1. Lab 2: Staining Bacterial Cells
*Burners: Be careful!
Pull back long hair…
rubber bands on computer
Remove dangling scarves
Put away everything but essential!
Be careful with dangling sleeves….

2. Why stain bacteria? Enables us to see them Aids with classification
Hay infusion: lots of bacteria, hard to see since they were not stained (clear MOs in clear liquid)
Aids with classification
No limbs or leaves to help us identify bacteria!
Use different results from staining to help ID

3. Smear Prep, Heat Fixing
Draw a LONG OVAL on your slide with a Gram stain pen, label with name of culture
Fill oval with bacteria using aseptic techniques (later)
BEFORE staining:
Slide must be air dried
only THEN heat fix:
pass slide through a flame: this keeps cells from being washed off and preserves their arrangement and morphology
Name

4. Types of stains
Simple stains: use ONE dye and ALL cells are stained the SAME color
Simple direct stain: cell is stained
Bacteria have a negative charge and dye has a positive charge; therefore, color is attracted to the cell (eg., Methylene Blue—actually a chloride salt)
Negative stain: stains the surroundings
Dye has a negative charge; therefore, color is repelled by the cell

5. Types of Stains (con’t)
Differential stains: more than one dye used and certain properties of bacteria allow them to stain different colors
Gram stain (today’s example)
Primary stain: Crystal Violet
All bacteria stain purple
Mordant: Gram’s Iodine
Acts to hold crystal violet in cell walls of Gram + cells
Decolorizer: Ethanol
Removes color from Gram – cells but Gram + cells remain purple
Counterstain: Safranin
Gram – cells stain red/pink and Gram + remain purple

7. Why do Gram + and Gram – cells react differently to decolorizing?
Cell wall contains a thick layer of peptidoglycan and teichoic acid
Thick peptidoglycan layer holds crystal violet
Gram –
Cell wall contains a thin layer of peptidoglycan and NO teichoic acid
Also contains a complex outer layer composed of lipopolysaccharide (LPS)
Layers not thick enough to hold purple dye

8. Cell walls

9. Morphology coccus (cocci) = spheres bacillus (bacilli) = rods
spirillum (spirilla) = spirals
Arrangement: (subjective!)
staphylo- = in bunches like grapes (Staphylococcus)
strepto- = in chains (Streptococcus)

10. Always use ASEPTIC TECHNIQUE
Flaming loop, flaming lip of tube, not touching or breathing on plates, loops, or inside of tubes, keeping caps on…
Lysol bench before and after use …
All transfers done to prevent unwanted microbes getting in, only the one you put there is there
Very Important, you will use all semester
We will demo aseptic technique for you

11. Lab Flow DEMOS of Techniques
Draw a large/Long oval in the center of 3 slides
Label slides: Mixed culture: tells you which side of slide contains bacteria
Apply loopfuls of culture until have a thin covering filling oval on all slides
Allow slides to air dry completely, only then heat fix all the slides
While waiting for slides to dry, view Simple stain (Methylene Blue) of Mixed culture Demo
Gram Stain
Choose 1 slide to Gram Stain
View and draw on 1000x total magnification
Have a TA look at your Gram stain technique
Repeat Gram staining using the TAs suggestions until you run out of time. Discard all slides in the lysol bin.
Get TA initials on your Gram stain (if its correct)