Lecture 2 Flashcards
Name the three advantages of observing unstained cells.
- Retention of natural shape.
2. Retention of characteristics arrangement of cells. 3. No alteration in size.
Why are unstained cells difficult to observe under ordinary light microscope?
Lack of contrast between the cells and the surrounding medium.
Describe a wet mount.
A drop of culture is placed on a glass slide and covered with a square glass coverslip.
Describe a hanging drop.
A special slide is used with a depression. A drop of culture is placed on a coverslip. The coverslip is then inverted over the depression. The coverslip is covered to prevent drying.
What is the best way to examine unstained microbial cells?
A light microscope called a phase contrast microscope. It creates contrast between the cells and the medium.
Dyes and stains are an organic compound that contain what colour-producing group and what salt producing group?
Chromophore and auxochrome
What group does the nitro and azo group belong to?
Chromophores
What group does the hydroxyl and amino group belong to?
Auxochromes
What is the function of the chromophores?
Colour of the dye
What is the function of the auxochromes?
It binds the colour to the cell
What types of charge does the basic dyes and acidic dyes have?
Basic= positive charge
Acidic=negative charge
What type of material does basic dyes have an affinity to and why?
Anionic material due to it’s positive charge. Opposite charges attract.
True or false. Acidic dyes have an affinity to cationic material?
True
Numerate the five sep process of staining a microbial stain.
- A loopful of smear is smeared on a film. If smear if solid, a small amount of water is added.
- The film dries.
- Fixation is performed using chemical or heat.
- The specimen is covered with the stain.
- The smear is gently rinsed with water to remove excess stain.
Describe a simple stain.
A simple stain consists of one dye and usually dissolves in water or ethanol.
Explain why most bacteria stain intensely with basic dyes.
Bacteria contain anionic (negative charge) material such as DNA, RNA and proteins. We know that basic dyes are positive in charge (unlike charges attract). Therefore, they stain intently with basic dies.
Give examples of basic dyes.
Methylene blue
Crystal violet
Carbol Fuschin
Name what simple stains serve for and what they are not used for.
They reveal the shape, arrangement and size of cells. They do not showcase the internal structure of the cell.
Define differential staining
A staining procedure that distinguishes structures within a cell or a cell type from another. Two different dyes are applied in order that a part of the cell structure appears in one colour and the remainder in another colour.
Name the two stain types.
Primary stain- first dye applied.
Secondary stain or counterstain- second dye applied.
Name the 2 most common types of differential stains
Gram stain
acid-fast stain
Explain the 4 steps to the Gram stain procedure.
- The smear is covered with crystal violet-then rinsed after 1 min.
- The smear is covered with Gram’s iodine for 1 min then rinsed. The iodine is a mordant that fixes the stain to the cell’s protoplasm.
- The smear is treated with a decolourizing agent such as 95% ethanol-then rinsed.
- The smear is counterstained with safarin (red). After 20-30 seconds, it’s rinsed.
Differential staining divides the bacteria into two classes. What are they? They appear what colour when stained?
- Gram positive bacteria- appears purple due to the crystal violet that is not removed by the decolourizing agent.
- Gram negative bacteria- appears pink due to the decolourized by the ethanol and then stained by the safrafin counterstain.
Explain why the Gram positive bacteria cells appear violet when stained?
The cell walls permeability plays a factor. The cell wall of the Gram positive cells get dehydrated from the decolourizing agent. This traps the crystal violet-iodine complex.
