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22 terms

Microbiology Lab: Biochemical Tests

Summary of the types of biochemical tests used in lab
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Anaerobic growth
Purpose: Differentiates microorganisms based on their ability to grow in the absence of oxygen.
Procedure: Inoculate TSA and streak for isolation; incubate 48h at 37C in an anaerobic jar.
Positive: see growth
Negative: no growth
BHI with 6.5% NaCl
Purpose: Differentiates microorganisms based on their ability to grow in 6.5% NaCl.
Positive: Turbidity (growth)
Negative: No growth (clear)
Bile esculin hydrolysis
Purpose: Differentiates based on ability to hydrolyze esculin to esuletin and dextrose. The esculetin reacts with ferric citrate in the medium to form a dark brown/black complex.
Positive: Blackening of the slant.
Negative: No color change.
Citrate
Purpose: Differentiates based on ability to utilize citrate as a sole carbon source. Uses a pH indicator called bromthymol blue. Organisms that are able to grow on the media will produce an alkaline reaction.
Positive: Growth with an intense blue color.
Negative: No growth/color change (remains green).
Coagulase
Purpose: Detection of the protein coagulase that is excreted by certain Staphylococcus species that causes fibrin clot formation. Inoculate a loop of organism into a coagulase (rabbit plasma) tube and incubate at 37 degrees. Positive: coagulation or clot formation (any degree).
Cystine Tryptic Agar (CTA) Sugars-Arabinose, Lactose, Mannitol, Raffinose, Salicin, Sucrose, Trehalose, Xylose
Purpose: Detection of carbohydrate utilization in microorganisms. Carbohydrate utilization is detected by the incorporation of the pH indicator, phenol red, in the medium. When the carbohydrate is metabolized, the medium is acidified resulting in a color change from red to yellow. Inoculate and incubate 48h at 37C. Positive: yellow color. Negative: no change in color.
Decarboxylation Reactions (Arginine, Lysine, Ornithine)
Principle: Dextrose, the amino acids arginine, lysine or ornithine and the pH indicator bromcresol purple are incorporated into the media. Following the fermentation of dextrose, there is a drop in the pH and a change of color from purple to yellow. If the organism can decarboxylate the amino acid, there is a rise in the pH due to the production of an amine that is an alkaline end product.
Arginine dihydrolase (decarboxylase)
Purpose: Detection of the enzyme arginine dihydrolase which hydrolyzes the amino acid arginine to ornithine, which is then decarboxylated to form the amine putrescine and carbon dioxide. Must put sterile mineral oil on the top of the medium after inoculating and cap should be tight during incubation. Positive: Purple color Negative: yellow
Lysine decarboxylase
Purpose: Detection of the enzyme lysine decarboxylase that degrades lysine to the diamine cadaverine and carbon dioxide. Mineral oil and cap tight as in previous test. Positive purple; negative yellow.
Ornithine decarboxylase
Purpose: Detection of the enzyme ornithine decarboxylase that degrades ornithine to the amine putrescine and carbon dioxide. Mineral oil and caps tight. Purple positive, yellow negative.
Hemolysis on SBA
Differentiates microorganisms based on their hemolytic activity on sheep blood agar. Alpha: incomplete hemolysis, greening of the media. Beta: complete hemolysis, clearing of the media. Gamma hemolysis: no hemolysis or discoloration of the media.
Methyl red
Purpose: Differentiates microorganisms based on their ability to produce and maintain stable acid end products from glucose fermentation. Inoculate an MRVP broth. Incubate 48-72 hours at 37C. Withdraw 1mL and transfer to a serological tube; add 5 drops of methyl red indicator. Positive: Immediate red color development. Negative: yellow color development.
Novobiocin
Differentiates microorganisms based on susceptibility to novobiocin. Inoculate an .85% saline tube with organism to a density equivalent to a .5 McFarland Standard. Dip a sterile swab into the suspension then inoculate a Mueller-Hinton agar plate with a lawn streak of the organism. Using sterile forceps apply a 5 microgram disc of novobiocin to the center of the plate. Incubate 37C for 24h. Susceptible: Zone of inhibition greater than 17mm. Resistant: zone less than 17mm.
O/F Dextrose (glucose) or trehalose (oxidation-fermentation reaction)
Differentiates microorganisms based on oxidative or fermentative metabolism of carbohydrates. Stab or inoculate two tubes per organism and leave on exposed to air (oxidative) and one with sterile mineral oil (fermentative). Oxidative is yellow color in open tube only, Fermentative is yellow in both tubes, and if it does neither, there will be a green or blue color in both tubes.
Optochin (P) disc
Differentiates microorganisms based on susceptibility to ethyl hydrocupreine hydrochloride. Steak organism evenly across the blood agar plate. Apply P disk to streaked area and incubate at 37C. Susceptible will have a zone of inhibition 14mm or larger.
Penicillin susceptibility
Same as all the other antibiotics-any zone of inhibition indicates that the organism is susceptible.
Production of red pigment at 25C
Just inoculate TSA and incubate at 25C for 48h. Positive will produce red pigment (S. marcescens is positive).
Purple broth fermentation reactions (arabinose, lactose mannitol, sucrose)
Differentiates members of Enterobacteriaceae based on carboydrate fermentation. Contains specific carbohydrates and the pH indicator bromcresol purple-when an organism ferments the carbohydrate, an acidic product is produced, which changes the indicator to yellow. A bubble in the tube indicates gas production.
SIM (Sulfur, indole, motility)
Differentiates microorganisms based on hydrogen sulfide production, indole production and motility. Stab the SIM tube about 2/3 of the way to the bottom and incubate for 48 with caps loose.
H2S production: blackening along stab line
Indole: after incubation, add 3-4 drops of Indole or Kovac's reagent-positive will show a red color.
Motility: positive will diffuse outward from the stab line.
Swarming motility
Differentiates based on ability to spread across a plate demonstrating a swarming type of motility.
Urea
Differentiates organisms based on urease production. The enzyme urease will hydrolyze urea yielding ammonia. Ammonia is alkaline an din the presence of phenol red, it will change the color from light orange to a deep reddish pink color.
Voges-Proskauer
Differentiates based on ability to produce a neutral end product, 2,3 butanediol from glucose fermentation. This end product is oxidized to form acetoin that is detected with the addition of the VP reagents alpha-napthol and KOH.
Inoculate MRVP with test organism and incubate 72 hours; add the contents of VPA and shake, then add 5 drops of VP B and shake. Positive will show a red color development within 15 minutes.