Tests To Determine Physiological Characteristics
Streak‐Plate Method
In order to identify bacteria, it is necessary to obtain a pure culture. This is done by using the streak‐plate method. Bacterial cells are spread over the surface of an agar plate in a continuous dilution, this allows the cells to be separated from each other. When the plate is incubated, those individual cells will grow into colonies that originated from a single cell.
Trypic Soy Agar Plates
• Trypticase soy agar is a bacterial growth medium.
• TSA is a general purpose media produced via enzymatic digestion of soybean meal and casein. • It is frequently the base media of other types of agar and it supports growth of many semi‐
fastidious bacteria
Growth Characteristics of EMB
• EMB agar is both a selective and differential agar. • EMB agar is inhibitory to gram‐positive bacteria, and is indicative of lactose fermentation. • It is selective because it contains bile salts and eosin and methylene blue dyes, which inhibit gram positive bacteria from growing. • It is also differential, in that lactose‐fermenting bacteria, will produce dark colonies with a green metalic sheen, while bacteria that cannot ferment lactose produce colorless colonies.
EMB Agar
Hektoen enteric agar (HEA)
• HEA contains dyes and bile salts which inhibit growth of Gram‐positives and high concentrations of three sugars: lactose, sucrose, and the plant glycoside salicin. dyes and bile salts prevent growth of Gram‐positive bacteria. • Acid production from any of the sugars results in color change of the dyes. Acid‐producers form orange or pink colonies, non‐fermenters of all three sugars form greenish colonies. If an organism ferments lactose, fermentation of the other two sugars cannot be detected.
• HEA contains sodium thiosulfate and ferric citrate. Some bacteria use thiosulfate anion as an electron acceptor, reducing it to sulfide. In this case a black precipitate of iron
sulfide forms in the centers of colonies.
Growth Characteristics of MAC
MacConkey agar is both a selective and differential agar it is inhibitory to gram‐positive bacteria, and is indicative of lactose fermentation. • It is selective because it contains bile salts and crystal violet dye, which inhibit gram positive bacteria from growing. • It is also differential, in that lactose‐fermenting bacteria, produce red colonies, while bacteria that cannot ferment lactose produce colorless colonies.
Motility Test
• The motility test is used to determine the presence of flagella
• Bacteria that are motile have flagella. • Non‐motile bacteria do not have flagella.
Motility Test
•
Procedure:
•
Inoculate an isolated colony into tryptic soy broth or brain heart infusion broth.
•
Incubate the culture for 24 hours
•
Place a drop of the broth onto a clean microscope slide, and cover with a cover slip.
•
Observe microscopically for the presence of motility.
•
A positive test result is indicated by individual bacterial cells moving in random directions.
Oxidative and Fermentative Tests
Fermentation Tests : determine if the unknown is capable of carrying out various fermentation reactions
Oxidative Tests: determines if the unknown carries out respiratory function
. Fermentation Tests
• O/F Glucose Test
• Durham Tube sugar fermentations
• Mixed‐ Acid Fermentation (methyl red [MR] test)
• Butanediol Fermentation (Voges‐
Proskauer [VP] Test)
• Citrate Test
O/F Glucose Test
• determines if the unknown organism is oxidative, fermentative or capable of both kinds of metabolism
‐ To perform this test an unknown organism is inoculated into a tube of O/F Glucose by stabbing with an inoculating needle. Mineral oil is added and the tubes are incubated at 37 degrees for 24 hours.
O/F Glucose Test
Interpretation
Result
Interpretation
Anaerobic Aerobic
Yellow Yellow
Oxidative and fermentatative
Green Yellow
Oxidative Metabolism
Green Green
Sugar not metabolized
(nonsaccharolytic)
• If it is determined by the O/F Glucose Test that the unknown bacteria is oxidative and not capable of fermenting sugars then the bacterium cannot be identified by fermentation tests
• If it is determined that the organism is fermentative in it’s metabolism it is important to which specific sugars are fermented or which fermentation pathways are used for growth
Durham Tube Sugar Fermentations
Phenol Red Fermentation
• If an organism ferments a sugar acid is usually produced and gas may be an end product. • To perform this test an unknown organism is inoculated into a Durham sugar tube. The carbohydrate tube is then incubated for 24 hours at 37 degrees. Durham Fermentation Tube
• The presence of an acid is indicated by a color change in the pH indicator (phenol red) from red (alkaline values) to yellow (acidic values)
• The production of gas such as hydrogen and carbon dioxide is revealed by the displacement of medium from the Durham Tube.
