Principles of Laboratory Tests Flashcards
What are the principles behind the DN’ase agar.
(4)
Some bacteria species produce DN’ase enzymes
DN’ase agar contains 0.2% DNA
After incubation and growth, the plate is flooded with 1M hydrochloric acid which precipitates DNA and turns the medium cloudy
DN’ase positive colonies will show a distinct clear zone around inoculum as they have broken down the DNA and is therefore no longer available for precipitation by HCL
What is the principle behind mannitol salt agar?
(3)
Selective and differential for Staphylococci
High salt concentration of salt (7.5%) selects for staphylococci
Mannitol and pH indicator phenol red is differential and will turn yellow if organism can ferment mannitol
What is the principle behind SAIDE (Chromid S. aureus Elite agar)?
(2)
AIDE agar is a chromogenic medium designed for the selective culture of S. aureus
Chromogenic substrates in the agar target specific enzymes in S. aureus to enable the differential growth of S. aureus colonies as pink colonies
What is the principle behind the Staphaurex Plus test?
(4)
Protein A is found on about 95% of Staph aureus strains
Protein A has the ability to bind to the Fc portions of IgG
Staphaurex Plus uses latex particles coated with porcine fibrinogen and rabbit IgG including specific polyclonal antibodies raised against capsular polysaccharides of S. aureus
When the reagent is mixed on a card with colonies of S, aureus, rapid agglutination occurs through the reaction between fibrinogen and clumping factor, Fc portion of IgG and Protein A, specific IgG and capsular polysaccharide
Why should Staphaurex Plus be used instead of a coagulase test?
(3)
Some strains of methicillin-resistant strains of S. aureus may express undetectable levels of clumping facto and protein A
These MRSA strains possess capsular polysaccharide
The capsule can mask both Protein A and the clumping factor thereby preventing agglutination
What is the principle behind novobiocin resistance for coag negative staph differentiation?
Saprophyticus is resistant to novobiocin while epidermidis is not
What is a positive novobiocin test
Resistance to novobiocin
What is a negative novobiocin test
Susceptibility to novobiocin
What species will give a positive novobiocin result
S. saprophyticus (resistant)
What species will give a negative novobiocin result
S. epidermidis (susceptible)
What is the principle of the tube coagulase test?
(5)
Designed to detect both free and bound coagulase
Done by emulsifying 1 colony of test organism in 1ml rabbit plasma and incubating at 37 degrees
Check for clot production at 4 hours and again after incubation
Positive = visible clot formation or lose web of fibrin
Negative = no clot formation, plasma flows freely
What is the principle behind the optochin susceptibility test
(2)
This test detects an organism’s susceptibility to the chemical optochin
Optochin tests the fragility of the bacterial cell membrane and causes S. pneumoniae to lyse due to changes in surface tension, as shown by a zone of inhibition around the optochin disc
What is the principle behind the lancefield grouping test
(4)
The majority of pathogenic strep possess specific carbohydrate antigens, which permit the classification of strep into groups
Strep group antigens can be extracted from bacterial cells and their presence demonstrated with latex particles previously coated with group-specific antibodies
These latex particles will agglutinate in the presence of homologous antigen, but will remain in smooth suspension if absent
The use of enzymatic extraction procedure considerably shortens the time required for antigen extraction and much improves the antigen yield
What is the principle behind Bacitracin susceptibility
(2)
Used to speciate group A, Beta haemolytic strep i.e. to confirm S. pyogenes
Group A strep are sensitive to bacitracin while other groups of strep are resistant
What is the principle behind MacConkey agar?
(5)
A selective and differential culture medium
Crystal violet and bile salts make it selective for Gram-negative and enteric (normally found in the intestinal tract) bacteria
Differential based on lactose fermentation.
Lactose fermenters turn red or pink on MacConkey agar, and nonfermenters do not change color.
The media detects lactose fermentation by enteric bacteria with the pH indicator neutral red.
What is the principle behind bile aesculin agar?
(4)
A selective and differential agar used to identify members of the Enterococcus genus
Bile salts are selective for Enterococcus
Aesculin is differential -> enterococcus hydrolyse aesculin to form aesculetin and dextrose
Aesculetin combines with ferric citrate in the medium to produce insoluble iron salts, resulting in the blackening of the medium which is a positive result
What is the principle behind the TAXO sugars test?
