Tests for gram negative bacteria

Colony isolation on TSA

• Gram negative

• Media: tryptic soy agar

• Tool: Inoculation loop

• Streaked by quad or t streak

• Results: Colony morphology

MacConkey Agar

• Gram negative

• Tool: inoculation loop

• Streak out bacteria (quad or t streak)

MacConkey agar Biochemical Basis

• Selective: Bile salts (and crystal violet) inhibit growth of all gram positive bacteria, gram negative cocci

• Only gram negative bacilli will grow

• Differential: Lactose is the sole carbon source available

• If bacteria ferments lactose, acidic metabolic products produced: pH decrease

• A pH indicator in the media (neutral red dye) will detect drop in pH and change the bacterial growth to pink/red

MacConkey agar results

• Positive for lactose fermentation=red bacterial growth/colonies

• Negative for lactose fermentation=white bacteria growth/colonies

DNAse

• Gram negative bacteria

• Media: DNAse agar plate

• Tool: Inoculation loop

• Inoculate 2 bacteria on each plate (in a single line)

DNAse Biochemical Basis

• Exoenzyme that hydrolyzes DNA

• Contains DNA

• If an organism is positive for DNAse enzyme, the enzyme will depolymerize the DNA in the media

• After incubation, flood plate with 1N HCL. HCL will complex with and form white precipitate with DNA

• If DNAse is produced and secreted by bacteria, it will clear the DNA in the area of the bacterial streak and no precipitate will form

DNAse results

• Positive for DNAse=clearing around bacterial streak

• Negative for DNAse= White precipitate around streak, confluent with the rest of the plate

Phenol red glucose fermentation

• Gram negative bacteria

• Media: Phenol red glucose broth (pink broth in a red-top tube with a small inverted glass durham tube in the broth)

• Inoculation: Swish bacteria in broth

Biochemical Basis of Phenol Red Glucose Fermentation

• If the bacteria has the ability to ferment the sugar glucose, it will produce acidic metabolic products, and the pH of the media will decrease. A pH indicator in the media (phenol red) will detect the drop in pH and change the color of the media to yellow

• If gas is produced during fermentation, a bubble of gas will trapped in durham tube

Results of Phenol Red Glucose Fermentation

• Positive results: glucose fermentation= yellow color change in the broth

• Gas production= Bubble in durham tube

• Negative results: Broth is reddish/orange, no visible bubble

Sulfur-indole-motility (sim test)

• Gram negative bacteria

• Media: SIM agar

• Tool: inoculation needle

• Stab the bacteria into the agar deep with the inoculation needle, and then draw the needle out along the same path

• After 48 hours incubation, add several drops of kovac’s reagent to the tube in order to read the indole portion of the test

Biochemical basis of SIM test

• S: sulfur reduction

• I: indole production

• M: motility

Sulfur reduction (SIM)

• Tests the ability of the bacteria to reduce sulfur via catabolism of the amino acid cysteine by the enzyme cysteine desulfurase. If hydrogen sulfide is produced, it will combine with iron in the medium to form ferric sulfide, a black precipitate

• Positive result= black precipitate

Indole production (SIM)

• Bacteria that have the enzyme tryptophanase are able to hydrolyze the amino acid tryptophan in the media, converting it to indole, pyruvic acid, ammonia. After 48 hours, kovac’s reagent is added which reacts with any indole that is present to produce a red quinoidal compound

• Positive result: Kovac’s turns bright red

• Negative result: Kovac’s is straw-colored

Motility (SIM)

• If bacteria is flagellated, and therefore motile, growth will radiate from the stab

• Positive result: Bottle brush appearance or the tube will be turbid/cloudy (or black if H2S is also present)

• Negative result: bacterial growth restricted to stab mark

Citrate test

• Gram negative bacteria

• Media: Simmons citrate agar (green agar slant)

