Staining Process - Midterm 1 ( and quiz 2 ) Flashcards
Why are microbes hard to see under microscope?
- Lack pigment (pretty much colorless)
2. Lightly Pigmented ( color so faint, you have hard time seeing it )
Whats the standard size of agar plate?
30-300 Colonies
Why does having more than 300 colonies provides more error
- Colonies cant be isolated from each other
- If organisms make large colonies, it’ll be harder for isolation
- Cells experiences competition for nutrients in augur plate, therefore smaller colonies form
Why does having smaller colonies forming on augur plate containing more than 300 colonies account for error?
some colonies are so small already so competing for nutrients in an augur plate makes them even smaller so that you might not be able to see them.
All media is…
Selective to some degree
Purpose of staining cell
To see cell organelles (capture the existence of it)
Why is it bad to stain cells that are alive?
Cells that are alive wont allow the stain to pass through their cell membrane for protection. therefore we have to kill the cell first.
What happens to dye once it penetrates the cell wall of the dead cell?
the dye form bonds with intracellular materials and resist being washed away.
How to make a smear?
- Take two drops of water
- Place cells into the drops of water via inoculating loop
- Mix and spread it in an area size of a nickel
Purpose of a smear?
- To see individual cells and
2. For dye to penetrate
Characteristics of a smear?
- Even spread
- Not clumped
- Thin
What are two methods of killing cells for staining purpose?
- Heat Fixation
2. Methanol Fixation
Why is Heat Fixation most common ?
Has advantage of requiring NO CHEMICALS and producing NO BIOLOGICAL WASTE
What is Methanol Fixation?
- Flood with methanol
- we dont use this because methanol is highly toxic
- It produces fewer distortions of cells
- produces better results upon staining particulary when working with broth cultures
What are the 4 types of basic simple stains?
- Crystal violet ( looks blue)
- Methylene blue ( blue)
- Safarin (looks red/pink)
- Basic Fuschian (looks red)
2 components of a stain?
- Colorless Inorganic ion (usually negative charge)
2. Colored Organic ion ( the dye)
If the colored organic ion is positively charged (cationic)..
they are basic dyes
If colored organic ion is negatively charged (anionic)
they are acidic dyes
example of anionic stain?
Nigrosin
Why are basic dyes used over acidic dyes in simple staining or gram staining?
- In neutral or alkaline environments, Cell membrane carries negative surface charge that attracts cationic dyes.
- Therefore its likely to enter and not be repelled by the cell
What are negative stains?
- Acidic dyes are prominent here
- Dye does not actually stain the cells
- Cells are identified as clear areas in a colored background since the negatively charged cell surface repels the negatively charged dye.
Categories of staining processes?
- Simple stains
- Differential stains
- Structural stains
What is Simple stain?
- it enters cell and stains everything.
- useful for determining morphological features
- provides no info on presence or function of specific cell structures
- basic and acidic dyes may be used
Differential Stain?
- Consists of two or more dyes used in combination
- Takes advantage of differences in cell structures (cell wall) to differentiate among cell types
- Gram stain and acid-fast stains are differential stains.
What are Structural stains?
Specifically stain particular structures and not the whole cell, and thus help identify the presence or absences of the structure in the cell
What is morphology?
Size, shape, and cell arangement of microbial strains
Significance of morphology?
- morphology of a cell is very specific to a particular genotype
- it can be used in identifying the microbes
- as well as in determining whether the cell is affected by changes in the environment/growth conditions
What are the Two major shape classes?
- Cylinder (rods and bacilli)
2. Spheres (Cocci)
Average Rod size in diameter?
0.5 uM - 1.0 uM
Average Rod size in length?
1.0 uM - 10 uM
Length of long bacillus megaterium
10 uM in length
Size of E. coli
Short rod with 1.0 uM - 3.0 uM
Cocci range in diameter?
0.5 uM to 1.0uM
What is Epulopiscium Fishelsoni?
Largest rod up to date
600 uM in length
80 uM in diameter
What does arrangement of cells dependent on?
- the number of planes of division and
2. whether or not the cells detach after division
Diplococci
Cocci that divide on a single plane and forms pairs
Streptococci
Cocci that divide on a single plane and form CHAINS
Tetrads
Cocci that divide in two planes
Sarcinae
Cocci that divide into three planes to form CUBOIDAL arrangements
Staphylococci
Cocci that divide in random planes to form SHEETs
Cell arrangements are highly characteristic of different bacterial strains but why are they harder to identify than shape?
Because they are easily disrupted during procedures such as making wet mounts or smears
What to avoid during smearing your cells?
- Avoid making exclusively circular motions.
- Avoid taking large amounts of cells from slant
- Avoid making tiny smears as the cells will not spread out sufficiently
Why shouldnt you heat fix the slide while its still wet?
itll cook your cells and cause distortion in shapes
How to heat fix cell?
- Hold slide at 45 degree angle with smear side up
2. pass it over flame quickly for 2-3 times
What are Gram-positive organisms?
Organisms that have thick cell walls
Composed of large amounts of peptidoglycan
What are Gram-negative organisms?
Organisms that have thinner cell walls
Composed of small amounts of peptidoglycan
What is peptidoglycan?
Its a porous layer and allows movement of small substrates, but it does slow the entry and exit of dyes from the cell
What is the outer membrane?
Specific to only gram-negative organisms
Additional layer outside cell wall
Composed of Phospholipids and lipopolysaccharides
What is periplasmic space?
Specific only to gram-negative organisms
Space between cytoplasmic membrane and outer membrane
What is the Mordant?
- Its grams iodine
- Strengthens bond between crystal violet dye and cell.
- does not effect color
What is the primary stain in gram stain?
Crystal violet
What is the Decolorizer?
- Solubilizes lipids
- dehydrating agent
- Mixture of EtOH and Acetone
How does Decolorizer affect Gram-negative bacteria?
- It dissolves the outer membrane
- then penetrates the peptidoglycan layer
- interacts with lipids of cytosolic membrane
- enters and exit cells.
- as it exits, it takes the CV-1 complex with it, destaining the cell
How decolorizer affect Gram-positive bacteria?
As decolorizer leaves cell, the CV-1 complex is partly trapped in the cell wall. the alcohol dehydrates the cells and shrinks the pores of the peptidoglycan, which slowers the influx of the decolorizer
End results after addition of decolorizer if done correctly?
Gram positive = Blue
Gram negative = no color
Critical steps in Gram stain method?
- Making the smear
2. Destaining process
What is the secondary stain?
The counterstain
Has to be a basic stain thats a contrasting color to the primary stain
Color: Safranin or Fushcin
Result colors after secondary stain is applied?
Gram positive cells are Blue/purple
Gram negative cells are pink/red
Where can you make the most common error in the gram-stain process?
decolorization step
What happens to gram-negative cells that have been destained for too short
it will appear purple due to retention of the primary dye
what happens to the gram-positive cells that have been over destained?
they will appear pink from having lost the primary stain and acquiring the secondary stain
What is gram variability?
when a cell that has aged produces a mixture both gram-negative and gram-positive coloring.
Mycobacterium cell walls are closely related to
gram-positive bacteria
