mlt 218 - biochemical ID methods

ALA test for porphyrin synthesis

purpose: used to determine the growth requirement for hemin (x factor) in the ID of haemophilus spp.

principle: organisms that do not require hemin produce porphobilinogen, a precursor to porphyrin (which is what is detected in the test)

bile esculin

principle: basis of this test is the hydrolysis of esculin into glucose and esculetin. when esculetin is produced, it reacts with an iron salt in the medium used to produce a dark brown or black color (positive result)

organisms to test on: B. fragilis, B. ovatus, and B. theraiotaomicron hydrolyze esculin whereas B. vulgatus does not

bile solubility

purpose: used to differentiate strepococcus pneumoniae from other alpha-hemolytic streptococcus spp.

QC organisms used: S. pneumoniae (bile soluble)

Enterococcus faecalis (bile insoluble)

this test is based on the observation that pneumococcal cells lyse when sodium desoxycholate (bile salt) is applied to the colony under specific conditions of time and temp but other strepococci do not lyse

CAMP

purpose: used to identify microorgamisms that can produce a thermostable, extracellular diffusible protein

useful in the ID of both S. agalactiae and many gram positive bacilli including Lisertia monocytogenes and Clostridum perfringens

catalase

principle: bacteria that synthesize this enzyme hydrolyze hydrogen peroxide into water into gaseous oxygen, which results in the release of gas bubbles

most useful in initial characterization of bacteria

reporting results: separates staphlococci (positive) from streptococci and enterococci (negative)

citrate utilization test

principle: growth within the medium is indicative of the utilization of this substance

when the bacteria metabolize this substance, the ammonium salts in the medium are broken down to ammonia, which increases alkalinity

this medium is recommended as part of differentiating the species within the enterobacterales

DNase test

principle: this substance are enzymes that hydrolyze DNA and release free nucleotides and phosphate

pos: pink, neg: no color change

organisms that produce a positive result: staphlococcus aureus, serratia spp., moraxella catarrhalis, stenotrophomonas maltophilia, aeromonas spp., vibrio cholerae, shewanella putrefaciens

gelatin liquefaction

principle: useful as a toxonomic aid in classification and identification of both gram neg and gram pos bacilli

hippurate hydrolysis rapid test

purpose: useful in the ID of Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis, and Strepococcus agalactiae by detecting the ability of the organism to hydrolyze sodium hippurate to benzoic acid and glycine by the action of the enzyme hippuricase

hydrogen sulfide production

principle: certain organisms can produce compounds of this substance

media used: triple sugar iron agar slants, hektoen enteric agar, XLD agar, salmonella-shigella agar

organisms tested: gram neg bacilli to determine if they can produce this substance, used to separate erysipelothrix (pos) from other gram pos bacilli, campylobacter (pos), and shewanella putrefaciens and shewanella algae (both pos)

indole test

purpose: ability of an organism to split this substance from the amino acid tryptophan is due to the presence of tryptophanase

QC organisms: E. coli (pos), Pseudomonas aeruginosa (neg)

kliger’s iron agar test and triple sugar iron agar test

purpose: both used to detect the production of acid from carbs, both contain glucose and lactose. only difference is that TSI has 1% sucrose included

observations: acid reaction - yellow, alkaline reaction - red, H2S production - black color, gas production - bubbles or cracks in media

leithinase and lipase detection

these are enzymes released by bacteria that destroy animal tissues

egg yolk agar (EYA)

motility test

purpose: used to detect the presence of flagella on bacteria, allowing them to travel in and out of the microscopic field

in the tube test method, semisolid motility medium is inoculated in a straight line down through the center of the tube

nitrate/nitrite reduction

purpose: determine if organism can reduce this substance to the other substance

organisms tested: gram neg bacilli and gram neg cocci, anaerobic organisms

ONPG test

principle: late lactose fermenters are very difficult to distinguish from lactose nonfermenters because both appear as colorless colonies on macconkey agar

organisms of note: N. lactamica

purpose: used to detect the enzyme beta galactosidase present in late lactose fermenters

oxidase test

principle: certain bacteria possess either cytochrome oxidase or indophenol oxidase, which catalyses the transport of electrons (hydrogen) from donor compounds (NADH) to electron receptors (usually oxygen) with the formation of water

used to ID gram neg bacilli that are not in the Enterobacterales

gram neg diolococci should be pos

phenylalanine deaminase test

used to determine the ability of a microorganism to oxidatively deaminate the amino acid L-phenylalanine to phenylpyruvic acid

PYR

principle: serves as a substrate for the detection of pyrrolindonyl peptidase. positive reaction results in a pink color

serves as a useful tool in the ID of Streptococcus pyogenes and Enterococcus spp. (both pos)

can also be used for the ID of E. coli

urease test

many organisms contain this enzyme which can split urea in the presence of water to release two molecules of ammonia and carbon dioxide

the ammonia combines with the carbon dioxide and water to form ammonium carbonate, resulting in an alkaline end-product, turning the indicator from its original orange-yellow color to bright pink

purpose: used as part of the ID of several genera and species of Enterobacterales, including Proteus, Klebsiella, and some Yersinia and Citrobacter species

directly, this test is used on gastric biopsy samples to detect the presence of H. pylori