STAINING

Wendy Grace Q. Almine, MA Bio

 Intro
• bacteria are microscopic
• colorless
• in order to visualize them to study
their structure, shape and structural
characteristics - make them visible.
• have to be contrasted to their
environvent
 Stain
• is a dye used to color the living or
dead organelles.
 Types of Dyes
• Acidic
• negativeely charged acid radicals
imparts color on eosin, acid
fuchsine, malachite
green,nigrosin,indian ink
 Types of Dyes
• Basic
• Positively charges radicals
combines with negatively charged
particlesin cytoplasm and gives
color
• ex. methylene blue, crystal violet
 Types of Dyes
• Neutral
• Both positively and negatively
charged imparts different colors to
different components.
• ex. Geimsa's stain, Leishman's stain
 Staining methods
• Simple Staining
• a stain which provides color contrast
but gives sme color to all bacteria
and cells
 Staining methods
• Differential Staining
• a stain which imparts different
colors to different bacteria
 Staining methods
• Negative Staining
• where cells remain clear and the
background is colored to create a
contrast to aid in the bteer
visualization of the image.
 Smear
• is a distribution of bacterial cells on
a slide for the purpose of viewing
under the microscope
Bact'l smear preparation
 Smear Fixation
• Heat fixation
• a. Pass air-dried smears through a flame
2-3 times. DO NOT OVERHEAT
 Purpose of heat fixation
• it kill the organims
• preserves their morphology (shape)
• at anchors the smear to the slide.
 Simple Staining
• to determinee the bacterial shape and
morphologic arrangement (e.g chains,
clusters, chains)
 Simple Staining
• is a method of staining in which bacteria
are stained using single stain

• methylene blue
• safranin
• malachite green
• basic fuchsin
• crystal violet
 Simple Staining

1. Smear loopful of microbes onto slide

2. Air dry
3.Drip methanol onto specimen
4. Flood slide with stain
5. Rinse with water and blot dry
6. Examine
Simple Staining
 Gram Staining

• most widely used differential

staining
• Use to differentiate “Gram
positive” “Gram negative”
bacteria
 Gram Positive
• the color of the • if the bacteria were
bacteria at the end of not decoulorize
the gram staining during
procedure depends decolorization step
on the chemical they will produce
composition of their blue to purple color -
cell wall “Gram positive”
 Gram Positive
• a thick layer of
peptidoglycan in the
cell walls of GP
bacteria makes it
difficult to remove
the crystal violet-
iodine during
decoulorization
 Gram Negative
• if on the other hand, • the thin layer of
the crystal violet was peptidoglycan in the
removed from cell cell walls of Gram
during negative bacteria
decoulorization, and makes it easier to
weere stain with remove the crystal
safranin - it will violet -iodine .
produce pink to red -
“Gram Negative”
 Steps in Gram Staining
• Heat fix your specimen to slide. Flood slide
with crystal violet solution; allow to act for 1
minute.
• Rinse the slide, then flood with iodine solution
; allow to act for 1 minute.
• Rinse off exces iodine, and decolourize with
acetone (5 secs)
• Wash slide with water
• Apply safranin counterstain for 30 seconds
• Wash with water, blot and air dry.
 methods
• primary stain
• mordant
• decolourizer
• counter stain
/
Differences between GP and GN

Gram Gram
Positive Negative
Bacteria bacteria
Color Blue - Purple Pink - Red
Peptidoglycan in Thick Layer Thin layer
cell walls
 Acid-fast Stain
• use to identify • Heat is necessary
Mycobacterium since the cell wall of
species mycobacterium
• In this process the contain waxes -
carbol fuchsin ( prevent stain from
bright red eye) is penetrating the cells.
drive into bacterial • heat- softens
cell using heat
 Acid-fast Stain
• decolorizing agent - • developed by Paul
used in an attempt to Ehrlich - German
remove the red color chemist.
from cells.
• because • differential staining
mycobacterium are together with Gram
not decolorized by Stain
acid- alcohol
treatment, they are
acid-fast stain.