• bacteria in nature usually are heterogeneous populations, even when in a normally sterile environment
• clinical microbiology designed to detect pathogen in complex sample by two-step process
• selective enrichment (conditions favoring selective growth)
• EMB plates (eosine-methylene blue) select for Gram negative organisms, like enterobacteriaceae
• mannitol-salt plates select for staphylococcus.
• isolation in pure culture (dilute by sequential streaking on agar, done twice to ensure colony is clonal)
• sterility must be maintained
• medium is non-enriching

Stains – necessary because of bacterial motion and lack of optical contrast

• streptococcus and staphylococcus are both Gram positive; differentiated by catalase test
• only staph possesses the enzyme catalase (H₂O₂  H₂O + O₂) and will fizz with added H₂O₂
• staphylococcus – grow as smooth, flat, pigmented colonies 2-3 mm in diameter (after 24 hours on blood agar)
• s. aureus (causes pyrogenic (feverish) infections) – coagulase positive
• s. epidermidis (causes infections of prosthetic devices) and s. sarpophyticus (cystitis) – coagulase negative
• other differentiating tests:
• s.aureus turns yellow (acid) on mannitol-salt plate (fermentation), s.epidermidis stays red (base)
• pigment on blood agar is cream or greyish for s.aureus, white for s.epidermidis
• streptococcus – grow as round, small, non-pigmented colonies 1-2 mm in diameter (after 24 hours)
• primary differentiation is by analysis of hemolysis (beta = complete, alpha = partial, gamma = none)
• beta include groups A, B, C, F, and G – only group A (s. pyrogenes) is inhibited by bacitracin
• differentiation by Lancefield grouping – coagulation with staph coated with specific antibodies
• alpha and gamma – tested in two ways
• bile-esculin medium – only enterococci and other type D strep will grow
• optochin inhibition – positive for s. pneumoniae
• some do not grow on bile esculin and are not inhibited by optochin – called "viridians"
• coliform group (also called Enterobacteriaceae, or colon-dysentery)
• includes Escherichia coli, salmonella, and shigella
• most live in mammalian gut, but some are free-living and found in soil (Enterobacter and Proteus)
• differentiation between fecal and free-living coliforms important for water/sewage bacteriology
• difficult to discriminate because of high degree of uniformity of the group
• all are small, Gram (-) catalase (+) rods which do not form spores
• flagella, when present, are always peritrichous (distributed over the entire cell surface)
• all produce acid from glucose and other carbohydrates
• all are facultatively anaerobic and possess a complete respiratory system
• nutrition is simple; most will grow on carbohydrate + inorganic salts without amino acids or vitamins
• they grow well on EMB
• spore formers – produce dry, gray-white spreading colonies that can potentially spread over the entire plate

• the more there is, the more likely it will be seen and be important
• positive assay does not necessary mean infection (unless it is from a sterile site), and negative does not preclude
• it is easier to see if it grows (by plating or by PCR)
• you need to know what you are looking for before you can look for it

• (1) Host Inflammatory or Immune Response (NOT the microbe itself)
• e.g., to distinguish bacterial from viral infection
• bacteria have greater increase in protein, greater decrease in glucose, and PMNs rather than lympho/monocytes

• cytology can detect nature of viral infections
• also, for chronic diseases, detection of host immune response can be helpful
• humoral – serum concentration of IgG against organisms which cannot be easily cultivated
• include many viral infections (HIV), mycoplasmal pneumonia, rickettsial infections, chlamydial infections, lyme disease, syphilis, leptospirosis, rheumatic fever, Legionnaires' disease, tularemia, brucellosis, histoplasmosis, coccidioidomycosis, and amebiasis
• rapid increase in IgG or presence of IgM suggests recent infection
• cellular – test for delayed-type hypersensitivity to mycobacterial or fungal antigens
• TB skin test (purified protein derivative, PPD)
• can get false negative if patient is anergic (no cell-mediated response) so must do positive control
• (2) Direct Visualization of the Organism via microscope (sputum, pus, urine; pleural, peritoneal, or cerebrospinal fluid)
• look at shape (cocci, bacilli, or coccobacilli) and gram staining properties
• e.g., diplococci (pairs of cocci) are pneumonia if gram (+) lancet-shaped, nesseria if gram (-) kidney shaped
• small gram (-) coccobacilli are hemophilus influenza
• some bacteria are "gram stain not helpful": mycobacteria, Legionairre’s disease, mycoplasma, chlamidia, virus, fungi, etc.
• sputum and pus must be "thinned" by pulling between two slides, CSF must be centrifuged

