrates such as phenylenediamine derivatives producing a dark product in bacteria
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