JOVE, JESSICA JANE B. March 4, 2012 ACETAMIDE UTILIZATION Purpose It is used for differentiating Pseudomonas aeruginosa from other non- glucose-fermenting, gram negative rods. Principle This test is used to determine the ability of an organism to use acetamide as the sole source of carbon. Bacteria that can grow on this medium deaminate acetamide to release ammonia. The production of ammonia results in a pH-driven color change of the medium from green to royal blue. Ingredients Magnesium sulphate 0.158 Sodium chloride 0.200 Sodium molybdate 0.005 Ferrous sulphate 0.0005 Dipotassium hydrogen phosphate 0.200 Acetamide Purpose of Ingredients It contains inorganic salts and acetamide as sources of carbon and nitrogen. Organisms growing in this medium metabolize acetamide, thereby liberating ammonia. This liberated ammonia can be detected by Nesslers reagent, which confirms Pseudomonas aeruginosa . Magnesium sulphate, ferrous sulphate and sodium molybdate are sources of ions that stimulate metabolism. Sodium chloride maintains osmotic equilibrium. Dipotassium hydrogen phosphate provides buffering to the medium. Considerations Do not stab the slant, since the test requires an aerobic environment. Do not inoculate from broth cultures, due to carryover of media. To avoid false-positive reactions, use a light inoculum to prevent carryover of substances from previous media. Quality control Positive: Pseudomonas aeruginosa Negative: Stenotrophomonas maltophilia Results Positive: Deamination of the acetamide resulting in a blue color (A) Negative: No color change (B)
Transcript
JOVE, JESSICA JANE B. March 4, 2012
ACETAMIDE UTILIZATIONPurpose
It is used for differentiating Pseudomonas aeruginosa from other non-glucose-fermenting, gram negative rods.
PrincipleThis test is used to determine the ability of an organism to use acetamide as the sole source of carbon. Bacteria that can grow on this medium deaminate acetamide to release ammonia. The production of ammonia results in a pH-driven color change of the medium from green to royal blue.
Purpose of IngredientsIt contains inorganic salts and acetamide as sources of carbon and nitrogen. Organisms growing in this medium metabolize acetamide, thereby liberating ammonia. This liberated ammonia can be detected by Nesslers reagent, which confirms Pseudomonas aeruginosa . Magnesium sulphate, ferrous sulphate and sodium molybdate are sources of ions that stimulate metabolism. Sodium chloride maintains osmotic equilibrium. Dipotassium hydrogen phosphate provides buffering to the medium.
ConsiderationsDo not stab the slant, since the test requires an aerobic environment. Do not inoculate from broth cultures, due to carryover of media. To avoid false-positive reactions, use a light inoculum to prevent carryover of substances from previous media.
ResultsPositive: Deamination of the acetamide resulting in a blue color (A)Negative: No color change (B)
http://himedialabs.com/TD/M1370.pdf
CETRIMIDEPurpose
It is used in the isolation and identification of Pseudomonas aeruginosa.Principle
It is the ability of an organism to grow in the presence of cetrimide, a toxic substance that inhibits the growth of many bacteria.
IngredientsEnzymatic Digest of Gelatin.................................................... 20 g Magnesium Chloride............................................................... 1.4 gPotassium Chloride................................................................. 10 gCetrimide (Cetyltrimethylammonium Bromide)........................ 0.3 gAgar ........................................................................................13.6 gGlycerol........10 mL
Purpose of IngredientsEnzymatic Digest of Gelatin provides the nitrogen, vitamins, and carbon in Cetrimide Agar. Magnesium Chloride and Potassium Chloride enhance the production of pyocyanin and fluorescein.7 Cetrimide (cetyltrimethylammonium bromide) is the selective agent. Cetrimide acts as a quaternary ammonium cationic detergent causing nitrogen and phosphorous to be released from bacterial cells other than Pseudomonas aeruginosa. Agar is the solidifying agent. Glycerol is supplemented as a source of carbon.
ConsiderationsIncomplete agar melting during preparation will invariably lead to significant inconsistency in the gel strength of the solidified agar, after sterilization and cooling.
This test is designed to differentiate bacteria on the basis of fermentative or oxidative metabolism of carbohydrates.
PrincipleIt is the ability of an organism to use carbohydrate substrates to produce acid byproducts.
IngredientsPeptone (tryptone) 2.0 gSodium chloride 5.0 gGlucose (or other carbohydrate)a 10.0 gBromthymol blue 0.03 g Agar 3.0 gDipotassium phosphate 0.30 gWater
Purpose of Ingredients Glucose is a sugar from which most common chemoheterotrophic bacteria can obtain energy by
fermentation and/or respiration. Glucose can also be utilized as a source of carbon, but these media include a large number of potential carbon sources (amino acids as well as glucose), and whether or not glucose is used as a carbon source cannot be directly determined from the reactions seen in these media.
