simple stains
staining procedure that only uses one stain and all bacteria are stained similarly
used for cell morphology, size, arrangement
differential stains (gram)
first step in identifying bacteria
uses two contrasting stains seperated by a declorizing agent
Acid fast
used to keep stain in place when washed with acid
Spore stain
used to observe endospores, capsules and flagella
types of staining techniques
aa
smears
speading a small amount of bacteria broth culture or solid inoculum mixed with a drop of distilled water on a clean slide allowing to dry
heat fixing a sample
passed over incommator several times or placed over a slide warmer
chemically fix the sample
cover smear with 95% methanol for 1 min
what is the purpose of heating/ fixing the smear?
to kill the bacteria and preserves with minimal distortion/shrinkage
simple stains
bacteria cell surfaces negatively charged and basic dyes are used as stains
use of a single basic dye is called a simple stain
doesnt tell you if its gram positive or negative
common basic dyes
1. methylene blue
2. crystal violet
3. safranin
4. malachite green
simple staining steps
1. use a clothespin to hold slide
2. cover slide with methylene blue and leave for 30-60 sec
3. wash off excess stain with distilled water
4. blot smear with paper towel and air dry
which bacteria is a rod?
bacillus megaterium
which bacterium is larger
bacillus megaterium
what value is a simple stain?
enhances the contrast between the bacterium and its surrounding material and permits greater clarity of detail
a
B
another method of fixing a smear?
chemically fixing
how does alcohol chemically fix the bacteria?
1. removes water (imp for mounting the cell)
2. denatures proteins (metabolism of the cell is stopped and cell dies)
3.it dissolves and removes lipids (cell membrane of bacteria is harmed by alcohol)
negative staining
does not stain bacteria stains background
dyes used for negative staining
india ink
nigrosin
steps in negative staining
1. add drop of nigrosin on clean slide
2.aseptically add organism
3.place second clean slide on the surface of the first slide and draw it back into the drop
4.when drop flows across the width of spreader slide
5. push spreader slide to other end
6. air dry
which cell is a rod?
bacillus subtilis
why is the size more accurate in a negative stain than in a direct stain?
No heat fixing or chemicals are used so there is no distortion of the cell
what microscopic technique gives the field similar appearance to that seen in a negative stain
dark field microscope
could any dye be used in place of nigrosin for negative staining?
no only a cell that does not penetrate the cell, india ink works
why can carbolfuchsin be used as a direct stain and as a negative stain?
ph is basic
Acidic congo red does not conatin suspended particiles like nigrosin but can give the appearance of a negative stain. what is the basis for this stain?
b/c it it acidic ( negative charge ) stain cannot bind to negatively charged bacteria
Gram staining steps
1. apply primary stain (crystal violet) 30 sec bacteria stained purple
(gram plus and minus are blue)
2. apply mordant (gram iodine) 10 sec (gram plus and minus blue)
3. apply decolorizing agent (ethanol)10-20 sec (gram plus blue gram minus colorless)
4. apply secondary stain or counter stain (safranin) 30sec the dye stains decolorized bacteria red (gram plus blue glam minus red
Colors
Crystal violet
Safranin
crystal violet-purple
safranin-red
use of mordant
grams iodine binds to the crystal violet preventing the compound from being clear thru the cell wall of the gram positive bacteria
gram positive cell wall
thick layer of peptidoglycan
gram negative cell wall
thin peptidoglycan layer covered by a lipopolysaccharide layer
...
gram negative bacteria
...
gram positive bacteria
...
mycobacteria
...
fungi
acid fast staining
differential stain
principal behind this staining
carbolfuschin binds to mycolic acids on the outside of the acid-fast bacteria which is not washed away with the acid-alcohol mixture that decolorizes all other bacteria
dyes used for acid fast staining
carbofuschin and methylen blue
acid fast steps
1. prepare fixed smear of culture
2. cover the smear with carbolfuchsin for 5 min
3. wash with distilled water
4. wash with decolorizer (acid alcohol) for 1 min
5. wash water
6. counterstain with methylene blue for 1 min
7. examine slide under microscope
Endospores
"resting bodies"
do not metabolize resistant to heat chemicals and harsh environments,
are formed when nutrients are not available
generally impermeable to stains so heat must be applied
capsule
increase virulence(disease causing ability)
decrease being phagocytiezed
Flagella
motility
location and presence are helpful in identifying bacteria
Steps in staining endospores
1. make smear let air dry, and heat fix
2. tear small piece of paper towel and place over smear
3.cover smear and paper with malachite green and steam the slife for 5 mins (keep wet)
4.remove paper and throw away, wash smear with h20
5.counterstain with safranin for 30 sec
6.wash with h20 30 sec
7.examine under microscope