Transcript Lab 2: Staining Bacterial Cells

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….
• Why stain bacteria?
– Enables us to see them
• 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
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)
Name
• 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
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
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
Why do Gram + and Gram – cells
react differently to decolorizing?
• Gram +
– 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
Cell walls
Morphology
•
•
•
•
•
coccus (cocci) = spheres
bacillus (bacilli) = rods
spirillum (spirilla) = spirals
Arrangement: (subjective!)
staphylo- = in bunches like
grapes (Staphylococcus)
• strepto- = in chains
(Streptococcus)
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
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)