Durham Fermentation Tube
Mixed Acid Fermentation
Methyl Red Test
• Important in differentiating some gram negative intestinal bacteria is that of mixed acid fermentation. • Some bacteria ferments glucose to produce a number of organic acids such as lactic, acetic, succinic and formic acids.
• CO2, H2 and ethanol are also produced in this fermentation
• A positive test, which turns red after Methyl red is added, indicates the organism carried out mixed‐acid fermentation.
Methyl Red Test
• To perform this test a glucose broth is used that has been buffered with peptone and dipotassium
phosphate is inoculated with an organism. The tube is then incubated at 37 degrees for 3‐5 days.
• 3‐4 drops of Methyl Red is then added to the tube
• If the tube becomes red immediately this is a positive result. If there is no change this is a negative result. Methyl Red Test
2,3‐ Butanediol Fermentation
Vogues‐Proskauer Test
• The Methyl Red Test and VP test are important tests in differentiating gram ‐
bacteria
• Some gram negative intestinal bacteria do not carry out mixed acid fermentation but rather they ferment glucose to produce limited amounts of some organic acids and primarily a more neutral end product, 2,3‐butanediol. Vogues‐Proskauer Test
• To perform this test a glucose broth is used that has been buffered with peptone and dipotassium
phosphate is inoculated with an organism. The tube is then incubated at 37 degrees for 3‐5 days.
• 1 ml of culture is removed with a pipette and mixed with 18 drops of Baritt’s Reagent A(alpha‐
naphthol) and 18 Drops of Barrit’s Reagent B (KOH).
• The tube is then vigorously shaken (to oxidize the 2,3‐butanediol to acetoin)and is left to stand for 30 minutes.
Vogues‐Proskauer Test
• Vogues Proskauer test after addition of Barritt’s A& B and waiting 20‐25 minutes.
• The red color on the right tube is a (+) test.
Vogues‐Proskauer Test Results
• A positive VP test will turn red to pink in color. • A negative VP test will remain unchanged
• If butanediol is produced it is VP positive and is usually Methyl Red negative Citrate Test
• Organisms that degrade citrate must use ammonium salts, that serve as a sole nitrogen source for growth, and in the process they produce ammonia that causes the medium to become alkaline.
• Under alkaline conditions the pH indicator turns the medium from dark green to a Persian blue
Citrate Test
• To perform this test an unknown organism is inoculated into a tube of Simmon’s Citrate by streaking the slant and then stabbing the medium with the inoculating needle. The tubes are incubated at 37 degrees for 24‐48 hours.
Citrate Test
Oxidative Tests
• Oxidase Test
• Catalase Test
• Nitrate reduction Oxidase Test
• The oxidase test assays for the presence of cytochrome oxidase. • Cytochrome oxidase occurs in bacteria that carry out respiration and where oxygen is the terminal electron acceptor
– The test differentiate between those bacteria that have cytochrom oxidase and use oxygen as a terminal electron acceptor and those that use oxygen as a terminal electron acceptor but have other types of terminal oxidases. Oxidase Test
• The enzyme is detected by the use of an artificial electron acceptor, which changes from yellow to purple when electrons are transferred from reduced cytochrome c to the artificial acceptor. Oxidase Test
• A swab of a bacteria from a TSA plate is taken by a sterile swab, alternatively a sample is also transferred from the plate to a piece of filter paper. Several drops of oxidase reagent are added to the cells on the paper. Oxidase Test
• Positive culture will cause the reagent to turn from yellow to purple in 10‐30 seconds (a)
• No change or a change after 30 seconds is considered a negative result (b)
Catlase Test
• This test is performed to detect for the presence of the enzyme catalase which degrades hydrogen peroxide into oxygen and water. • Strict anaerobes and aerotolerant bacteria lack this enzyme. • The presence of catalayse is one way to determine aerobes and facultive areobes which have this enzyme.
• Organisms containing the catalase enzyme will form oxygen bubbles when exposed to hydrogen peroxide.
Catalase Test
• To perform this test a wooden stick is used to transfer a small amount of growth to a slide. Hydrogen peroxide is then added. • If catalase is produced there will be vigorous bubbling due to the breakdown of hydrogen peroxide and the production of oxygen gas. – A sample cannot be taken from blood agar
– A wire loop cannot be used Catalase Test
Positive
Negative
Nitrate Reduction • Some Facultative anaerobes use nitrate as a terminal electron acceptor in a type of anaerobic respiration called nitrate respiration.
