Chapter 9: Biochemical tests for gram-negative bacteria

What is lactose?

• a disaccharide, made up of glucose+galactose

How is lactose degraded by bacteria?

• β-galactoside Permease: transports lactose across the cell wall

• β-galactosidase: breaks down lactose into glucose and galactose

What happens if a bacteria has β-galactosidase but not the permease?

• It will be a delayed lactose fermenter (dLF)

What is the fermentation pathway?

• gycolysis pathway that converts glucose to pyruvate anaerobically

• uses oxygen in an organic molecule such as NO3

• this process produces acid products, pH indicator detects acid

What is the oxidation pathway?

• glycolysis pathway to convert glucose to pyruvate aerobically

• pyruvate is fully oxidized to CO2

• produces weak acid, neutralized by peptones in agar and it shows a neutral

O/F Basal Medium (Oxidation/Fermentation)

• contains low conc of peptone

  • increased sugar content to detect small amounts of acid

• uninoculatred medium is green

• turns yellow with acid

• turns blue with alkaline

O/F Medium

1. Both tubes are yellow, producing acid, so this is a fermenter

2. Oil overlay tube is negative while open tube is acidic, so oxidizer/nonfermenter

3. Both tubes are unchanged, so nonfermenter/nonoxidizer

What sugars does a TSI agar (Triple Sugar Iron) contain?

• Glucose, lactose and sucrose

What sugars does a Kligler Iron Agar (KIA) contain?

• Glucose and lactose

TSI & KIA

• both contain ferrous sulfate and sodium thiosulfate

  • detects H2S production

• phenol red: yellow=acid and red=base

• slant is aerobic

• butt is anaerobic

How do you read a TSI/KIA agar?

• read as slant/butt

  • alkaline/alkaline = K/K

  • acid/acid = A/A

• H2S is noted based on bubbles/splitting and black color

What is the purpose of the ONPG test?

• To determine if the organism is a dLF

• Ortho-nitrophenyl-β-D-galactopyranoside structurally resembles glucose, and will permeate the plasma membrane easily

ONPG Test

• A positive result is yellow, because the bacteria hydrolyzes ONPG into galactose and O-nitrophenol

• A negative result is clear

What is the Embden-Meyerhof pathway?

• fancy way to say glycolysis

• glucose —> pyruvate, and further degradation to mixed acids

What are the two seperate pathways that bacteria use to further degrade pyruvate?

1. Mixed acid pathway

2. Butylene glycol pathway

What test is used to detect the Mixed acid pathway?

• Methyl Red (MR) test

  • broth contains glucose and methyl red

  • negative = yellow

  • positive = red

What test is used to detect the Butylene Glycol pathway?

• Voges Proskauer (VP) pathway

  • glucose broth with added 40% KOH & 5 % α-Naphthol

  • Negative = Yellow

  • Positive = Red

• Red because acetoin is oxidized to diacetyl; this diacetyl reacts with the KOH and a-naphthol to form a red complex

What are the enzymes that bacteria can degrade for energy?

1. lysine

2. ornithine

3. arginine

What is the process for the lysine decarboxylase test?

• lysine + lysine decarboxylease —> cadaverine + CO2

What is the process for the ornithine decarboxylase test?

• ornithine + ornithine decarboxylase —> putrescine + CO2

What is the process for the arginine dihydrolase test?

• Two step process

  1. Arginine + Arginine dihydrolase—> Agmatine + CO2

  2. Agmatine —> Putrescine + Urea

What are the characteristics of the Moeller Decarboxylase test?

• Standard agar is purple with a pH of 6

  • contains two indicators, small amount of glucose, peptones and 1% specific amino acid

• Initial fermentation drops pH, turns agar yellow

• For decarboxylation to occur, two conditions must be met

  1. Anaerobic environment (mineral oil on top0

  2. Acidic environment (due to fermentation of glucose)

• Decarboxylation of AA causes alkaline pH shift, turning it back to purple

• Positive = Purple

• Negative = Yellow

What are the characteristics of the Phenylalanine Deaminase Test? (PAD)

• Bacteria can also remove the NH2 group of AA for energy

• Tests if bacteria have enzyme to convert phenylalanine —> phenylpyruvic acid

• Slant is normally clear/slight yellow with small amount of phenylalanine

• Colony is innoculated onto the slant, allowed to grow, then 10% Ferric Chloride is added after time has passed

• Green = Positive (Phenylpyruvic acid is present)

• Not green = Negative

Which 3 bacteria of the Enterobacterales are positive for PAD?

