Lab Exam 2: Staining Labs Flashcards
What is a bacterial smear?
Thin film of cells that can be stained for enhancing visability.
What is the purpose of staining bacterial cells?
Enhance visibility of the cell and its structures.
Define “simple stain”
Utilizes a single dye
What information about bacteria can be determined from a simple stain? Provide examples.
Shape (cocci, bacilli, coccobacilli), size, and arrangement (single cell, pairs, chains, clusters).
Smear preparation
1) Flame loop and place 2-3 loopfuls of tap water onto a clean side.
2) flame loop again and aseptically remove a small amount of growth form the surface of an agar slant by touching it with one side of the loop.
3) mix the cells with the loopfuls of water, create a dime size smear.
4) allow slide to air dry.
5) heat fix by passing through the flame 2-3 times.
Simple stain procedure
1) Palace slides on staining rack positioned over the sink.
2)Flood smears with crystal violet for 30-60s.
3) Rinse slide quickly under tap water by allowing the force of the water stream to run down the back of your hand onto the slide. Do not let the stream directly hit the slide or the smear may wash off.
4) Shake off excess water and allow to air dry.
What happens when you have too much bacteria on a slide?
You get clumping. Unable to properly confirm what the true grouping of the bacteria is.
What is a differential stain and how does it compare to a simple stain?
Differential stain uses more then one dye to differentiate between different types of microorganisms. Simple stain only uses one dye.
List the three characteristics of a bacterial species provided by a Gram stain and the common outcomes fore each.
Gram Status (+=purple or -=pink)
Cell morphology (bacilli=rods, cocci=spheres, spirill=spirals)
Arrangement (single, doubles, chain, clusters)
Which step of the Gram stain procedure is most critical? Why?
Decolonization, because too much will remove the crystal violet from the gram poss and gram neg but too little will not remove enough crystal violet from the gram neg for it to dye pink.
Gram stain procedure
1) flood smear with crystal violet, primary stain, all cells purple. 30-60s and rinse w/ tap water
2) flood smear with Iodine, mordant, cells remain purple. 60s and rinse w/tap water
3) allow Alcohol to drip onto slide, continue drip by drip for about 5 seconds. Decolorizer, Gram + purple, Gram - colorless. 5-10s rinse very well w/tap water
4) flood smear Safranin, counterstain, Gram + stays purple, Gram - pink/red. 1-2min rinse and dry
Think back on both the simple stain and the gram stain. Some cell arrangements that you observe might be artifacts resulting from the smear preparation. Can you explain how this might happen? Give examples.
Result of clumping due to smear being too thick. Cells are close together but its a result of technique not true arrangement.
If the iodine step were omitted, what color would you expect a Gram-negative microorganism to be? A Gram-positive? Explain.
All cells would end up pink. The iodine step is the mortar that locks in the crystal violet into the gram positive. Without this step the alcohol will remove the stain from all cells.
A student accidentally uses water instead of the decolorizer. What color would you expect a Gram-negativve organism to be? A gram-positive? Explain.
Everything would be purple since the stain is not removed from the gram negative.
Why is it important to use only young cultures for Gram staining?
decreased cell wall integrity in older gram + cells means it will destain more easily and end up pink.
With both the simple stain and the gram stain both staph and strep appear blue at the end of the staining process. Can you assume an organism is Gram positive by its appearance after applying a simple stain? Why? Why not?
No. Simple stain is not differential, all cells will be the same exact color regardless of its type. Only one dye is being used to stain.
Can you rely on a cell arrangement to differentiate staphylcocci form streptococci? Explain.
Strep occurs in pairs or chains while staph occurs in clusters and pairs. So yes you can use the pairs vs. cluster to help figure it out.
Suppose you Gram stained a sample from a pure culture of bacteria and observed a filed of red and purple cocci. Adjacent cells were not always the same color. What were the possible explanations?
Assuming the culture really is pure and there is no contamination, then a likely explanation for both red and purple cocci being found adjacent to one another is that the sample of cells is too old and consists of Gram positive cells.
