Simmons Citrate Agar: PM 12042 Practical No.10

• These are the study notes for Department of Plantation Management Agricultural Microbiology (PM 12042) Practical No.10

Simmons Citrate Agar

• Useful biochemical medium aids in the differentiation and identification of certain genera and species of gram-negative bacilli, particularly Enterobacteriaceae members.
• Detects the ability of certain organisms to utilize citrate as the sole carbon source in a medium containing inorganic ammonium salts as the nitrogen source.
• Sodium citrate is a simple organic compound found as one of the metabolites in the Kreb's cycle.
• Bacteria utilize citrate as a carbon source to get energy.
• Koser devised a basal broth medium in which ammonium phosphate supplied the nitrogen
• Simmons converted Koser's liquid medium to a solid by adding agar and an indicator system with bromthymol blue.
• The alkaline reaction happens when excess CO2 is generated when citrate is cleaved to form oxaloacetate that is decarboxylated to pyruvic acid and CO2.
• Excess CO2 combines with sodium and water to form sodium carbonate.
• Bacteria utilizing citrate extract nitrogen from the ammonium phosphate results in ammonia production, which combines with water to form NH4OH.
• The indicator turns from green to blue when alkalinity exceeds pH 7.6.
• The components, per liter of deionized filtered water, are:
• Magnesium Sulfate 0.20 g
• Monoammonium Phosphate 1.00
• Dipotassium Phosphate 1.00
• Sodium Citrate 2.00
• Sodium Chloride 5.00
• Agar 15.00
• Bromthymol Blue 0.08
• The Final pH is 6.9 ± 0.2 at 25°C

Motility Test Medium

• Used to detect motility of gram-negative enteric bacilli.
• Bacterial motility can be observed via examination of the tubes.
• Growth spreads out from the inoculation line in motile organisms and throughout the tube in highly motile organisms.
• Nonmotile organisms only grow along the stab line.
• The approximate formula per Liter:
• Beef Extract 3.0 g
• Pancreatic Digest of Casein 10.0 g
• Sodium Chloride 5.0 g
• Agar 4.0 g
• Inoculate tubes with a pure culture by stabbing the medium column center to greater than half the depth, then incubate for 24-48 hours at 35 ± 2°C in aerobic conditions.

Indole test

• A biochemical test determines the ability of an organism to split indole from the amino acid tryptophan via the system "tryptophanase."
• Indole generated by reductive deamination from tryptophan via indolepyruvic acid.
• Amine (-NH2) group is removed from the tryptophan molecule.
• Final products of the reaction: indole, pyruvic acid, ammonia (NH3), and energy, where Pyridoxal phosphate is required
• Color change after reaction with an added reagent indicates the results of an indole test.
• Grow the pure bacterial culture in sterile tryptophan or peptone broth for 24-48 hours before testing.
• Add 5 drops of Kovac's reagent (isoamyl alcohol, p-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) to the culture broth after incubation.
• Ehrlich's reagent (ethyl alcohol in place of isoamyl alcohol) is used when performing tests on nonfermenters and anaerobes.
• A positive result is shown by the presence of red or red-violet color in the surface alcohol layer of the broth
• A negative result appears yellow.
• A variable result showing an orange color results from skatole presence, which is methyl indole or methylated indole.
• Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas hydrophilia, Aeromonas punctata, Bacillus alvei, most Citrobacter sp., Edwardsiella sp., Escherichia coli, Flavobacterium sp., Haemophilus influenzae, most Proteus sp. (not P. mirabilis), Plesiomonas shigelloides, Pasturella multocida, Pasturella pneumotropica, Streptococcus faecalis, and Vibrio sp.
• Bacteria which give negative results for the indole test include: Actinobacillus spp., Aeromonas salmonicida, Alcaligenes sp., most Bacillus sp., Bordtella sp., Enterobacter sp., Lactobasillus spp., most Haemophilus sp., most Klebsiella sp., Neisseria sp., Pasturella haemolytica, Pasturella ureae, Proteus mirabilis, Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp.
• Tryptone Water formula per Liter:
• Tryptone 10.0 g
• Sodium Chloride 5.0 g
• For determination using pure cultures:
• Inoculate Tryptone Water with a light inoculum of an 18-24 hour pure culture.
• Incubate the tubes at 35 ± 2°C with loosened caps for 18-24 hours.
• Add 0.5 mL of Indole Reagent (Kovacs) directly to the tube and agitate. Allow tubes to stand for 5-10 minutes.