• (3) Detection of Microbial Antigens (by immunofluorescence)
• allows rapid specific identification, often in less than one hour

• (4) Detection of Microbial Nucleotide Sequences (by PCR)
• very sensitive – theoretically from a single DNA molecule, realistically from 10-20 (other techniques require 10⁶ or more)
• useful for HIV, Treponema pallidum, Hepatitis C
• so sensitive that false positives appear more often than with other tests
• (5) Isolation of Organism in Culture
• useful for bacterial isolation, to quantify bacterial growth, or to identify fastidious organisms(grow only on specific media)
• potential problems
• contamination (e.g., saliva in sputum or non-pathogenic commensals in non-sterile sites like vagina or pharynx)
• inadequate culture conditions (e.g., anaerobic organisms that do not on aerobic cultures)
• Gram stain helpful to identify bacteria not present in cultures
• due to ease of other methods of detection, many agents are not frequently cultured in clinical settings
• viral isolation – expensive and laborious, requires immediate placement in viral transport medium
• Rickettsia, Chlamydia, and Mycoplasma – immune methods much easier
• fungi, mycobacteria – need weeks to grow (including Mycobacterium tuberculosis and Histoplasma capsulatum)

• Begin by performing a Gram stain:
• (1) allow a drop of culture to dry and fix with Bunsen burner
• (2) flood the slide with crystal violet for 30 seconds, then rinse
• (3) flood the slide with Gram’s iodine solution for 30 seconds, then rinse
• (4) flood the slide with 95% ethanol twice for five seconds each time, then rinse
• (5) flood the slide with safranine, rinse, then allow to dry
• Gram positive will be purple, gram negative will be red.
• Note: Spore staining uses the same protocol, with malachite green instead of crystal violet; spores will be green, and vegetative cells will be red.
• Gram negative, catalase positive (e.g., reactive to H₂O₂) might be enterobacteria; distinguishing them requires a kit.
• Gram positive cocci can be identified accoring to the following scheme:

• First one must identify which organisms have been isolated.
• Second one needs to quantitate how much drug one needs to achieve inhibition of the organism.
• the next question is: "Can this concentration of drug be reached in the human without negative consequences?"
• clinical efficacy is affected by:
• the sensitivity of the organism to the antibiotic
• the absorption of the antibiotic by the route of administration chosen
• diffusion of the antibiotic into the infected area
• the toxicity of the antibiotic
• allergies of patients to the antibiotic
• site and severity of infection
• state of host defenses (it is ultimately host defenses that take the bugs out)
• Disc Diffusion
• Antibiotics are placed on discs and then placed on an
• Agar plate with a lawn of organism. The area around the discs is a measurement of the susceptibility of the organism to the antibiotic.
• The Zone Diameter determines whether the organism is Resistant, Intermediate, or Susceptible to the antibiotic.
• Susceptible – organism is readily inhibited by levels of antibiotics attained in the blood on the usual dosage
• Intermediate – organism is inhibited only by blood levels achieved with high dosage IV drug administration or by urinary levels in patients with lower urinary tract infections (UTI’s) and normal renal function, with drugs that are concentrated in the urine.
• Resistant – organism is resistant to usually achievable levels
• Dilution Testing
• The minimal inhibitory concentration MIC of doubling dilutions of an antibiotic which will inhibit growth of a defined bacterial population is determined after overnight incubation. Minimal bactericidal concentration MBC is the lowest concentration producing a kill rate of 99.9%. this can be performed in test tubes, microdilution plates, or by a gradient diffusion method called an E-test.
• The E-test is a strip of plastic impregnated with antibiotic in varying concentrations. The level at which there is no longer inhibition is the MIC.
• the reproducibility of MIC’s are within one dilution away. ~95% confidence
• b - Lactamase Detection
• direct detection of b -lactamase can be done using isolates of Staphylococci, Haemphilus influenza, and Neisseria gonorrhea. This is accomplished by placing a disc containing yellow cephalosporin in a plate of the organism in addition to a disc of b -lactam. If the yellow disc turns red, it indicates the production of b -lactamase.
• Bacteriostatic and Bactericidal activity of serum and body fluids
• An old test to see how well an antibiotic is working in a person. Replaced by MIC’s
• Direct Drug Measurement
• Measurement of blood and body fluids to check toxicity of certain agents (e.g. Vancomycin, Chloramphenicol)
• CSF levels need to be monitored in Meningitis, not the blood levels, because they are not the same.