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Principles of Test used by the team

Laboratory test Principles

Dear Mr Loh, my group has compile the principles of laboratory testings which are to done my the group members for the identification of the microorganism

MacConkey agar
MacConkey agar is a selective, differential platingmedium. It selects enterobacteriaceae and other gram-negative bacteria and differentiates them into lactose fermenters and non-lactose fermenters. The presence of bile salts and crystal violet will inhibit the growth of gram-positive microorganism. The incorporation of lactose serves as the sole carbohydrate source. Gram-negative bacilli which ferments lactose produces a dark red to pink colonies while non-lactose fermenting gram negative bacilli produces colourless or transparent colonies> Image search> MacConkey agar

Eosin-Methylene Blue Agar
Eosin-methylene blue agar is a selective, differential medium used for the isolation and identification of gram-negative bacteria. Eosin Y and methylene blue dyes inhibit the growth of gram-positive bacteria and allows the growth of gram-negative bacteria. Lactose and sucrose are incorporated to allow differentiation of isolates based on lactose fermentation. Fermentation is detected by colour changes and precipitation of dye such as pH drops. Sucrose is incorporated as it serves as another alternative source of carbohydrates for slow lactose fermenters.> Image search> Eosin-methylene blue agar

Blood Agar
Blood agar is used to cultivate a wide variety of fastidious bacterium. An infusion agar of tryptic soy agar base can be enriched by the addition of 5-10% of sheep’s, rabbit’s blood. The incorporation of blood not only provides enrichment for growth but also allows the detection and characterization of haemolytic patterns


-alpha-hemolysis represented by alpha sign
alpha-hemolysis is the partial clearing of the blood around the colony that results in a green discoloration pf the medium

-Beta-hemolysis represented by beta sign
Beta-hemolysis is the complete clearing of blood around the bacterial colonies. This is due to the complete haemolysis of the surrounding RBCs

-Gamma-hemolysis represented by gamma sign
Gamma-hemolysis occurs when the RBCs surrounding the colonies are unaffected and there is no change in the colour of the bacterial colonies

Mannitol Salt Agar
Differential test for mannitol fermentors and non-fermentators; S. aureus ferments mannitol producing yellow colonies.

Left: Non- fermented mannitol Right: Fermented mannitol (S. aureus)

Triple Sugar Iron Agar Test
TSI contains glucose, lactose and sucrose as well as a pH-sensitive color indicator. It also contains an iron ingredient for detecting hydrogen sulfide production, which blackens the medium if it occurs. Fermentation of the sugars by the test organism is interpreted by color changes in the butt
and the slant of the mediums.

Acid (yellow) butt – alkaline (pink) slant (K/A)
- glucose fermented
- sucrose or lactose not fermented

acid butt - acid slant (A/A)
- glucose fermented
- lactose and/or sucrose fermented

alkaline butt - alkaline slant (K/K)
- neither glucose, lactose or sucrose fermented
- organism probably not an enteric bacillus

Gas production - indicated by bubbles in the butt. With large amounts of gas production, the agar may be broken or pushed upward.
Hydrogen sulfide production - indicated by any amount of blackening in the butt.

TSI agar > > faculty>TBarta > fermentation > TSI agar

Indole test
Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid and ammonia. Indole production is an important characteristic in the identification of many species of microorganisms being particularly useful in separating E. coli (positive) from members of the Klebsiella-Enterobacter-Hafnia-Serratia group (mostly negative) > >johnson >labtools >biochem

Interpretation Positive test: bright pink colour at the interface between the broth and the reagent Negative test: No colour change or a slight yellow ring

Methyl Red test
Methyl red is an acid sensitive dye that is yellow at pH above 4.5 and red at pH below 4.5. This test is used to indicate whether glucose has been broken down completely to highly acidic end products or only partially to less acidic end products. > >johnson >labtools >biochem

Positive - bright red color, indicates pH of 4.5 or less
Negative - yellow or orange color, indicates pH of above 4.5

Voges Proskauer
The Voges-Proskauer test is used to detect the presence of acetylmethylcarbinol, one of the end products of glucose metabolism. > >johnson >labtools >biochem

Positive - strong red color will develop
Negative - no color change

Citrate test
Organisms that are capable of utilizing citrate as their sole carbon source also utilize ammonium salts as their sole nitrogen source. This results in an alkaline environment that turns the indicator bromothymol blue to an intense blue when pH is above 7.6. This characteristic can used to differentiate members of the family Enterobacteriaceae and other gram-negative rods. > >johnson >labtools >biochem

InterpretationPositive test: growth or a deep blue colour of the agarNegative test: no growth or no colour change of the agar (green)
Right tube- negative results; Left tube- positive results

Urease Test
Detects the enzyme urease, which breaks down urea into ammonia. Ammonia is a base and thus will raise the pH of the media if it is present. This change in pH is indicated by a pH indicator called phenol red which is present in the media.