MRVP (methyl red-Vogues Proskauer)

Methyl red test

• Methyl red, also called C.I. Acid Red 2, is an indicator dye that turns red in acidic solutions, of which is an azo dye, and is a dark red crystalline powder.
• Methyl red is red in pH under 4.4, yellow in pH over 6.2, and orange in between, with a pKa of 5.1.
• Methyl red identifies bacteria producing stable acids by mixed acid fermentation of glucose (cf. Voges-Proskauer (VP) test).
• The methyl red test is the "M" portion of the four IMViC tests used to characterize enteric bacteria, which identifies based on glucose metabolism.
• All enterics initially produce pyruvic acid from glucose metabolism.
• Methyl-red-positive enterics subsequently use the mixed acid pathway to metabolize pyruvic acid to lactic, acetic, and formic acids, including Escherichia coli and Proteus vulgaris.
• Methyl-red-negative enterics subsequently use the buytylene glycol pathway to metabolize pyruvic acid to neutral end-products, including Serratia marcescens and Enterobacter aerogenes.
• Procedure:
• Inoculate an isolate into a tube with a sterile transfer loop, and incubate at 35°C for 2-5 days.
• After incubation, transfer 2.5ml of the medium to another tube.
• Add five drops of the pH indicator methyl red to this tube.
• Gently roll the tube between the palms of the hands to disperse the methyl red.
• Enterics that metabolize pyruvic acid to other acids lower the pH of the medium to 4.2, turning methyl red red, for a positive test.
• Enterics that metabolize pyruvic acid to neutral end-products lower the pH of the medium to 6.0, turning methyl red yellow, for a negative test.

Voges-Proskauer Test

• Determines if an organism can produce acetylmethylcarbinol (acetoin) from fermentation of glucose, by incubating the culture in Clark and Lubb's medium.
• One half of this medium is then used for the methyl red test and the other half for a Voges-Proskauer test.
• The the medium will turn pink-reddish in color if Alpha-naphtol (5%) and potassium hydroxide (40%) are added to the medium and acetoin is present .

1. Uninoculated medium.
2. Positive reaction: Klebsiella.
3. Negative reaction: Escherichia coli.

Triple Sugar Iron or TSI

• Roughly named for its ability to test microorganism's ability to ferment sugars and to produce hydrogen sulfide.
• Often used in the selective identification of enteric bacteria including but not limited to Salmonella and Shigella.
• The TSI slant contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate.
• The slanted shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying degrees (an aerobic environment) or not exposed to air (an anaerobic environment).
• TSI agar medium was developed with sucrose in Kligler's iron agar
• Bacteria fermenting any of the three sugars in the medium produce byproducts; Usually acids, which change the color of the red pH-sensitive dye (phenol red) to a yellow color.
• Position of the color change distinguishes the acid production associated with glucose fermentation from the acidic byproducts of lactose or sucrose fermentation.
• Bacteria that ferment sugars in the anaerobic butt of the tube are enterobacteria.
• Bacteria utilize thiosulfate anion as a terminal electron acceptor, reducing it to sulfide
• Hydrogen sulfide (H2S) reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a black precipitate.
• Examples of sulfide-producing bacteria include Salmonella, Proteus, Citrobacter and Edwardsiella species.
• The blackening of the medium is almost always observed in the butt (bottom) of the medium.
• All lactose fermenters result in yellow slant/yellow butt (acid/acid reaction), whereas non-lactose fermenters may result in pink/yellow or yellow/yellow (if sucrose is fermented).
• Blackening of the butt due to H2S production may mask the acid reaction (yellow) in the butt.
• Salmonella enterica serovar Typhi may result in blackening of the medium at the interface of butt and slant.
• Under anaerobic conditions bacteria use H+ as an electron acceptor and reduce it to hydrogen gas, that is not very soluble
• Hydrogen may lift the agar from the butt of the tube or fracture the agar and carbon dioxide may not show as bubbles because it is far more soluble in the medium.

