Diagnostic Laboratory Tests
Students should be familiar with the following diagnostic laboratory tests, media, and
procedures that were discussed in lecture and/or demonstrated in the laboratory and
how they are used in the identification of bacteria. Consult the Laboratory Manual and
textbook for further information
Gram stain – includes heat fixation, crystal violet, iodine, decolorizer, and safranin.
Gram + stain purple, contain thick peptidoglycan layer, Gram – stain red, contain thin
peptidoglycan layer and outer membrane.
Kinyoun acidfast stain – stained with carbolfuchsin, decolorized with acid-alcohol
solution, counterstained; positive acid-fast stain reacton corresponds to higher infectivity,
confirms mycobacterial infection.
Auramine-Rhodamine Stain - also known as the Truant fluorochrom stain, used to
visualizee acid-fast bacilli using fluorescence microscopy, notably species in the
Mycobacterium genus. Acid-fast organisms display a reddish-yellow fluorescence. AR
stain is a mixture of Auramine O and Rhodamine B.
Alpha, beta, gamma hemolysis – alpha: colony surrounded by zone of intact, but
discolored erythrocytes that have a green or brownish-green color; beta: colonies
surrounded by clear zone in which few or no intact erythrocytes are visible; gamma:
colonies produce no change in blood surrounding the colony.
Catalase test – place drop of 3% H2O2 on slide containing colony, oxygen bubbles
indicate a positive test.
Coagulase test – coagulase clots plasma; use kit with latex particles coated with
fribrinogen to detect bound coagulase (“clumping factor”) and IgG which detects protein
A, which binds the Fc region. Positive = white clumps. Also can be performed by
adding organisms to rabbit plasma.
Optochin test – ethylhydrocupreine hydrochloride; pure culture of organism form lawn
on blood agar, filter disk paper placed on plate, incubated overnight in a CO2 incubator.
Zone of inhibition >/= 14mm indicates susceptibility. Alpha and non-hemolytic
streptococci are not susceptible.
Bacitracin test – antibiotic that impairs cell wall synthesis in some gram + bacteria, any
degree of inhibitory zone surrounding a filter paper disk indicates susceptibility.
Novobiocin test – a narrow spectrum toxic antimicrobial agent that inhibits bacterial
DNA gyrase, zone of inhibition of </= 12mm around disk on sheep blood agar indicates
resistance.
Nitrocefin (Cefinase) test – Beta lactamase production detection, disks contain
chromogenic cephalosporin, nitrocefin, place drop of water on disk on slide, smear
colony on disk, beta lactamase producing strains hydrolyze nitrocefin changing it from
yellow to red as amide bond in beta-lactam ring is hydrolyzed.
Oxidase test – used to aid in identification of gram – rods and Neisseria. Neisseria are
oxidase positive, all members of Enterobacteriacae are oxidase negative. Other gram –
rods, such as Pseudomonas aeruginosa, camplybacter and vibrio are oxidase +. The
oxidase reagent, 1% tetramethyl-p-phenylenediamine dihydrochloride, detects
cytochrome oxidase enzyme system. Drop of reagent on swab, use swab to collect
colonies from blood or chocolate agar plate; positive = dark purple color within a few
seconds.
PYR test – based on hydrolysis of pyrrolidonyl-α-napthylamide by organisms that
contain enzyme pyrrolidonyl arylamidase. Identifies group A strep from Enterococcus,
which are PYR-positive. PYR substrate on disk on slide, scrape unknown organism onto
disk, incubate at room temp 1 min; red color = positive.
Indole test – tryptophanase catalyzes the reaction to metabolize the amino acid
trypophan to produce indole, pyruvic acid and ammonia; this test identifies oxidase
negative, lactose fermenting gram – rod (like E.coli). Add drop or two of indole reagent
to swab, collect colonies from blood agar plate on swab, observe for blue color within
seconds.
Bile esculin test – growth in presence of bile and hydrolysis of esculin to produce a
black color in nutrient agar; distinguishes Enterococcus from streptococci, except
groupD and O organisms; use 6.5% NaCl to distinguish enterococci from group D.
