BIOD171 Lab 7 Notebook

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School

Santa Fe College *

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171

Subject

Biology

Date

Dec 6, 2023

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pdf

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Uploaded by CommodoreSummer12164

Lab 7 Notebook Back to Home Page Title: KD07 Secondary Assays for Bacterial Characterization Objective: To profile bacterial populations based on additional biochemical approaches to better determine growth /metabolic characteristics. Procedure: Indole Test: Biochemical test to determine a bacterial species ability to convert (breakdown) tryptophan into indole. Test #1 (4 quadrants on a card) 1. Using a sterile loop pick an isolated colony from BAP 2. Smear directly onto the reaction area of the slide 3. Examine test are for color change within 20 seconds *color is not always perfect; may get a slightly different/not as strong color Test #2 (liquid culture) 1. Using a sterile loop pick an isolated colony from BAP 2. Inoculate bacteria in media broth 3. Grow at 37 ° C overnight 4. Add ~ 5 drops of Kovac’s reagent to culture TSI Test: (Triple Sugar Iron) agar is used to assess ability of bacteria to ferment sugars and/or produce hydrogen sulfide

1. Use a sterile loop to inoculate slant from liquid culture 2. Press loop into the agar slant 3. While removing loop, streak slanted slide of agar with loop 4. Grow at 37 ° C overnight 5. Next day examine samples API Test: Broad, multi-panel test used for rapid characterization of bacterial species 1. Fill all tubes with bacteria culture · Fill CIT, VP, and GEL entirely · Add mineral oil ADH, LDC, ODC, H2S, and URE = anaerobic 2. Add 5 mL distilled water into tray, add API strip and cover with lid 3. Incubate at 37 ° C overnight 4. Next day examine samples Notes: Indole Test #1: (breaks down or converts tryptophan into indole) Indole negative = yellow color Indole positive = red/pink orange color Indole Test #2: *cloudiness culture has grown *bright pink band indole was produced (strong +) *light pink band indole was produced but weaker (weak +) *help to know bacterial physiology – such as spore formation/drug resistance: if they are able to breakdown tryptophan into indole more resistant to drugs (antibiotics) and survive in harsh conditions (spore formation)

TSI Test: (ph indicator slants are red; if it stays red then it is negative) *if bacteria is able to ferment sugars (glucose, fructose, sucrose) the color will change; if it produces hydrogen sulfide gas then the tube turns dark *yellow to pink then it is a positive test API Test: *blue arrow needs to cover in mineral oil to create anaerobic environment Results: Indole Test: breaks down tryptophan down into indole Positive test color changes to pink Negative test color doesn’t change stays yellow 1. E. coli (gram negative) is positive for indole (turned pink) 2. Pseudomonas (gram negative) is negative for indole (stayed yellow) 3. Salmonella (gram negative) is negative for indole (stayed yellow) TSI Test: breaks down (ferments) glucose and/or produces hydrogen sulfide gas Control is red – change to yellow then positive for glucose fermentation Turns black then positive for production of hydrogen sulfide gas 1. E. coli (gram negative) yellow, solid slant, positive for fermenting glucose (3 sugars); negative for hydrogens sulfide gas (but did produce a gas) 2. Salmonella (gram negative) yellow slant in bottom positive for fermenting glucose; turned black in top of slant positive for producing hydrogen sulfide ASI Test: broad test Sample 1: 5045550

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ONPG POSITIVE AHD NEGATIVE LDC POSITIVE OCD NEGATIVE CIT NEGATIVE H2S NEGATIVE URE NEGATIVE TDA NEGATIVE IND POSITIVE VP POSITIVE GEL NEGATIVE GLU POSITIVE MAN POSITIVE INO NEGATIVE SOR POSITIVE RHA POSITIVE SAC NEGATIVE MEL POSITIVE AMY NEGATIVE ARA NEGATIVE Sample 2: 2206002 ONPG NEGATIVE AHD POSITIVE LDC NEGATIVE OCD NEGATIVE CIT POSITIVE

H2S NEGATIVE URE NEGATIVE TDA NEGATIVE IND NEGATIVE VP NEGATIVE GEL POSITIVE GLU POSITIVE MAN NEGATIVE INO NEGATIVE SOR NEGATIVE RHA NEGATIVE SAC NEGATIVE MEL NEGATIVE AMY NEGATIVE ARA POSITIVE

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