Catalase Test

• Catalase is a common enzyme that functions to catalyze the decomposition of hydrogen peroxide to water and oxygen.
• Catalase converts millions of molecules of hydrogen peroxide to water and oxygen per second.
• Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long that contains four porphyrin heme (iron) groups
• Human catalase optimum pH is approximately 7 and the human catalase optimum temperature is at 37°C.
• Reaction of catalase in the decomposition of hydrogen peroxide is: 2 H2O2 → 2 H2O + O2
• General rate of the reaction can be determined by the Michaelis-Menten equation and also oxidize different toxins.
• Any heavy metal ion (such as copper cations in copper(II) sulfate) and Cyanide act as noncompetitive and competitive inhibitors on catalase, respectively.
• To prevent damage from hydrogen peroxide, catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules.
• Mice genetically engineered to lack catalase are phenotypically normal indicating that this enzyme is dispensable in animals under some conditions.

Distribution among organisms

• All known animals use catalase in every organ, with particularly high concentrations occurring in the liver.
• Bombardier beetle mixes hydroquinones and hydrogen peroxide, causing oxygen to be liberated from hydrogen peroxide which oxidizes the hydroquinones and acts as the propellant.
• The oxidation reaction is very exothermic (ΔΗ = −202.8 kJ/mol) which rapidly heats the mixture to the boiling point.
• Catalase is also universal among plants, and many fungi are also high producers of the enzyme.
• Streptococcus species are an example of aerobic bacteria that do not possess catalase.
• Catalase Test identifies bacteria species using hydrogen peroxide
• The presence of catalase enzyme in the test isolate is detected using hydrogen peroxide
• Small amount of bacterial isolate is added to hydrogen peroxide and bubbles of oxygen are observed
• The Catalase Test is used to differentiate between bacterial species in the lab
• Place a drop of hydrogen peroxide on a microscope slide
• Use an applicator stick
• Then smear a colony sample into the hydrogen peroxide drop.
• If bubbles or froth forms, the organism is catalase-positive, i.e. Staphylococci and Micrococci.
• If negative, the organism is catalase-negative, i.e. Streptococci and Enterococci
• Presence of catalase in bacterial cells depends on both the growth condition and the medium used to grow the cells.

Carbohydrate Utilization

• Bacteria produce acidic products when they ferment certain carbohydrates, which lowers pH of the medium and will cause the pH indicator (phenol red) to turn yellow.
• The carbohydrate tests are the:
• Glucose (Dextrose) test
• Lactose test
• Sucrose test
• Fermentation results from left to right:
• Left tube shows less acid formation than far right tube, but gas is still made
• Center shows no carbohydrate utilization to produce acid or gas
• Right tube shows acid was produced as evidenced by the yellow color, and gas was made (look at the bubble in the Durham tube)

Gelatin Utilization

• Tests if bacteria can digest the protein gelatin by making gelatinase.
• Use a transfer needle to stab the gelatin in this media.
• After incubating the inoculated media for at least 48 hrs, transfer the tube into a refrigerator.
• The tube should be completely chilled prior to observation.
• If the media is solid after refrigeration, then the bacteria did not digest gelatin.
• If the media is liquefied even after refrigeration, the bacteria is able to digest gelatin.

Starch hydrolysis

• Detects the enzyme amylase, which breaks down starch by using Gram's iodine
• If starch has been hydrolyzed (broken down) then there is a reddish color or a clear zone around the bacterial growth
• If it has not been hydrolyzed then there is a black/blue area indicating the presence of starch.
• Inoculating loop spreads bacteria onto plate surface
• After the bacteria have grown, add a few drops of Gram's iodine to the plate and look for the color immediately after adding the iodine.