6.5% NaCl test – separates enterococci from streptococci, especially bile-esculine
positive S. bovis, S. equines. Todd-hewitt broth (used to grow strep) with NaCl added to
final conc of 6.5%; bile-esculin + gram +, catalase – cocci that grow are enterococci, bile
esculin + gram + catalase – cocci that do not grow are in bovis-equinus group.
CAMP test – distinguishes group A streptococci from group B, which secrete CAMP
factor that enhances the hemolytic action of beta lysin on blood agar. Unknown
organism streaked perpendicular to S. aureus, “arrow head” of hemolysis indicates
group B strep.
Motility test – semisolid agar inoculated by “stabbing”, motile organisms will spread out
form the point of inoculation; Listeria monocytogenes forms “umbrella” pattern, nonmotile orgs grow only at the site of inoculation.
Sheep blood agar – nutrient agar + 5% sheep blood, supports the growth of most
pathogens; almost always used when initially plating out bacteriologic specimen.
MacConkey agar – contains lactose, peptone; bile salts and crystal violet to inhibit
Gram + bacteria. Indicator dye, neutral red is taken up by colonies fermenting lactose
(usually non-pathogenic), non-colored colonies are formed by important stool pathogens
such as Salmonella and Shigella. Also used to control swarming of Proteus sp.
Xylose-lysine-desoxycholate agar – contains sucrose, lactose and xylose as carb
sources, lysine added to detect lysine decarboxylase rxn, bile salts (desoxycholate)
selectively inhibits gram + but not coliforms. Sodium thiosulfate and ferric ammonium
citrate added to permit detection of hydrogen sulfide gas production. Phenol red is pH
indicator. Salmonella colonies appear red due to elevation of pH (lys decarboxylation)
and contain black centers (hydrogen sulfide production in presence of ferric ammonium
citrate). Shigella appear colorless or red, E. coli and Enterobacter appear yellow due to
carb utilization.
Chocolate agar – blood agar heated to 80 degrees C for 15 min until color chocolate
brown; lyses RBC releasing hemoglobin and growth factors needed by fastidious
bacteria, heat also coagulates proteins, helping absorb fatty acids and amino acids that
inhibit some bacteria. N. gonorrhoeae, H. influenzae cultivable on chocolate but not
ordinary blood agar.
Mannitol salt agar – pink agar, yellow zone indicates acid production.
Mueller-Hinton agar – contains starch and casamino acids, fastigioius pathogen
Neisseria grow well on it, starch binds traces of free fatty acid that would otherwise
inhibit growth; also widely used for antibiotic and sulfonamide sensitivity tests of staph,
enteric gram – rods, and strep when supplemented with 5% blood.
Thayer Martin agar – selective for meningococci and gonococci; nutrient rich agar
supplemented with hemoglobin, vitamins and other known growth factors for Neisseria,
contains antibiotic vancomysin, colistin and nystatin, which inhibit practically all normal
flora
Sabouraud dextrose agar – used for isolation, identification and maintenance of
pathogenic and non-pathogenic fungi. Contains dextrose, a nitrogen source, agar and
acid pH of 5.6, which inhibits many bacteria present in mycological specimens. Various
inhibitory agens may be incorporated to restrict growth of other fungi/bacteria.
Lowenstein-Jensen agar – used to culture Mycobacterium
Selective media – basic or enriched medium to which antibiotics, growth inhibitory dyes
or other chemicals such as bile salts have been added to select for growth of organisms
that are resistant to these factors. MacConkey agar contains crystal violet that inhibits
gram positive bacteria and selects for gram negative.
Differential media – can be nutritive or enriched and additional substrates such as
carbohydrates have been added to detect specific metabolic characteristics of
organisms that set them apart from similar organisms. Mannitol salt agar allows
differentiation of staphylococci that can ferment mannitol and those that cannot because
there is a pH indicator incorporated that shows color change when the sugar is
converted to acid. Mannitol salt agar also has a high NaCl content, which discourages
growth of organisms found on skin.
