1. Staining bacteria cells
General Microbiology
Staining bacteria cells

2. These characteristics can be use for primary bacterial identification
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

3. Staining bacteria: outline of the procedure
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
3. Differential staining
4. Microscopic observation
Gram
Malachite green
Acid-fast stain

4. Aseptic transfer and the Bunsen burner flame

5. Aseptic transfer and the Bunsen burner flame

6. Aseptic transfer and the Bunsen burner flame
oxidant flame
inner cone
unburt gas
outer cone
Hottest part of the flame
reductant flame

7. Staining bacteria cells:
simple staining

8. 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

9. Staining bacteria cells:
differential stain

10. Differential stains, such as the
Gram stain
Ziehl–Neelsen (acid-fast stain)
Malachite stain
differentiate bacteria based on the chemical composition of their cell wall.
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.

11. Overview of a bacterial
staining procedure
Figure: 04-03
Caption:
Staining cells for microscopic observation.

12. 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

13. S. epidermidis (G+)
Escherichia coli (G-)
Simple stain with crystal violet
blu-violet

14. GRAM STAIN PROCEDURE

15. 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

16. 1
Figure: 04-04a-01
Caption:
The Gram stain. (a) Steps in the Gram stain procedure.

17. Figure: 04-04a-02
Caption:
The Gram stain. (a) Steps in the Gram stain procedure.

18. Gram positive and Gram negative reactions

19. G. Dehò, E. Galli     Biologia dei Microrganismi    
Copyright 2012 C.E.A. Casa Editrice Ambrosiana

20. Gram stain of a mixture of Staphylococcus aureus and Escherichia coli

21. Neisseria gonorrhoeae
Gram stain of yogurt
Neisseria gonorrhoeae
Gram Stain of pus smear

22. GRAM STAIN PROCEDURE ?

23. Agar Sangue
Mannitol Salt Agar

24. 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%)

25. 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.

26. 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 ± 25°C
Positive controls:
Expected results
Staphylococcus aureus ATCC® *
Good growth; yellow colonies with yellow halo.
Staphylococcus epidermidis ATCC® *
Good growth; pink colonies with pink medium.
Negative controls:
Escherichia coli ATCC® 8739 *
No Growth

27. Proteus mirabilis (G-)
Escherichia coli (G-)
Proteus mirabilis (G-)
Bacillus clausii (G+)
Cled Agar ?

28. 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

29. Sabouraud Glucose Agar

31. CATALASE TEST Smear a bacterial colony or suspension
add a drop of 30% hydrogen peroxide

32. 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)

33. ENDOSPORES https://www.youtube.com/watch?v=UHsqFjP1dZg

34. ENDOSPORES Mother cell releases spore
DNA replicates and extends into an axial filament
Septum forms near one pole, separating forespore from mother cell. Each gets a chromosome
Mother cell engulfs the forespore, surrounding it with a second membrane
Chromosomes of mother cell disintegrate
Mother cell releases spore
Forespore develops a cortex layer of peptidoglycan
Dipicolinc acid is synthesized and calcium is incorporated into the spore coat

36. G. Dehò, E. Galli Biologia dei Microrganismi Copyright 2012 C. E. A
G. Dehò, E. Galli   Biologia dei Microrganismi     Copyright 2012 C.E.A. Casa Editrice Ambrosiana

37. 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

40. 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