Lecture 3B Microscopy and Staining Flashcards
(38 cards)
Preparation of Specimens for Light Microscopy
- Smear
- Fixing
- Staining
Smear: Spread a __ of material containing microorganisms over the slide surface. Allow to __
- thin film
- air dry
Fixing: Process that __ microorganisms and __ them to a microscope slide. Fixing __ and __distortion of cells.
- kills
- attaches
- preserves
- minimizes
Staining: Coloring microorganisms with a dye that emphasizes certain structures. Before staining a sample, it must be __. Stains are __composed of __
- fixed
- salts
- 2 components: positive ion (cation) and negative ion (anion)
The colored ion in staining is called the __
chromophore
Two types of dyes
- basic dyes
- acidic dyes
A type of dye
- Chromophore is in __ ions.
Most commonly used dyes.
- Bacteria are slightly __ charged at pH 7, therefore they stain with basic dyes.
- Give examples
BASIC DYE
- positive
- negatively
- crystal violet, methylene blue, safranin (red/pink)
A type of dye
- Color is in __ ions.
- Stain the background: __
- Bacteria __ stain with acidic dyes.
- Used to observe __
- Minimal distortion because heat fixing is not necessary and dye is not taken up by cells.
- Give examples
ACIDIC DYE
- negative
- negative staining
- do not
- cell shape, size, and capsules
Examples:
- eosin (yellow)
- nigrosin (black/dark gray)
- india ink (black/dark gray)
cell wall __ the acidic dye
repels
Types of staining methods
- Simple Stains
- Differential Stains
a. Gram Stain
b. Acid-Fast Stain (Ziehl-Nielsen Stain) - Special Stains
a. Endospore Stain
b. Capsule Stain
c. Flagella Stain
A type of staining method
- Aqueous or alcohol solution of a single basic dye.
- Primary purpose is to stain the entire organism to view cell shape and basic structures.
SIMPLE STAINS
Procedure of simple staining:
- stain is applied for a certain time, then washed off
- slide is dyed and examined
A type of staining method
- React differently to different types of bacteria.
- Can be used to distinguish among different groups of bacteria.
Differential stains
two important differential stains used in microbiology
- gram stain
- acid fast stain (Ziehl-Nielsen Stain)
Who developed gram stain?
Hans Gram (1884), a Danish microbiologist
A type of staining method
- The most useful staining procedure in medical microbiology.
- culture must be young to undergo this stain
- provides useful information for disease treatment
- Distinguishes bacteria of two large and medically important groups:
__ bacteria
__ bacteria
Gram stain (Differential stain)
what type of bacteria have thick cells walls
gram-positive bacteria
Steps of gram stain
- Primary stain
- Mordant
- Decolorizing
- Counterstain
Steps of gram stain:
1. __ stain: Cover a heat fixed smear with a basic dye (__).
2. __: After smear is rinsed with water, an __ solution is applied
3. Decolorizing:
Gram negative cells will be __.
Gram-positive cells will remain __.
4. Counterstain:
Gram-negative cells are stained __.
Gram-positive cells remain __.
- primary
- crystal violet
- mordant
- iodine mordant
- decolorized
- purple
- pink/red
- purple
slide is washed with alcohol which will remove stain from gram-negative cells but not from gram-positive cells
decolorizing
alcohol is rinsed off. safranin is applied, which will stain the cells that were decolorized
counterstaining
- Gram-positive cells have very __ peptidoglycan cell walls, whereas gram-negative cells have very __ cell walls. Crystal violet easily penetrates both cell types.
- Because of its larger size, the crystal violet-iodine complex [CV-I] is not easily removed from gram- __ cells, due to their thick cell wall. The CV-I complex is readily washed out of gram-__cells with alcohol.
- Counterstain (this refers to __ name of stain) only colors gram-__ cells.
- thick
- thin
- positive
- negative
- safranin
- negative
Application of Gram stain
- Gram-positive cells with their very thick peptidoglycan cell walls, are __ to penicillins and cephalosporins.
- Gram-negative cells with their thin cell walls and lipopolysaccharide layer are __ to these antibiotics.
CHEMOTHERAPY
- susceptible
- resistant
Limitations of Gram stain
- Not all bacterial cells stain well with the Gram stain.
- Gram-stain only works well on young bacterial cultures, that are actively growing. Therefore it is best to use cultures 18 to 24 hours old.
- Older cultures (over 24-48 hours), are often gram-variable.