Peptone is a commonly-used medium ingredient which mainly supplies amino acids (sources of nitrogen, carbon, sulfur and energy for many bacteria). A minimum amount of peptone should be incorporated in the medium, as overabundance of ammonium (alkaline) released from deamination of amino acids can neutralize the acids.
pH indicator: Brom-thymol blue turns yellow with a net acidic pH and blue with a net alkaline pH.Considerations
The recommended incubation condition is at 35°C for 48 hours for most gram-negative rods. Slow growing bacteria may take 3 to 4 days before results can be observed.
ResultPositive: Acid production (A) is indicated by the color indicator changing to yellow in the carbohydrate containing deep.Negative: Red or alkaline color in the deep with carbohydrate equal to the color of the inoculated control tube. No change (NC) or neutral (N): there is growth in the media, but neither the carbohydrate-containing media nor the control base turn alkaline (red).
Determine whether a gram negative rod utilizes glucose and lactose or sucrose fermentative and forms hydrogen sulfide
PrincipleThe TSI is a two reaction chamber with an aerobic slant portion and an anaerobic deep portion. The slant of the tube is exposed to atmospheric oxygen and will become alkaline due to oxidative decarboxylation of peptides and amino acids. The slant tends to become and remain alkaline (red). Amino acid degradation is minimal in the deep (anaerobic) portion, and thus a small quantity of acid produced can be detected because few amines are being formed from amino acids.
Purpose of ingredientsEnzymatic Digest of Casein, Enzymatic Digest of Animal Tissue, and Yeast Enriched Peptone provide the nitrogen, carbon, and vitamins required for organism growth. Triple Sugar Iron Agar contains three carbohydrates, Dextrose, Lactose and Sucrose. When the carbohydrates are fermented, acid production is detected by the Phenol Red pH indicator. Sodium Thiosulfate is reduced to hydrogen sulfide, and hydrogen sulfide reacts with an iron salt yielding the typical black iron sulfide. Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator. Sodium Chloride maintains the osmotic balance of the medium. Agar is the solidifying agent.
ConsiderationsStore sealed bottle containing the dehydrated medium at 2 - 30°C. Once opened and recapped, place container in a low humidity environment at the same storage temperature. Protect from moisture and light by keeping container tightly closed.
ResultsAn alkaline slant-acid butt (red/yellow) indicates fermentation of dextrose only. An acid slant-acid butt (yellow/yellow) indicates fermentation of dextrose, lactose and/or sucrose. An alkaline slant-alkaline butt (red/red) indicates dextrose or lactose were not fermented (non-fermenter). Cracks, splits, or bubbles in medium indicate gas production. A black precipitate in butt indicates hydrogen sulfide production.
To determine the presence/absence and location of flagella on various microorganismsPrinciple
A wet mount technique for staining bacterial flagella is simple and is useful when the number and arrangement of flagella are critical in identifying species of motile bacteria.
IngredientsSolution I - 10 ml of 5% aqueous solution of phenol, 2 g of tannic acid, and 10 ml of saturated aqueous solution of aluminum potassium sulfate-12 hydrateSolution II - 0.6% Crystal Violet in Ethanol, 2.0% Tannic Acid, 2.5% Phenol and 5.7% Aluminum Potassium Sulfate.
Purpose of IngredientsSolution I – act as a mordantSolution II - remove coarse precipitate.
ConsiderationsBe careful not to overheat sample, as excess heat will destroy the flagella.
ResultsPresence or absence of flagellaNumber of flagella per cellLocation of flagella per cell
http://jcm.asm.org/content/27/11/2612.full.pdf
PSEUDOMONAS ISOLATION AGARPurpose:
It is recommended for the selective isolation and differentiation of Pseudomonas aeruginosa.Principle:
The medium is designed to enhance pyocyanin production, thereby improving the differentiation of pseudomonads. The modified formula contains a low phosphorous content, glycerol, magnesium chloride and potassium sulfate, all of which promote pyocyanin production. Pyocyanin production is unique to Pseudomonas aeruginosa and is noted as a blue-green water soluble pigment that imparts a greenish color into the media.
Purpose of Ingredients:Glycerol acts as an energy source, while peptone provides nutrients necessary for bacterial growth. Irgasan is incorporated into the medium to inhibit the growth of many gram-positive and gram-negative microorganisms other than Pseudomonas spp.
Considerations:Upon receipt store at 2-8 degrees C. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.