• Tool: Loop

• Glide loop with bacteria up slant

Citrate test biochemical basis

• Bacteria will only grow on simmons citrate agar if they have the ability to utilize exogenous citrate as its sole carbon source. This property is dependent upon two enzymes in the bacterial cell

  • Citrate permease: allows for transport of citrate into the cell

  • Citrase (citrate lyase) ultimately converts citrate into alkaline carbonates

• Citrate (a Krebs cycle intermediate) is generated by many bacteria; however, utilization of exogenous citrate requires the presence of citrate transport proteins (permeases).  Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO₂. The CO₂ that is released will subsequently react with water and the sodium ion in the medium to produce sodium carbonate, an alkaline compound that will raise the pH.  The media contains Bromothymol blue as a pH indicator.  Bromothyol blue is normally green at the neutral pH of uninoculated media, but will turn royal blue at more alkaline pH above 7.6. 

Results of the citrate test

• Positive result: bacterial growth and media color change to royal blue

• Negative result: no bacterial growth and media remains green

Urease test

• Media: Urea agar (orange colored agar slant)

• Tool: loop

• Inoculation: Glide loop with bacteria up slant

Biochemical basis of urease test

• Used to identify bacteria capable of hydrolyzing urea using the enzyme urease

• Urease hydrolyzes urea in the media, forming the weak base, ammonia as one of its products

• Alkaline ammonia raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink

Results of urease test

• Weak positive result: Bright pink growth

• Negative result: Orange color of original media, sometimes yellow

• Strong positive result: Entire tube turns bright pink

Triple Sugar Iron (TSI) test

• Media: TSI slant (pink agar slant)

• Tool: inoculation needle

• Inoculation: Inoculate both the butt and slant of the agar, first stab the bacteria into the butt of the agar deep. Next as you withdraw the needle, streak the slant with the bacteria as well

Biochemical basis of TSI: Sulfur reduction

• Incorporates the same principles as the phenol red fermentation test, as well as the sulfur reduction portion of the SIM test

• Tests the ability of the bacteria to reduce sulfur by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase.  If hydrogen sulfide is produced, it will combine with iron in the medium to form ferric sulfide, a black precipitate.   Positive Result = black precipitate 

Biochemical basis of TSI: Sugar fermentation

• As with other phenol-red based sugar fermentation tests, fermentation is indicated by a yellow color change in the medium. TSI media contains the sugars lactose, sucrose, and small amounts of glucose. If the bacteria can only ferment glucose, the small amount of glucose in the media fermented relatively quickly, and following this fermentation the reaction in the aerobic environment of the slant produces an alkaline environment.  The media contains phenol red as a pH indicator, and therefore if only glucose fermentation occurs, the butt will appear yellow, and the slant will appear red.  If the bacteria is able to ferment lactose or sucrose in addition to glucose, this will occur throughout the tube, and both the butt and slant will appear yellow.    

TSI test results

• Yellow or black butt: positive for glucose fermentation

• Yellow slant: positive for lactose or sucrose fermentation

• Black precipitate: positive for H2S production (sulfur reduction)

Oxidase test

• Reagent: Oxidase reagent soaked on white filter paper

• Tool: Sterile wooden dowel

• Inoculation: Pick up a substantial sample of bacteria on the wooden dowel and transfer it to the filter paper that has been soaked with oxidase reagent.  Wait no more than 10 seconds to observe color change to dark purple.   A color change after 10 seconds is not a valid result 

Biochemical basis of oxidase

Tests for: the enzyme cytochrome oxidase as part of the electron transport chain 

• In the cell, cytochrome oxidase transfers electrons from the ETC to oxygen, reducing oxygen to water 

• Oxidase test utilizes artificial electron donors and acceptors 

• When the electron donor is oxidized by cythochrome oxidase, it turns dark purple (positive) 

Results for oxidase test

• Positive result: Dark purple spot on the filter paper forms immediately

• Negative result: No color change