Unknown Project Day 1 Material

How to do the lactose and sucrose fermentation test

*hint- these are the ones with the Durham tube inside

1. Using sterile technique, perform a solid to liquid media transfer using your unknown organism plate culture and the lactose broth. 

2. Be sure not to disturb the durham tube when performing the inoculation

*make sure to gently touch the colony on the agar, and to touch a blank surface to make sure inoculating loop is cool

How to do the SIM media inoculation

1. Sterilize the inoculating needle

2. Aseptically pick up one colony of your unknown culture with the inoculating needle

3. Transfer to the SIM agar deep media via single stab inoculation (make sure you only stab directly into the agar deep in a straight line once)

4. Re-sterilize the inoculating needle

*stab only 2/3rd of the way through (DON’T TOUCH THE BOTTOM)

How to do the MR/VP test

Perform a solid to liquid media transfer using your unknown organism plate culture and the MR/VP broth.

*this will all go in one tube on Day 1, then split on Day 2

How to do the Citrate test

Perform a solid to slant culture transfer to inoculate the Simmons Citrate media.

*use inoculating loop

How to do the Urease test

Perform a solid to liquid media transfer using your unknown organism plate culture and the urease broth.

How to complete the catalase test

1. Divide your unknown culture plate into two sections by drawing a line down the middle on the bottom of the plate 

2. Add two or three drops of hydrogen peroxide (H2O2) to the surface of the growth of the unknown organism on the LEFT side of the plate.

3. Observe and record results (see if there are bubbles or no bubbles).

How to complete the oxidase test

1. Add two or three drops of the p-aminodimethylaniline oxalate to the surface of the growth of the unknown organism on the RIGHT side of the plate.

2. Observe color change if any within 10 to 30 seconds, record results.

How to do the streak plate

1. Quadrant 1: Dip sterile swab in broth, squeeze against tube wall, and streak 1/4 of the plate. Discard swab.

2. Quadrant 2: Flame loop, cool it. Drag twice through Q1, then streak Q2. Flame loop.

3. Quadrant 3: Flame loop, cool it. Drag twice through Q2, then streak Q3. DO NOT FLAME.

4. Quadrant 4: Drag once through Q3, then make wide "S" motions to fill the rest of the plate. Flame loop.

How to label test tube and agar plate

Include organism name (e.g., E. coli), date, your initials, and group number.

How to do Gram stain

1. Prepare Smear & Fix: Spread a thin layer of bacteria on a slide, air dry, and heat-fix by passing it over a flame 3–4 times.

2. Primary Stain (Crystal Violet): Flood the slide with crystal violet for 1 minute. Rinse gently with water. Cells are now purple.

3. Mordant (Iodine): Flood with Gram’s iodine for 1 minute. Rinse gently with water. Cells remain purple, iodine forms complex with dye.

4. Decolorize (Alcohol/Acetone): Hold the slide at an angle and add decolorizer for 5–10 seconds (or until runoff is clear). Crucial step: Too long will make positive look negative. Rinse immediately.

5. Counterstain (Safranin): Flood with safranin for 30–60 seconds. Rinse gently with water.

6. Dry and View: Blot dry with bibulous paper and view under 100x oil immersion.