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