• Enzymes involved in nitrate reduction are inducible and are only produced if nitrate is present a anaerobic conditions exist for growth.
Nitrate Reduction Procedure
• 2‐3 drops of Reagent A and 2‐3 drops of Reagent B is added to A nitrate broth culture with Durham tubes of the unknown organism. • If a deep red color fails to appear the organism did not reduce nitrate or it may have produces a nongaseuos
nitrate reduction. • If negative Zinc is then added to the culture and then shaken. If a red color develops in the tube the nirate
was reduced by the zinc indicating a negative test for Nitrate reduction. If not color develops a nongaseous end product may have been formed, which means the unknown reduced nitrate.
Hydrolytic and Degradative Reactions
• Starch Hydrolysis
– Bacteria that hydrolyzes starch produce amalses
that degrade the starch molecule into molecules of maltose, glucose, sucrose and dextrins.
– Starch hydrolysis is detected by adding iodine to starch medium. • Iodine complexes with the starch macromolecule and causes the medium to turn blue
• If the starch has been degraded the medium adjacent to the bacterial growth will be clear after iodine is added
Starch Hydrolysis
Hydrolytic and Degradative Reactions
Casein Hydrolysis Test • Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. • Casein is a large protein that is responsible for the white color of milk. • This test is conducted on milk agar which is a complex media containing casien, peptone and beef extract. Casein Hydrolysis Test • If an organism can produce casein, then there will be a zone of clearing around the bacterial growth. Hydrolytic and Degradative Reactions
Fat Hydrolysis/Spirit Blue Agar • Fat hydrolysis is achieved when the glycerol molecule in a triglyceride is cleaved from the adjoining fatty acids. This reaction is catalyzed by the exoenzyme lipase. • Spirit blue agar contains tributyrin as a substrate for lipase and a pH indicator that becomes colorless as the pH of the medium is lowered in response to the build‐up of fatty acids due to the fat‐hydrolyzing action of lipase • You must confirm the negative result by confirming the shiny appearance of the agar • A dull, matte appearance or a clearing of the blue color in the media is considered a positive result for fat hydrolysis. Hydrolytic and Degradative Reactions
Tryptophan Degradation
• Tryptophan is degraded by tryptophanase
into indole, ammonia, pyruvic acid. • Pyruvic acid is then involved in metabolic pathway so that ATP energy for bacterial cell is generated. While other products like NH3 and Pyruvate is metabolized, indole is not. And it stays in the medium.
Tryptophan Degradation
• Upon addition of Kovac’s reagent, deep red ring at the top of the agar/ broth is formed when Kovac’s reagent reacts with indole. This is the (+) result.
Hydrolytic and Degradative Reactions
Urea Hydrolysis
• The medium used to perform this test is a Urea broth that contains a phenol red indicator • Urea, is the end product of amino acid metabolism. Some organisms contain urease, which allows them to break down urea to form CO2 and ammonia. Ammonia reacts with water to form ammonium hydroxide. Urea Test
• Broth becomes red‐purple color if test is positive due to production of ammonium hydroxide. • If negative, broth remains orange.
Hydrolytic and Degradative Reactions
Phenylalanine deaminase • Phenylalanine deaminase medium tests the ability of an organism to produce the enzyme deaminase. This enzyme removes the amine group from the amino acid phenylalanine and releases the amine group as free ammonia. As a result of this reaction, phenylpyruvic acid is also produced. • It is used to differentiate members of the genera Proteus, Morganella, and Providencia
from other Enterobacteriaceae. Phenylalanine Deaminase Test
• After incubation, 10% ferric chloride is added to the media; • if phenylpyruvic acid was produced, it will react with the ferric chloride and turn dark green.
• If the medium remains a straw color, the organism is negative for phenylalanine deaminase production.
Phenylalanine Deaminase Test
• Positive Result Negative Result
Multiple Test Media
IMViC Tests
• IMViC is an acronym that stands for indole, methyl red, Voges‐Proskauer, and citrate • Enterobacteriaeae are Gram‐negative bacteria that grow in the intestinal tract of humans and other animals. The IMViC tests are frequently employed for identification of this group of microbes. Multiple Test Media
Indole Test
• The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan. Indole positive bacteria such as produce tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products. • The indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs.
