Download Staining bacteria cells - E-Learning per i corsi di Studi in Biologia

General Microbiology
Staining bacteria cells
Staining bacteria cells for microscopic
examination makes it possible:
- to define their:
• cell size
• shape
• arrangement
- to study their:
• chemical properties
• structures.
These characteristics can be use for
primary bacterial identification
Staining bacteria: outline of the procedure
1. Preparing cells for staining
Fixation: cells are dead and made to adhere to a slide
by chemical or heat treatment
2. Simple stain
crystal violet
Gram
3. Differential staining Malachite green
Acid-fast stain
4. Microscopic observation
Aseptic transfer and the Bunsen burner flame
Aseptic transfer and the Bunsen burner flame
Aseptic transfer and the Bunsen burner flame
Hottest part of the flame outer cone
inner cone
unburt gas
reductant flame
oxidant flame
Staining bacteria cells:
simple staining
Simple stains use a single basic dye to color
bacterial cells so that their size, shape and
arrangement can be observed
- crystal violet,
- methylene blue,
- safranin
Staining bacteria cells:
differential stain
• Differential stains, such as the
- Gram stain
- Ziehl–Neelsen (acid-fast stain)
- Malachite stain
differentiate bacteria based on
composition of their cell wall.
the
chemical
• Differential stain use two dyes instead of one: the
first stain is the primary stain, the second is the
counterstain.
• A decolorization step can occur between the
application of the primary stain and counterstain.
Overview of a bacterial
staining procedure
SIMPLE STAIN PROCEDURE
1. Stain with crystal violet 2% (e.g)………...1 min.
2. Wash off with tap water
3. Blot dry with bibulous paper
S. epidermidis (G+)
Escherichia coli (G-)
Simple stain with crystal violet
blu-violet
GRAM STAIN PROCEDURE
http://www.youtube.com/watch?v=OQ6C-gj_UHM&feature=related
GRAM STAIN PROCEDURE
1. Stain with crystal violet 2%…… ……….….1 min.
2. Gram’s iodine (Lugol)………………………1 min.
3. Wash off with tap water
4. Decolorizer (Alcohol 50%-Acetone 50%)…20 sec.
5. Wash off with tap water
6. Safranin 0,25%………………………………1 min.
7. Wash off with tap water
8. Blot dry with bibulous paper
1
Gram positive and Gram negative reactions
G. Dehò, E. Galli
Biologia dei Microrganismi
Copyright 2012 C.E.A. Casa Editrice Ambrosiana
Gram stain of a mixture of Staphylococcus
aureus and Escherichia coli
Gram stain of yogurt
Neisseria gonorrhoeae
Gram Stain of pus smear
GRAM STAIN PROCEDURE
?
Agar Sangue
Mannitol Salt Agar
AGAR SANGUE
Formula tipica
Triptone 14.5 g
Peptone di soia 5.0 g
Sodio cloruro 5.0 g
Agar agar 14.0 g
Fattori di crescita 1.5 g
Sangue defibrinato di cavallo o montone 50 ml(5%)
Su piastre di agar sangue di montone, i microrganismi coltivano con le seguenti
caratteristiche:
Streptococchi b emolitici : colonie più grandi (2-4 mm) circondate da una zona di
trasparenza (β-emolisi) (Streptococcus pyogenes (A), Streptococcus agalactiae (B))
Streptococchi alfa emolitici : colonie (1-2 mm) circondate da un alone di colore
verde (α-emolisi) (streptococchi viridanti)
Pneumococchi: normalmente colonie larghe, mucose, piatte, circondate da una zona
di colore verde (α- emolisi)
g emolisi: Streptococcus faecalis
Stafilococchi: colonie bianche o giallo-oro con o senza alone di beta emolisi
Alcune specie di Haemophilus danno reazioni beta emolitiche e possono essere
confuse con gli streptococchi beta emolitici.
MANNITOL SALT AGAR or CHAPMAN
Typical Formula* gm/litre
`Lab-Lemco’ powder 1.0
Peptone 10.0
Mannitol 10.0
Sodium chloride
75.0
Phenol red 0.025
Agar 15.0
pH 7.5 ± 0.2 @ 25°C
Positive controls:
Expected results
Staphylococcus aureus ATCC® 25923 *
Good growth; yellow colonies with yellow
halo.
Staphylococcus epidermidis ATCC®
12228 *
Good growth; pink colonies with pink
medium.
Negative controls:
Escherichia coli ATCC® 8739 *
No Growth
Escherichia coli (G-)
Proteus mirabilis (G-)
Bacillus clausii (G+)
Cled Agar
?
MacConkey Agar
•selettivo:
inibisce la crescita dei batteri Gram+. Sali biliari, cristalvioletto.
•differenziale:
differenzia i lattosio fermentanti, dai non-fermentanti. Lattosio (fonte di
carbonio)
•indicatore di Ph:
Neutral red
Sabouraud Glucose Agar
CATALASE TEST
- Smear a bacterial colony or suspension
- add a drop of 30% hydrogen peroxide
ENDOSPORES
• cell differentiation
• genetic program
• most endospore forming bacteria are found in soil or
aquatic
environments,
including:
Clostridium
perfringens, C. botulinum and C. tetani are the causative
agents of gas gangrene, botulism and tetanus,
respectively; or Bacillus anthracis and Bacillus cereus
are the causative agents of anthrax and a self limiting
food poisoning, respectively.
• endospores may be located in the middle of the
bacterium (central), at the end of the bacterium (terminal)
and near the end of the bacteria (subterminal)
ENDOSPORES
https://www.youtube.com/watch?v=UHsqFjP1dZg
http://www.wwnorton.com/college/biology/microbiology2/ch/04/animations.aspx
ENDOSPORES
DNA replicates and
extends into an axial
filament
Septum forms near
one pole, separating
forespore
from
mother cell. Each
gets a chromosome
Dipicolinc
acid
is
synthesized and calcium is
incorporated into the spore
coat
Forespore develops
a cortex layer of
peptidoglycan
Mother cell engulfs the
forespore, surrounding
it
with
a
second
membrane
Chromosomes of mother cell
disintegrate
Mother
releases
spore
cell
G. Dehò, E. Galli Biologia dei Microrganismi
Copyright 2012 C.E.A. Casa Editrice Ambrosiana
Malachite Green (Shaeffer e Fulton)
1. Stain with malachite green 5%…………….5-6
min.
heating allows malachite green to enter the through spore coat of endospores
cooling traps the dye inside the spore coat (spore coats, like acid-fast cell
walls are resistant to most staining reagents);
vegetative cells take up malachite green as well.
2. Wash off with tap water
3. Safranin 0,25%………………………………1 min.
4. Wash off with tap water
5. Blot dry with bibulous paper
Ziehl-Neelsen procedure
1. Stain with carbolfuchsin…………….3 min.
heating allows carbolfuchsin to enter the through wall of bacteria rich of
mycolic acids and lipids.
2. Wash off with tap water
3. Decolorizer (Alcohol -Acid solution)…2 min
4. Wash off with tap water
5. methylene blue ……………………….2 min.
6. Wash off with tap water
7. Blot dry with bibulous paper