Labs 5-8 Flashcards
Goals in Preparing a Good Smear
- Causes the cells to adhere to the microscope slide
- Ensures shrinkage of cells does not occur
- Prepare thick smears so that individual cells are visible
Steps for Preparing a Smear
- Wash slide(s)
- Label slide(s) and make a target circle
- Flame wire loop and then use it to transfer specimen to slide
- Flame rim of test tube (if used) and wire loop
- If using slant culture, add two small drops of water to the specimen
- Spread organism over target circle
- Allow slide(s) to dry by normal evaporation
- After completely dry, pass slide over the flame of a Bunsen Burner several times, but avoid prolonged heating
Reasons for Heating a Smear
- Causes bacteria to stick to the slide (denatures protein)
- Kills bacteria so they are no longer hazardous
Simple Staining
The use of single stain to color a bacterial cell; creates a contrast between a cell and its background
Simple Staining Procedure
- Use positively charged (cationic) dyes (methylene blue, basic fuchsin, crystal violet), which have color-bearing ions or chromophores
- Bacteria are negatively charged so dye is attracted and sticks to them
- Time ranges from 30 seconds to 2 minutes depending on the type of dye used
- After required time, smear is washed off, blotted dry, and examined under oil immersion
Obtainable Information from a Simple Stain
- Shape and sometimes size (shrinkage possible)
- Surface structures are sometimes visible
- Arrangement of cells: can help identify type
Differential Staining
Takes advantage of the fact that cells or structures within cells display dissimilar staining reactions that can be distinguished by the use of different dyes
Gram Stain
Helps identify cell wall:
- Gram positive: thick, peptidoglycan cell wall; stains purple
- Gram negative: thin, peptidoglycan cell wall; stains pink
Gram Staining Procedure
- Primary (initial) Stain: crystal violent for 20 seconds
- Wash slide with water
- Mordant: helps stain stick better to gram positive cells; Gram’s iodine for 1 minute
- Decolorization: washes stain, removing any color from gram negative cells; Ethyl alcohol until colorless (20-30 seconds)
- Wash slide briefly with water
- Counterstain: stains gram negative cells without changing gram positive stain; Safranin for 1 minute
- Wash off slide with water and allow slide to dry
Acid Fast Stain (Ziehl Neelsen Method)
A differential stain used for identifying mycolic acid containing mycoplasms - Mycobacterium and some Nocardia
Mycolic Acid
Waxy cell wall material; complex lipid composed of fatty acids and fatty alcohols that have hydrocarbon chains up to 80 carbons in length; significantly affects the staining properties of mycoplasms and prevents them from being stained by many routinely used stains
Aced Fast Staining Procedure
- Primary Stain: Carbolfuchsin (mixed with phenol, necessary for dye to penetrate waxy cell wall) heated for 5 minutes
- Heat acts as a mordant (makes stain complex more permeable to the mycolic acid)
- Wash with water
- Decolorization: Acid-alcohol for 1 minute; does not remove the entrapped stain (acid-fast cells); cells not containing mycolic acid are easily decolorized (non-acid-fast)
- Rinse breifly with water
- Counterstain: methylene blue for 30 seconds; makes non-acid-fast cells visible
- Wash briefly with water then allow to dry
- acid fast appear deep red; non-acid-fast appear blue
Gram Stain Results
- B. subtilis: rod-shaped gram positive
- S. epidermidis: round gram positive; staphylococcus
- S. marcesens: rod-shaped gram negative
- S. aureus: gram-positive staphylococcus
- E. coli: gram-negative bacillus
Acid Fast Results
- S. epidermidis: acid-fast negative staphylococcus
- S. aureus: acid-fast negative staphylococcus
- M. smegmatis: acid-fast positive bacillus
