of Identification Bacteria: Unknowns
Students should add 10-15 drops of each reagent directly to the test tubes for the MRVP tests. Students should be sure to vortex the media for each test after the reagents have been added and prior to reading the test results. For the MRVP tests: Methyl Red for the Methyl Red test. Barritt's Reagents: -Naphthol and then KOH for the Voges-Proskauer test
Day#4: Hydrolysis Tests
Materials: (per student) 1 Urea broth (5ml) (Ex. 5-15) 1 Tryptone broth (5ml) (Ex. 5-20) 1 Spirit Blue Agar plate (25ml) (Ex. 5-19) 1 Milk Agar plate (25ml) (Ex. 5-16) 1 Starch Agar plate (25ml) (Ex. 5-13) TSB cultures of: S. aureus E. coli E. tarda
Students will be inoculating controls on the agar plates opposite their unknown as indicated in the figure below. Controls will not be inoculated for the Urea and Tryptone broths. All tube media will be incubated at 37oC for 48 hours. All agar plates will be incubated at 37oC for 24 hours.
Positive Control Unknown Negative Control
Hydrolysis Tests:
Urea Hydrolysis: Tests for: the presence of urease which can hydrolyze urea into ammonia and CO2 Reagent: - pH indicator: Phenol Red for detecting ammonia production; pH of media will rise as ammonia is produced; (red for pH >7 and yellow for pH <7) Results: - positive: red (fuschia) color change - negative: yellow (orange) color Tryptone Hydrolysis: Tests for: presence of tryptophanase which can hydrolyze tryptophan into indole and pyruvic acid Reagent: - Kovac's Reagent (p-dimethylaminobenzaldehyde); used for detecting indole Results: - positive: red ring at the top of the media - negative: no red ring
Hydrolysis Tests:
Fat Hydrolysis (Spirit Blue Agar): Tests for: the presence of lipases which can hydrolyze fats into glycerol and fatty acids Reagent: - media: Spirit Blue Agar (which contains lipids) Results: - positive: dark precipitate on the growth; depletion of the lipids around the line of inoculation - negative: no dark precipitate on growth; no depletion of lipids
Casein Hydrolysis (Milk Agar): Tests for: the presence of caseinase which can hydrolyze casein (the predominant protein in milk that gives it its white color) Reagent: - media: Milk Agar Results: - positive: clear zone around growth - negative: no zone round growth (remains milky-white)
1
Hydrolysis Tests:
Starch Hydrolysis: Tests for: the presence of amylases which can hydrolyze starch Reagent: - media: starch agar - iodine Results: - positive: clear zone around growth - negative: no clear area around growth and media is stained brownish-blue by the starch-iodine complex
Ex 5-29: API 20E Identification System for Enterobacteriaceae and Other Gram-Negative Rods (pgs. 220-228) The API 20E system is a miniaturized version of many of the conventional tube tests that we have used in the identification of your Bacterial Unknown (a Gram-negative rod that is a member of the Enterobacteriaceae). The uses system a plastic test strip with 20 microtubes and cupules. Dehydrated media is present in each microtube, that is rehydrated and inoculated with a bacterial suspension. After incubation, the reactions are recorded. Test reagents are added, where necessary, and the results are recorded on the result sheet as + or and then tabulated to reveal a 7-digit code.
Materials: (per group of students with the same unknown) TSA plate of bacterial unknown (use the student plate that has the most isolated colonies) 0.85% saline blank (9.9ml) API 20E Test strip Inoculation of the Test strip: 1. Using the water bottle on your bench, fill the depressions in the tray of the incubation box to create a humid environment. Do not overfill the tray or water will wash over the test strip and contaminate your results. 2. Using an inoculating loop, obtain several isolated colonies and prepare a saline suspension of your bacterial unknown. The suspension should be rather turbid. 3. Using a sterile pasteur pipet, fill all of the tubes in the test strip with your bacterial suspension. Be sure not to introduce any bubbles into the tube that may prevent the bacteria from rehydrating the media in the bottom of the tube. For tests CIT, VP, and GEL: fill both the tube and cupule with the bacterial suspension For tests ADH, LDC, ODC, H2S, and URE: using another sterile pasteur pipet, and fill the cupule with sterile mineral oil to provide an anaerobic environment. 4. Place the test strip in the tray and place the lid on the incubation box. 5. Place the test strip in the basket on the TA bench for incubation.
2
Recording results of test strip: 1. Record the results of the spontaneous reactions referring to Table 5-34 (pgs204-205) of your lab manual. Place a + on the results sheet where a positive reaction occurred and a on the results sheet where a negative reaction occurred. 2. 3. Add one drop of ferric chloride to the TDA tube. A positive reaction is a brown-red color. A negative reaction is a yellow color. Add one drop of Barritt's reagents (-naphthol and KOH) to the VP tube. A positive reaction is a pink or red color that appear within 10 minutes. Add one drop of Kovac's reagent to the IND tube. A positive reaction is a red ring within 2 minutes. Add two drops of sulfanilic acid and N,N dimethyl-1-naphthylamine to the GLU tube. A positive reaction is a red color within 2 to 3 minutes. Confirm the negative through the addition of zinc. Add all of the positive results within each group and enter the number in the circle on the results sheet. Using the Analytical Profile Index, locate your seven-digit number to determine the identification of your bacterial unknown.
4. 5.
6. 7.
Hydrolytic Reactions: Inoculations:
Urea Broth
Tryptone Broth
S. aureus (+) Unknown E. coli (-)
E. tarda (+) Unknown E. coli (-)
E. tarda (+) Unknown E. coli (-)
Spirit Blue Agar
Starch Agar
Milk Agar
3