Staining: Bacteria and Stain

Title: Staining

Introduction:

Microorganisms are small and colorless, invisible to unaided eyes. When observing them under microscopes, we use various methods to make microbes apparent. One of the most important methods is staining. Staining techniques play an essential role in the studying of microorganisms; they help to reveal characteristics of microbes, such as their morphologies, sizes, arrangements, chemical components and many more. The purposes of the experiments are to be familiar with each staining technique, to understand the differences between the techniques, to able to read results and to have the knowledge for future use in various fields.

-Simple stain helps with determining the sizes, shapes and arrangements of microorganisms.

-Gram stain assists in identifying the type of microbes, whether they are gram positive or negative, which in turn tells us what kind of cell wall the microbes have.

-Endospore stain tells whether microorganisms make endospores.

-Acid-fast stain is helpful in recognizing the existence of mycolic acid in cell walls.

-Capsule stain reveals the existence of capsules.

Methods:

Simple Stain

For this experiment, I used E. coli to make a heat fix smear. I placed a drop of water in middle of the slide, then put in small amount of E. coli and mixed well. The slide was allowed to air dry. When it was completely dry, I passed the slide over the flame for 3 times to fix the cells to the slide. For the staining part, I put a drop of methylene blue onto the smear for 30 second, rinsed with water, blotted dry and observed the slide using oil immersion.

Gram Stain

For comparison purpose, I used two bacteria for this experiment. They are E. coli, a gram negative bacteria and Staphylococcus, a gram positive bacteria. I prepared a heat fixed smear of both bacteria. First, I used crystal violet as my primary stain, put few drops of it on the smear and let it sit for 30 seconds. Then, I used iodine as mordant, covered the smear with it for 60 seconds. The next step was to decolorize the slide with acetone alcohol. It was important to let the decolorizer only sit for 1 to 2 seconds and rinse off with water. Last step was to counter stain with Safranin for 30 seconds, follow by rising with water. The slide was air dried and observed using oil immersion.

Endospore Stain

Endospore stain is a time consuming procedure because it is hard to penetrate the thick wall of the endospores. Therefore, I started off with a wet mount with bacteria Clostridium observing existence of endospores. Once confirmed there were endospores, I made a heat fixed smear. Covering the smear with a small piece of paper towel, I saturated the paper towel with malachite green. Then I passed the slide constantly over flame for about two minutes. The goal was to have the smear remain copper sheen for two minutes. Remove the paper towel, I rinsed the slide with water for 30 seconds. Last few steps were to counterstain with safranin for 30 seconds, rinsed with water, blotted dry and observed using oil immersion.

Acid-Fast Stain

For this technique, a control group was used along with bacteria sample. I made a heat fixed smear of Saphyloccoccus spp. and Mycobacterium. Covering the smear with a small piece of paper towel, I added few drops of carol-fuchsin as primary stain. Then I passed the slide in and out of flame for about two minutes, added stain when needed. Next step was to decolorize the smear with acid alcohol for 20 seconds, then rinsed with water. I used methylene blue to counter stain the smear for 30 seconds, rinsed with water, blotted dry and observed using oil immersion.

Capsule Stain

Capsule stain is different from other stain techniques because it does not start with a heat fixed smear. I placed a drop of congo red on one end of the slide, and well mixed Klebsiella with the ink. Using another slide’s edge, I pulled the ink and bacteria across the entire slide. After letting the smear air dry, I covered it with crystal violet for 30 seconds, rinsed with water, blotted dry and observed using oil immersion.

