Lab Practical Review Flashcards
Spore forming pathogens are most likely Gram + or Gram -? Rods or Cocci?
Gram + Rods
Gram Stain Steps
Drop of distilled water on slide first
Than mix in bacteria
Allow to air dry
Heat fix over burner (3-4 passes over flame)
Once completely dry begin the staining portion
Crystal Violet - 30 seconds
RINSE
Gram’s Iodine (Mordant) - 30 seconds
RINSE
Decolorizing Alcohol - 10 drops/until its clear
RINSE
Safranin Counterstain - 30 seconds
RINSE
Use Bibulous paper to dry
- In the Gram stain, a bacterial smear is dried and then heat-fixed to cause it to adhere to the glass slide. It is then stained with crystal violet dye, the primary stain, which is rinsed off and replaced with Gram’s iodine. The iodine acts as a mordant - that is, it binds the dye to the cell. The smear is then decolorized with alcohol and counterstained with safranin. Gram (+) organisms take on the crystal violet stain which gets locked in by the Gram’s iodine. They are dark blue/purple in color and are dense. Gram (-) organisms with their fatty cell walls do not take on the first two steps of the Gram stain, but the decolorizing alcohol allows the counterstain to absorb. They are red and more translucent.*
What color does a Gram + pathogen stain in a gram stain?
- Blue.
- Gram (+) organisms take on the crystal violet stain which gets locked in by the Gram’s iodine.
- They are dark blue/purple in color and are dense.
What color does a Gram - pathogen stain a gram stain?
- Red.
- Gram (-) organisms with their fatty cell walls do not take on the first two steps of the Gram stain, but the decolorizing alcohol allows the counterstain to absorb.
- They are red and more translucent.
What does a Gram Stain tell us?
- Bacterial Shape & Arrangement
- If the bacteria is Gram (+) or Gram (-)
- Gram (+) cell walls contain peptidoglycan which is easy to stain and hard to decolorize. - Gram (-) cell walls are made up of phospholipids which are difficult to stain, but easy to decolorize
Do you heat fix a Gram Stain Slide?
YES
What does a Negative Stain tell us?
Morphology only. The shape & arrangement of the microbe
Do you heat fix a Negative Stain?
NO
What type of stain is this?
Gram Stain
What type of stain is this?
Negative Stain
What does an Endospore Stain tell us?
Determines whether the microbe is a spore forming bacteria or not
What is the most common spore forming bacteria?
Gram + Rods
Explain the Negative Stain steps
Procedure:
1. Flame and cool your loop and place a loopful of India ink in the center of a clean microscope slide
2. Flame and cool your loop and mix a small amount of bacteria into the stain
3. Mix and spread the bacteria out
4. Let AIR DRY - Do NOT heat fix as this will distort the shape
5. examine under oil immersion
Explain the Endospore Stain steps
- Add water to a beaker and bring to a boil
2 Prepare a smear on a clean slide and heat fix - Place your slide on the beaker. Place a piece of paper towel over smear - this will help prevent the dye from running off the slide
- Place drops of Malachite green dye on the paper towel and steam for 5 minutes. Continue to add stain to prevent the dye from drying on the slide
- Decolorize with water for about 30 seconds. The vegetative cells lose the dye, but the endospores retain the dye
- Counterstain with safranin for 30 seconds. Rinse with water. Blot dry with bibulous paper
- Observe under oil immersion. The endospores appear green and the vegetative cells appear pink
When a bacteria is grown on separate agar plates at different temperatures (degree celsius) what does that tell us?
This experiment shows us that different bacteria have different temperatures in which they prefer to grow.
What can we observe from these plates?
From left To right:
10 C
20 C
30 C
40 C
50 C
1. E. coli
2. Pseudomonas fluorescens
3. Bacillus stearothermophilus
Define: Obligate Aerobe
Grows only in the presence of oxygen
Define: Obligate Anaerobe
Will not grow in the presence of oxygen.
What type of stain & bacteria shape is seen?
Negative Stain - cocci
What type of stain & bacteria shape is seen?
Negative Stain - rods
Special Notes to consider to Improve Your Gram Stain?
- Gram (+) organisms can lose their ability to retain the crystal violet complex as they age. This can cause the smear to appear Gram variable - with both colors present. It is interesting to note that Gram (+) organisms can appear Gram (-), but Gram (-) organisms never appear Gram (+).
- Gram (-) cocci are really, really, really, really, really, really, really rare.
- The most important step to the Gram stain is the decolorizing alcohol. You can decolorize too much and cause a Gram (+) organism to appear Gram (-).
- Thick smears do not stain well. Spread your smears out and look towards the thinner edges.
What type of stain & bacteria shape is seen?
Gram Stain. Gram (+) rods
What type of stain & bacteria shape is seen?
Gram Stain. Gram (+) cocci
What type of stain & bacteria shape is seen?
Gram Stain. Gram (-) rods