Day 3 5/16

SO MANY STAINS

Gram Staining

The purpose of a gram stain is to identify  the bacteria by dividing them into either gram-positive or gram-negative groups. And as always, we did this to observe the cell shape, arrangement, and size of the bacteria.

The materials we used were:

slide with fixed smear of bacteria
crystal violet stain
Gram's iodine
95% ethanol
safranin
staining rack of sink
wash bottle with deionized water
biblulous paper
forceps
compound microscope
immersion oil

Fixing the smear

In our experiment, we placed a series of liquids over our fixed smear slide until it turned black. We don't think that was supposed to happen but this is science, where things happen that you don't expect. Anyways, we started by fixing the smear using the aseptic technique, and after letting it cool, they party really got started.


First, we placed the slide on the staining rack so it wouldn't fall into the abyss of the sink. Then, after covering the bacteria with crystal violet stain for 20 seconds, we rinsed the slide with water to remove the excess stain. 

Next, we covered it with Gram's iodine for 1 minute and rinsed that off too. Our bacteria just isn't ready for that kind of commitment. Then we decolorized the bacteria with 95% ethanol by holding the slide at a 45 degree angle. we added the decolorizing agent drop by drop until the color stopped running. Again, we immediately rinsed off the slide to remove the decolorizing agent.

We then covered the smear with safranin for one minute and then proceeded to rinse the excess off. 

After blotting with bibulous paper and marking the area with a China marker, we observed it under the microscope with the immersion oil.

After observation, our bacteria showed results of being gram-negative. We described it as reddish in color and consisting of small rounded rods.

Capsule Staining:

The purpose of this staining technique was to view the bacterial capsules or slime layers of the unknown bacteria. In this experiment, we had to prepare a smear of bacteria in nigrosin to use for the capsule staining, so we'll mention how to do that first.

This method required our unknown bacteria (from the petri plate to be specific), nigrosin stain (the black stuff), 2 microscope slides (clean please), and an inoculating loop (sterilized).

 In order to prepare the bacteria smear in nigrosin, we first placed a drop of the nigrosin on one end of the microscope slide. Following the aseptic technique, we transferred a small amount of bacteria from the petri plate into the nigrosin drop. We mixed it as well as we could within such a small parameter. Next, we took the second microscope slide and held it at a 45 degree angle in the bacteria-nigrosin drop. But we didn't stop there. we used the second slide to smear the drop along the first microscope slide until  the smear was a thin film with a feathered edge at the trailing end. Then, we waited for the smear to completely air dry.

As for the real deal (the capsule staining), we needed a few more materials to carry out this process.

More Materials:

safranin

staining rack over a sink

wash bottle with deionized water

compound microscope

immersion oil

After the smear air dried, we covered it with the safranin and tender loving care. Then, we gently rinsed off any excess stain with the water, making sure not to over rinse. 

We blotted the slide with the bibulous paper and examined the smear under the microscope

After much struggle trying to focus our microscope, Dr. P helped us in our quest to find and understand the capsules of our bacteria. We found that our bacteria have very small round capsules.

Endospore Staining

The purpose of this stain was to view the bacterial endospores under the microscope and to observe the location of an endospore in a sporulating cell. Endospores are formed by a few types of gram-positive bacteria. The purpose of endospores is to ensure the survival of a particular kind of bacteria. They are specialized for survival in harsh conditions. Equipped with a thick protein coat, endospores are resistant to most staining. However, the use of heat helped to permeate the malachite green stain into the spore coat.

The materials used were:

 

slide with fixed smear of bacteria

malachite green stain

safranin

staining rack 

steaming water in a large beaker

hot plate for heating the water

piece of filter paper

wash bottle with deionized water

bibulous paper

forceps

microscope

immersion oil

In this experiment, we placed the staining rack over the steaming beaker and then placed the fixed smear slide on the rack. Next, we placed the filter paper on top of the slide and saturated in with the malachite green stain.

 
We stained the bacteria for 5-6 minutes and added additional stain as it evaporated. Afterwards, we used the forceps to remove the filter paper from the slide and placed it in a biohazard bag (proper disposal is no joke, kids).

After allowing the slide to cool, we rinsed the slide with water for about 30 seconds to removed the excess malachite green stain. We then covered the smear with safranin for about 75 seconds and rinsed with with water yet again to remove the excess stain. We blotted the water from the slide with the bibulous paper before viewing it under the microscope with immersion oil.

 We observed that our bacteria had very small, round spores within the bacteria.

 Acid-Fast Staining

The purpose of this last and final stain of the day was to distinguish bacteria based on the lipid content of their cell walls: acid-fast and non-acid-fast. We did not get to use our beloved "N" unknown bacteria, but instead Dr. P provided us with the bacteria that we were to use.

This experiment required:

microscope slide with fixed smear of bacteria

Ziehl-Neelsen carbolfuchsin

acid-alcohol

methylene blue

hot plate

large beaker filled with water

wash bottle with deionized water

filter paper

staining rack

bibulous paper

forceps

compound microscope

immersion oil

perseverance

After boiling the water on the hot plate, we placed the staining rack on the beaker and the bacteria smear on top of that. We placed the filter paper on top of the slide and saturated it with the Zeihl-Neelsen carbolfuchsin, making sure it didn't dry out. We stained the bacteria for 3-5 minutes. Next, we used the forceps to remove the paper from the slide and, once again, disposed of it properly in a biohazard bag. We allowed the slide to cool before going forth with other shenanigans.

After cooling, we placed the staining rack over the sink and rinsed the slide with water to remove any excess stain. We then used acid-alcohol to decolorize the bacteria by holding it at a 45 degree angle and adding drop by drop until the color stopped running. We immediately rinsed the slide to remove the deolorizing agent. We covered the smear with methylene blue for 2 minutes but inevitably rinsed that off as well. After blotting the slide with bibulous paper, we examined the smear under the microscope with immersion oil.

 The results were inconclusive because much of this process washed off the small amount of bacteria on the slide.