Diagnostic Media, Part II

BIOL 3702L - Diagnostic Media II Fall 2020

Purpose

• Understand the underlying mechanisms of BIOL 3702L: the various diagnostic tests presented; Diagnostic • Properly conduct these diagnostic tests; Media II • Accurately interpret the results of these tests; and • In future experiments, use critical thinking skills in concert with these skills to identify unknown specimens.

http://www.aviano.af.mil/News/Article- https://fems-microbiology.org/international-womens-day-8-march- Display/Article/724709/mdss-airmen- 2017/microbiologist-holding-a-petri-dish-with-bacteria-2/ Copyright © C. R. Cooper, Jr. ensure-success-through-support/ Copyright © C. R. Cooper, Jr. 1 2

Diagnostic Tests for This Week Triple Sugar Iron Test

• Triple Sugar Iron • Triple Sugar Iron (TSI) is a differential • Urease Test medium that determine a microbe’s ability • Citrate Utilization to ferment sucrose, glucose, and lactose in The latter tests, when addition to produce gas and/or hydrogen combined with the sulfide.

Tube A: positive indole test; Tube B: positive methyl red test; indole and the MR-VP Tube C: negative Voges-Proskauer test; Tube D: negative • TSI also uses phenol red to detect acid citrate result. tests are collectively Kamel and Jarjes. 2005. Essentials of Bacterioogy and production. Immunology termed the IMViC tests. • TSI contains ferrous ammonium sulfate

which forms a black precipitate with H2S. https://fems-microbiology.org/international-womens-day-8-march- https://fems-microbiology.org/international-womens-day-8-march- Copyright © C. R. Cooper, Jr. 2017/microbiologist-holding-a-petri-dish-with-bacteria-2/ Copyright © C. R. Cooper, Jr. 2017/microbiologist-holding-a-petri-dish-with-bacteria-2/ 3 4

Triple Sugar Iron Test (cont.) Triple Sugar Iron Test (cont.)

• TSI is inoculated by streaking the slant and stabbing the agar butt. • After incubation, observe the following: • Color of slant – (designated ‘K’); (designated ‘A’) • Color of butt – (designated ‘K’); (designated ‘A’) • Gas production – bubbles or cracks in agar or agar medium raised from bottom of tube Uninoculated TSI agar slant • H2S production – in agar https://fems-microbiology.org/international-womens-day-8-march- https://fems-microbiology.org/international-womens-day-8-march- Copyright © C. R. Cooper, Jr. 2017/microbiologist-holding-a-petri-dish-with-bacteria-2/ Copyright © C. R. Cooper, Jr. 2017/microbiologist-holding-a-petri-dish-with-bacteria-2/ 5 6

Dr. Cooper 1 BIOL 3702L - Diagnostic Media II Fall 2020

Triple Sugar Iron Test (cont.) Triple Sugar Iron Test (cont.)

Slant = ; Butt = ; Gas = bubbles; H2S= Slant = ; Butt = ; Gas = bubbles/cracks; H2S = none

https://fems-microbiology.org/international-womens-day-8-march- https://fems-microbiology.org/international-womens-day-8-march- Copyright © C. R. Cooper, Jr. 2017/microbiologist-holding-a-petri-dish-with-bacteria-2/ Copyright © C. R. Cooper, Jr. 2017/microbiologist-holding-a-petri-dish-with-bacteria-2/ 7 8

Triple Sugar Iron Test (cont.) Urease Test

• Detects microbes capable of breaking down urea into ammonia and carbon dioxide • Urease detected by a color change of the incorporated pH indicator • When urea is hydrolyzed, ammonia accumulates creating an alkaline (increased pH) environment • Change to a deep pink or purple red (cerise) color indicates a positive test Slant = ; Butt = ; Gas = none; H2S = none • Yellow color appears, then test is negative

Urease Test (cont.) Urease Test (cont.)

• Two tests to be conducted: • Urease Wee-Tabs • Urease/Indole

• Urease Wee-Tab Procedure • Place a tab in 1 ml water • Inoculate heavily • Incubate at 37°C for 1-24 hours Positive (right image) and negative (left • Observe for color change image) urease test results

Dr. Cooper 2 BIOL 3702L - Diagnostic Media II Fall 2020

Urease Test (cont.) Citrate Test

• Urease/Indole Procedure • Simmons Citrate Agar differentiates among • Place 0.5 ml water per tube various Gram-negative enteric bacilli based • Inoculate heavily upon their ability to utilize citrate. • Incubate at 37°C for 1-6 hours • This medium contains ammonium • Observe for color change for up to 6 hours dihydrogen phosphate (nitrogen source) • Test for indole production by adding 1-2 drops of Kovacs’ reagent and sodium citrate (sole carbon source). • Positive result is formation of a red layer • Also, this medium contains the pH indicator • Negative result is no red layer formation bromothymol blue, which is in color under alkaline conditions.

Citrate Test (cont.) Citrate Test (cont.)

• This test detects growth and citrate utilization as a carbon source. • Metabolism of citrate produces an alkaline carbonate. • As a result, the pH of the medium increases, i.e., becomes more alkaline. • The medium turns due The left image depicts a negative citrate reaction, whereas the right to pH change in the presence of image shows growth and a positive citrate utilization reaction. bromothymol blue.

https://www.huffingtonpost.com/barbara- jacoby/asking-questions-is-really-hard_b_7052722.html Lab Report Expectations

• Complete each of the report sheets for each given exercise. • Share data where appropriate, but do not Questions? plagiarize! • Answers to discussion questions must be clear, concise, and grammatically sound. • Retain these laboratory exercise reports. Your laboratory instructor may not call for them at this time, but may do so in the near future.

Dr. Cooper 3