How do you Gram stain unknown bacteria?
When Gram staining an unknown, the best method is to make three bacterial smears on the slide. One should be a known Gram positive organism like Staphylococcus aureus. The other should be a known gram negative organism like Escherichia coli . In the middle, make a smear of your unknown organism.
How do you know if a Gram stain is positive or negative?
When the stain combines with bacteria in a sample, the bacteria will either stay purple or turn pink or red. If the bacteria stays purple, they are Gram-positive. If the bacteria turns pink or red, they are Gram-negative.
How do you make a Gram stain smear?
Below are some guidelines for preparing a smear for a Gram-stain.
- Place one needle of solid bacterial growth or two loops.
- If working from a solid medium, add one drop (and only one drop)
- Now, with your inoculating loop, mix the specimen with the water.
- Place the slide on a slide warmer and wait for it to dry.
What are the two types of smear preparation?
Four different types of smear preparation methods (conventional method, blood film method, drop and rest method, and water-wash method) were carried out according to the standard reference as described below.
How do you prepare a fixed smear sample?
In order to heat fix a bacterial smear, it is necessary to first let the bacterial sample air dry. Then either place the slide in the slide holder of a microincinerator, or pass the dried slide through the flame of a Bunsen burner 3 or 4 times, smear side facing up. Once the slide is heat fixed, it can then be stained.
How is Gram staining used to differentiate bacteria?
Gram staining is a differential staining technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction.
What causes Gram positive bacteria to stain Violet?
Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.
How does a Gram positive bacteria look in a microscope?
If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope.
How does a counterstain color a Gram positive cell?
The thick cell wall of gram-positive cells is dehydrated by the decolorizing step, causing them to shrink and trapping the stain-iodine complex inside. After the decolorizing step, a counterstain is applied (usually safranin, but sometimes fuchsine) to color the bacteria pink.