(3)
Used for identification of gram negative cocci
Different species utilise (ferment) different sugars
Discs containing different sugars, dextrose, maltose, sucrose and lactose are placed on the culture
If the organism results in a drop in pH, upon the addition of phenol red indicator there will be a resulting colour change
What is the principle behind the Catarrhalis disk test?
(2)
This is based on the ability of M. catarrhalis to hydrolyse tributyrin
The Remel Catarrhalis disk test is a reagent impregnated tributyrin hydrolysis disc test for the presumptive ID of catarrhalis
What is the principle behind PEMBA agar
(5)
B. cereus selective agar
Agar contains a peptone level of 0.1% and the addition of sodium pyruvate improves egg yolk precipitation and enhances sporulation
Bromothymol blue is added as a pH indicator to detect mannitol utilisation
The medium is made selective by addition of Polymyxin B
B. cereus = peacock blue colony with no clearance
What is the principle behind the motility test
(3)
Through a hanging drop preparation
Essential to distinguish between motility and Brownian motion
Bacteria move in a definite direction but Brownian = continuous purposeless undirected agitation
What is the principle behind the trehalose test
(5)
Peptone water ‘sugars’ comprise of a simple peptone medium plus 0.5% ‘sugar’ and Phenol Red indicator
Peptones yield alkaline products on breakdown, so a change in colour of the indicator only occurs when acid produced from fermentation of the ‘sugar’ exceeds alkali from the peptones
If gas is produced is collected in an inverted tube (Durham tube)
Peptone water sugars can be used to test for fermentation of a wide range of sugars including glucose, lactose, maltose, mannitol, sucrose, dulcitol and trehalose
Trehalose is pink, if fermented it turns yellow
What is the principle behind urease test?
(4)
Certain microbes e.g. Proteus mirabilis or Helicobacter pylori produce the hydrolytic enzyme urease
Urease decomposes urea to ammonia and carbon dioxide
Urea is a neutral substance and its decomposition is accompanied by the production of alkli
Phenol red indicator in the medium changes from a yellow-orange shade at pH 6.8 to pink at alkaline pH8.1
What is the principle behind the Tinsdale Medium
(5)
Tinsdale medium contains serum, tellurite, cystine and blood
Tinsdale differentiates C. diphtheriae and the diphtheroids found in the upper respiratory tract
This differentiation was based on the ability of C. diphtheriae to produce black colonies, surrounded by a brown/black halo
The dark halo is due to the production of H2S from cystine, interacting with the tellurite salt
Diphtheroids do not produce this halo
What is the principle behind using lactose gelatin medium
Some bacteria demonstrate proteolytic activity that results in the liquidification of gelatin
Lactose is added to the medium to investigate fermentation
Used to identify C. perfringens
What is the principle behind the egg yok agar - Naglar reaction?
(4)
Used to detect lecithinase and lipase production and proteolytic activity of anaerobes -> recommended in the Naglar Test for the ID of C. perfringens
Egg yolk agar is used which acts as a supply of lecithin and free fats which allows detection of lecithinase and lipase production and proteolytic activity
Lecithin break down results in a white opaque zone of precipitation that spreads beyond the edge of the colony
Lipase activity forms an iridescent sheen on the surface of the agar
What is egg yolk agar?
(3)
A non-selective medium
Supplemented with a suspension of egg yolk and enriched with haemin and vitamin K
Haemin and vitamin K enhance the growth of anaerobic microorganisms
What is the principle behind the Naglar reaction?
A test used to differentiate C. perfringens from other perfringens -> detects the alpha toxin (lecithinase) produced by it
Works by neutralising lecithinase C produced through a specific antitoxin
It works by exploiting the lecithinase ability of the organism already seen on egg yolk agar -> antitoxin is added only to one half of the plate
What is the principle behind the anaerobic and metronidazole susceptibility test for anaerobic GNBs
(3)
We need to prove that the bacilli are in fact anaerobic
Need to put up an aerobic and anaerobic plate -> the organism must grow on the anaerobic plate
Anaerobic GNBs are susceptible to metronidazole so a disc should be put up on the anaerobic plate to prove its an anaerobic GNB
What is the principle behind the MID8 Identification Mastring Test?
