Principles of Laboratory Tests

Question 1

What are the principles behind the DN’ase agar.
(4)

Answer 1

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

Question 2

What is the principle behind mannitol salt agar?
(3)

Answer 2

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

Question 3

What is the principle behind SAIDE (Chromid S. aureus Elite agar)?
(2)

Answer 3

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

Question 4

What is the principle behind the Staphaurex Plus test?
(4)

Answer 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

Question 5

Why should Staphaurex Plus be used instead of a coagulase test?
(3)

Answer 5

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

Question 6

What is the principle behind novobiocin resistance for coag negative staph differentiation?

Answer 6

Saprophyticus is resistant to novobiocin while epidermidis is not

Question 7

What is a positive novobiocin test

Answer 7

Resistance to novobiocin

Question 8

What is a negative novobiocin test

Answer 8

Susceptibility to novobiocin

Question 9

What species will give a positive novobiocin result

Answer 9

S. saprophyticus (resistant)

Question 10

What species will give a negative novobiocin result

Answer 10

S. epidermidis (susceptible)

Question 11

What is the principle of the tube coagulase test?
(5)

Answer 11

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

Question 12

What is the principle behind the optochin susceptibility test
(2)

Answer 12

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

Question 13

What is the principle behind the lancefield grouping test
(4)

Answer 13

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

Question 14

What is the principle behind Bacitracin susceptibility
(2)

Answer 14

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

Question 15

What is the principle behind MacConkey agar?
(5)

Answer 15

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.

Question 16

What is the principle behind bile aesculin agar?
(4)

Answer 16

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

Question 17

What is the principle behind the TAXO sugars test?
(3)

Answer 17

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

Question 18

What is the principle behind the Catarrhalis disk test?
(2)

Answer 18

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

Question 19

What is the principle behind PEMBA agar
(5)

Answer 19

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

Question 20

What is the principle behind the motility test
(3)

Answer 20

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

Question 21

What is the principle behind the trehalose test
(5)

Answer 21

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

Question 22

What is the principle behind urease test?
(4)

Answer 22

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

Question 23

What is the principle behind the Tinsdale Medium
(5)

Answer 23

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

Question 24

What is the principle behind using lactose gelatin medium

Answer 24

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

Question 25

What is the principle behind the egg yok agar - Naglar reaction?
(4)

Answer 25

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

Question 26

What is egg yolk agar?
(3)

Answer 26

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

Question 27

What is the principle behind the Naglar reaction?

Answer 27

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

Question 28

What is the principle behind the anaerobic and metronidazole susceptibility test for anaerobic GNBs
(3)

Answer 28

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

Question 29

What is the principle behind the MID8 Identification Mastring Test?

Answer 29

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

Question 30

What is the principle behind the X and V factor test for Haemophilus
(3)

Answer 30

Haemophilus species are all dependant on one or both of the growth factors, X and V

X = haemin

V = nicotinamide adenine dinucleotide (NAD)

Question 31

Explain why Haemophilus species can grow on chocolate agar
(3)

Answer 31

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

Question 32

What is the principle behind King’s A Medium
(3)

Answer 32

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

Question 33

What is the principle behind Brilliance Agar used to grow enterobacterales

Answer 33

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

Question 34

What is the principle behind the citrate test?

Answer 34

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

Question 35

What is the principle behind the indole test
(2)

Answer 35

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

Question 36

What is the principle behind the the MR test?
(3)

Answer 36

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

Question 37

What is the principle behind the VP test?
(3)

Answer 37

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

Question 38

What is the principle behind the amino acid decarboxylase test?
(5)

Answer 38

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

Question 39

What is the principle behind New York City agar/GC?
(6)

Answer 39

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

Question 40

What is the principle behind Hoyle’s Medium?
(4)

Answer 40

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

Question 41

What is the principle behind Fraser broth?
(3)

Answer 41

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

Question 42

What is the principle behind Listeria Chromogenic Agar Base?
(3)

Answer 42

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.

Question 43

What is the principle behind PLET
(5)

Answer 43

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.

Question 44

What is the principle behind the Deoxycholate citrate agar?
(3)

Answer 44

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)

Question 45

What is the principle behind XLD media?
(7)

Answer 45

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

Question 46

What is the principle behind STEC Chromagar

Answer 46

Chromagar specific for shiga-toxin E.Coli serotype, these appear as a mauve colour while other enterobacteraciae appear colourless, blue or are inhibited.

Question 47

What is the principle behind Chrom GTI for Shigella?

Answer 47

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)

Question 48

What is the principle behind selenite broth?
(5)

Answer 48

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.

Question 49

What is the principle behind Bismith Sulphate Agar
(4)

Answer 49

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

Question 50

What is the principle behind Baird-Parker Media
(3)

Answer 50

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.

Question 51

What is the principle behind CCD agar
(3)

Answer 51

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.