Negative indirect or background staining.

• Used to observe bacterial morphology without the use of harsh chemicals or heat fixing
techniques.
• Uses an acid stain
○ Nigrosin
○ India ink
○ Eosin
• The stain does not penetrate and stain the bacterial cell due to repulsion between negative
charge of the stain and the negatively charged bacterial wall.

Simple Staining.

• To observe bacterial details.

• Cell shape, size and arrangement.
• Basic Dyes
○ Crystal violet
○ Carbolfuchsin
○ Methylene blue

Gram Staining

• First step involves staining with a basic dye crystal violet. (Primary stain).
• Iodine solution which functions as a mordant, which increases the interaction between the
dye so that the dye is more tightly bound and the cell is more strongly strained.
• stain is then decolorized by washing with an agent such as 95% ethanol or isopropanol-
acetone.
• Gram positive will retain the crystal violet while gram negative will lose the crystal
violet.
• Stain is then counter stained with basic dye, called safrin which colors the bacteri pink.

Acid –Fast staining.

• Heating with carbolfuchsin

• Acid fastness is due to the high lipid content.
• Organisms will appear red

Cultivation of anaerobic bacteria.

• Commercial anaerobe broth- meat medium and thioglycolate.

• Wrights tube: anaerobic conditions are created using pyrogallol and NAOH.
After streaking the anaerobe on the agar slant, cotton plug is pushed into the
culture, pyrogallol crystals are added and 1ml of 10% NAOH is added. The tube
is closed with a rubber stopper and immediately inverted.
• Brewers’s anaerobe petri dish.
 ○ Inexpensive method to grow anaerobes.
○ Brewers special cover
○ Contains high concentration of thioglycolllic acid.
○ The free sulfhydryl group of thioglycolate reduces any oxygen present.
• Gas pack anaerobe system
○ Palladium catalyst (catalyzes the formation of water from hydrogen and
oxygen.)

Bacterial transformation.

• B-subtilis donor stain ans Sb100 recipient

• Phospahet buffer
• 0.5 ml of lysozyme weakens the walls
• Chloroform to lyse the bacteria

SB100

50ml of difco antibiotic assay.

Dilution in MM1 and MM2