InterpretationPositive test: A color change from yellow to bright pinkish-redNegative test: No colour change

Oxidation-Fermentation Test
The oxidative-fermentative approach permits the separation of bacteria by a simple method that differentiates anaerobic and aerobic degradation of carbohydrates in an environment responsive to weak production of acids. Agar is added to give a semisolid consistency so that acids that form on the surface of the agar may permeate throughout the medium, making interpretation off the pH shift of the indicator bromothymol blue easier to visualize.

Positive test: medium becomes yellow at the top indicating acid productionNegative test: medium remains green if no acid formed

Catalase Test
The catalase test is used to differentiate between bacterial species in the lab. The test is done by placing a drop of hydrogen peroxide on a microscope slide. Using an applicator stick, the colony is smeared into the hydrogen peroxide drop. If bubbles or froth form, the organism is said to be catalase-positive; if not, the organism is catalase-negative

Catalase positive reaction indicating S. aureus infection>html>stitch.php?s=72880656991974&id=39006255044608

Oxidase Test
Use to determine the presence of oxidase enzymes. An oxidase paper is touched onto colony, in the presence of oxidase, paper changes to purple indicating Pseudomonas aeruginosa. The absence of oxidase result in no colour change, and is indicative of Enterobacteriaceae family

Coagulase Test
Coagualse test is performed to test the ability of the microorganism to clot plasma by the action of coagulase

Rabbit plasma is incubated with staphylococcus colony If staphylococci isolated from infection are able to clot blood, then they are coagulase positive S. aureus.

Coagulase test differentiates S. aureus, from other staphylococcus species such as S. epidermidis and S. saprophyticus present in the normal flora.

Right: Coagulase negative; Left: Coagulase positive


Bile esculin Test
The medium contains esculin and peptone that is to provide nutrition to the microorganism while bile inhibits Gram-postivie bacteria other than Group D streptococci and enterococci, and sodium azide to inhibit the Gram-negative bacteria. Ferric citrate is added as a color indicator.

Positive test: An agar slant that is more than half darkened after incubation.
Negative test: Less than half the slant has darkened.

Optochin Test
Optochin (ethylhydrocupreine hydrochloride), a quinine derivative, has a detergent-like action and causes selective lysis of pneumococci. A sterile disk impregnated with optochin is placed on the first sector of an isolation plate before incubation. A zone of inhibition (area with no growth) of 14 mm. or more in diameter will be seen around the disk after incubation if the organism is Streptococcus pneumoniae. Other alpha-hemolytic streptococci are resistant to (not killed by) optochin. Their colonies will thus grow right up to the disk of optochin or have zones of inhibition less than 14 mm. in diameter.

Bile Solubility Test
Bile will selectively lyse colonies of Streptococcus pneumoniae while other strep are immune to bile's activity. When a bile salt such as desoxycholate is added directly to Streptococcus pneumoniae growing on an agar plate or in a broth culture the bacteria will lyse and the area become clear. Other alpha-hemolytic streptococci are resistant to (not lysed by) bile and will stay visible or turbid (cloudy).

Bile-esculi agar
Bile-esculi agar is a selective, differential agar used to isolate and identify group D streptococci and enterococci. Bile salts is a selective component and esculin is the differential ingredient. All group D streptococci and enterococci hydrolyzes esculin. The products of esculin hydrolysis react with ferric citrate in the medium to produce insoluble iron slats. This results in the blackening of the agar.


6.5% NaCl Test
NaCl is sued to differentiate those gram-positive cocci that will grow in 6.5% NaCl from those that are inhibited by this salt concentration. Enterococcus species can withstand higher salt concentration than other gram-positive cocci

Egg Yolk Agar
Egg yolk agar is a differential medium used in the detection of lecithinase, lipase and protease activity. The egg emulsion in the agar provides the lecithin, lipase and proteins to be degraded by these enzymes. A given microorganism may produce one or all of the above enzymes.

Left plate: Positive test for lecithinase
Right plate: Negative for lecithinase>kkiser>Lecithpn.jpg

DNase Test
Extacellular DNases are only present in a few bacterial species such as Serratia marcescens, Pseudomonas aerugionsa. The biochemical and morphology of other Enterbacteriaceae are similar especially in Klebsiella-Enterobacter supp. The only difference which separates S. marcescens from Klebsiella-Enterobacter supp is the DNases that is produced.

8% sodium chloride broth
Test is done to differentiate vibrio cholerae and vibrio parahaemolyticus.
Unlike vibrio parahaemolyticus, vibrio cholerae cannot grow at 8% sodium chloride broth.

Spore stain (Bartholomew and mitters)
In spore stain test, hot malachite green is used to stain spores, while the safranin is used as a counterstain. In this test, spores would be stain green and the rest of the cell appear pink/red

References> Optochin sensitivity & bile solubility > search > catalase > search > oxidase

Mahon C. R. and Manuselis G, Jr (1995). Textbook of Diagnostic Microbiology. W.B. Saunders Company

MacFaddin J. F. (2000). Biochemical Test for Indentification of Medical Bacteria. Lippincott Williams & Wilkins


Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

med>topic2422/">>med>topic2422 > >labpix > E.coli > biochemical test > > Labs > Enterobacteriaceae > biochemical test


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