1. Proteus

2. Morganella

3. Providencia

What are the characteristisc of the Lysine Iron Agar Slant?

• Bacteria can decarboxylate or deaminate lysine

• slanted agar contains lysine, glucose, ferric ammonium citrate and sodium thiosulfate

• Decarboxylation of lysine occurs anaerobically

  • Positive = Dark purple butt, H2S production turns butt black

• Deamination can also occur

  • Positive = yellow butt (glucose fermentation) and reddish-purple or plum color

Which are the two tests that can help differentiate Proteus, Morganella and Providencia from the other Enterobacterales?

1. Phenylalanine Deaminase Test (PAD)

2. Lysine Iron Agar (LIA)

What is unique about the way Proteus, Morganella and Providencia metabolize amino acids?

• These organisms deaminate the amino aicd instead of decarboylate

  • remove -NH2 instead of -COOH for energy

What are the characteristics of the citrate utilization test?

• Checks to see if an organism can use sodium citrate as a sole carbon source

• Media contains citrate, ammonium salts and pH indicator

• If the bacteria can utilize citrate as carbon source, then they will also utilize ammonium as nitrogen source

• Breakdown of ammonium salts produces ammonia, which turns the pH indicator blue due to alkaline shift

What are the characteristics of the Dnase test?

• most Dnase are endonucleases, but some are exonucleases that can be produced by bacteria such as Staph aureus and Serratia marascens.

• Test media is innoculated with DNA, then streaked with bacteria

• After incubation, 1N HCL added to plate.

• Clear zone around streak = positive, because broken down DNA is soluble in HCL

• Precipitate = negative result, because whole DNA is insoluble in HCL

What are the characteristics of the Indole test?

• Checks for the presence of bacteria that contain the enzyme to deaminate tryptophan

  • leads to byproducts Indole, pyruvic acid and ammonia

• Bacteria are inoculated in a peptone and tryptophan broth, then incubated

• PDAB is added to broth

• Positive = Red

What is the purpose of the nitrate—>nitrite reduction test?

• to check and see if an organism can reduce nitrate to nitrite, and then further nitrite to N2 gas

What is the reaction of the nitrate and nitrite reduction test?

• KNO3 + nitrate reductase —> nitrite

  • two reagents can be added to detect nitrite, produce red color

• Nitrite + Salfanillic acid + N,N-Dimethyl-a-naphylamine—>diazo red dye

What are the two possible outcomes if the no color is seen on the nitrate/nitrite reduction test?

1. Organism cannot reduce nitrate

2. Nitrate has been reduced passed Nitrite to N2, NO or N2O

What is the purpose of zinc dust in the nitrate/nitrite reduction test?

• zinc dust will reduce nitrate to nitrite

  • therefore after addition of zinc to negative result, if a red color is produced, nitrate is reduced. this means that the organism is a true negative and cannot reduce nitrate.

  • If there is no color change after the addition of zinc dust, this indicates a positive result. This means that nitrate was originally present, and reduces all the way down to a gas.

What is the purpose of the oxidase test?

• To check and see if an organism contains the enzyme cytochrome C oxidase

• Most enterobacterales are oxidase negative

How is the oxidase test performed?

• Kovac’s reagent and rapid spot oxidase tests are used

• filter paper is impregnated with reagent, dropped onto bacterial colony, and a positive result turns the paper purple

Which enterobacterales organism is oxidase positive?

• Plesiomonas

What is the purpose of the Urease test?

• to see if an organism can hydrolyze urea to produce ammonia

  • (NH2)2CO + H2O —> 2NH3 + CO2

• surface of agar (Christensen’s agar) is inoculated, not stabbed

• phenol red is indicator

• Positive = Bright pink color becuase alkaline pH

• Negative = Yellow color because acidic pH