Labs 5-8 Flashcards Preview

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Flashcards in Labs 5-8 Deck (14):
1

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

2

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

3

Reasons for Heating a Smear

- Causes bacteria to stick to the slide (denatures protein)
- Kills bacteria so they are no longer hazardous

4

Simple Staining

The use of single stain to color a bacterial cell; creates a contrast between a cell and its background

5

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

6

Obtainable Information from a Simple Stain

- Shape and sometimes size (shrinkage possible)
- Surface structures are sometimes visible
- Arrangement of cells: can help identify type

7

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

8

Gram Stain

Helps identify cell wall:
- Gram positive: thick, peptidoglycan cell wall; stains purple
- Gram negative: thin, peptidoglycan cell wall; stains pink

9

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

10

Acid Fast Stain (Ziehl Neelsen Method)

A differential stain used for identifying mycolic acid containing mycoplasms - Mycobacterium and some Nocardia

11

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

12

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

13

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

14

Acid Fast Results

- S. epidermidis: acid-fast negative staphylococcus
- S. aureus: acid-fast negative staphylococcus
- M. smegmatis: acid-fast positive bacillus