Anaerobic Identification Discs
A commercial system for the identification of gram negative anaerobic bacteria based on the susceptibility to six antimicrobials
Erythomycin, Eifampicin, Colistin sulphate, Penicillin, Kanamycin, Vancomycin
What is the principle behind the X and V factor test for Haemophilus
(3)
Haemophilus species are all dependant on one or both of the growth factors, X and V
X = haemin
V = nicotinamide adenine dinucleotide (NAD)
Explain why Haemophilus species can grow on chocolate agar
(3)
Horse blood agar contains X factor normally
Horse blood agar contains a NADase enzyme which inactivates factor V(NAD)
However when the blood is heated to 80 degrees (chocolate agar) the NADase activity is destroyed and thus makes the V factor available
What is the principle behind King’s A Medium
(3)
Contains magnesium chloride and potassium sulphate which enhances pigment profuction
Selective supplement = antimicrobial cocktail comprising centrimide (antiseptic) and nalidixic acid (antibiotic)
P. aeruginosa is resistant to both these selective agents
What is the principle behind Brilliance Agar used to grow enterobacterales
Contains specific chromogenic substrates (Red Gal) which are cleaved by the B-galactosidase enzymes prouced by E. Coli and coliforms, resulting in pink colonies
Medium also contains phenylalanine and tryptophan, as an indication of tryptophan deaminase activity, indicating the presence of Proteus species, morganella and providencia species which appear brown
What is the principle behind the citrate test?
Test for an organisms ability to grow with citrate as its sole carbon source
Utilisation of citrate releases alkaline bicarbonate ions that cause the medium pH to increase above 7.4
Incorporated bromothymol blue indicator turns green to blue under alkaline conditions
What is the principle behind the indole test
(2)
Test for the hydrolysis of aa tryptophan, which leads to formation of indole
If indole is produced then the addition of 8 drops kovaks reagent a cherry red/pink colour change will be seen
What is the principle behind the the MR test?
(3)
Test for production of acids from the metabolism of glucose to produce a pH of 4.4 in the medium
These acids are not further metabolised and are said to be stable acids
At a pH of 4.4. or less the methyl red indicator will stay red
What is the principle behind the VP test?
(3)
Detection of products of glucose metabolism -> further breakdown of acids found in MR test -> this produces neutral end products e.g. acetoin and 2,3-butanediol
These neutral products raise the pH from 4.4 to create a negative MR test
acetoin and 2,3-butanediol react with a-naphthol to produce a mahogany red colour
What is the principle behind the amino acid decarboxylase test?
(5)
Test for the production of decarboxylase enzyme which removes the carboxyl group from an amino acid
Decarboxylase broth contains nutrients, dextrose, pyridoxal, bromocresol purple and cresol red indicators, plus a single aa e.g. lysine or arginine
Bromocresol purple turns purple at an alkaline pH and turns yellow at an acidic pH
No colour change if organism cannot ferment dextrose
If dextrose fermenter -> acidic byproducts are formed -> low pH -> medium turns yellow -> decarboxylation begins -> alkaline byproducts produced -> pH raised = purple
What is the principle behind New York City agar/GC?
(6)
A transparent, highly selective medium for the isolation of Neisseria gonorrhoeae from clinical specimens.
It contains peptone, corn starch agar base buffered with phosphates and supplemented with horse plasma, horse haemoglobin, dextrose, yeast autolysate and antibiotics.
Phosphate buffers the medium and sodium chloride provides electrolytes
Starch neutralises the toxic metabolites produced by Neisseria
Yeast fulfils the CO2 requirements needed to enhance CO2 growth
The cocktail of antibiotics prevents the growth of organisms other than Neissera
What is the principle behind Hoyle’s Medium?
(4)
Hoyle’s agar is a selective medium that uses tellurite to differentially select Corynebacterium diphtheriae from other upper respiratory tract flora.