Results:

Simple Stain

Shape: bacilli Arrangement: single Size: Length 3 um, width 1um Color: blue Magnification: oil immersion 1000X

Gram Stain

Shape: bacilli (E. coli) cocci (Staphylococcus) Arrangement: single (E. coli) grape like cluster (Staphylococcus) Size: length 3 um, width 0.7 -1 um (E. coli) diameter 0.7 um (Staphylococcus) Color: gram negative, red/pink (E. coli) gram positive, purple (Staphylococcus) Magnification: oil immersion 1000X

Endospore Stain

Shape: bacilli (Clostridium) oval (endospores) Arrangement: single (Clostridium) single (endospores) Size: length 2.8-3 um, width 0.8 um (Clostridium) diameter 1.1 um (endospores) Color: dark red (Clostridium) green (endospores) Magnification: oil immersion 1000X

Acid-Fast Stain

Shape: bacilli (Mycobacterium) cocci (Staphylococcus) Arrangement: single (Mycobacterium) grape like cluster (Staphylococcus) Size: length 2.5-4 um, width 0.2-0.4 um (Mycobacterium) diameter 0.8 um (Staphylococcus) Color: fuchsia (Mycobacterium) dark blue/black (Staphylococcus) Magnification: oil immersion 1000X

Capsule Stain

Shape: bacilli (Klebsiella) Arrangement: single Size: length 2-3 um, width 0.4 um Color: reddish background, cells are purple Magnification: oil immersion 1000X

Discussion:

Simple Stain
Methylene blue stained the bacteria and turned all the cells blue. This definitely help in identifying the shape, size and arrangement of E. Coli. The staining method is helpful because it is permanent and tells a lot about the cell.

Gram Stain
Gram stain helps to identify the type of cell wall bacteria have. Bacteria all have peptidoglycan on their cell wall, but the amount of it determines if the bacteria are gram negative or gram positive. For gram negative bacteria, they have only 10% peptidoglycan in the cell wall, therefore when stained with crystal violet and iodine, they do not retain the color after acetone alcohol wash. For gram positive bacteria, 90% of their cell wall are peptidoglycan, which can hold on to crystal violet color even after the acetone alcohol wash. From the experiment, it is clear that E. coli is a gram negative bacteria since it obtained the reddish pink color from Safranin and staphylococcus is a gram positive bacteria because it held on to the purple color from crystal violet.

Endospore Stain
Bacteria that make endospores are harder to kill because endospores have thick walls that are difficult to penetrate. So it is important to determine if specific bacteria make endospores so proper prevention/treatment can be implemented. From the experiment, there were a lot of endospores present but not a lot of bacteria. Also the endospores were not within the bacteria. This is maybe because the Clostridium colonies I used were more than 48 hours old and there were not enough food for the bacteria from the agar, so the bacteria started to die and ejected endospores, leaving them behind. With proper nutrients, these endospores will form new bacteria.

Acid-Fast Stain
The use of control group was really helpful in determining which cells contain mycolic acid.
Mycolic acid is a waxy molecule in the cell wall. Because of its waxy property, the cell wall is hard to penetrate and normally stains can not get in. Carbol-fuchsin, the primary stain of acid fast, is able to get through the cell wall because of heat. So the heating step is extremely important for the final outcome. After the acid alcohol wash, control group Staphylococcus lose the fuchsia color, but Mycobacterium hold on to fuchsia because of mycolic acid. At this step, counterstain with methylene blue is necessary so that Staphylococcus is visible.

Capsule Stain

Most of the above staining methods require a heat fix smear, but not for capsule stain. I had to do this staining twice because I mistakenly heat fixed the slide the first time. This altered the result because heat broke down the capsules and they were no longer visible under the microscope. The use of congo red was to create a background color. It does not stain the cell. The only staining factor is crystal violet which stains bacteria purple. The second time I followed the procedures correctly and my result met my expectation. There were clear oval rings surround purple rod shape bacteria. The clear rings were the capsules.

Conclusion:
Various staining techniques help greatly in observing microorganisms and many important information reveal, such as microbes’ morphology, size, arrangement, chemical make up and etc. With all these useful information, scientists have a better understand of these living tiny organisms. Scientists are able to come up with methods to prevent and treat diseases caused by bacteria based on their unique characteristics.