Indole Test
• When Kovac's reagent (is added to a broth with indole in it, a dark pink color develops. Multiple Test Media
Kligers Iron Agar (KIA) Test
• This is a differential medium. It tests for organisms’ abilities to ferment glucose and lactose to acid and acid plus gas end products. It allows for identification of sulfur reducers which binds with iron to form a black precipitate. • Glucose fermentation will create acidic byproducts that will turn the phenol red indicator in the media yelllow. KIA Test
• With your needle pick material from a colony and stab through the center of the agar to the bottom of the tube. • Then streak the surface of the slant. This double inoculation allows growth of your unknown organism on the aerobic surface of the slant and in the largely anaerobic butt of the tube. • Incubate your Kligler slants, which must be interpreted after incubation for 24 hours.
KIA results
• Lactose (+) or (‐). Thus lactose (+) organisms yield a yellow slant and lactose (‐) organisms yield a red slant.
• Glucose (+) or (‐) glucose (+) organisms yield a yellow butt. Fermentation of lactose . all lactose‐positive organisms are also glucose‐positive. If a black color from iron sulfide obscures the color of the butt you can presume it is yellow.
• H2S (+) or (‐). If an organism forms H2S, the lower portion of the tube will turn black, due to formation of iron sulfide.
• Gas formation (+) or (‐). If an organism forms gas from glucose or lactose the agar in the butt will show bubbles or cracks.
Positive Negative
Lactose yellow slant red slant
Glucose yellow butt red butt
KIA results
Multiple Test Media
Sulfur Indole Motility (SIM) • This is a differential medium. It tests the ability of an organism to : reduce sulfur, produce indole and be motile. • Sulfur can be reduced to H2S either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. • If hydrogen sulfide is produced, a black color forms in the medium. Sulfur Indole Motility (SIM)
• SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate from the stab mark and make the entire tube appear turbid. • Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color (indole +). • If hydrogen sulfide is produced, a black color forms in the medium. SIM results
Negative
for indole
production
.
Positive for
indole
production
Positive for
motility and
sulfur
reduction
Positive
for motility
and
negative
for sulfur
reduction
Multiple Test Media
Litmus Milk
• To differentiate bacteria based on various reactions that occur in skim milk supplemented with a litmus pH indicator. • Bacterial enzymes alter the media and may bring about various changes. Litmus is added to the medium to detect pH changes that may occur as a result of these enzymatic reactions. Above a pH of 8.3 litmus is blue, while below a pH of 4.5 litmus is red. Possible Results:
• A. Acid/Reduction/Curd
B. Reduction/Curd (arrow denotes gas pocket)
C. Uninoculated Control
D. Acid Formation
E. Proteolysis of casein
F. Alkaline Reaction
Decarboxylase, Lysine, Aginine, Ornithine Decarboxylase
• Decarboxylase broth tests for the production of the enzyme decarboxylase, which removes the carboxyl group from an amino acid. Decarboxylase broth contains nutrients, dextrose, pyridoxal, and the pH indicators bromcresol purple and cresol red.
• Bromcresol purple turns purple at an alkaline pH and turns yellow at an acidic pH. Decarboxylase, Lysine, Aginine, Ornithine Decarboxylase
• If the inoculated medium is yellow, or if there is no color change, the organism is decarboxylase‐negative for that amino acid. If the medium turns purple, the organism is decarboxylase‐positive for that amino acid.
• Mineral oil is used to promote fermentation by locking out oxygen, and it also prevents false alkalinization at the surface
of the medium. • Ornithine decarboxylation yields putrescine.
• Lysine decarboxylation results in cadaverine. • These byproducts are sufficient to raise the pH of the media so that the broth turns purple
Lysine
• positive reaction as the medium has turned purple displays a
negative result
Gelatin Hydrolysis(Liqefication) Test
• Nutrient gelatin is a differential medium that tests the ability of an organism to produce an exoenzyme, called gelatinase, that hydrolyzes gelatin. • Gelatinase allows the organisms that produce it to break down gelatin into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the organism. Gelatinase Test
• When gelatin is broken down, it can no longer solidify. If an organism can break down gelatin, the areas where the organism has grown will remain liquid even if the gelatin is refrigerated.
Gelatin hydrolysis
the tube on the the left is positive for gelatinase production, as evidenced by the liquidation of the media.
Whereas the tube on the right is negative