Lab 4 Lab 3 Goals and Objectives Learn methods of differential staining: Use two or more stains and categorize cells into groups Gram Staining Separates bacteria in two different groups – Gram positive and Gram negative Important first test for bacterial identification Acid – Fast Staining Detects the high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria Mycolic acids are long fatty acids found in the cell walls of a group of bacteria that includes Mycobacterium tuberculosis. Important diagnostic tool

Gram stain – determine bacterial cell wall structure Figure 15.2 Figure 15.1

GRAM STAIN 1. Crystal Violet = Primary Stain: stains all cell walls purple 2. Iodine = Mordant: combines with crystal violet to create large insoluble dark purple complex 3. Alcohol = Decolorizer: solubilizes Gram negative outer membrane flushing iodine/crystal violet complex out of thin wall, thick Gram positive cells resist decolorization 4. Safranin = Counterstain: stains cell walls pink (obscured by purple walls of Gram positive, but Gram negative, who had become colorless, now are pink)

2 loops each organism Wax Pencil mixed together ***Use wax pencils (China Marker): Sharpie will wash off with alcohol! Be sure to resuspend broth cultures completely! Exercise 15: Gram Stain: Each pair make 2 slides each mix and stain: 1. Staphylococcus aureus & Pseudomonas aeruginosa: two loops of each broth mixed 2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed 2 loops each organism mixed together Wax Pencil

Gram Staining Add step: (3.5) Wash 2 seconds immediately!!! Fig 15.3 pg 110 Add step: (3.5) Wash 2 seconds (until water runs clear) (until water runs clear) immediately!!!

Gram stain Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Bacillus subtilis Kelbsiella pneumonia

Exercise 17: Acid Fast Staining 1. Carbolfuchsin – primary stain Staphylococcus aureus 2. Aid-alcohol – decolorizer 3. methylene blue – counter-stain Mycobacterium smegmatis Each pair make minimum one acid fast slide with: Staphylococcus aureus (use 2-4 loops of broth on slide first) Mycobacterium smegmatis (slant, use needle, get tiny amount and mix into broth on slide very well: break up all clumps) 2 loops each organism mixed together Wax Pencil

Fig 17.1 pg 118 Acid Fast Stain 30sec

***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!! ***Make all smears on slides first so they can dry before heat fixing!!! Split the work to save time but make sure everyone gets to see all slides! Each pair needs for lab 4: 1 broth culture Staphylococcus aureus 1 broth culture Pseudomonas aeruginosa 1 broth culture Bacillus subtilis (replaces B. megaterium) 1 broth culture Escherichia coli 1 wet slant 5 day culture Mycobacterium smegmatis Crystal violet Gram’s iodine 95% Alcohol (Ethanol) Safranin Methylene blue Acid-alcohol Carbolfuchsin ***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!!