MMG Lab 4
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- What is the most important differential staining procedure used in microbiology?
- -gram stain
- What are differential stains?
- -staining that uses more than one dye and used to distinguish between different groups of bacteria
- Who developed the gram stain?
- -Danish physician, Hans Christian Gram in 1884
- What does gram reaction depend on?
- -structurally intact organisms
- Describe gram positive cells.
-
-retain primary crystal violet dye when complexed with iodine
-appear purple-blue
-thick walls with one layer and have a very high conc. of peptidoglycan
-low lipid concentation - Describe a Gram negative cell.
-
-unable to retain crystal violet-iodine complex when washed with alcohol
-retain Safranin and appear red
-two or more cell wall layers, small amount of peptidoglycan
-high lipid concentration - What is peptidoglycan?
-
-found only in bacteria
-high molecular weight
-has repeating carbohydrate polymer linked by AA bridges
-forms backbone of cell wall - What occurs during decolorization?
-
-alcohol removes lipids in cell walls causing them to become porous
-alcohol shrinks peptidoglycan (locking in dye in Gr +) - How old should cultures be before they should be discarded?
- -16 to 24 hours if possible
- How does acidity affect gram cell stain?
- -cells grown in acidic environments can lose their ability to retain the CV-I complex resulting on erroneous Gram reactions
- What happens in a flase Gram negative?
- -slide is overdecolorized and appear pink (though should be blue)
-
What color do yeasts and fungi sometimes APPEAR to stain?
Animal cells? -
-blue
-red
*Gram staining is only differential for bacteria! - What are the four basic cellular characteristics of a bacterium that should be described when identifying it?
-
-Gram stain reaction
-Size
-Shape
-Spatial Arrangement - What is the substitute dye that can replace safranin for colorblind people?
- -Bismarck Brown
- What is a primary stain?
- -the initial stain used which enters both types of cells: crystal violet
- What is a mordant?
-
-makes the staining solution work more intensely
-wherever it meets dye, it forms water insoluble pools (slightly soluble in alcohols and acetone) - Review problems with Gram staining.
- Appendix E, pg 27 and 28
- What are 6 things TA's look for to grade a stained slide?
-
-cellular damage from heat fixing
-density
-distribution
-stain precipitation
-Gram reaction
-Quality of counterstain