General Microbiology Staining bacteria cells Staining bacteria cells

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 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 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.

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=OQ 6 C-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

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Gram positive and Gram negative reactions

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

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 p. H 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 ?

Mac. Conkey 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=UHsq. Fj. P 1 d. Zg http: //www. wwnorton. com/college/biology/microbiology 2/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 cell releases spore

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