Enriched media – general medium to which additional growth supplements have been
added, such as blood or serum products, eggs, and/or vitamins and minerals essential
for fastidious organisms; e.g. sheep blood agar
Nutritive media – basic medium with simple nutrients such as protein extracts (peptone,
tryptone) derived enzymatically from meat, milk or soybean; supports growth of
nonfastidious organisms; e.g. trypiticase soy agar (TSA), nutrient agar, brain heart
infusion agar, Mueller-Hinton agar
Oxidation reaction – obligate aerobes and facultative anaerobes generate energy by
oxidation. Reaction is performed in a test tube containing semisolid glucose medium
with a pH indicator. Each strain is placed in 2 tubes with one overlaid with mineral oil to
create an anaerobic environment. Fermentative organisms will utilize carbohydrates and
produce acid in both aerobic and anaerobic environments; oxidative organisms will only
use carbohydrates in the aerobic environment. Asaccharolytic organisms do not use
carbs for energy and will not grow in either tube.
Fermentation reaction – fermentation is anaerobic and is done by obligate and
facultative anaerobes. See above. Discussion on p. 34 of lab manual
API Biochemical strip – identify gram-negative bacteria based on the differential
abilities of species to utilize carbohydrates or to react with certain enzymatic substrates.
Agar disk diffusion test – test antibiotic susceptibility; procedure: apply paper antibiotic
discs of variable known concentration to colonized plate and measure zone of inhibition
(include disc diameter); zone diameter is inversely proportional to the activity of the drug
against the bacterium; this method does NOT measure MIC (minimum inhibitory
concentration)
Agar gradient diffusion test – set up is the same as for agar disk diffusion except that
instead of a filter paper disk with antimicrobial, a plastic strip called the E-test is used.
The E-test has the antimicrobial incorporated in a continuous gradient. Determine MIC
by looking at the intersection of the zone of growth inhibition with the numerical reading
on the E-test strip.
Agar dilution test – antibiotic concentrations are diluted in agar plates that are then
inoculated with bacteria; determines MIC
Microbroth dilution test – antibiotics are diluted in broth, usually in well of a microtiter
plate, bacteria are added and determine the lowest concentration of antibiotic needed to
prevent bacterial growth; determines MIC
Minimum inhibitory concentration – lowest concentration of antimicrobial agent that
inhibits growth of a bacterium
Satellitism – Haemophilus needs NAD (V factor) to grow, so if S. aureus is plated on
blood agar and releases NAD from hemolysis, the Haemophilus will grow around it.
Method can also be done with strips containing X and V factor and seeing which
Haemophilus species will grow around them
X and V Factor test – Haemophilus species can be determined by their requirements
for exogenous hemin (X factor) and/or NAD (V factor), both of which are in chocolate
agar. H. influenzae will grow on blood agar by cross streaking with S. aureus or on
unsupplemented agar with added X and V factors.
Sterility test with biological indicator – following incomplete sterilization (autoclaving),
the purple media turns yellow upon incubation, due to organism growth
Enzyme immunoassay – detect the presence of an antibody or an antigen in a sample;
unknown amount of antigen is affixed to a surface, and then a specific antibody is
washed over the surface so that it can bind to the antigen. This antibody is linked to an
enzyme, and in the final step a substance is added that the enzyme can convert to some
detectable signal
Complement fixation –patient’s serum is reacted with antigen and excess complement.
Ag-Ab complexes bind, activate and fix complement. Residual complement is assayed
through lysis of rbc’s coated with antibody
Latex agglutination – detect antibody or soluble antigen. Virus-specific antibody
causes latex particles coated with viral antigens to clump. Antibody-coated latex
particles can also be used to detect viral antigen. Like hemagglutination.
Darkfield microscopy – like a regular light microscope, except a condenser is used that
prevents transmitted light from directly illuminating the specimen. Only oblique light hits
the specimen, which allows it to be brightly lit against a dark background. Resolving
power is better that with brightfield (light) microscopy, which makes it possible to see thin
bacteria, like Treponema and Borrelia. Disadvantage: cannot view internal structures
because light does not pass through the organism.