Oxidase test

• Determines if a bacterium produces certain cytochrome c oxidases
• Uses disks impregnated with a reagent such as N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator.
• The reagent is a dark blue to maroon color when oxidized, and colorless when reduced.
• Strains may either be oxidase positive (OX+) or negative (OX-).
• Strains that are OX+ normally means that the bacterium contains cytochrome c oxidase and can therefore utilize oxygen for energy production with an electron transfer chain.
• Typically the Pseudomonadaceae are OX+
• Another example is the preliminary identification of Neisseria and Moraxella genera
• Many Gram-negative spiral curved rods also includes Helicobacter pylori, Vibrio cholera, and Campylobacter jejuni.
• Legionella pneumophila is oxidase positive.
• Strains that are OX- normally means that the bacterium does not contain cytochrome c oxidase and therefore cannot utilize oxygen for energy production with an electron transfer chain.
• Typically Enterobacteriaceae are OX-.
• Procedures
• Wet each disk with about 4 inoculating loops of de-ionized water.
• Use a loop to aseptically transfer a large mass of pure bacteria to the disk.
• Observe the disk for up to 3 minutes.
• A color change does not occur within three minutes is negative.
• Live bacteria cultivated on trypticase soy agar plates may be prepared using sterile technique with a single-line streak inoculation and incubated at 37°C for 24-48 hours to establish colonies.
• Two-to-three drops of the reagent DMPD is added to the surface of each organism to be tested.
• A positive test (OX+) will result in a color change to pink, through maroon and into black, within 10-30 seconds.
• A negative test (OX-) will result in a light pink coloration or absence of coloration.

Phenylalanine (PA) Deaminase Test

• Bacteria are smeared on phenylalanine agar slant.
• Phenylalanine is an aromatic amino acid (has ring structure associated)
• If bacteria possess phenylalanine deaminase, it can remove amino group, leaving phenylpyruvic acid.
• To complete test (after bacterial growth), 5% solution of ferric chloride is added to slant.
• If test is positive, slant will turn olive green on surface and if negative, no change
• Members of the genus Proteus are generally positive for this test.

Urease Test

• Urea broth (orange), which contains phenol red indicator in low concentration is the medium.
• Urea is usually toxic, is the end product of amino acid metabolism.
• Some organisms contain urease, which allows them to break down urea to form CO2 and ammonia which reacts with water to form ammonium hydroxide.
• Broth becomes red-purple color if test is positive due to production of ammonium hydroxide; If negative, broth remains orange.

Coagulase test

• Transfer suspect S. aureus colonies into small tubes containing 0.2-0.3 ml BHI broth and emulsify thoroughly.
• Incubate BHI culture suspension and slants 18-24 h at 35°C
• Add 0.5 ml reconstituted coagulase plasma with EDTA (B-4, above) to the BHI culture and mix thoroughly. Incubate at 35°C and examine periodically over 6 h period for clot formation.
• Only firm and complete clot that stays in place when tube is tilted or inverted is considered positive for S. aureus.

Serological methods [agglutination test, ELISA, Western blot]

• Utilize antibodies in the Serological methods
• Serological methods using antibodies include: agglutination tests, ELISAs, Western blots
• Antibodies are highly selective in terms of the proteins (or other cell structures) by distinguishing the proteins from one species to another.

Bacteria Identification Flow Chart( gram negative bacteria)

• Lactose Fermentation
• positive = coliforms
• Citrate test
  • positive
• H2S test
• positive = Citobacter - negative - motility test
  • positive = Enterobacter
  • negative = Klebsiella
  • negative (also Indole Positive, motility positive, H2S negative) = Escherichia coli.
• negative = noncoliforms Urease test
  • positive
  • Phenylalinine deaminase test
  • positive = Proteus
  • negative
  • Phenylalinine deaminase test
  • negative
    • Motility test
    • negative = Shigella (also produces Acid with glucose)
      • positive = Salmonella (also produces Acid and Gas with glucose) Oxidase positive = Pseudomonis

Vocabulary

• Serovar [serotype]: strain differentiated by serological means
• Biovar [biotype]: strains that are differentiated by biochemical or other non-serological means.
• Morphovar [morphotype]: strain which is differentiated on the basis of morphological distinctions.
• Isolate ('i-so-lit): pure culture derived from a heterogeneous, wild population of microorganisms.