Week 2, Day 2

May 21, 2013

Today in lab we started with endospore staining and capsule staining. After entering the lab, getting our lab coats, washing our hands, and cleaning our stations, we retrieved our samples and gathered the blank slides needed for the staining. While the water needed for preparing our endospore stain was set up to boil, we began our capsule stain. The capsule stain is similar in preparation to the negative stain. We prepared a blank slide with a very small drop of nigrosin and mixed some of our bacteria sample into the drop with an inoculating loop.

 We smeared the drop across the slide using the edge of another blank slide.

Smearing Nigrosin

Nigrosin Stained Slide

 We let the smeared slide air-dry before covering it with safranin. We left the safranin on for a little over 60 seconds and then gently washed off the excess stain with water.

Applying Safranin to Slide

Rinsing Excess Safranin

 The slide was blotted with bibulous paper before being observed with immersion oil under the microscope. The capsule stain is used to determine if a specimen has a capsule or slime layer. We observed rod-shaped bacteria with no visible capsule.

Capsule Stain (No Capsules)

By this point the water was boiling for our endospore stain preparation. We placed a previously fixed slide over the beaker containing the boiling water. A piece of bibulous paper was placed over the slide and was saturated with malachite green. We left the malachite green on the slide for about 5 minutes, adding additional malachite green drops to the paper to prevent the stain from drying.

Bibulous over Fixed Slide

Saturated with Malachite Green

 Using forceps, we removed the slide from the heat and threw away the piece of stained paper in the biohazard bag. We waited until the slide had cooled before rinsing the slide with water for 30 seconds to remove excess malachite green. We covered the smear with safranin for 70 seconds and rinsed the excess off with water afterwards, After blotting the slide with bibulous paper, we examined it under the microscope with immersion oil. Our bacteria had no discernible endospores.

Blotting with Bibulous 

Rinsing Excess Malachite Stain

Applying Safranin to Malachite Green Stain

Our professor brought out broth tubes for our groups. We labeled our tube and transferred some of our bacteria into the broth tube with an inoculating needle, by stabbing the needle a little past halfway down the tube. We then placed the broth in the 25° C incubator. We will be returning to this sample later to check the motility of our bacteria.

Our professor also brought out starch agar plates, skim milk agar plates, nutrient gelatin deep tubes,  tributyrin agar plates, and litmus milk tubes. We transferred some of our bacteria to each of these containers. For the plates we streaked snake-like lines down the surface, for the gelatin tube we stabbed an inoculating needle into it like in our motility preparation, and for the litmus milk tube we transferred some sample on an inoculating loop. The starch, skim milk, gelatin, and tributyrin samples are to test whether or not our bacteria specimen has the ability to hydrolyze starch, casein, gelatin, and triglycerides, respectively. The litmus milk sample is to test whether or not our bacteria utilizes lactose, protein, and litmus in the litmus milk. These are all means we will use to identify our bacteria later. Once we were finished preparing our samples, we stored the plate samples (which we shared half-and-half with another group) in the 30° C incubator. We stored the tube samples (which only contained our sample bacteria) in the 25° C incubator. We also replaced our slant, streak plate, and broth cultures in the fridge for storage. We then cleared our benches, washed our hands, and removed our lab coats for the day.

Starch Plate with Streak

Skim Milk Plate with Streak