The medium appears cream to yellow colored, and takes the form of a free-floating powder. It is a modification of Neill’s medium.[1]
Hoyle’s tellurite agar contains beef extract, peptones, sodium chloride, blood, tellurite and agar
C. diptheria grows with grey colonies
What is the principle behind Fraser broth?
(3)
Fraser broth can also be used for the selective enrichment of Listeria.
It contains acriflavine and nalidixic acid and ferric ammonium citrate which makes it selective
What is the principle behind Listeria Chromogenic Agar Base?
(3)
A selective agar for the growth of Listeria. Contains peptones, salts, chrogenic substrate lithium chloride and antibiotics. B-glucosidase is common to all Listeria species and gives the colonies their blue colour.
L. monocytogenes has lipase which causes an opaque white halo around blue colonies.
Incubate at 37 degrees for 24 hours and incubate negative strains for another 24 hours.
What is the principle behind PLET
(5)
Polymyxin-lysozyme-EDTA-thallous acetate agar for the selective growth of Bacillus anthracis
Contains: beef heart infusion from solids and tryptose provide the carbonaceous and nitrogenous compounds necessary for growth whereas sodium chloride (NaCl) provides the osmotic equilibrium.
Thallous acetate, polymyxin, lysozyme (FD185) are inhibitory agents allowing growth of B.anthracis while inhibiting contaminants.
Lysozyme specifically suppresses the growth of gram-negative contaminants.
Colonies of B.anthracis appear in 36-40 hours after incubation at 37°C. Roughly circular, creamy-white colonies with a ground-glass texture.
What is the principle behind the Deoxycholate citrate agar?
(3)
Selective and differential medium for non-lactose fermenting enterobacterales.
Selective agents are sodium deoxycholate and sodium citrate.
Differential agents are lactose and sodium ferric chloride. Shigella appears as a pale coloured colony (NLF), lactose fermenters/commensals are pink, non-lactose fermenters are colourless/pale, Salmonella often have a dark centre (H2S positive)
What is the principle behind XLD media?
(7)
Xylose lysine decarboxylase
Selective and differential media for shigella flexneri
Sodium deoxycholate is the selective agent
Xylose, Lysine and H2S are the differential agents
Lactose Fermenters/Commensals yellow
Shigella = red (xylose Lysine)
Salmonella red with black centres
What is the principle behind STEC Chromagar
Chromagar specific for shiga-toxin E.Coli serotype, these appear as a mauve colour while other enterobacteraciae appear colourless, blue or are inhibited.
What is the principle behind Chrom GTI for Shigella?
Proteus mirabilis are pale pink colonies with tan centres, salmonella enteric is teal coloured colonies with black centres, E. Coli are pink to violet colonies, Shigella sonnei are teal coloured colonies (non H2S producing salmonellas look the same)
What is the principle behind selenite broth?
(5)
Broth which enhances culture yield by reducing growth of faecal coliforms and faecal streptococci.
Used for the selective enrichment for the cultivation of Salmonella species.
The broth is used by taking a sub-culture of broth from the upper third of the broth column at least 5cm in depth.
Its used for the better enrichment of Salmonella Typhi.
Incubate for 37 hours for not longer than 18 hours.
What is the principle behind Bismith Sulphate Agar
(4)
Selective Media for Salmonella Typhi
Bismuth sulfite indicator along with brilliant green inhibits the intestinal gram-positive and gram-negative bacteria.
S. Typhi grow as black colonies with a surrounding metallic sheen resulting from hydrogen sulfide production and reduction of sulfite to black ferric sulfide.
Salmonella Paratyphi A grows as light green colonies
What is the principle behind Baird-Parker Media
(3)
Used for the selective isolation of Staphylococci species.
It contains lithium chloride and tellurite to inhibit the growth of alternative microbial flora, while the included pyruvate and glycine promote the growth of Staphylococci.
Staphylococcus colonies show up black in colour with clear zones produced around them.
What is the principle behind CCD agar
(3)
Charcoal cefoperazone deoxycholate (CCD) agar-modified
An improved version of the original CCD agar.
Modified CCD agar is developed for the direct isolation of campylobacters from human and animal feces and its major constituents are charcoal, cefoperazone, and sodium deoxycholate.
